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BACKGROUND: Oxidative stress plays a critical role in intervertebral disc degeneration. As a reducing material with good biocompatibility, black phosphorus quantum dots have the potential to resist oxidative stress and retard intervertebral disc degeneration. OBJECTIVE: To evaluate the effect of black phosphorus quantum dots on scavenging reactive oxygen species in the microenvironment of an intervertebral disc through in vivo and in vitro experiments, and further explore the role of black phosphorus quantum dots in Nrf2/ARE pathway and intervertebral disc inflammation. METHODS: Black phosphorus quantum dots were prepared by a liquid exfoliation technique. (1) In vitro experiment: The nucleus pulposus cells of SD rats were isolated and extracted, and the passages 2-4 nucleus pulposus cells were cocultured with different solutions, including F12-DMEM medium (blank group), black phosphorus quantum dot solution, hydrogen peroxide solution, hydrogen peroxide+black phosphorus quantum dot solution, hydrogen peroxide+black phosphorus quantum dot+Nrf2 specific inhibitor ML385 solution. Cell live/dead staining and intracellular reactive oxygen species, mitochondrial membrane potential and western blot assay were performed respectively. (2) In vivo experiment: Thirty SD rats were randomly divided into sham operation, puncture and puncture + black phosphorus groups, with 10 rats in each group. A Co7-10 intervertebral disc degeneration model was established using intervertebral disc puncture in the puncture group and the puncture+black phosphorus group. Black phosphorus quantum dot solution was injected in the intervertebral disc after a puncture in the puncture+black phosphorus group. The intervertebral disc tissue imaging and histological staining were evaluated at 4 and 8 weeks after surgery. RESULTS AND CONCLUSION: (1) In vitro experiment: Live/dead staining revealed that the black phosphorus quantum dots had good biocompatibility, were non-toxic to cells, and had a protective effect on nucleus pulposus cells under oxidative stress. Intracellular reactive oxygen species and JC-1 fluorescent probes showed that black phosphorus quantum dots could regulate the reduction of mitochondrial membrane potential caused by oxidative stress in nucleus pulposus cells and protected cells from hydrogen peroxidation-induced intracellular oxidative stress. Western blot analysis showed that compared with the blank group, the protein expressions of Nrf2, heme oxygenase 1, quinone oxidoreductase and type II collagen were decreased in the hydrogen peroxide group (P < 0.05), while the protein expressions of tumor necrosis factor α, interleukin 1β, matrix metalloproteinase 13 and p65 were increased (P < 0.05). The addition of black phosphorus quantum dots could reverse the inhibitory effect of hydrogen peroxide on the Nrf2 pathway and reduce the inflammatory response caused by oxidative stress, but NrF2-specific inhibitors could cancel this effect. (2) In vivo experiment: X-ray and MRI demonstrated that at 4 and 8 weeks after surgery, the intervertebral disc height and water content of nucleus pulposus in the puncture group were lower than those in the sham operation group (P < 0.05), and the intervertebral disc height and water content of nucleus pulposus in the puncture+black phosphorus group were higher than those in the puncture group (P < 0.05). Histological staining exhibited that the degree of intervertebral disc degeneration in the puncture+black phosphorus group was less than that in the puncture group, and the expression of heme oxygenase 1 protein was higher than that in the puncture+black phosphorus group. (3) Our results have indicated that black phosphorus quantum dots can exert an antioxidant effect and delay intervertebral disc degeneration by regulating Nrf2/ARE pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Ischemic postconditioning is one of the effective ways to reduce ischemia-reperfusion injury and has been more and more widely used in clinical practice in recent years, but its specific molecular mechanism has yet to be studied. OBJECTIVE: To investigate the role and mechanism of piRNA-005854 in the aging cardiomyocytes caused by hypoxic postconditioning. METHODS: In vitro, cardiomyocytes were administered 8 mg/mL D-galactose for 9 days to induce their aging. β-Galactosidase staining was used to observe the aging of cardiomyocytes. Senescent cells were treated with hypoxia/reoxygenation and hypoxic postconditioning. ELISA was utilized to detect changes in myocardial injury markers creatine kinase isoenzyme MB and lactate dehydrogenase levels. Western blot assay was applied to detect the expression changes of autophagy-related proteins LC3II, p62, ULK1 and phosphorylated ULK1 in aging cardiomyocytes. qRT-PCR was employed to determine the expression level of piRNA-005854. piRNA-005854 inhibitor and piRNA-005854 mimics were transferred into aging cardiomyocytes and followed with hypoxic postconditioning. Western blot assay was used to examine the expression of LC3II, p62, ULK1 and phosphorylated ULK1. RESULTS AND CONCLUSION: (1) D-galactose induced obvious senescence of cardiomyocytes 9 days later. (2) Compared with the normoxia group, creatine kinase isoenzyme MB and lactate dehydrogenase levels increased in the hypoxia/reoxygenation group (P < 0.01); LC3 II/I expression was increased; p62 expression was decreased; ULK1 phosphorylation level was increased, and piRNA-005854 expression was increased (P < 0.01). (3) Compared with the hypoxia/ reoxygenation group, creatine kinase isoenzyme MB and lactate dehydrogenase levels significantly reduced in the hypoxic postconditioning group (P < 0.01); LC3 II/I expression significantly decreased (P < 0.05); p62 expression increased (P < 0.01); ULK1 phosphorylation level decreased (P < 0.05), and piRNA-005854 expression decreased (P < 0.01). (4) After transfection of piRNA-005854 inhibitor, LC3II/I expression was decreased (P < 0.01); the expression of p62 was increased significantly (P < 0.05); the phosphorylation level of ULK1 was decreased significantly (P < 0.01). After transfection of piRNA-005854 mimics, LC3II/ I expression was increased significantly; the expression of p62 was decreased, and the phosphorylation level of ULK1 was increased significantly (P < 0.01). (5) The results show that piRNA-005854-mediated reduction of ULK1-dependent autophagy level is a possible mechanism that hypoxic postconditioning exerts its protective effect on aging cardiomyocytes. [ABSTRACT FROM AUTHOR]

Objectives Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation–associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein. Methods We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n  = 8) and serum (n  = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using western blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains. Results All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein. Conclusions Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade. [ABSTRACT FROM AUTHOR]

