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While hyperthermia has been shown to induce a variety of cytotoxic and sensitizing effects on cancer tissues, the thermal dose–effect relationship is still not well quantified, and it is still unclear how it can be optimally combined with other treatment modalities. Additionally, it is speculated that different methods of applying hyperthermia, such as water bath heating or electromagnetic energy, may have an effect on the resulting biological mechanisms involved in cell death or in sensitizing tumor cells to other oncological treatments. In order to further quantify and characterize hyperthermia treatments on a cellular level, in vitro experiments shifted towards the use of 3D cell spheroids. These are in fact considered a more representative model of the cell environment when compared to 2D cell cultures. In order to perform radiofrequency (RF)-induced heating in vitro, we have recently developed a dedicated electromagnetic field applicator. In this study, using this applicator, we designed and validated an experimental setup which can heat 3D cell spheroids in a conical polypropylene vial, thus providing a reliable instrument for investigating hyperthermia effects at the cellular scale. [ABSTRACT FROM AUTHOR]

The smooth muscle cells (SMCs) located in the vascular media layer are continuously subjected to cyclic stretching perpendicular to the vessel wall and play a crucial role in vascular wall remodeling and blood pressure regulation. Mesenchymal stem cells (MSCs) are promising tools to differentiate into SMCs. Mechanical stretch loading offers an opportunity to guide the MSC-SMC differentiation and mechanical adaption for function regeneration of blood vessels. This study shows that cyclic stretch induces the expression of SMC markers α-SMA and SM22 in MSCs. These cells exhibit contractile ability in vitro and facilitate angiogenesis in the Matrigel plug assay in vivo. The contraction of SMCs requires remodeling of their energy metabolism. However, the underlying mechanism in the differentiation of MSCs into SMCs remains to be revealed. Cyclic stretch training promotes glycolysis, oxidative phosphorylation, and mitochondrial fusion and modulates mitochondrial dynamics-related proteins (MFN1, MFN2, DRP1) expression, thereby contributing to MSCs differentiation. Yes-associated protein (YAP) affects mitochondrial dynamics, oxidative phosphorylation, and glycolysis to regulate stretch-mediated differentiation into SMCs. Additionally, Piezo-type mechanosensitive ion channel component 1 (Piezo1) impacts energy metabolism and MSCs differentiation by regulating intracellular Ca 2+ levels and YAP nuclear localization. It indicates that YAP can integrate stretch force and energy metabolism signals to regulate the differentiation of MSCs into SMCs., (© 2025 Federation of American Societies for Experimental Biology.)

G-Protein Coupled Receptor, Class C, Group 5, Member A (GPRC5A) has been extensively studied in lung and various epithelial cancers. Nevertheless, its role in the skin remains to be elucidated. In this study, we sought to investigate the function of this receptor in skin biology. Our research demonstrated that its expression responds to mechanical substrate changes in human primary keratinocytes. Furthermore, we observed the reinduction of GPRC5A during wound healing at the leading edges in an ex vivo burn model, coinciding with the translocation of its C-terminal region into the nucleus. We identified the cleavage site of GPRC5A by N-TAILS analysis, and cathepsin G was characterized as the protease responsible for proteolysis in cultured cells. In order to gain a deeper understanding of the role of GPRC5A in keratinocytes, we performed a GPRC5A knockdown in N/TERT-1 cells using short-hairpin RNA. Our findings indicate a strong association between GPRC5A and adhesion regulation pathways. Additionally, our results demonstrate that GPRC5A KD enhances cell adhesion while reducing cell migration and differentiation. It is noteworthy that these effects were reversed by the addition of a recombinant polypeptide that mimics the C-terminal region of GPRC5A. In conclusion, our study reveals that GPRC5A plays an unexpected role in regulating keratinocyte behavior, with implications for its C-terminal region translocation into the nucleus. These results offer promising avenues for future research in the field of wound healing., (© 2025 Federation of American Societies for Experimental Biology.)

Ethnopharmacological Relevance: Astragali Radix-Angelicae Sinensis Radix is an important traditional Chinese medicine used for the treatment of cardiovascular diseases. Our previous studies have shown that Astragali Radix-Angelicae Sinensis Radix can inhibit vascular intimal hyperplasia and improve the blood vessel wall's ECM deposition, among which six main active components can be absorbed into the blood, suggesting that these components may be the main pharmacodynamic substances of Astragali Radix-Angelicae Sinensis Radix against vascular intimal hyperplasia., Aim of the Study: A mouse model of atherosclerosis was used to study the relationship between the anti-intimal hyperplasia effect of Astragali Radix-Angelicae Sinensis Radix and the inhibition of VAF activation and ECM synthesis. Furthermore, an in vitro rat VAF activation model was used. The effects of the main active ingredients of Astragali Radix-Angelicae Sinensis Radix on the proliferation, migration and ECM synthesis of VAF were observed. The mechanism of its action was investigated by focusing on TGF-β1/Smads signaling pathway., Materials and Methods: Male ApoE -/- mice were used to establish an AS model. Observe the morphological changes of blood vessels, the expression of Vimentin, α-SMA, ECM-related factors and TGF-β1/Smads signaling pathway-related proteins. Ang Ⅱ was used to induce the VAF activation model. The cell activity, cell proliferation, cell migration, cell phenotypic markers, ECM-related factors, cell cycle regulation-related proteins and TGF-β1/Smads signaling pathway-related proteins were determined. On this basis, TGF-β1/Smads signaling pathway agonists and inhibitors were used to study the effects of the compatibility of six active components on TGF-β1/Smads signaling pathway., Results: Astragali Radix-Angelicae Sinensis Radix can reduce aortic intimal hyperplasia, inhibit the expression of aortic α-SMA, Vimentin, ECM components, TGF-β1, p-Samd2 and p-Samd3. Cell experiments showed that the six active ingredients could inhibit the proliferation and migration of VAF to varying degrees, inhibit the expression of α-SMA, cell cycle promoters, ECM components, up-regulate the expression of Vimentin, P21, MMP2 and MMP9. The above effects were enhanced after the combination of the six components. The 6 components and their combinations could inhibit the expression of TGF-β1/Smads signaling pathway-related proteins and up-regulate the expression of Samd7 to varying degrees. The above effects were enhanced after the combination of the 6 components. TGF-β1/Smads signaling pathway inhibitor LY2157299 showed similar effects with the six components. The inhibitory effects of the six active ingredients on TGF-β1/Smads signaling pathway-related proteins and the promotion of Smad7 expression were attenuated when agonists were added into the six active ingredient combinations. However, adding TGFβ1/Smads signaling pathway inhibitor EGF to the six active ingredient combinations had no effect on the above effects., Conclusion: Astragali Radix-Angelicae Sinensis Radix can inhibit intimal hyperplasia, VAF activation, and ECM synthesis in atherosclerosis. The six active ingredients may be the main pharmacological substances of Astragali Radix-Angelicae Sinensis Radix to inhibit the activation of VAF, and the combination of six ingredients can enhance their effects, which may be mediated by inhibiting the activation of the TGF-β1/Smad2/3 signaling pathway., Competing Interests: Declaration of competing interest All authors disclosed no relevant relationships., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In China, Osteoking is a commonly used treatment and preventive measure for osteoporosis. The pathophysiology of osteoporosis is closely associated with apoptosis; however, it remains unclear whether the role of Osteoking in promoting bone formation is linked to apoptosis., Aim of Study: This study aims to investigate whether Osteoking inhibits apoptosis of BMSCs in osteoporotic rats via the PI3K/AKT signaling pathway and to conduct a detailed exploration of this mechanism. The goal is to provide a theoretical basis for the clinical application of Osteoking in osteoporosis treatment., Methods: A rat model of osteoporosis was established through bilateral ovariectomy (OVX), followed by treatment with Osteoking. After ten weeks of therapy, BMD was evaluated. The biomechanics of the left tibia were measured, the left femur was sequenced, and the right tibia was stained using histomorphometric and Masson's staining methods. Peripheral serum was collected to measure bone-related markers, including E2, PINP, and CTX. RNA-Seq results were verified using the remaining bone samples. Comparative analysis demonstrated the efficacy of Osteoking in treating osteoporosis and provided preliminary insights into the underlying mechanisms. Primary BMSCs were cultured using bone marrow apposition. CCK8 assays were conducted to screen the intervention conditions of Osteoking and LY294002. Various concentrations of Osteoking-containing serum and LY294002 were tested separately to determine the optimal intervention concentration for drug delivery. The impact of Osteoking on lipid formation was also evaluated. Following treatment of BMSCs from OVX rats with Sham serum, OVX serum, OVX + LY294002 serum, and Osteoking + LY294002 serum, the expression of PI3K/AKT/mTOR, osteogenesis-related regulatory factors, and apoptosis-related regulatory factors was assessed. Flow cytometry was employed to evaluate apoptosis in BMSCs., Results: Osteoking significantly improved whole-body BMD and bone biomechanical indices in OVX rats. It also significantly elevated the serum levels of E2 and PINP while reducing the level of CTX, which significantly improved bone microstructure and promoted new bone formation. RNA-seq analysis indicated that the therapeutic mechanism involved the PI3K/AKT signaling pathway. Osteoking increased the expression of RUNX2 and decreased the expression of PPAR-γ, a marker of lipogenesis, in OVX rats. Extraction of BMSCs for subsequent studies revealed a significant reduction in proliferation and osteogenic differentiation, along with an increase in lipogenic differentiation, in the OVX group. Osteoking treatment inhibited the expression of PPAR-γ and increased the expression of RUNX2 in BMSCs. Additionally, Osteoking reversed the LY294002-mediated inhibition of PI3K/AKT/mTOR signaling pathway activation, increased the expression of the apoptosis-protecting protein Bcl2, and decreased the expression of apoptosis-associated proteins Caspase3 and Bax., Conclusion: Osteoking markedly improved bone microstructure, biomechanics, and bone density in OVX rats. Osteoking-containing serum reversed the imbalance in lineage differentiation in OVX rats, characterized by reduced osteogenic differentiation and increased lipid differentiation of BMSCs. Furthermore, Osteoking-containing serum significantly increased BMSC proliferation and prevented apoptosis in OVX rats through the PI3K/AKT signaling pathway., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024. Published by Elsevier B.V.)

Ethnopharmacological Significance: The therapeutic efficacy of Ershen Zhenwu Decoction (ESZWD)-a famous formulation from Xin'an for patients with chronic heart failure heart-kidney Yang deficiency (CHF-HKYD)-is well established. Still, the underlying molecular mechanism is not clear., Aim of the Study: This study investigated mechanisms by which ESZWD suppresses cardiac pathology, including myocardial fibrosis, in CHF-HKYD model rats and Ang II-stimulated cardiac fibroblasts (CFs)., Materials and Methods: The components in ESZWD were analyzed by ultra-high-performance liquid chromatography coupled with Quadrupole Time-Of-Flight mass spectrometry (UHPLC-Q-TOF-MS). CHF-HKYD model was established in the male Sprague-Dawley rats through bilateral thyroidectomy and intraperitoneal administration of 0.02% doxorubicin (DOX), twice weekly for 3 weeks. Subsequently, the CHF-HKYD model rats were randomly categorized into the Model, ESZWD-L (3.96 g/kg/d ESZWD), ESZWD-M (7.92 g/kg/d ESZWD), ESZWD-H (15.84 g/kg/d ESZWD), and Sac/Val (68 mg/kg/d sacubitril/valsartan) groups and treated daily for 4 weeks. As a control, the sham surgery group (Sham) was used. Primary cardiac fibroblasts (CFs) were categorized into Control, Model, ESZWD, and Sac/Val groups. Then, the CFs were stimulated with Ang-II. The ESZWD and Sac/Val groups were incubated with different concentrations of drug-containing sera and their effects on CF viability were assessed via the CCK-8 assay. The ESZWD and Sac/Val groups received drug-containing serum concentrations determined by CCK-8 assay results. The cardioprotective effects of ESZWD were determined using echocardiography, Hematoxylin & Eosin (H&E) staining, Masson staining, and Sirius red staining, and the Enzyme Linked Immunosorbent Assay (ELISA). ESZWD's effects on the Ras Homolog Gene Family Member A (RhoA)/Rho-Associated Coiled-Coil Kinases (ROCKs) signaling pathway and myocardial fibrosis were assessed by Western blotting and Quantitative Real-Time PCR (qRT-PCR) analyses. Immunofluorescence was used to observe fibrotic markers in CFs., Results: ESZWD treatment improved cardiac function in the CHF-HKYD rats by significantly reducing myocardial fibrosis and ventricular remodeling. ESZWD treatment increased the rats' body temperature (T b ) and 24-h urine volume, left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS), and decreased LV internal systolic diameter (LVIDs), LV internal diastolic diameter (LVIDd), and heart weight/body weight (HW/BW) compared to the Model group. In comparison to the model rats, ESZWD treatment decreased serum levels of B-type natriuretic peptide precursor (NT-proBNP), tumor necrosis factor-alpha (TNF-α), interleukin-11 (IL-11), and IL-17A. ESZWD treatment significantly down-regulated the protein and mRNA expression levels of collagen I A1, α-SMA, RhoA, ROCK1, and ROCK2 in the heart tissues of the CHF-HKYD rats and the Ang II-stimulated CFs., Conclusion: ESZWD significantly improved cardiac function and attenuated myocardial fibrosis and inflammation in the CHF-HKYD rats by inhibiting the RhoA/ROCKs signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Traditional Chinese medicine (TCM) is frequently used to treat osteoarthritis (OA). Duhuo Jisheng decoction (DHJSD), a Chinese patent medicine, was commonly used Chinese herbal formula for the treatment of OA. In Western medicine, dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme has been proved to be a promising strategy to treat inflammatory diseases with reduced side effects., Aim of the Study: To elucidate the dual action mechanism of DHJSD targeting COX-2 and 5-LOX against OA., Materials and Methods: DHJSD, containing 1495 compounds was screened using a virtual screening approach based on molecular docking. The inhibitory effect of hit compounds against COX-2 and 5-LOX was validated using enzyme-based assays. In vitro, rat chondrocytes were treated with IL-1β (10 ng/mL) for 24 h to induce OA model in vitro. The chondrocyte viability was evaluated using an CCK-8 assay. ELISA was used to detect inflammatory factors expression. Immunofluorescence was used to assess the expression level of collagen II and MMP-13. In addition, a rat cartilage explants culture model was established, and safranin O and HE staining analysis were carried to assess cartilage matrix degradation and cartilage damage, respectively. In vivo, carrageenan-induced paw edema assay was used to examine anti-inflammatory activity, and the gastric ulcerogenic effect was further detected. Finally, Molecular dynamics simulations and binding free energy analysis were carried to explore the binding mechanism., Results: 13 compounds from DHJSD were identified as promising candidates by a virtual screening approach. Among these candidates, three hits 7,4'-dimethoxyisoflavone, genistein, and fraxetin displayed dual inhibition of COX-2 and 5-LOX. Further in vitro assay indicated that 7,4'-dimethoxyisoflavone, genistein, and fraxetin could inhibit PGE2, LTB4, TNF-α, IL-6, or NO production in IL-1β-induced chondrocytes. In addition, the three compounds reduced IL-1β-induced degradation of collagen II and expression of MMP-13 in rat chondrocytes. The results of anti-inflammatory activity of the three compounds in vivo showed that the highest anti-inflammatory activity with edema inhibition percentages of 50.00%, 56.00%, and 51.00% after 3 h, respectively. Moreover, it was found that 7,4'-dimethoxyisoflavone, genistein, and fraxetin have a superior gastric safety profile comparable to indomethacin. Finally, molecular dynamics simulations, binding free energy analysis, and detailed interaction mode demonstrated that 7,4'-dimethoxyisoflavone, genistein, and fraxetin interacted well with both COX-2 and 5-LOX., Conclusions: 7,4'-dimethoxyisoflavone, genistein, and fraxetin from DHJSD with excellent anti-inflammatory effects and no gastric ulceration effects, which helps to explain the dual action mechanism and potential material basis of DHJSD in treating OA and provide evidence to support DHJSD's clinical use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Qifu yixin prescription (QYP), an effective traditional Chinese medicine formula, has been utilized in the clinical treatment of cardiovascular diseases for over two decades and has been granted a national invention patent in China. It has demonstrated the ability to improve clinical symptoms in patients with heart failure. However, its precise effects and underlying molecular mechanisms remain unclear., Aim of the Study: To evaluate the efficacy of QYP in treating HF and the underlying mechanisms., Materials and Methods: The heart failure (HF) model in mice was established using transverse aortic constriction (TAC), while neonatal rat cardiac fibroblasts (CFs) were utilized for in vitro experiments. The bioactive compounds in QYP were identified through high-performance liquid chromatography (HPLC). Cardiac hypertrophy, function, and fibrosis were assessed using morphological observations, echocardiography, and histomorphometric analyses. To investigate the underlying mechanisms by which QYP alleviates HF, transcriptomic analysis was conducted, and network pharmacology was employed to explore its potential mechanisms of action. Mechanistically, the expression levels of sGC, PKG, ERK, and p-ERK were analyzed using western blotting, immunohistochemistry, and immunofluorescence. Molecular docking was conducted to assess the binding affinity of the compounds of QYP to sGC. Additionally, the effects of QYP on CFs were investigated through cell-based assays., Results: We identified 33 bioactive compounds in QYP. Histomorphometric and transcriptomic analyses indicated that QYP alleviates cardiac fibrosis in HF. Network pharmacological analysis suggested that the sGC/cGMP/PKG and MAPK pathways are key mechanisms underlying the effects of QYP on cardiac fibrosis. The findings confirmed that QYP activates sGC, leading to the inhibition of ERK phosphorylation. Molecular docking revealed that the compounds of QYP exhibit strong binding affinity to sGC. Additionally, cell-based experiments demonstrated that QYP effectively suppresses CFs activation by stimulating sGC., Conclusions: These results indicate QYP improves cardiac fibrosis in HF by activating sGC to inhibit ERK phosphorylation. We propose that QYP is a potential treatment for HF with anti-fibrotic properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Periostracum Cicadae (PC), the molted exoskeleton of the cicada Cryptotympana pustulata Fabricius, is frequently employed in Chinese herbal medicine. Based on traditional therapies and pharmacological studies, PC appears to have immunomodulatory activity. However, the specific impact of PC on immunomodulation, particularly its effect on dendritic cells (DCs), remains unknown. DCs act professionally as antigen-presenting cells that trigger adaptive immune responses, making them critical for immunomodulation., Materials and Methods: The DCs derived from mouse bone marrow were used to examine the suppressive effect of PC extract on DC activation and maturation. The in vivo suppressive effect was evaluated using a mouse model of contact hypersensitivity (CHS) responses. The determination of the substances in the sample was performed by Liquid chromatography-mass spectrometry/mass spectrometry., Results: The ethyl acetate extract of PC (PCEA) significantly decreased the expressions of proinflammatory cytokines (IL-12, interleukin [IL]-6, as well as tumor necrosis factor [TNF]-α) and surface markers CD80 and CD86 in lipopolysaccharide-stimulated DCs. In the 2,4-dinitro-1-fluorobenzene-induced CHS mouse model, PCEA treatment dramatically attenuated the severity of symptoms. This was evidenced by the alleviation of ear swelling and a reduction in the count of infiltrating CD3 + T cells in the tested ears. In addition, N-acetyldopamine dimer and trimer were identified as major components., Conclusion: This study is the first to show that components derived from PCEA inhibit the activation and maturation of DCs as well as CHS responses, indicating they have the potential for treating delayed-type hypersensitivity or DC-related immune disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Prunella vulgaris L.(PV) and Tussilago farfara (TF) are perennial herbs rich in flavonoids and phenolic compounds with immense medicinal value. PV extract (PV-E) possesses potent antipyretic, anti-inflammtory, antioxidant, antiseptic, anti-cancer and immune stimulatory properties and have been traditionally known for the treatment of wounds, ulcers and sores. TF extract (TF-E) has been known for antibacterial, antioxidant, anti-inflammatory, anti-viral, anti-diabetic, anti-cancer, anti-obesity and wound healing effects. Additionally, TF-E infusions have been used for asthma, cough, and bronchopneumonia treatments., Aim of the Study: The therapeutic efficacy of transplanted human adipose stem cells (hASCs) is abrogated under the deteriorating effects of heat stress offered by burn wounds. Earlier researches has documented antioxidant priming as an effective strategy to enhance stem cell performance. As both PV-E and TF-E are known for their potent antioxidant effects. The present study aims to examine the cryoprotective effects of PV-E and TF-E priming on hASCs against in-vitro heat-induced thermal stress. Moreover, we determined the anti-inflammatory potential of both PV-E and TF-E on rabbits., Methods: Antioxidant capacity of both PV-E and TF-E is examined via DPPH assay and anti-inflammatory activity is assessed in rabbits using carrageen-induced paw edema model of inflammation. Next, we investigate the efficacy of different doses (1.25-100 μg/ml) of PV-E and TF-E on hASCs; MTT, LDH, calcein AM staining, and wound scratch assay were used to assess cell viability, cytotoxicity, proliferation ability and cell migration potential in the cells. Then, hASCs were pretreated for 24 h with optimum doses of PV-E and TF-E determined from MTT assay results and were subsequently exposed to in-vitro thermal injury (51 °C,10 min). The cytoprotective effects of both PV-E and TF-E priming under thermal stress were investigated via MTT, LDH, annexin-V staining and gene expression analysis., Results: Both PV-E and TF-E extracts demonstrated potent antioxidant and effective anti-inflammatory activities, with a clear reduction in inflammation. Study on hASCs exhibited improved cell viabilities, enhanced cell proliferation and migration abilities of both extracts. While heat stress data revealed that PV-E (2.5 μg/ml) and TF-E (5 μg/ml) pretreatment significantly ameliorated effects of thermal-injuries in hASCs as depicted by significantly enhanced cell viabilities, low LDH release profile, and lower annexin-V expression and regulated gene expression of the pretreated cells., Conclusion: PV-E and TF-E priming effectively enabled hASCs to combat thermal injury by significantly promoting cell survival than untreated cells. Hence, these findings suggest that PV-E and TF-E priming could be used to attain improved cellular responses and enhanced therapeutic efficacy in burnt tissue., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Jianpi Qingre Chubi prescription primarily consists of a compound formula, also known as Huangqin Qingre Chubi Capsules (HQC), which strengthens the spleen and resolves dampness, clear heat, and collaterals. Long-term clinical use has shown that HQC improves joint swelling and pain in patients with osteoarthritis. Mechanistically, we demonstrated that HQC inhibits inflammatory responses, extracellular matrix degradation, and delays chondrocyte senescence., Aim: To determine the bioactivity and mechanism of action of Jianpi Qingre Tongluo prescription (HQC) on osteoarthritis (OA)., Materials and Methods: First, the chondroprotective effects of HQC were assessed using histopathology, immunohistochemical staining and protein blotting in an OA rat model. Additionally, we identified key targets for crucial targets of HQC in OA using the Network Pharmacology and Gene Expression Omnibus (GEO) dataset (GSE98918 and GSE152805). In vitro conditions, IL-1β-treated chondrocytes served to study the impact of HQC on OA development and the senescence-associated secretory phenotype (SASP). This was evaluated using a series of approaches, such as flow cytometry assays, and immunofluorescence staining, and then verified by rescue experiments., Results: Therapy with HQC attenuated the severity of osteoarthritis (demonstrated by histopathology, OARSI grading scores, and Mankin scores) and SASP factors (as indicated by IL-1β, IL-6, IL-4, IL-37, MMP13, ADAMTS5, COL2A1, and ACAN levels, and apoptotic cell death). HQC might treat osteoarthritis via four important targets (STAG1, TP53, P21, and P16), with the p53 signalling pathway representing one of the main pathways. The HQC acts primarily on chondrocyte clusters. In vitro experiments indicated that STAG1 overexpression accelerates chondrocyte apoptosis, promotes SASP factor expression and extracellular matrix (ECM) degradation, and facilitates OA progression. HQC-containing serum suppressed the expression of the STAG1/TP53/P21 pathway, regulated SASP factors, and restored ECM balance., Conclusion: Jianpi Qingre Tongluo prescription modulated SASP factors by regulating the STAG1/TP53/P21 signal transduction axis and decelerating cartilage senescence and degradation in patients with OA. Jianpi Qingre Tongluo may be an effective drug candidate., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest to report regarding the present study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The clinical application of Doxorubicin (DOX) is constrained due to its cardiotoxic side effects. Oxidative stress and inflammation are crucial mechanisms driving doxorubicin-induced cardiotoxicity (DIC). Peroxiredoxin 5 (Prx5) is central to these inflammatory responses. However, the specific role of Prx5 in DIC remains unclear. This study aims to investigate the impacts of Prx5 on DIC and the underlying mechanisms., Methods: A cardiac-specific Prx5-overexpressing mice was used to establish a doxorubicin (DOX)-induced cardiotoxicity (DIC) model. Neonatal mouse cardiomyocytes (NMCMs) were cultured and stimulated with DOX. Prx5 overexpression or knockdown in cardiomyocytes was achieved using a Prx5-overexpressing adenovirus or small interfering RNA (siRNA), respectively. Echocardiography, histopathological assessments, and molecular techniques were employed to examine the effects and mechanisms of Prx5 on DIC., Results: Prx5 expression is upregulated in cardiac tissues following DOX administration. In DOX-exposed mice, overexpression of Prx5 significantly improved cardiac function and reduced myocardial injury. It inhibited myocardial hypertrophy and fibrosis, and diminished oxidative stress and inflammatory responses. Conversely, Prx5 knockdown in vitro aggravated DOX-induced cardiomyocyte inflammation and oxidative stress. Mechanistically, overexpression of Prx5 also resulted in the downregulation of Toll-like receptor 4 (TLR4) and phosphorylated P65 expression. Moreover, the protective effects of Prx5 were significantly abrogated by a TLR4 agonist., Conclusion: Prx5 overexpression could protect against DOX-induced cardiac oxidative stress and inflammation. Mechanistically, Prx5 overexpression potentially inhibits the TLR4/NF-κB signaling pathway to improve DOX-induced myocardial injury. These findings strongly suggest that Prx5 could be a potential candidate target for the treatment of DOX-induced myocardial injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Microglia, the primary immune cells of the central nervous system, play a crucial role in the neuroinflammatory processes following ischemic stroke. Targeting neuroinflammation is a promising strategy to enhance the outcomes of ischemic stroke. Benzydamine (BA), a well-known non-steroidal anti-inflammatory drug, has demonstrated potential in inhibiting pro-inflammatory cytokines across various disease models. However, the potential role of BA in microglial activation and post-stroke neuroinflammation remains unclear. Our study reveals that BA effectively suppresses the lipopolysaccharide (LPS)-stimulated pro-inflammatory responses of primary microglia, with high-dose BA (10 μM) suppressing LPS-induced inflammatory markers by up to 59.1 % in the mRNA levels of IL-1β. Furthermore, BA mitigated ischemic brain injury in experimental stroke mice. BA treatment also significantly attenuated neuroinflammatory responses and attenuates ischemic brain injury in experimental stroke mice. Further investigation revealed that BA reduces the release of the LPS-stimulated pro-inflammatory factors and activation of primary microglia by directly binding to and inhibiting the activity of cathepsin S (CTSS). In conclusion, our study identifies BA as a promising CTSS inhibitor with potential to suppress neuroinflammation following ischemic stroke. Our findings provide a theoretical basis for developing new neuroprotective strategies., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Kawasaki disease (KD), characterized by systematic vasculitis, is a leading cause of pediatric heart disease. Although recent studies have highlighted the critical role of deubiquitinases in vascular pathophysiology, their specific contribution to KD remains largely unknown. Herein, we investigated the function of the deubiquitinase USP7 in both KD patients and a CAWS-induced KD murine model. USP7 expression level is increased both in HCAECs induced by KD sera and cardiac CD31 + endothelial cells of KD mice. Whereas knockout of USP7 increases the cellular proportion of endothelial cells and potentially attenuates the elevated EndoMT, fibrosis, and inflammation in cardiac tissue of KD mice, consistently with the in vitro experiment observed in HCAECs induced by TGF-β2. Mechanistically, USP7 interacts with SMAD2/3, enhancing their protein stability by removing the K48 ubiquitin chain from both proteins and preventing their proteasome degradation, thus increasing the p-SMAD2 levels and nuclear entry. Importantly, intraperitoneal injection of USP7 inhibitor, P22077 elicited a robust anti-EndoMT and anti-vascular inflammation effect in KD model mice. Therefore, our study uncovered a previously unrecognized function of increased USP7 in KD by augmenting TGFβ2/SMAD2/SMAD3 signaling, thus facilitating the transcription of genes implicated in the EndoMT, cardiac fibrosis, and vascular remodeling. Our finding suggests that USP7 could serve as a potential therapeutic target for the prevention and treatment of coronary artery lesions in KD and related vascular diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The exposure of humans and animals to environmental compounds is rarely restricted to a single chemical. Unfortunately, very few studies were conducted to determine cadmium and lead combined effect. The aim of this study was to clarify the neurotoxicity induced by combined exposure to lead and cadmium and its mechanism of action. Sprague Dawley (SD) rats were randomly divided into control, lead, cadmium, and combined lead and cadmium groups, and Y-maze was used to detect the learning and memory ability of the rats, and the hippocampal tissue was subjected to HE pathology staining. Detection of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) and inflammatory factors IL-1β, TNF-α levels, immunofluorescence for microglia marker Iba1, and western blot for ferroptosis-related proteins SLC7A11, GPX4, and FTH1. Primary rat microglia were extracted, the combined dose was determined by CCK8, and the differentially expressed proteins were identified by 4D-DIA quantitative proteomics technology. The differentially expressed proteins were analyzed by KEGG analysis software for bioinformatics, and the results were verified by immunofluorescence. The results showed that combined exposure to lead and cadmium decreased the learning and memory ability of the rats, and the levels of lead and cadmium in the blood and the hippocampus increased, and the expression of microglia marker Iba1 in the hippocampus was elevated, and the expression of inflammatory factors IL-1β and TNF-α was elevated, combined exposure decreased GSH and SOD levels, increased ROS and MDA levels, increased iron content, and decreased expression of iron death-related proteins SLC7A11, GPX4 and FTH1. Extracted rat primary microglia, CCK8 to determine the co-toxicity dose after proteomics testing, found that iron metabolism-related protein SLC39A8 was down-regulated, STEAP3 was up-regulated, and validation results were consistent with the proteomics results. Conclusion, combined lead and cadmium exposure may exacerbate neurotoxicity by mediating microglia ferroptosis via STEAP3, SLC39A8., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Citrullinated vimentin (cVIM) triggers the immune response and is the primary autoantigen of rheumatoid arthritis (RA). Ginsenoside compound K (CK), which exerts significant anti-inflammatory effects, was the objective of this study. We aimed to investigate the role and mechanism of CK in regulating presentation of citrullinated peptides., Methods: In RA fibroblast-like synoviocytes (RA-FLS), the expression of autoantigen cVIM, antigen presentation-related molecules, autophagy-related proteins, and autophagic flux were investigated. The effect of CK on the antigen presentation capability of FLS was also examined under conditions of autophagy induction and inhibition. Finally, Wistar rats were immunized with cVIM to evaluate the therapeutic effect of CK in an RA model., Results: In RA-FLS, CK mitigated the expression of cVIM, autophagy-associated proteins, and antigen presentation-related molecules. This regulatory effect was associated with autophagy. cVIM-immunized rats exhibited more severe arthritis and higher levels of anti-CCP antibodies than those with adjuvant- and vimentin (VIM)-induced arthritis. CK significantly alleviated arthritis inflammation in cVIM-immunized rats., Conclusions: CK alleviates cVIM-induced arthritis symptoms, with the regulation of autophagy presenting a key cellular event involved in cVIM generation and RA-FLS antigen-presenting ability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