Objective Autoantibodies against MDA5 (melanoma differentiation-associated protein 5) serve as a biomarker for DM (dermatomyositis) and indicate a risk factor for interstitial lung disease (ILD). MDA5 is a protein responsible for sensing RNA virus infection and activating signalling pathways against it. However, little is known about the antigen epitopes on MDA5 autoantibodies. We aimed to determine the interaction of the MDA5 autoantibody–antigen epitope. Methods Cell-based assays (CBAs), immunoprecipitation-immunoblot assays, and various immunoblotting techniques were used in the study. Results We demonstrated that DM patient autoantibodies recognize MDA5 epitopes in a native conformation–dependent manner. Furthermore, we identified the central helicase domain （3Hel） formed by Hel1, Hel2i, Hel2, and pincer as the major epitopes. As proof of principle, the purified 3Hel efficiently absorbed MDA5 autoantibodies from patient sera through immunoprecipitation-immunoblot assay. Conclusion Our study uncovered the nature of the antigen epitopes on MDA5 and can provide guidance for diagnosis and a targeted therapeutic approach development. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer (PaC) is a highly malignant gastrointestinal tumor with invasive and metastatic characteristics. Interleukin-6 (IL-6), a negative prognostic marker, contributes to PaC progression. However, the mechanism of IL-6 in PaC is not yet fully understood. Methods: miR-455-5p levels were first tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in PaC tissues or cells. Subsequently, PaC cell-related functions were identified through CCK-8, Transwell, and Western blotting. Changes in miR-455-5p and IGF-1R expression were confirmed using RT-qPCR and Western blotting. miR-455-5p methylation was assessed by bisulfite sequencing PCR. Results: The authors discovered that miR-455-5p was expressed at low levels in PaC tissues and cells, and miR-455-5p expression was observably reduced by IL-6 in PaC cells. In addition, IL-6 dramatically induces miR-455-5p methylation in PaC cells. Functionally, the data revealed that IL-6 could facilitate the malignant properties of PaC cells, including proliferation, epithelial–mesenchymal transition, and metastasis. The authors found that miR-455-5p could suppress the progression of PaC cells by downregulating IGF-1R in PaC cells. Mechanistically, IL-6 downregulated miR-455-5p and upregulated IGF-1R, and miR-455-5p reduced IGF-1R expression through targeted binding. Conclusions: The authors demonstrated that the miR-455-5p/IGF-1R axis is necessary for the induction of IL-6 in PaC progression. The results here may provide a theoretical basis for the application of the IL-6/miR-455-5p/IGF-1R axis in the clinical therapy of PaC. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer is a common male malignancy and the leading cause of cancer death in men. Long non-coding RNAs (lncRNAs), microRNA (miRNAs) and mRNAs networks mediate prostate cancer progression. Herein, we investigated the functions of lncRNA AC008972.1 and its regulatory mechanism in prostate cancer. Materials and Methods: The expression levels of lncRNA AC008972.1, miR-143-3p, and TAOK2 were detected in prostate cancer tissues and cell lines by reverse transcription–quantitative polymerase chain reaction. PC3 and LNCaP cells were used to establish lncRNA AC008972.1-knockdown, miR-143-3p-overexpressing, and thousand-and-one-amino acid 2 kinase (TAOK2)-downregulated cells. Cell viability was examined by MTT assays and cell proliferation was detected by clone formation assay. Cell migration and invasion were detected by wound scratch assay and transwell chamber assay. The apoptosis rate was analyzed by flow cytometry. The protein expression was detected by Western blot assay. The RNA interaction was explored and validated by RNA binding protein immunoprecipitation (RIP) assay and dual luciferase activity assay. A mouse xenograft model was established to investigate the effect of lncRNA AC008972.1 on prostate cancer progression. Results: High expression of lncRNA AC008972.1 was associated with low overall survival in prostate cancer patients. Downregulation of lncRNA AC008972.1 suppressed prostate cancer progression by inhibiting cell viability, proliferation, migration, and invasion, in addition to the EMT process, whereas cell apoptosis was significantly promoted. LncRNA AC008972.1 bound with miR-143-3p and negatively regulated miR-143-3p expression. MiR-143-3p overexpression suppressed prostate cancer malignant behaviors in vitro. TAOK2 expression was decreased by miR-143-3p through the complementary targeting of TAOK2 mRNA. Downregulation of lncRNA AC008972.1 mitigated prostate cancer malignant behaviors in vitro based on miR-143-3p/TAOK2 node. Furthermore, the data of xenograft model experiment showed that inhibition of lncRNA AC008972.1 suppressed tumor growth in vivo. Conclusions: Knockdown of lncRNA AC008972.1 inhibits prostate cancer cell growth via downregulation of TAOK2 induced by miR-143-3p. LncRNA AC008972.1 acts as an oncogene in the progression of prostate cancer and may provide a novel therapeutic target for prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) play critical roles in the tumorigenesis and radiosensitivity of multiple cancers. Nevertheless, the biological functions of circRNA periostin (circ‐POSTN) in esophageal cancer (EC) progression and radiosensitivity have not been well elucidated. Methods: The expression of circ‐POSTN, microRNA‐876‐5p (miR‐876‐5p), and proto‐oncogene tyrosine‐protein kinase (FYN) was analyzed by quantitative reverse transcription PCR (RT‐qPCR). Cell proliferation was assessed by MTT, colony formation, and 5‐ethynyl‐2′‐deoxyuridine (EDU) assays. All protein levels were detected by western blot assay. Cell apoptosis and invasion were assessed by flow cytometry analysis and transwell assay, respectively. Dual‐luciferase reporter and RNA immunoprecipitation (RIP) assays were used to validate the interaction between miR‐876‐5p and circ‐POSTN or FYN. The role of circ‐POSTN in vivo was explored by establishing mice xenograft model. Results: Circ‐POSTN was overexpressed in EC tissues and cells. Knockdown of circ‐POSTN inhibited cell proliferation and invasion and elevated apoptosis and radiosensitivity in EC cells. MiR‐876‐5p was a direct target of circ‐POSTN, and its knockdown reversed the role of sh‐circ‐POSTN in EC cells. FYN was a direct target of miR‐876‐5p, and FYN elevation weakened the effects of miR‐876‐5p overexpression on the progression and radiosensitivity of EC cells. Moreover, circ‐POSTN acted as a miR‐876‐5p sponge to regulate FYN expression. Circ‐POSTN interference also suppressed tumor growth and enhanced radiosensitivity in vivo. Conclusion: Circ‐POSTN knockdown inhibited proliferation and invasion, but increased apoptosis and enhanced radiosensitivity in EC cells via modulating miR‐876‐5p/FYN axis, which might be a potential diagnostic and therapeutic target for EC. [ABSTRACT FROM AUTHOR]