NEK2 (NIMA-related kinase 2) has recently gained attention for its potential role in osteoarthritis (OA) chondrocytes, however, its specific involvement remains unclear. This study aimed to investigate the role of NEK2 in OA progression and the underlying molecular mechanisms. Primary mouse knee chondrocytes were stimulated with IL-1β to establish an in vitro OA model, followed by the knockdown of NEK2 or ATF2. The results indicated that silencing NEK2 or ATF2 impeded the IL-1β-induced decrease in cell proliferation and increase in inflammation, extracellular matrix (ECM) degradation, and apoptosis in chondrocytes. NEK2 or ATF2 knockdown restored IL-1β-induced autophagy defects. Mechanistically, NEK2 interacts with ATF2 to reduce its ubiquitylation level and enhance its stability by phosphorylating ATF2 at Ser-112. Consistently, ATF2 overexpression reversed the protective effect of NEK2 silencing on IL-1β-induced autophagy defects and chondrocyte injury. Additionally, a mouse OA model was established using medial meniscus destabilization (DMM) surgery, and NEK2 was knocked down by intra-articular injection of an adenovirus-mediated NEK2 interference vector. Downregulation of NEK2 mitigated cartilage degradation andautophagy defects ina mouse OA model. In conclusion, NEK2 promoted OA progression by enhancing ATF2 stability by phosphorylating it at Ser-112., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Trained immunity represents a functional state of the innate immune response, characterized by enduring epigenetic reprogramming of innate immune cells. This phenomenon facilitates a sustained and advantageous reaction of myeloid cells to subsequent challenges. Bletilla striata polysaccharide (BSP) is the primary active component of Bletilla striata, mainly consisting of mannose and glucose in its chemical structure. Previous studies have demonstrated BSP's immunomodulatory properties, highlighting its effectiveness in enhancing the immune response. In the present study, we demonstrated that BSP administration induced the trained innate immunity, as evidenced by the BSP-induced generation of mature myeloid cells, especially neutrophils in the peripheral circulation in amouse model of chemotherapy-induced myelosuppression. Furthermore, we showed that BSP-induced trained immunity acts at the level of hematopoietic stem and progenitor cells (HSPCs) and induces the expansion of HSPCs. Mechanistically, our study demonstrated that BSP induced the activation of Notch signaling in HSPCs, and Notch signaling is indispensable for the BSP-mediated generation of trained HSPCs. Collectively, our data demonstrated for the first time that BSP induced trained immunity by regulating theexpansion of the myeloid progenitors. Harnessing trained immunity could be a promising strategy for protection from chemotherapy-induced myelosuppression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Extracellular vesicles from Rheumatoid arthritis (RA) derived synovial fibroblasts (EVs RASF ) have been implicated in the pathogenesis of RA, acting as mediators of cell-to-cell communication. This study aimed to elucidate the role of the chemokine receptor CCR5 and EVs positive for CCR5 (EVs RASF ) in RA, focusing on their impact on cartilage destruction and bone erosion in a rat model of Adjuvant-induced arthritis (AIA). In vivo experiments were conducted using AIA rats, treated with either EVs RASF , EVs RASF without CCR5 (EVs RASF-CCR5 ), or EVs M which encapsulated the CCR5 antagonist Maraviroc. The results demonstrated that EVs RASF-CCR5 reversed the catabolic effect of EVs RASF on hRA-CHs. EVs RASF accelerated cartilage destruction and bone erosion in the AIA rats, as evidenced by increased arthritis scores, joint damage, and NF-κB activation. In contrast, EVs RASF-CCR5 and EVs M treatment mitigated these effects, suggesting a detrimental role of CCR5 in EVs RASF -mediated RA pathogenesis. These findings highlight the critical role of CCR5 in mediating the pro-inflammatory and destructive effects of EVs RASF in RA, suggesting that targeting CCR5 may represent a novel therapeutic strategy for RA management. In conclusion, this study provides valuable insights into one of the molecular mechanisms underlying RA pathogenesis, emphasizing the importance of EVs RASF and CCR5 in mediating synovial inflammation and joint destruction. The results underscore the potential of CCR5 as a therapeutic target, opening avenues for the development of targeted interventions in RA treatment with synovial fibroblast derived EVs serving as a convenient, stabilizing vehicle for delivering Maraviroc into the RA affected joint tissues., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Li Yin reports was provided by The Education Department Of Henan Province. Li Yin reports financial support was provided by Health Commission of Henan Province. Shushan Li reports financial support was provided by Health Commission of Henan Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mast cells (MC) are crucial effectors in immediate allergic reactions. Monomeric IgE sensitizes MC and triggers various signaling responses. FcεRI/IgE/antigen crosslinking induces the release of several mediators, including bioactive lipids, but little is known about endocannabinoids (eCBs) secretion. Here, we studied the effects of IgE-induced sensitization and FcεRI crosslinking on anandamide (AEA) synthesis and release in bone marrow-derived mast cells (BMMC). Our results showed that mIgE induced AEA secretion through phospholipase C activation. Secreted AEA contributed to p38 phosphorylation induced by mIgE sensitization. Prolonged mIgE sensitization promoted the formation of long-lasting CB2-GPR55 heteromers. FcεRI crosslinking also caused AEA production. Notably, CB2 deficiency increased IL-2 and IL-3 cytokine expression in response to FcɛRI crosslinking. CB2 and GPR55 agonists reduced IL-2 and IL-3 mRNA expression caused by FcεRI activation. Our findings suggest that a) IgE binding to FcɛRI and its antigen-dependent activation leads to an AEA-dependent autocrine regulatory loop that contributes to intracellular signaling in MC and that b) CB2 and GPR55 receptors play a critical role in modulating the effector phase of MC activation by specifically regulating cytokine expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Inflammation and apoptosis contribute to the development of dry eye disease (DED) and meibomian gland dysfunction (MGD). This study aimed to investigate the effect of caffeine on the ocular surface and tear inflammatory cytokines through clinical, in vivo, and in vitro experiments., Methods: In the clinical study, comprehensive ophthalmic examinations of participants in the control and the caffeine groups were compared, including ocular surface and tears inflammatory cytokines. For in vitro study, rat meibomian gland epithelial cells (RMGECs) and human corneal epithelial cells (HCECs) were pretreated with or without caffeine and then stimulated with lipopolysaccharide (LPS). Inflammatory responses, apoptosis, and differentiation in cells were analyzed. In vivo study, apolipoprotein E knockout (ApoE -/- ) mice were given caffeine-diet or no caffeine-diet, and their meibomian glands (MGs) and corneal tissue were compared., Results: Participants in the caffeine group exhibited significantly healthier ocular surface, lower tears inflammatory cytokines and a reduced prevalence of DED compared to the control group. In vitro study, caffeine pretreatment attenuated inflammatory responses, apoptosis and differentiation in LPS-induced RMGECs. Meanwhile, caffeine also markedly suppressed inflammatory responses and apoptosis in LPS-induced HCECs. In vivo study showed that ApoE -/- mice with caffeine-diet had more normal morphology of MGs and corneas compared to those without caffeine-diet, along with reduced inflammatory responses, cells apoptosis and ductal keratinization. Both in vitro and in vivo studies indicated that caffeine treatment was observed to inactivate of nuclear factor kappa B (NF-κB) phosphorylation., Conclusions: Our study indicated that caffeine may be a protective potential of ocular surface, providing a new perspective on clinical treatment for DED and MGD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cyclophosphamide is a widely used immunosuppressive and chemotherapeutic agent in clinics. Previous studies have indicated that cyclophosphamide treatment induces cellular senescence in patients, although the underlying mechanisms remain elusive. Here, we reported that cyclophosphamide induced T cell senescence in the spleen of mice. Meanwhile, phosphoramide mustard cyclohexanamine (PM), an active metabolite of cyclophosphamide, triggered human T cell senescence. RNA sequencing analysis revealed that PM promoted senescence of primary human T cells through the activation of the p53 signaling pathway. Moreover, PM strongly increased the acetylation of p53 lysine 373 in T cells. Compared with the wild-type p53, mutating lysine 373 of p53 to arginine markedly reduced the p21 and p16 expression in PM treated Jurkat cells. Notably, we found that theophylline, an activator of histone deacetylase HDAC, ameliorated the senescence phenotype of both primary human T cells induced by PM and splenic T cells induced by cyclophosphamide in mice. Together, the results from current study could provide a potential strategy against cyclophosphamide-induced T cell senescence by inhibiting p53 acetylation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The role of extracellular vesicles (EVs) derived from inflammatory chondrocytes in EV-based therapy for osteoarthritis (OA) has received little attention. We examined the effects of EVs derived from both normal rat chondrocytes (nEVs) and IL-1β-treated rat chondrocytes (iEVs) on IL-1β-treated rat chondrocytes, macrophages, and osteoblasts, alongside mRNA-seq and miRNA-seq analyses of both them. Additionally, nEVs and iEVs were administered intra-articularly in the joints of rat models subjected to anterior cruciate ligament transection (ACLT), and the morphological alterations across the joints were assessed. These findings indicated that iEVs, compared with nEVs, significantly enhanced collagen II synthesis in IL-1β-treated chondrocytes, accompanied by marked increases in ER stress and autophagy. In comparison to nEVs, iEVs exhibited a greater effect on facilitating M2-type macrophage polarization while simultaneously diminishing M1-type polarization, a process likely mediated by the downregulation of chemotactic cytokines such as Cxcl10, Ccl5, Cxcl9, Cxcl1, and Cxcl11. iEVs exerted a more pronounced influence on the phenotypic characteristics of IL-1β-treated osteoblasts than nEVs. In the ACLT-rat model, iEVs, akin to nEVs, effectively mitigated articular cartilage degradation. However, there was no significant difference in OARSI Scores between the two groups, despite iEVs exerting a greater effect on increasing hyaline cartilage thickness and proteoglycan content. iEVs were superior to nEVs in attenuating synovium inflammation and promoting trabecula formation in the femur subchondral bone. Consequently, iEVs, akin to nEVs, significantly alleviated OA-induced damage. Moreover, iEVs outperformed nEVs in certain aspects, notably in augmenting hyaline cartilage, reducing synovium inflammation, and promoting trabecular formation in the subchondral bone during the early stage of OA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) induces significant neuroinflammation, primarily driven by microglia. Neonatal microglia (NMG) may have therapeutic potential by modulating the inflammatory response of damaged adult microglia (AMG). This study investigates the influence of NMG on AMG function through extracellular matrix (ECM) remodeling and the formation of tunneling nanotubes (TnTs), with a focus on the role of Serpina3n. We established an in vitro TBI model using a 3D Transwell system, co-culturing damaged AMG with NMG. Viral vector transfection was employed to manipulate Serpina3n expression in NMG. Quantitative real-time PCR, Western blotting, and ELISA were utilized to assess inflammatory markers, ECM remodeling proteins, and TnTs-related proteins. Co-culturing with NMG significantly inhibited M1 polarization of AMG and reduced the release of pro-inflammatory cytokines while promoting M2 polarization and increasing the production of anti-inflammatory cytokines. NMG expressed higher levels of Serpina3n, which played a crucial role in reducing Granzyme B, matrix metalloproteinase (MMP) 2 and MMP9 expression, thereby mitigating ECM remodeling. Inhibition of Serpina3n in NMG increased pro-inflammatory markers and decreased TnTs formation proteins, whereas overexpression of M-sec in AMG counteracted these effects. This highlights the importance of TnTs in maintaining microglial function and promoting an anti-inflammatory environment. In conclusion, NMG improve the function of damaged AMG by modulating ECM remodeling and promoting TnTs formation through the action of Serpina3n., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