DNA damage response (DDR) is the principal mechanism regulating genomic stability and cell cycle checkpoint activation by coordinating DNA repair and apoptotic pathways. Ataxia telangiectasia and Rad3-related protein (ATR) play a significant role in the DDR due to its capability to detect a wide spectrum of DNA damage. Therefore, targeting DDR, specifically ATR, is a promising therapeutic strategy in cancer treatment. Furthermore, the inhibition of ATR sensitizes cancer cells to radiotherapy (RT). Herein, we, for the first time, investigated the synergistic effects of Elimusertib (BAY-1895344) as a highly potent selective ATR inhibitor with RT combination in triple-negative breast cancer (TNBC), in vitro. MDA-MB-231 TNBC cells were firstly treated with different concentrations of Elimusertib for 24 h and then exposed to 4 and 8 Gy of X-ray irradiation. After post-irradiation for 72 h, WST-1, Annexin V, cell cycle, acridine orange/propidium iodide, mitochondria staining and western blot analysis were conducted. Our findings showed that 4 Gy irradiation and lower doses (especially 2 and 4 nM) of Elimusertib combination exerted a considerable anticancer activity at 72 h post-irradiation through apoptotic cell death, marked nuclear and mitochondrial damages and the suppression of ATR-Chk1 based DDR mechanism. ATR inhibition by Elimusertib in combination with RT may be a promising new treatment strategy in the treatment of TNBC. However, further experiments should be performed to elucidate the underlying molecular mechanisms of the therapeutic efficacy of this combination treatment and its association with DNS repair mechanisms in TNBC, in vitro and in vivo. [ABSTRACT FROM AUTHOR]

It is well recognized that mitochondrial dynamics plays a vital role in cartilage physiology. Any perturbation in mitochondrial dynamics could cause disorders in cartilage metabolism and even lead to the occurrence of cartilage diseases such as osteoarthritis (OA). TGF‐β3, as an important growth factor that appears in the joints of OA disease, shows its great potential in chondrocyte growth and metabolism. Nevertheless, the role of TGF‐β3 on mitochondrial dynamics is still not well understood. Here we aimed to investigate the effect of TGF‐β3 on mitochondrial dynamics of chondrocytes and reveal its underlying bio‐mechanism. By using transmission electron microscopy (TEM) for the number and morphology of mitochondria, western blotting for the protein expressions, immunofluorescence for the cytoplasmic distributions of proteins, and RNA sequencing for the transcriptome changes related to mitochondrial dynamics. We found that TGF‐β3 could increase the number of mitochondria in chondrocytes. TGF‐β3‐enhanced mitochondrial number was via promoting the mitochondrial fission. The mitochondrial fission induced by TGF‐β3 was mediated by AMPK signaling. TGF‐β3 activated canonical p‐Smad3 signaling and resultantly mediated AMPK‐induced mitochondrial fission. Taken together, these results elucidate an understanding of the role of TGF‐β3 on mitochondrial dynamics in chondrocytes and provide potential cues for therapeutic strategies in cartilage injury and OA disease in terms of energy metabolism. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer (OVCA) is prevalent in female reproductive organs. Despite recent advances, clinical outcomes remain poor, warranting fresh treatment avenues. Honokiol has an inhibitory effect on proliferation, invasion, and survival of cancer cells in vitro and in vivo. Therefore, this study intended to explore specific molecular mechanism by which honokiol affected OVCA progression. Methods: Bioinformatics analyzed the drug honokiol that bound to OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2). Cellular thermal shift assay (CETSA) verified the binding relationship between honokiol and OTUB2. Cell counting kit 8 (CCK‐8) tested the IC50 value and cell viability of OVCA cells after honokiol treatment. Corresponding assay kits determined malonic dialdehyde (MDA) and Fe2+ levels in OVCA cells. Flow cytometry measured reactive oxygen species levels. Western blot detected OTUB2, SLC7A11, and transcriptional co‐activators Yes‐associated protein (YAP) expression, and quantitative polymerase chain reaction (qPCR) detected OTUB2 expression. Immunohistochemistry (IHC) detected the expression level of Ki67 protein in tumor tissues. Results: Honokiol was capable of inducing ferroptosis in OVCA cells. CETSA confirmed that honokiol could bind to OTUB2. Further cell functional and molecular experiments revealed that honokiol induced ferroptosis in OVCA cells via repression of YAP signaling pathway through binding to OTUB2. In addition, in vivo experiments have confirmed that honokiol could inhibit the growth of OVCA. Conclusion: Honokiol induced ferroptosis in OVCA cells via repression of YAP signaling pathway through binding to OTUB2, implicating that OTUB2 may be an effective target for OVCA treatment, and our study results may provide new directions for development of more effective OVCA treatment strategies. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting. [ABSTRACT FROM AUTHOR]

Aim: Prolonged high‐fat diet (HFD) consumption has been shown to impair cognition and depression. The combined effects of HFD and lipopolysaccharide (LPS) administration on those outcomes have never been thoroughly investigated. This study investigated the effects of LPS, HFD consumption, and a combination of both conditions on microglial dysfunction, microglial morphological alterations, synaptic loss, cognitive dysfunction, and depressive‐like behaviors. Methods: Sixty‐four male Wistar rats were fed either a normal diet (ND) or HFD for 12 weeks, followed by single dose‐subcutaneous injection of either vehicle or LPS. Then, cognitive function and depressive‐like behaviors were assessed. Then, rats were euthanized, and the whole brain, hippocampus, and spleen were collected for further investigation, including western blot analysis, qRT‐PCR, immunofluorescence staining, and brain metabolome determination. Results: HFD‐fed rats developed obese characteristics. Both HFD‐fed rats with vehicle and ND‐fed rats with LPS increased cholesterol and serum LPS levels, which were exacerbated in HFD‐fed rats with LPS. HFD consumption, but not LPS injection, caused oxidative stress, blood–brain barrier disruption, and decreased neurogenesis. Both HFD and LPS administration triggered an increase in inflammatory genes on microglia and astrocytes, increased c1q colocalization with microglia, and increased dendritic spine loss, which were exacerbated in the combined conditions. Both HFD and LPS altered neurotransmitters and disrupted brain metabolism. Interestingly, HFD consumption, but not LPS, induced cognitive decline, whereas both conditions individually induced depressive‐like behaviors, which were exacerbated in the combined conditions. Conclusions: Our findings suggest that LPS aggravates metabolic disturbances, neuroinflammation, microglial synaptic engulfment, and depressive‐like behaviors in obese rats. [ABSTRACT FROM AUTHOR]

Aims: Sodium‐glucose cotransporter 2 (SGLT2) inhibitors such as canagliflozin (CANA) have emerged as an effective adjuvant therapy in the management of diabetes, however, past observations suggest CANA may alter skeletal muscle mass and function. The purpose of this work was to investigate the effects of CANA on skeletal muscle metabolism both with and without insulin resistance. Methods: C2C12 myotubes were treated with CANA with or without insulin resistance. Western blot and qRT‐PCR were used to assess protein and gene expression, respectively. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Mitochondrial, nuclei and lipid content were measured using fluorescent staining and microscopy. Results: CANA decreased mitochondrial function and glycolytic metabolism as did insulin resistance, however, these changes occurred without significant alterations in gene expression associated with each pathway. Additionally, while insulin resistance reduced insulin‐stimulated pAkt expression, CANA had no significant effect on insulin sensitivity. Conclusions: CANA appears to reduce mitochondrial and glycolytic metabolism without altering gene expression governing these pathways, suggesting a reduction in substrate may be responsible for lower metabolism. [ABSTRACT FROM AUTHOR]

Fibroblast activation protein (FAP) has been indicated to express in cancer‐associated fibroblasts (CAFs) in most cancers. This work was dedicated to exploring FAP's effects on hepatocellular carcinoma (HCC). The data were extracted from The Cancer Genome Atlas, Gene Expression Omnibus, ImmPort, and Reactome databases. The correlation between FAP and HCC patients' prognosis was explored via survival analysis. The qRT‐PCR and western blot analysis were used to analyze the FAP mRNA and protein expression levels, respectively. The cell proliferation and apoptosis were determined using the cell counting kit‐8 assay kit and Annexin V‐FITC/PI apoptosis kit, respectively. The HCC patients with FAP overexpression displayed a worse prognosis. The FAP expression was positively associated with the infiltration levels of tumor purity, B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, and dendritic cell. The optimal nine immune related genes were screened between two groups (FAP high vs. low). Moreover, we identified 24 energy metabolism related genes (FAP high vs. low) and these 24 genes were highly expressed in the high FAP expression group. The FAP expression had a significant positive correlation with the expression of PD‐1, CTLA4, PDL‐1, and PDL‐2. The FAP overexpression promoted proliferation and migration while inhibiting the apoptosis of HCC cells. The FAP overexpression promoted the progression of HCC by regulating the immunity to affect the prognosis of HCC patients, thereby serving as a poor prognostic marker for HCC patients. [ABSTRACT FROM AUTHOR]

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T‐cell‐specific adapter protein (TSAd), interacts with the non‐receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C‐terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline‐rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST‐pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co‐expressed and may interact with Fyn, we were unable to co‐precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd‐Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co‐expression of competing binding partners. [ABSTRACT FROM AUTHOR]

Currently, the prognosis of osteosarcoma (OS) remains discouraging, especially in elderly/metastatic OS patients. By impairing the antitumor effect of immune cells, tumor immune microenvironment (TIME) provides an environment conducive to tumor proliferation, which highly requires accelerated nicotinamide adenine dinucleotide (NAD+) metabolism for energy. Recently, many genes involved in the sustained production of NAD+ in malignant tumors have been verified to be possible prognostic indicators and therapeutic targets. Therefore, the current study was to probe into the association of NAD+ metabolism‐related genes with TIME, immunotherapeutic response, and prognosis in OS. All OS data for the study were acquired from TARGET and GEO databases. In bioinformatics analysis, we performed Cox analysis, consensus clustering, principal component analysis, t‐distributed stochastic neighbor embedding, uniform manifold approximation and projection, gene set enrichment analysis, gene set variation analysis, Lasso analysis, survival and ROC curves, nomogram, immune‐related analysis, drug sensitivity analysis, and single‐cell RNA sequencing (scRNA‐seq) analysis. Cell transfection assay, RT‐qPCR, western blot analysis, as well as cell wound healing, migration, and invasion assays were performed in vitro. Bioinformatics analysis identified A&B clusters and six NAD+ metabolism‐related differentially expressed genes, constructed risk model and nomogram, and performed immune‐related analysis, drug susceptibility analysis, and scRNA‐seq analysis to inform the clinical treatment framework. In vitro experiment revealed that CBS and INPP1 can promote migration, proliferation as well as invasion of OS cells through TGF‐β1/Smad2/3 pathway. Based on bioinformatics analysis and in vitro validation, this study confirmed that NAD+ metabolism affects TIME to suggest the prognosis of OS. [ABSTRACT FROM AUTHOR]

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague–Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell‐substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F‐actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho‐focal adhesion kinase (FAK), FAK, phospho‐paxillin, paxillin, and F‐actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F‐actin distribution. Lidocaine reduced F‐actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho‐FAK, F‐actin, and phospho‐paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F‐actin, phospho‐FAK, and phospho‐paxillin expression when cells are treated with lidocaine. [ABSTRACT FROM AUTHOR]