Sevoflurane impairs learning and memory of the developing brain. However, strategies to mitigate these detrimental effects have been scarce. Herein, we investigated whether tetramethylpyrazine pretreatment could alleviate the impairment of learning and memory and its underlying mechanism in sevoflurane-exposed neonatal rats. Postnatal 7-day Sprague-Dawley (SD) rats or primary hippocampal neurons were pretreated with tetramethylpyrazine and then exposed to sevoflurane. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and lactate dehydrogenase (LDH) assays were used to detect neuronal injury. Learning and memory function were evaluated by novel object recognition and Morris water maze tests. Long-term potentiation (LTP) was recorded to evaluate synaptic plasticity electrophysiologically in the hippocampal slices. Golgi-Cox staining or PSD95 immunochemistry was used to detect the morphology of dendritic spines. Western blotting was employed to assess the expressions of cleaved Caspase-3, PSD95, N-methyl-D-aspartate receptor (NMDAR) subunits NMDAR1, NMDAR2A and NMDAR2B in the hippocampus or cultured neurons. It was found that neonatal exposure of sevoflurane impaired learning and memory, increased neuronal apoptosis, altered the morphology of dendritic spines, upregulated the expressions of NMDAR2A and PSD95, and induced LTP deficits. Pretreatment with tetramethylpyrazine not only alleviated impairment of learning and memory, but also improved sevoflurane-induced changes in neuronal damage, dendritic spine morphology, NMDAR2A and PSD95 expressions, as well as LTP. These findings indicated that pretreatment with tetramethylpyrazine alleviated the impairment of learning and memory induced by sevoflurane through improvement of hippocampal synaptic plasticity in neonatal rats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

Background: Sepsis-associated encephalopathy (SAE) often results from neuroinflammation. Recent studies have shown that brain platelet-derived growth factor receptor β (PDGFRβ) cells, including pericytes, may act as early sensors of infection by secreting monocyte chemoattractant protein-1 (MCP-1), which transmits inflammatory signals to the central nervous system. The erythroblast transformation-specific (ETS) transcription factor Friend leukemia virus integration 1 (Fli-1) plays a critical role in inflammation by regulating the expression of key cytokines, including MCP-1. However, the role of pericyte Fli-1 in neuroinflammation during sepsis remains largely unknown., Methods: WT and pericyte-specific Fli-1 knockout mice were subjected to endotoxemia through LPS injection or sepsis via cecal ligation and puncture (CLP). In vitro, Fli-1 was knocked down using small interfering RNA in cultured mouse brain pericytes, followed by LPS stimulation., Results: Elevated Fli-1 levels were observed in isolated brain pericytes 2 h after LPS administration, in brain tissues 4 h after CLP, and in cultured mouse brain pericytes 2 h after LPS stimulation in vitro. In endotoxemic mice, pericyte-specific Fli-1 knockout reduced expression of MCP-1 and IL-6 in brain tissue 2 h after LPS injection. At 24 h post-LPS administration, protein levels of MCP-1 and IL-6, and microglia activation were suppressed in pericyte-Fli-1 knockout mice. Additionally, Fli-1 deficiency in pericytes significantly reduced MCP-1 and IL-6 mRNA levels in the brain tissue 4 h after CLP. Moreover, in cultured brain pericytes, Fli-1 knockdown markedly decreased MCP-1 and IL-6 levels after LPS stimulation. Notably, LPS stimulation increased Fli-1 levels via TLR4-Myd88 signaling, which subsequently led to elevated production of MCP-1 in brain pericytes., Conclusions: Fli-1 in pericytes may serve as a crucial mediator of neuroinflammation during sepsis by directly regulating pivotal cytokines such as MCP-1 and IL-6. Therefore, Fli-1 has the potential to serve as a therapeutic target in SAE and other neuroinflammatory disorders., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethical approval: The protocol for all animal studies was approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina., (© 2025. The Author(s).)