• This study revealed that Asn124, Asn135 and Asn492 in OATP1A2 and Asn176 and Asn538 in OATP2B1 are N -glycosylation sites. • The effect of deletion of N -glycans on the transport activity of OATPs was evaluated based on the kinetics parameters per single molecules using the expression level of OATPs on the plasma membrane quantified by LC-MS/MS. • Among the three N -glycans of OATP1A2, those at Asn124 and Asn135 are especially important for the expression on the plasma membrane and transport activity. • Deletion of each N -glycan of OATP2B1 reduces the expression on the plasma membrane and enhances substrate affinity, and these effects becomes stronger when both N -glycans are disrupted. Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N -glycosylation sites, but their influence remains unclear. This study aimed to identify the N -glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N -glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N -glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N -glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function. [ABSTRACT FROM AUTHOR]

Herbal supplements containing several types of plant sterols, vitamins, and minerals, are marketed for prostate health. In the majority of these supplements, the most abundant plant sterol is saw palmetto extract or its' principal component, beta-sitosterol. In terms of prostate health, previous work almost exclusively focused on the effects of beta-sitosterol on prostatic epithelium, with little attention paid to the effects on prostatic stroma. This omission is a concern, as the abnormal accumulation of collagen, or fibrosis, of the prostatic stroma has been identified as a factor contributing to lower urinary tract symptoms and dysfunction in aging men. To address whether beta-sitosterol may be promoting prostatic fibrosis, immortalized and primary prostate stromal fibroblasts were subjected to immunoblotting, immunofluorescence, qRT-PCR, ELISA, and image quantitation and analysis techniques to elucidate the effects of beta-sitosterol on cell viability and collagen expression and cellular localization. The results of these studies show that beta-sitosterol is nontoxic to prostatic fibroblasts and does not stimulate collagen production by these cells. However, beta-sitosterol alters collagen distribution and sequesters collagen within prostatic fibroblasts, likely in an age-dependent manner. This is a significant finding as prostate health supplements are used predominantly by middle aged and older men who may, then, be affected disproportionately by these effects. [ABSTRACT FROM AUTHOR]

The Beige and Chediak‐Higashi (BEACH) domain‐containing, Neurobeachin‐like 2 (NBEAL2) protein is a molecule with a molecular weight of 300 kDa. Inactivation of NBEAL2 by loss‐of‐function mutations in humans as well as deletion of the Nbeal2 gene in mice results in functional defects in cells of the innate immune system such as neutrophils, NK‐cells, megakaryocytes, platelets and of mast cells (MCs). To investigate the detailed function of NBEAL2 in murine MCs we generated MCs from wild type (wt) and Nbeal2−/− mice, and deleted Nbeal2 by CRISPR/Cas9 technology in the murine mast cell line MC/9. We also predicted the structure of NBEAL2 to infer its function and to examine potential mechanisms for its association with interaction partners by using the deep learning‐based method RoseTTAFold and the Pymol© software. The function of NBEAL2 was analysed by molecular and immunological techniques such as co‐immunoprecipitation (co‐IP) experiments, western blotting, enzyme‐linked immunosorbent assay and flow cytometry. We identified RPS6 as an interaction partner of NBEAL2. Thereby, the NBEAL2/RPS6 complex formation is probably required to control the protein homeostasis of RPS6 in MCs. Consequently, inactivation of NBEAL2 leads to accumulation of strongly p90RSK‐phosphorylated RPS6 molecules which results in the development of an abnormal MC phenotype characterised by prolonged growth factor‐independent survival and in a pro‐inflammatory MC‐phenotype. [ABSTRACT FROM AUTHOR]

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity. [ABSTRACT FROM AUTHOR]

Background: Aberrant expressions of desmoglein 2 (Dsg2) and desmocollin 2(Dsc2), the two most widely distributed desmosomal cadherins, have been found to play various roles in cancer in a context-dependent manner. Their specific roles on breast cancer (BC) and the potential mechanisms remain unclear. Methods: The expressions of Dsg2 and Dsc2 in human BC tissues and cell lines were assessed by using bioinformatics analysis, immunohistochemistry and western blotting assays. Wound-healing and Transwell assays were performed to evaluate the cells' migration and invasion abilities. Plate colony-forming and MTT assays were used to examine the cells' capacity of proliferation. Mechanically, Dsg2 and Dsc2 knockdown-induced malignant behaviors were elucidated using western blotting assay as well as three inhibitors including MK2206 for AKT, PD98059 for ERK, and XAV-939 for β-catenin. Results: We found reduced expressions of Dsg2 and Dsc2 in human BC tissues and cell lines compared to normal counterparts. Furthermore, shRNA-mediated downregulation of Dsg2 and Dsc2 could significantly enhance cell proliferation, migration and invasion in triple-negative MDA-MB-231 and luminal MCF-7 BC cells. Mechanistically, EGFR activity was decreased but downstream AKT and ERK pathways were both activated maybe through other activated protein tyrosine kinases in shDsg2 and shDsc2 MDA-MB-231 cells since protein tyrosine kinases are key drivers of triple-negative BC survival. Additionally, AKT inhibitor treatment displayed much stronger capacity to abolish shDsg2 and shDsc2 induced progression compared to ERK inhibition, which was due to feedback activation of AKT pathway induced by ERK inhibition. In contrast, all of EGFR, AKT and ERK activities were attenuated, whereas β-catenin was accumulated in shDsg2 and shDsc2 MCF-7 cells. These results indicate that EGFR-targeted therapy is not a good choice for BC patients with low Dsg2 or Dsc2 expression. Comparatively, AKT inhibitors may be more helpful to triple-negative BC patients with low Dsg2 or Dsc2 expression, while therapies targeting β-catenin can be considered for luminal BC patients with low Dsg2 or Dsc2 expression. Conclusion: Our finding demonstrate that single knockdown of Dsg2 or Dsc2 could promote proliferation, motility and invasion in triple-negative MDA-MB-231 and luminal MCF-7 cells. Nevertheless, the underlying mechanisms were cellular context-specific and distinct. [ABSTRACT FROM AUTHOR]