Background: Endocrine-disrupting chemicals (EDCs) interfere with the endocrine system and negatively impact reproductive health. Biochanin A (BCA), an isoflavone with anti-inflammatory and estrogen-like properties, has been identified as one such EDC. This study investigates the effects of BCA on transcription, metabolism, and hormone regulation in primary human granulosa cells (GCs), with a specific focus on the activation of bitter taste receptors (TAS2Rs)., Methods: Primary human GCs from 60 participants were treated with 10 µM BCA, and selective antagonists were used to block TAS2R activation. The study assessed the expression of TAS2R14 and TAS2R43, and analyzed the impact on StAR and CYP17A1 gene expression. Intracellular calcium levels, lipid droplet size, and mitochondrial network complexity were measured to evaluate cellular metabolism and energy dynamics., Results: BCA treatment significantly upregulated TAS2R14 and TAS2R43 expression, leading to a 70% increase in StAR mRNA levels and a twofold increase in CYP17A1 expression (p < 0.05). These effects were reversed by TAS2R antagonists. Additionally, BCA treatment decreased intracellular Ca 2+ levels (p < 0.01) and reduced lipid droplet size (p < 0.001), both of which were counteracted by antagonists. Enhanced mitochondrial network complexity (p < 0.001) was also observed, suggesting increased mitochondrial fusion and improved cellular energy dynamics., Conclusion: The findings indicate that BCA modulates transcriptional and metabolic processes in GCs through the activation of TAS2Rs, highlighting their role in endocrine regulation. The statistically significant results emphasize the relevance of further exploring the effects of EDCs like BCA on reproductive health. Collaborative research efforts are essential to address and mitigate the adverse impacts of EDCs on fertility., Competing Interests: Declarations. Ethics approval and consent to participate: Written informed consent was obtained from all participants prior procedure and was approved by the University of Siena’s ethical committee. (CEAVSE, Protocol number 18370, 2 October 2020). Consent for publication: Written informed consent for publication was obtained from all participants. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Cerebral ischemia-reperfusion injury (CIRI), which stays unresolved in the clinic, occurs after recanalization of blood vessels serving brain tissues in acute ischemic stroke patients and can result in massive brain cell death, and cell ferroptosis contributes greatly to this process. Our research firstly found that TNFSF9 expression harbored diagnostic value on CIRI patients and intended to further investigate its regulatory mechanism in CIRI, which might facilitate its diagnostic and therapeutic application in the clinic. The level of TNSF9 mRNA was augmented in the plasma of CIR patients, and its silence impeded ferroptosis, apoptosis, and release of inflammatory mediators of BMECs with OGD/R treatment. Besides, SP1 positively regulated TNFSF9 expression as one of its transcription factors, and TNFSF9 overexpression reversed SP1 silence-mediated inhibition on ferroptosis, apoptosis, and release of inflammatory mediators in OGD/R-treated BMECs. In addition, silencing SLC3A2 could neutralize the benefit effects of TNFSF9 downregulation on BMECs under OGD/R context in vitro, and silencing TNFSF9 neutralized necrotic volumes in rat brain induced by CIRI via modulating SLC3A2 expression in vivo. TNFSF9 regulated by SP1 aggravated CIRI via boosting ferroptosis, apoptosis, and release of inflammatory mediators of BMECs under OGD/R situation by suppressing SLC3A2 expression in vitro and in vivo., Competing Interests: Declarations. Ethics Approval and Consent to Participate: Written informed consents were obtained from all participants, and this study was permitted by the Ethics Committee of the Second Affiliated Hospital of Zhejiang Chinese Medical University. Consent for Publication: Not applicable. Conflict of Interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Interfacial heterogeneity is widely explored to reveal molecular mechanisms of force-mediated pathways due to biased tension. However, the influence of cell density,, curvature, and interfacial heterogeneity on underlying pathways of mechanotransduction is obscure., Methods: Polydimethylsiloxane (PDMS)-based stencils were micropatterned to prepare the micropores for cell culture. The colonies of human mesenchymal stem cells (hMSCs) were formed by controlling cell seeding density to investigate the influences of cell density, curvature and heterogeneity on mechanotransduction. Immunofluorescent staining of integrin, vinculin, and talin-1 was conducted to evaluate adhesion-related expression levels. Then, immunofluorescent staining of actin, actinin, and myosin was performed to detect cytoskeleton distribution, especially at the periphery. Nuclear force-sensing mechanotransduction was explained by yes-associated protein (YAP) and laminA/C analysis., Results: The micropatterned colony of hMSCs demonstrated the coincident characters with engineered micropores of microstencils. The cell colony obviously developed the heterogeneous morphogenesis. Heterogeneous focal adhesion guided the development of actin, actinin, and myosin together to regulate cellular contractility and movement by integrin, vinculin, and talin-1. Cytoskeletal staining showed that actin, actinin, and myosin fibers were reorganized at the periphery of microstencils. YAP nuclear translocation and laminA/C nuclear remodeling were enhanced at the periphery by the regulation of heterogeneous focal adhesion (FA) and cytoskeleton arrangement., Conclusions: The characters of the engineered clustering colony showed similar results with prepared microstencils, and colony curvature was also well adjusted to establish heterogeneous balance at the periphery of cell colony. The mechanism of curvature, spreading, and elongation was also investigated to disclose the compliance of FA and cytoskeleton along with curvature microarrays for increased nuclear force-sensing mechanotransduction. The results may provide helpful information for understanding interfacial heterogeneity and nuclear mechanotransduction of stem cells., Competing Interests: Declarations. Ethics approval and consent to participate: All animal maintenance and procedures were performed in accordance with the Basel declaration and the approval of the Animal Care and Ethics Committee of Shanghai University (no. ECSHU 2021-207). There were no human subjects. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Icariin (ICA) serves as the primary biologically active compound in traditional Chinese medicine Epimedium, while Icariside II (ICSII) represents one of its gastrointestinal metabolites. Although ICA and ICSII have demonstrated osteogenic differentiation- promoting effects on BMSCs, there is limited literature comparing their effects and underlying mechanisms. This study aimed to compare the osteogenic effects of Icariin and Icariside II, along with their respective osteogenic mechanisms. In this study, we initially determined the optimal concentrations of Icariin (10 -5 mol/L) and Icariside II (10 -6 mol/L) for inducing rBMSCs osteogenic differentiation using CCK8, ALP activity assay, and flow apoptosis assay. Subsequently, we compared the vascularization and osteogenic capacity of the two groups through alizarin red staining assay, Western Blot, and RT-PCR. Subsequently, we assessed the phosphorylated and non-phosphorylated expression of JNK, ERK1/2, P38, and AKT at different time intervals. We observed their phosphorylated expression and the expression of angiogenic/osteogenic markers after blocking with their corresponding inhibitors. It was observed that both the Icariin and Icariside II groups promoted the expression of osteogenic/angiogenic markers RUNX2, OCN, OPN, VEGF, and ANG1. While there was no significant difference in their osteogenic abilities, ICSII exhibited a stronger promotion of angiogenic differentiation markers, VEGF, compared to ICA. Additionally, it was observed that both ICA and ICSII could activate ERK1/2 phosphorylation, thereby further promoting the osteogenic/angiogenic differentiation of rBMSCs through the activation of the MAPK/ERK1/2 signaling pathway., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Diabetes treatments by the control of sodium-glucose cotransporter 2 (SGLT2) is commonly conducted while there are still uncertainties about the mechanisms for the SGLT2 overexpression in kidneys with diabetes. Previously, we have reported that glomeruli and proximal tubules with diabetic nephropathy express toll-like receptor TLR2/4, and that the TLR ligand lipopolysaccharide (LPS) of periodontal pathogens have caused nephropathy in diabetic model mice. Recently, many researchers suggested that the periodontal pathogenic bacteria Fusobacterium (F.) nucleatum has the TLR4-associated strong activator of the colorectal inflammation and cancer. The present study aimed to investigate the possibility of F. nucleatum as an exacerbation factor of diabetes through the renal SGLT2 induction., Methods: The induction of the SGLT2 by F. nucleatum LPS (Fn-LPS) were investigated in the streptozotocin-induced diabetic mouse renal tissue and cultured renal proximal epithelial cells. The changes of blood glucose levels and survival curves in diabetic mice with Fn-LPS were analyzed. The Fn-LPS-induced SGLT2 production in the diabetic mouse renal tissue and in the cultured proximal epithelial cells was examined by ELISA, quantitative RT-PCR, and immunohistochemical analysis., Results: The SGLT2 expression in the cultured mouse tubular epithelial cells was significantly increased by TNF- or co-culture with Fn-LPS-supplemented J774.1 cells. The period to reach diabetic condition was significantly shorter in Fn-LPS-administered diabetic mice than in diabetic mice. All Fn-LPS-administered-diabetic mice reached humane endpoints during the healthy period of all of the mice administered Fn-LPS only. The promotion of the SGLT2 expression at the inner lumen of proximal tubules were stronger in the Fn-LPS-administered-diabetic mice than in diabetic mice. The renal tissue SGLT2 mRNA amounts and the number of renal proximal tubules with overexpressed SGLT2 in the lumen were more in the Fn-LPS-administered-diabetic mice than in diabetic mice., Conclusions: This study suggests that F. nucleatum causes the promotion of diabetes through the overexpression of SGLT2 in proximal tubules under the diabetic condition. Periodontitis with F. nucleatum may be a diabetic exacerbating factor., Competing Interests: Declarations. Ethical approval: All methods were performed in accordance with the relevant guidelines and regulations. The animal experimental procedures were prepared following the ARRIVE guidelines. The protocol of the experiments for animal use was approved by the Animal Experiment Committee of Fukuoka Dental College (No. 19010). For studies with human subjects, include the statement “All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and/or with the Helsinki Declaration of 1964 and later versions. Consent to participate: Not applicable. Consent to publish: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Myelination is a key biological process wherein glial cells such as oligodendrocytes wrap myelin around neuronal axons, forming an insulative sheath that accelerates signal propagation down the axon. A major obstacle to understanding myelination is the challenge of visualizing and reproducibly quantifying this inherently three-dimensional process in vitro. To this end, we previously developed artificial axons (AAs), a biocompatible platform consisting of 3D-printed hydrogel-based axon mimics designed to more closely recapitulate the micrometer-scale diameter and sub-kilopascal mechanical stiffness of biological axons. First, we present our platform for fabricating AAs with tunable axon diameter, stiffness, and inter-axonal spacing. Second, we demonstrate that increasing the Young's modulus E or stiffness of polymer comprising the AAs increases the extent of myelin ensheathment by rat oligodendrocytes. Third, we demonstrate that the responses of oligodendrocytes to pro-myelinating compounds are also dependent on axon stiffness, which can affect compounds efficacy and the relative ranking. These results reinforce the importance of studying myelination in mechanically representative environments, and highlight the importance of considering biophysical cues when conducting drug screening studies., Competing Interests: A. J. and K. K. are the founders and stock holders of Artificial Axon Labs Inc., which is commercializing artificial axons platform. K. J. V. V holds scientific advisor position at Artificial Axon Labs. A. J. and K. J. V. V., are inventors of patent # 11535826, “Engineered 3D-printed artificial axons”, Daniela Espinosa-Hoyos, Anna E. Jagielska, Huifeng Du, Nicholas X. Fang, Krystyn J. Van Vliet, which covers the materials and assay used in this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2025 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Retinopathy of prematurity (ROP) is a major cause of childhood blindness worldwide, highlighted by retinal neovascularization. Ubiquitin is present throughout the retina. The deubiquitinating enzyme ubiquitin-specific protease 39 (USP39) has been reported to be involved in angiogenesis. Here, this study aimed to investigate the effects of USP39 on ROP and its associated mechanism., Methods: Hypoxia-induced human retinal microvascular endothelial cells (hRMECs) were adopted for functional analyses. Detection of mRNA and protein was conducted using quantitative real-time PCR and western blotting. Cell migration, invasion and angiogenesis were evaluated using transwell and tube formation assays. Protein interaction was determined by immunoprecipitation assay. Oxygen-induced retinopathy (OIR) mouse models were used for in vivo analysis., Results: USP39 level was higher in hypoxia-induced hRMECs, functionally, USP39 silencing reversed hypoxia-induced migration, invasion and angiogenesis in hRMECs. In further mechanism analysis, we found that USP39 stabilized SIRT2 protein expression in hRMECs by inducing SIRT2 deubiquitination. Moreover, SIRT2 up-regulation abated hypoxia-evoked migration, invasion and angiogenesis in hRMECs. Besides that, the inhibitory effects of USP39 silencing on hypoxia-induced metastatic and angiogenic behaviors were abolished after SIRT2 overexpression. In addition, USP39 silencing blocked the activation of phosphoinositide 3-kinase (PI3K)/protein kinase B pathway (AKT) by regulating SIRT2. In vivo assay showed that levels of USP39, SIRT2, matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9 and Vascular endothelial growth factor A (VEGFA) were increased in the retinas of OIR mice, while intravitreal injection of USP39 short hairpin RNA (shRNA) could reduce their expression., Conclusion: USP39 stabilized SIRT2 expression by deubiquitination and promoted hypoxia-induced metastatic and angiogenic behaviors of RMECs in vitro, as well as retinal angiogenesis in vivo., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

α-Synuclein (αS) is a 140 amino-acid neuronal protein highly enriched in presynaptic nerve terminals. Its progressive accumulation in Lewy bodies and neurites is the hallmark of Parkinson's disease (PD). A growing number of studies highlights a critical interplay between lipid metabolism and αS biology. Some of these works postulate a physical interaction between αS and lipid droplets (LDs), but further clarity is needed, not least because typically exogenous αS and/or heterologous systems have been studied. Here, we investigated the effects of acute LD accumulation on endogenous wild-type αS in primary rat cortical neurons. To induce robust LD accumulation within hours, we inhibited the neuronal triacylglycerol hydrolase DDHD2, a phospholipase, using the compound KLH45. KLH45-induced LD accumulation did not affect total levels, phosphoserine-129 status, or solubility of αS, and no co-localization between LDs and αS was observed under these conditions. These findings suggest that a "second hit" and/or a specific LD lipid composition may be necessary for lipid excess to affect αS homeostasis. Our work thus contributes to the debate on αS structure and lipid interaction., Competing Interests: Declarations. Ethics approval: As indicated in the description of experimental procedures, all animal procedures were approved by the Institutional Animal Care and Use Committee at BWH (Protocol # 2016N000305). Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aim: Human dental pulp stem cells (hDPSCs) constitute a promising alternative for central nervous system (CNS) cell therapy. Unlike other human stem cells, hDPSCs can be differentiated, without genetic modification, to neural cells that secrete neuroprotective factors. However, a better understanding of their real capacity to give rise to functional neurons and integrate into synaptic networks is still needed. For that, ex vivo differentiation protocols must be refined, especially to avoid the use of fetal animal serum. The aim of our study is to improve existing differentiation protocols of hDPSCs into neuron-like cells., Methods: We compared the effects of the (1) absence or presence of fetal serum during the initial expansion phase as a step prior to switching cultures to neurodifferentiation media. We (2) improved hDPSC neurodifferentiation by adding retinoic acid (RA) and potassium chloride (KCl) pulses for 21 or 60 days and characterized the results by immunofluorescence, digital morphometric analysis, RT-qPCR and electrophysiology., Results: We found that neural markers like Nestin, GFAP, S100β and p75 NTR were expressed differently in neurodifferentiated hDPSC cultures depending on the presence or absence of serum during the initial cell expansion phase. In addition, hDPSCs previously grown as spheroids in serum-free medium exhibited in vitro expression of neuronal markers such as doublecortin (DCX), neuronal nuclear antigen (NeuN), Ankyrin-G and MAP2 after neurodifferentiation. Presynaptic vGLUT2, Synapsin-I, and excitatory glutamatergic and inhibitory GABAergic postsynaptic scaffold proteins and receptor subunits were also present in these neurodifferentiated hDPSCs. Treatment with KCl and RA increased the amount of both voltage-gated Na + and K + channel subunits in neurodifferentiated hDPSCs at the transcript level. Consistently, these cells displayed voltage-dependent K + and TTX-sensitive Na + currents as well as spontaneous electrophysiological activity and repetitive neuronal action potentials with a full baseline potential recovery., Conclusion: Our study demonstrates that hDPSCs can be differentiated to neuronal-like cells that display functional excitability and thus evidence the potential of these easily accessible human stem cells for nerve tissue engineering. These results highlight the importance of choosing an appropriate culture protocol to successfully neurodifferentiate hDPSCs., Competing Interests: Declarations. Ethics approval and consent to participate: Human third molars were obtained from healthy donors of between 18 and 45 years of age and tooth samples were obtained by donation after written informed consent in compliance with the 14/2007 Spanish directive for Biomedical research. The study protocol was approved on date 03/02/2021 by the Ethics Committee of the University of the Basque Country UPV/EHU and the competent authority (Administración Foral de Bizkaia) regarding the use of human cells with CEISH M10/2020/172 and M10/2023/025 entitled “Matrices de anclaje nanoestructuradas basadas en grafeno y polímeros biodegradables para inducir la neurodiferenciación de células madre y regenerar el tejido nervioso”. The study was conducted in accordance with the Declaration of Helsinki. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Intervertebral disc degeneration disease (IVDD) is a major cause of disability and reduced work productivity worldwide. Annulus fibrosus degeneration is a key contributor to IVDD, yet its mechanisms remain poorly understood. Autophagy, a vital process for cellular homeostasis, involves the lysosomal degradation of cytoplasmic proteins and organelles. This study aimed to investigate the role of autophagy in IVDD using a hydrogen peroxide (H 2 O 2 )-induced model of rat annulus fibrosus cells (AFCs)., Methods: AFCs were exposed to H 2 O 2 to model oxidative stress-induced degeneration. Protein expression levels of collagen I, collagen II, MMP3, and MMP13 were quantified. GEO database analysis identified alterations in miR-2355-5p expression, and its regulatory role on the mTOR pathway and autophagy was assessed. Statistical tests were used to evaluate changes in protein expression and pathway activation., Results: H 2 O 2 exposure reduced collagen I and collagen II expression to approximately 50% of baseline levels, while MMP3 and MMP13 expression increased twofold. Activation of autophagy restored collagen I and II expression and decreased MMP3 and MMP13 levels. GEO analysis revealed significant alterations in miR-2355-5p expression, confirming its role in regulating the mTOR pathway and autophagy., Conclusions: Autophagy, mediated by the miR-2355-5p/mTOR pathway, plays a protective role in AFCs degeneration. These findings suggest a potential therapeutic target for mitigating IVDD progression., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Enteroviruses can infect various human organs, causing diseases such as meningitis, the common cold, hand-foot-and-mouth disease, myocarditis, pancreatitis, hepatitis, poliomyelitis, sepsis, and type 1 diabetes. Currently, there are no approved treatments for enterovirus infections. In this study, we identified a synergistic combination of orally available, safe-in-man pleconaril, AG7404, and mindeudesivir, that at non-toxic concentrations effectively inhibited enterovirus replication in human cell and organoid cultures. Importantly, the cocktail did not alter glucose and insulin levels in the culture medium of pancreatic β-cells and preserved the contraction rhythm of infected heart organoids. These findings highlight a promising drug cocktail for further preclinical studies and clinical trials targeting a broad range of enterovirus-mediated diseases., Competing Interests: Declarations. Ethics approval: The study involved commercially available human cell lines, WT virus strains, which we isolated from patient samples or obtained via collaborations. All experiments with viruses were performed in BSL2 laboratories in compliance with the guidelines of the national authorities, and under appropriate ethical and safety approvals. All experiments conducted with pancreatic cancer organoids have been approved by the Helsinki University Hospital ethical board, HUS/74/2023, HUS/23/2024. The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for study on lung organoids (METC 2012 − 512). Competing interests: The authors have no relevant financial or non-financial interests to disclose., (© 2025. The Author(s).)