Erythrocytes are impressive tools for drug delivery, especially to macrophages. Therefore, berberine was loaded into erythrocytes using both hypotonic pre-swelling and endocytosis methods to target macrophages. Physicochemical and kinetic parameters of the resulting carrier cells, such as drug loading/release kinetics, osmotic fragility, and hematological indices, were determined. Drug loading was optimized for the study using Taguchi experimental design and lab experiments. Loaded erythrocytes were targeted to macrophages using ZnCl2 and bis-sulfosuccinimidyl-suberate, and targeting was evaluated using flow cytometry and Wright–Giemsa staining. Differentiated macrophages were stimulated with lipopolysaccharide, and the inflammatory profiles of macrophages were evaluated using ELISA, western blotting, and real-time PCR. Findings indicated that the endocytosis method is preferred due to its low impact on the erythrocyte's structural integrity. Maximum loading achieved (1386.68 ± 22.43 μg/ml) at 1500 μg/ml berberine treatment at 37 °C for 2 h. Berberine successfully inhibited NF-κB translation in macrophages, and inflammatory response markers such as IL-1β, IL-8, IL-23, and TNF-α were decreased by approximately ninefold, sixfold, twofold, eightfold, and twofold, respectively, compared to the LPS-treated macrophages. It was concluded that berberine-loaded erythrocytes can effectively target macrophages and modulate the inflammatory response. [ABSTRACT FROM AUTHOR]

Background: Cell division cycle associated 5 (CDCA5) plays ontogenetic role in various human cancers. However, its specific function and regulatory mechanism in ccRCC remain uncertain. Methods: Immunohistochemistry and western blots were performed to investigate the expression of CDCA5 in ccRCC tissues. Genetic knockdown and upregulation of CDCA5 were performed to investigate its functional roles in ccRCC proliferation, migration, apoptosis and sunitinib resistance. Furthermore, Co-IP assay and LC–MS/MS were performed to investigate the underlying mechanisms. Results: We found that CDCA5 expression is frequently upregulated in ccRCC tumors and is associated with poor prognosis of ccRCC patients. Functionally, CDCA5 promotes proliferation, migration, and sunitinib resistance, while inhibiting apoptosis in ccRCC cells. In vivo mouse xenograft model confirms that silencing of CDCA5 drastically inhibits the growth of ccRCC. Mechanistically, we discovered that CDCA5 interacts with Eukaryotic Translation Elongation Factor 1 Alpha 1 (EEF1A1) to regulate mTOR signaling pathway, thereby promoting ccRCC progression. Conclusions: Taken together, our results demonstrate the significant role of CDCA5 in ccRCC progression. The findings may provide insights for the development of new treatment strategies targeting CDCA5 for ccRCC patients. [ABSTRACT FROM AUTHOR]

Background: PIEZO1 works differently in different cancers and at different stages of development. The objective of the current study was to explore the function and underlying mechanism of PIEZO1 in lung adenocarcinoma (LUAD) cells. Methods: Different LUAD cell lines were treated with PIEZO1 inhibitor (GsMTx4) and agonist (Yoda1), and the expression of PIEZO1 in LUAD cells was detected using real‐time quantitative PCR (RT‐qPCR) and western blotting. The effects of PIEZO1 on invasion, migration and epithelial‐mesenchymal transition (EMT) markers protein expression of LUAD cells were detected using the MTT assay, flow cytometry, transwell assay, wound‐healing assay, and western blotting. Reactive oxygen species (ROS) agonists (BAY 87–2243) and inhibitors (NAC) and Wnt/β‐catenin pathway inhibitors (iCRT3) were selected to treat A549 cells to investigate the mechanism of PIEZO1 on ROS production and Wnt/β‐catenin expression in A549 cells. Results: In A549, NCI‐H1395, and NCI‐H1975 cells, GsMTx4 promoted cell proliferation, invasion, migration, upregulated EMT‐related marker protein expression, and inhibited cell apoptosis, while Yoda1 exerted effects opposite to those of GsMTx4. In A549 cells, GsMTx4 can reduce ROS production, it also inhibited ROS production, apoptosis, and downregulated proapoptotic markers induced by BAY 87–2243. Importantly, BAY 87–2243 blocked the effect of GSMTX4‐induced Wnt/β‐catenin overexpression. Similarly, Yoda1 can reduce the effect of NAC. In addition, iCRT3 can block the upregulation of EMT‐related marker proteins by GsMTx4, and increase apoptosis and decrease cell invasion and migration. Conclusion: In summary, PIEZO1 acts as a cancer suppressor by regulating the ROS/Wnt/β‐catenin axis, providing a new perspective on the role of mechanosensitive channel proteins in cancer. [ABSTRACT FROM AUTHOR]

As a gasotransmitter, carbon monoxide can induce tumor cell apoptosis by reversing the Warburg effect. Carbon monoxide-releasing molecules (CORMs) are a promising option for CO administration, but how to deliver the threshold concentration of CO into the tumor tissue remains a challenge. A series of novel targeted CORMs combining glycyrrhetinic acid (GA) derivative conjugates with a lipophilic mitochondriotropic triphenylphosphonium cation (TPP+) moiety were synthesized and evaluated for their antitumor activities. Among the complexes, complex 2 possessed excellent antitumor activities in HepG2 cells with IC50 values of 12.18 ± 4.66 μM compared with 5-FU. It caused the production of reactive oxygen species and reduced the mitochondrial membrane potential against HepG2 cells. Western blot analysis showed that complex 2 up-regulated the expression of the Bax protein and down-regulated the expression of Bcl-2, activated caspase-3, and HO-1. The docking study demonstrated that complex 2 can interact with the Bcl-2 protein through hydrophobic interaction and hydrogen bonds. The aforementioned results indicated that complex 2 triggered cell apoptosis through a mitochondrial pathway. These results suggest that the complexes may be promising anticancer agent candidates. [ABSTRACT FROM AUTHOR]