Cell micro-patterning controls cell fate and function and has potential for generating therapeutically usable mesenchymal stromal cell (MSC) populations with precise functions. However, to date, the micro-patterning of human cells in a translational context has been impossible because only ruminant media supplements, e.g. fetal bovine serum (FBS), are established for use with micro-patterns (MPs). Thus, there are currently no good manufacturing practice (GMP)-compliant media available for MPs. This study tested a xenogenic-free human plasma and platelet lysate (hP + PL) medium supplement to determine its compatibility with MPs. Unfiltered hP + PL medium resulted in significant protein deposition, creating a 'carpet-like' layer that rendered MPs ineffective. Filtration (3×/5×) eliminated this effect. Importantly, quantitative comparison using droplet digital PCR revealed that human MSCs in all media types exhibited similar profiles with strong myogenic Calponin 1/Transgelin 2 (TAGLN2) and weaker osteogenic alkaline phosphatase/Runt-related transcription factor 2 marker expression, and much weaker adipogenic (lipoprotein lipase/peroxisome proliferator-activated receptor gamma) and chondrogenic (collagen type II/aggrecan) expression, with profiles being dominated by myogenic markers. Within these similar profiles, an even stronger induction of the myogenic marker TAGLN2 by all hP + PL- compared to FBS-containing media. Overall, this suggested that FBS can be replaced with hP + PL without altering differentiation profiles. However, assessing individual MSC responses to various MP types with defined categories revealed that unfiltered hP + PL medium was unusable. Importantly, FBS- and 3× filtered hP + PL media were comparable in each differentiation category. Summarized, this study recommends 3× filtered hP + PL as a xenogenic-free and potentially GMP-compliant alternative to FBS as a culture medium supplement for micro-patterning cell populations in both basic and translational research that will ensure consistent and reliable MSC micro-patterning for therapeutic use., (© 2025 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

An inflammatory response is related to different inflammatory mediators generated by immune and endometrial cells. The links between lipopolysaccharide (LPS), cytokines, and leukotrienes (LTs) in endometrial stromal cells remain unclear. This study aimed to examine the influence of LPS, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4 and IL-10 on 5-lipooxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA and protein abundances, and LTB4 and cysteinyl (cys)-LTs release including LTC4, by the cultured pig endometrial stromal cells, as well as on cell viability. 24-hour exposure to LPS, TNF-α, IL-4 and IL-10 up-regulated 5-LO mRNA and protein abundances. LPS increased LTAH mRNA abundance, while TNF-α, IL-1β and IL-10 augmented LTAH mRNA and protein abundances. TNF-α and IL-4 increased LTCS mRNA and protein abundances. In addition, LTCS mRNA abundance was enhanced by LPS and IL-4, while LTCS protein abundance was increased by IL-1β. Cells responded to LPS, TNF-α, IL-1β and IL-10 with increased LTB4 release. TNF-α, IL-1β and IL-4 stimulated LTC4 release. Cys-LTs release was up-regulated by LPS, TNF-α, IL-1β and IL-4. All studied cytokines augmented cell viability. In summary, LPS, TNF-α, IL-1β, IL-4 and IL-10 are potential LTs immunomodulatory agents in endometrial stromal cells. These functional interactions could be one of the mechanisms responsible for local orchestrating events in inflamed and healthy endometrium., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Intervertebral disc degeneration (IDD) is a leading cause of low back pain, often linked to inflammation and pyroptosis in nucleus pulposus (NP) cells. The role of Periostin (POSTN) in IDD remains unclear., Objective: This study aims to investigate the influence of POSTN on pyroptosis and NLRP3 inflammasome activation in NP cells during IDD., Methods: IVD samples were collected from patients undergoing spinal surgery and classified according to the Pfirrmann grading system. Human NP cells were cultured and treated with IL-1β to induce a pyroptotic phenotype. Western blotting, Immunofluorescence (IF), and immunohistochemistry (IHC) assessed the expression levels of relevant proteins. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified the binding of IRF2 to the POSTN and GSDMD promoters and evaluated the activation levels of target genes. The severity of IDD was evaluated using MRI and histological analysis., Results: Deletion of POSTN significantly alleviated IDD by suppressing NLRP3 inflammasome activity and pyroptosis in NP cells. POSTN was found to aggravate NP cell pyroptosis by activating the NLRP3 inflammasome through the NF-κB (P65) and cGAS/STING signaling pathways. Furthermore, POSTN interacted with Notch1 to induce NLRP3 expression. IRF2 was identified as a regulator of POSTN at the transcriptional level, contributing to NLRP3 activation and NP cell pyroptosis. IRF2 also directly induced the transcriptional expression of GSDMD, mediating pyroptosis in NP cells. Chemical screening identified Glucosyringic acid (GA) as a direct inhibitor of POSTN, which delayed IDD progression., Conclusion: The study elucidates the pivotal role of POSTN in mediating NP cell pyroptosis through the NLRP3 inflammasome and highlights GA as a promising therapeutic candidate for IDD. These findings provide new insights into the molecular mechanisms of IDD and potential avenues for treatment., Competing Interests: Declarations. Ethics approval and consent to participate: All experimental designs and protocols involving animals and human samples were approved by the Animal Ethics Committee and Medical Ethics Committee of Lanzhou University Second Hospital, Gansu, People’s Republic of China (approval D2023-303 and 2023 A-258) and complied with the recommendations of the academy’s animal research guidelines. Consent for publication: All authors gave their consent for publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Caspases are known to mediate neuronal apoptosis during brain development. However, here we show that nonapoptotic activation of caspase-3 at presynapses drives microglial synaptic phagocytosis. Real-time observation and spatiotemporal manipulation of synaptic caspase-3 in the newly established, mouse-derived culture system demonstrate that increased neuronal activity triggers localized presynaptic caspase-3 activation, facilitating synaptic tagging by complements. High-resolution live imaging reveals that caspase-3 activation promotes synapse-selective complement-dependent microglial phagocytosis without axonal shearing. Furthermore, activity-dependent caspase-3 activation at inhibitory presynapses induces microglial phagocytosis in mice and increases seizure susceptibility. This increased susceptibility is reversed by genetic depletion of microglial complement receptors. Thus, localized, nonapoptotic caspase activity guides complement-dependent microglial synaptic phagocytosis and remodels neuronal circuits., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The cAMP-response element-binding protein (CREB) transcription factor controls the expression of the neuronal immediate early genes c -fos , Arc , and Bdnf and is essential for long-lasting synaptic plasticity underlying learning and memory. Despite this critical role, there is still ongoing debate regarding the synaptic excitation-transcription (E-T) coupling mechanisms mediating CREB activation in the nucleus. Here we employed optical uncaging of glutamate to mimic synaptic excitation of distal dendrites in conjunction with simultaneous imaging of intracellular Ca 2+ dynamics and transcriptional reporter gene expression to elucidate CREB E-T coupling mechanisms in hippocampal neurons cultured from both male and female rats. Using this approach, we found that CREB-dependent transcription was engaged following dendritic stimulation of N -methyl-d-aspartate receptors (NMDARs) only when Ca 2+ signals propagated to the soma via subsequent activation of L-type voltage-gated Ca 2+ channels resulting in activation of extracellular signal-regulated kinase MAP kinase signaling to sustain CREB phosphorylation in the nucleus. In contrast, dendrite-restricted Ca 2+ signals generated by NMDARs failed to stimulate CREB-dependent transcription. Furthermore, Ca 2+ -CaM-dependent kinase-mediated signaling pathways that may transiently contribute to CREB phosphorylation following stimulation were ultimately dispensable for downstream CREB-dependent transcription and c-Fos induction. These findings emphasize the essential role that L-type Ca 2+ channels play in rapidly relaying signals over long distances from synapses located on distal dendrites to the nucleus to control gene expression., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Eph/ephrin signaling is crucial for organizing retinotopic maps in vertebrates. Unlike other EphAs, which are expressed in the embryonic ventral retina, EphA4 is found in the retinal ganglion cell (RGC) layer at perinatal stages, and its role in mammalian visual system development remains unclear. Using classic in vitro stripe assays, we demonstrate that, while RGC axons are repelled by ephrinB2, they grow on ephrinB1 stripes through EphA4-mediated adhesion. In vivo, retinal axons from EphA4-deficient mice from either sex show impaired arborization in the medial, but not lateral, regions of the superior colliculus that express ephrinB1. Gain-of-function experiments further reveal that ephrinB1-mediated adhesion depends on EphA4 tyrosine kinase activity but it is independent of its sterile alpha motif. Together, our findings suggest that EphA4/ephrinB1 forward signaling likely facilitates adhesion between retinal axon terminals and cells in the medial colliculus, contributing to the establishment of proper connectivity within the visual system., (Copyright © 2024 the authors.)

The extracellular matrix (ECM) stores signaling molecules and facilitates mechanical and biochemical signaling in cells. However, the influence of biomimetic "rejuvenation" ECM structures on aging- and degeneration-related cellular activities and tissue repair is not well understood. We combined physical extrusion and precise "on-off" alternating cross-linking methods to create anisotropic biomaterial microgels (MicroRod and MicroSphere) and explored how they regulate the cell activities of the nucleus pulposus (NP) and their potential antidegenerative effects on intervertebral discs. NP cells exhibited aligned growth along the surface of the MicroRod, enhanced proliferation, and reduced apoptosis. This suggests an adaptive cellular response involving adhesion and mechanosensing, which causes cytoskeletal extension via environmental cues. NP cells maintain nuclear membrane integrity through the YAP/TAZ pathway, which activates the cGAS-STING pathway to rectify the aging mechanisms. In vivo, MicroRod carries NP cells and reduces inflammatory factor and protease secretion in degenerated intervertebral discs, inhibiting degeneration and promoting NP tissue regeneration. Our findings highlight the role of mechanical stress in maintaining cellular activity and antiaging effects in harsh environments, providing a foundation for further research and development of antidegenerative biomaterials.

Bone marrow mesenchymal stromal cells (BM-MSCs) are integral components of the bone marrow microenvironment, playing a crucial role in supporting hematopoiesis. Recent studies have investigated the potential involvement of BM-MSCs in the pathophysiology of acute lymphoblastic leukemia (ALL). However, the exact contribution of BM-MSCs to leukemia progression remains unclear because of conflicting findings and limited characterization. In this study, we compared BM-MSCs derived from pediatric ALL patients with those from matched healthy donors (HDs). Our results indicate that while both ALL-MSCs and HD-MSCs meet the criteria established by the International Society for Cellular Therapy, they exhibit significant differences in proliferation and differentiation capacity. ALL-MSCs displayed markedly lower proliferation rates and reduced osteogenic/adipogenic differentiation potential compared to HD-MSCs. Furthermore, co-culture experiments revealed that MSCs enhance the survival of leukemic blasts through both soluble factors and direct cell-cell interactions, underscoring their anti-apoptotic properties. Importantly, our findings demonstrate that interactions with leukemic cells activate the Wnt/β-catenin signaling pathway in MSCs, suggesting a potential target for therapeutic intervention. Overall, this study enhances our understanding of the role of BM-MSCs in leukemia and highlights β-catenin as a promising target for future therapies., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethical approval: The Medical Ethics Committee of Ankara Pediatric Hematology and Oncology Hospital approved the isolation of MSCs from healthy donors and ALL patients (2014–063). Informed consent was obtained from pediatric patients, their families, and donors., (© 2025. The Author(s).)

Neurons derived from induced pluripotent stem cells (h-iPSC-Ns) provide an invaluable model for studying the physiological aspects of human neuronal development under healthy and pathological conditions. However, multiple studies have demonstrated that h-iPSC-Ns exhibit a high degree of functional and epigenetic diversity. Due to the imprecise characterization and significant variation among the currently available maturation protocols, it is essential to establish a set of criteria to standardize models and accurately characterize and define the developmental properties of human neurons derived from iPSCs. In this study, we conducted comprehensive cellular and network level analysis of the functional development of human neurons, generated from iPSCs obtained from healthy young female peripheral blood mononuclear cells by BDNF and GDNF treatment. We provide a thorough description of the maturation process of h-iPSC-Ns over a 10-week in vitro period using conventional whole-cell patch clamp and dynamic clamp techniques, alongside with morphometry and immunocytochemistry. Additionally, we utilized calcium imaging to monitor the progression of synaptic activity and network communication. At the single cell level, human neurons exhibited gradually decreasing membrane resistance in parallel with improved excitability. By the fifth week of maturation, firing profiles were consistent with those of mature regular firing type of neurons. At the network level, fast glutamatergic and depolarizing GABAergic synaptic connections were abundant together with synchronized network activity from the sixth week of maturation. Alterations in the expression of GABA A receptor subunits were also observed during the process of maturation. The sequence of differentiation events was consistent, providing a robust temporal framework to execute experiments at defined stages of neuronal maturation as well as to use a specific set of experiments to assess a culture's maturation. The uncovered progression of differentiation events provides a powerful tool to aid the planning and designing of targeted experiments during defined stages of neuronal maturation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Retinal diseases often lead to degeneration of specific retinal cell types with currently limited therapeutic options to replace the lost neurons. Previous studies have reported that overexpression of ASCL1 or combinations of proneural factors in Müller glia (MG) induce regeneration of functional neurons in the adult mouse retina. Recently, we applied the same strategy in dissociated cultures of fetal human MG and although we stimulated neurogenesis from MG, our effect in 2D cultures was modest and our analysis of newborn neurons was limited. In this study, we aimed to improve our MG reprogramming strategy in a more intact retinal environment. For this purpose, we used an in vitro culture system of human fetal retinal tissue and adult human postmortem retina. To stimulate reprogramming, we used lentiviral vectors to deliver constructs with a glial-specific promoter (HES1) driving ASCL1 alone or in combination with additional developmental transcription factors (TFs) such as ATOH1 and NEUROD1 . Combining IHC, scRNA-seq, and electrophysiology, we show that human MG can generate new neurons even in adults. This work constitutes a key step toward a future clinical application of this regenerative medicine approach for retinal degenerative disorders., Competing Interests: Competing interests statement:Some of the research described is included in a patent owned by the University of Washington and licensed to Tenpoint Biopharma. T.A.R. has equity in Tenpoint. U.S. Patent Application Serial No. 17/050,999 filed October 27, 2020 for methods and compositions to stimulate retinal regeneration. T.A.R. receives consulting fees from Tenpoint Biopharma.

This study aimed to investigate the effects of CpG oligodeoxynucleotide (CpG-ODNs)-coated chitosan nanoparticles (CNP) on the phenotype of murine macrophages and their proinflammatory cytokine profile in vitro. CNP-CpG-ODNs loaded with FITC-scrambled siRNA were prepared using the ionotropic gelation method. Peritoneal macrophages were isolated and exposed to CNP-CpG-ODNs. Treated macrophages were assessed for uptake capacity. Flow cytometry was used to evaluate the expression levels of MHC-II, CD40, and CD86 costimulatory molecules in treated macrophages. Furthermore, the secretion levels of proinflammatory cytokines (TNF-α and IL-6) and the release of nitric oxide (NO) were measured in the culture supernatant of treated macrophages using sandwich ELISA and the Griess reaction, respectively. These in vitro studies showed that CNP-CpG-ODNs had no cytotoxic effect on macrophages and were efficiently taken up by them. Additionally, CNP-CpG-ODNs significantly increased the production of TNF-α, IL-6, and NO in the culture supernatant compared to CNP alone. Moreover, CNP-CpG-ODNs enhanced the expression of MHC-II, CD40, and CD86 costimulatory molecules on macrophages. These findings indicate that incorporating CpG-ODNs into CNPs promotes macrophage maturation and a proinflammatory phenotype. Therefore, CNP-CpG-ODNs may serve as an effective system for targeted gene delivery to macrophages, enhancing immune responses., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In Brief: PI3K-AKT signaling activates steroidogenesis by inducing estradiol and progesterone production, while MEK-ERK1/2 signaling regulates steroidogenesis by inhibiting estradiol and inducing progesterone production in granulosa cells (GCs). Both pathways are essential for glycolytic and mitochondrial metabolism in these cells., Abstract: The PI3K-AKT and MEK-ERK1/2 signaling pathways are integral to fundamental cellular processes, such as proliferation, viability and differentiation. In GCs, these pathways are activated by follicle-stimulating hormone (FSH) and IGF1 through respective receptors. We investigated the comparative transcriptome changes induced by the AKT and ERK (ERK1/2) pathways using corresponding inhibitors in GCs. GCs isolated from antral follicles showed positive signals for phospho-AKT and phospho-ERK proteins. Treatment of cultured GCs with FSH and IGF1 induced phospho-AKT and phospho-ERK levels. Transcriptome analysis revealed 1436 genes regulated by AKT and 654 genes regulated by the ERK pathway. Among these, 94 genes were commonly downregulated and 11 genes were commonly upregulated in both datasets, while 110 genes were oppositely regulated. Bioinformatics analysis revealed that the inhibition of the PI3K-AKT and MEK-ERK pathways downregulates key reproductive processes and upstream molecules. Notably, AKT inhibition affected FSH, ESRRG and HIF1 pathways, while ERK inhibition impacted CG, FOS, TGFβ, EGR1 and LH pathways. Transcriptome data showed that genes related to estradiol production were inhibited by ERK and induced by the AKT pathway. This was verified by radioimmunoassays, and mRNA and protein analysis of CYP19A1 and STAR genes. In addition, transcriptome data suggested the downregulation of glucose metabolism in GCs. Using validation experiments, we confirm that both pathways are essential for glucose uptake, lactate production and mitochondrial activity in GCs. These data provide a resource for informing future research for analyzing various novel candidate genes regulated by the AKT and ERK pathways in GCs and other cell types.

For cultured meat to effectively replace traditional meat, it is essential to develop scaffolds that replicate key attributes of real meat, such as taste, nutrition, flavor, and texture. However, one of the significant challenges in replicating meat characteristics with scaffolds lies in the considerable gap between the stiffness preferred by cells and the textural properties desired by humans. To address this issue, we focused on the microscale environment conducive to cell growth and the macro-scale properties favored by humans. This led to the development of the adaptive bio-orchestrating anisotropic scaffold (ABS), which satisfies both cellular and human requirements. The ABS is produced using the anisotropic freeze-initiated ion coordination method, which sequentially aligns and enhances the fibril structure of food-derived proteins, effectively bridging the gap between cellular and culinary perspectives. Notably, the microenvironments of the ABS exhibited exceptional myoblast cell differentiation, with macro-scale 3D mechanical textures that are consistent regardless of the chewing direction, due to the aligned fibril and cell structure. The ABS containing bovine myotubes demonstrated a mechanical texture nearly identical to that of beef sirloins.