Background: Diabetic kidney disease (DKD) is a primary microvascular complication of diabetes with limited therapeutic effects. Delving into the pathogenic mechanisms of DKD and identifying new therapeutic targets is crucial. Emerging studies reveal the implication of ferroptosis and immune dysregulation in the pathogenesis of DKD, however, the precise relationship between them remains not fully elucidated. Investigating their interplay is pivotal to unraveling the pathogenesis of diabetic kidney disease, offering insights crucial for targeted interventions and improved patient outcomes. Methods: Integrated analysis, Consensus clustering, Machine learning including Generalized Linear Models (GLM), RandomForest (RF), Support Vector Machine (SVM) and Extreme Gradient Boosting (xGB), Artificial neural network (ANN) methods of DKD glomerular mRNA sequencing were performed to screen DKD-related ferroptosis genes.CIBERSORT, ESTIMATE and ssGSEA algorithm were used to assess the infiltration of immune cells between DKD and control groups and in two distinct ferroptosis phenotypes. The ferroptosis hub genes were verified in patients with DKD and in the db/db spontaneous type 2 diabetes mouse model via immunohistochemical and Western blotting analyses in mouse podocyte MPC5 and mesangial SV40-MES-13 cells under high-glucose (HG) conditions. Results: We obtained 16 differentially expressed ferroptosis related genes and patients with DKD were clustered into two subgroups by consensus clustering. Five ferroptosis genes (DUSP1,ZFP36,PDK4,CD44 and RGS4) were identified to construct a diagnostic model with a good diagnosis performance in external validation. Analysis of immune infiltration revealed immune heterogeneity between DKD patients and controls.Moreover, a notable differentiation in immune landscape, comprised of Immune cells, ESTIMATE Score, Immune Score and Stromal Score was observed between two FRG clusters. GSVA analysis indicated that autophagy, apoptosis and complement activation can participate in the regulation of ferroptosis phenotypes. Experiment results showed that ZFP36 was significantly overexpressed in both tissue and cells while CD44 was on the contrary.Meanwhile,spearman analysis showed both ZFP36 and CD44 has a strong correlation with different immune cells,especially macrophage. Conclusion: The regulation of the immune landscape in DKD is significantly influenced by the focal point on ferroptosis. Newly identified ferroptosis markers, CD44 and ZFP36, are poised to play essential roles through their interactions with macrophages, adding substantial value to this regulatory landscape. [ABSTRACT FROM AUTHOR]

This study aims to investigate the role and mechanism of tubiquitin-conjugating enzyme E2 C (UBE2C) in acute myeloid leukemia (AML). Initially, UBE2C expression in leukemia was analyzed using the Cancer Genome Atlas database. Further, we silenced UBE2C expression using small-hairpin RNA (sh-RNA). UBE2C expression was detected via the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis. Apoptotic events and reactive oxygen species (ROS) levels were detected by flow cytometry. A xenograft model of leukemia cells were established, and the protein levels of UBE2C, KI-67, and cleaved-caspase 3 were detected by immunohistochemistry. We reported an overexpression of UBE2C in leukemia patients and cell lines (HL60, THP-1, U937, and KG-1 cells). Moreover, a high expression level of UBE2C was correlated with a dismal prognosis in AML patients. UBE2C knockdown inhibited the viability and promoted apoptosis in AML cells by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Furthermore, UBE2C knockdown increased cellular Fe2+ and ROS levels, and enhanced erastin-induced ferroptosis in a proteasome-dependent manner. UBE2C knockdown also suppressed the tumor formation of AML cells in the mouse model. In summary, our findings suggest that UBE2C overexpression promotes the proliferation and inhibits ferroptosis in AML cells by activating the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Aim of the study: To evaluate the therapeutic effect of SYNC in diarrhea irritable bowel syndrome (IBS-D) and explore its underlying mechanism through transcriptomic sequencing (RNA-Seq). Materials and methods: A rat model of IBS-D was constructed to elucidate the effects of SYNC. Abdominal withdrawal reflex (AWR), fecal water content (FWC), and recording body weight were calculated to assess visceral sensitivity in rats. Histopathological changes in the colon and alterations in mast cell (MC) count were determined. Immunohistochemistry was employed to assess mast cell tryptase (MCT) expression in rat colons. Serum levels of corticotropin-releasing Hormone (CRH), interleukin-6 (IL-6), calcitonin gene-related peptide (CGRP), and 5-hydroxytryptamine (5-HT) were quantified using ELISA. RNA-Seq of colon tissue was performed, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Western blot analysis was conducted to quantify the expression levels of key proteins in the Nr4a3 pathway in the colon and hypothalamus tissues of rats. Results: SYNC alleviated visceral hypersensitivity and mood disorders in rats with IBS-D. Moreover, it was positively correlated with its dosage and the observed effects, such as the enhancement of the colon's mucosal lining condition and reduction in the number and activation of MCs within the model group. SYNC reduced the expression levels of factors related to the brain-gut axis and inflammatory markers in the bloodstream. RNA-Seq analysis indicated that SYNC down-regulated the expression of Nr4a3 and PI3K. These SYNC-targeted genes primarily played roles in immune regulation and inflammatory responses, correlating with the modulation of Nr4a3 and the PI3K/AKT pathway. Western blot analysis further confirmed SYNC's influence on inflammation-related MC activation by downregulating key proteins in the Nr4a3/PI3K pathway. Conclusions: SYNC inhibited mast cell activation and attenuated visceral hypersensitivity in the colon tissues of IBS-D rats. These effects were mediated by the Nr4a3/PI3K signaling pathway. [ABSTRACT FROM AUTHOR]

Silibinin is a flavonoid compound extracted from the seeds of Silybum marianum (L.) Gaertn. It has the functions of liver protection, blood-lipid reduction and anti-tumor effects. However, the potential molecular mechanism of silibinin against tumors is still unknown. This study aimed to assess the anti-tumor effects of silibinin in adenoid cystic carcinoma (ACC2) cells and Balb/c nude mice, and explore its potential mechanism based on network pharmacology prediction and experimental verification. A total of 347 targets interacting with silibinin were collected, and 75 targets related to the tumor growth process for silibinin were filtrated. Based on the PPI analysis, CASP3, SRC, ESR1, JAK2, PRKACA, HSPA8 and CAT showed stronger interactions with other factors and may be the key targets of silibinin for treating tumors. The predicted target proteins according to network pharmacology were verified using Western blot analysis in ACC2 cells and Balb/c nude mice. In the pharmacological experiment, silibinin was revealed to significantly inhibit viability, proliferation, migration and induce the apoptosis of ACC2 cells in vitro, as well as inhibit the growth and development of tumor tissue in vivo. Western blot analysis showed that silibinin affected the expression of proteins associated with cell proliferation, migration and apoptosis, such as MMP3, JNK, PPARα and JAK. The possible molecular mechanism involved in cancer pathways, PI3K-Akt signaling pathway and viral carcinogenesis pathway via the inhibition of CASP3, MMP3, SRC, MAPK10 and CDK6 and the activation of PPARα and JAK. Overall, our results provided insight into the pharmacological mechanisms of silibinin in the treatment of tumors. These results offer a support for the anti-tumor uses of silibinin. [ABSTRACT FROM AUTHOR]

Energy metabolism plays a pivotal role in the pathogenesis of endometriosis. For the initial stages of the disease in adolescents, this aspect remains unexplored. The objective of this paper was to analyze the association of cellular and endosomal profiles of markers of glycolysis, mitochondrial biogenesis, apoptosis, autophagy and estrogen signaling in peritoneal endometriosis (PE) in adolescents. We included 60 girls aged 13–17 years in a case–control study: 45 with laparoscopically confirmed PE (main group) and 15 with paramesonephric cysts (comparison group). Samples of plasma and peritoneal fluid exosomes, endometrioid foci and non-affected peritoneum were tested for estrogen receptor (Erα/β), hexokinase (Hex2), pyruvate dehydrogenase kinase (PDK1), glucose transporter (Glut1), monocarboxylate transporters (MCT1 and MCT2), optic atrophy 1 (OPA1, mitochondrial fusion protein), dynamin-related protein 1 (DRP1, mitochondrial fission protein), Bax, Bcl2, Beclin1, Bnip3, P38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor 1 (Hif-1α), mitochondrial voltage-dependent anion channel (VDAC) and transforming growth factor (TGFβ) proteins as markers of estrogen signaling, glycolysis rates, mitochondrial biogenesis and damage, apoptosis and autophagy (Western-Blot and PCR). The analysis identified higher levels of molecules associated with proliferation (ERβ), glycolysis (MCT2, PDK1, Glut1, Hex2, TGFβ and Hif-1α), mitochondrial biogenesis (OPA1, DRP1) and autophagy (P38, Beclin1 and Bnip3) and decreased levels of apoptosis markers (Bcl2/Bax) in endometrioid foci compared to non-affected peritoneum and that in the comparison group (p < 0.05). Patients with PE had altered profiles of ERβ in plasma and peritoneal fluid exosomes and higher levels of Glut1, MCT2 and Bnip3 in plasma exosomes (p < 0.05). The results of the differential expression profiles indicate microenvironment modification, mitochondrial biogenesis, estrogen reception activation and glycolytic switch along with apoptosis suppression in peritoneal endometrioid foci already in adolescents. [ABSTRACT FROM AUTHOR]

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and β-glycerophosphate (βGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 μg.mL−1/2 mM Asc/βGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastomas are the most aggressive brain tumors, and affected patients still only have an extremely poor prognosis with today's therapeutic options. Further developments are still necessary, particularly in therapeutic approaches. Orlistat can act as an antitumor agent as it inhibits fatty acid synthase, decreases tumor cell proliferation, and stimulates tumor cell apoptosis. Investigations conducted on breast, pancreatic, hepatic, and colorectal tumors showed that FASN—fatty acid synthase, a protein that catalyzes the de novo synthesis of long-chain fatty acids—is strongly upregulated. Using an animal model, we tested whether the drug's effects could be demonstrated visually, quantitatively, and by texture analysis based on MR studies. Histology and immunochemistry were used as references. The key results of the present study are as follows: Firstly, a significant difference was found between orlistat-treated and untreated tumors in MRI studies based on morphology and texture analyses. Secondly, the expression of FASN was reduced in the orlistat group, which, however, did not result in a higher apoptosis rate in the treatment group. Our findings suggest that some effects of orlistat on tumor cell proliferation must have taken place during therapy. Further studies should investigate the effects of FASN inhibition when combined with targeted therapies. Background: This study aimed to investigate the effects of tetrahydrolipstatin (orlistat) on heterotopic glioblastoma in mice by applying MRI and correlating the results with histopathology and immunochemistry. Methods: Human glioblastoma cells were injected subcutaneously into the groins of immunodeficient mice. After tumor growth of >150 mm3, the animals were assigned into a treatment group (n = 6), which received daily intraperitoneal injections of orlistat, and a control group (n = 7). MRI was performed at the time of randomization and before euthanizing the animals. Tumor volumes were calculated, and signal intensities were analyzed. The internal tumor structure was evaluated visually and with texture analysis. Western blotting and protein expression analysis were performed. Results: At histology, all tumors showed high mitotic and proliferative activity (Ki67 ≥ 10%). Reduced fatty acid synthetase expression was measured in the orlistat group (p < 0.05). Based on the results of morphologic MRI-based analysis, tumor growth remained concentric in the control group and changed to eccentric in the treatment group (p < 0.05). The largest area under the receiver operating curve of the predictors derived from the texture analysis of T2w images was for wavelet transform parameters WavEnHL_s3 and WavEnLH_s4 at 0.96 and 1.00, respectively. Conclusions: Orlistat showed effects on heterotopically implanted glioblastoma multiforme in MRI studies of mice based on morphologic and texture analysis. [ABSTRACT FROM AUTHOR]

Simple Summary: Although the most well-known molecular mechanism in essential thrombocythemia (ET) is the activation of JAK2/STAT, the study of mechanisms that are independent of JAK2/STAT activation related to the disease is on the rise. Previous studies in a C. elegans model revealed that the overexpression of nhr-2 counterparts could be a JAK2/STAT-independent mechanism, derived from CALR mutations in ET patients. In this work, we evaluated the expression of potential orthologs of nhr-2 in human cell lines and in mononuclear cells from ET patients with CALR or JAK2 mutations. The results show that mutant calreticulin is associated with an overexpression of RXRA in patients with ET, which could be relevant in the disease, pointing to the need for future research testing retinoids and other drugs targeting RXRα for the treatment of these patients. Essential thrombocythemia (ET) is a blood cancer caused by mutations in JAK2 and CALR. It is widely recognized that both mutations lead to the constitutive activation of JAK2/STAT signaling, although other JAK/STAT-independent pathogenic mechanisms triggered by these alterations have also been described in ET. In an attempt to study JAK2/STAT-independent mechanisms derived from CALR mutations, our research group created a C. elegans model with patient-like mutations in calreticulin that lacks JAK counterparts. The introduction of patient-like mutations in the calreticulin of C. elegans leads to an increase in the transcriptional expression of nhr-2, independently of JAK2/STAT activation. In the present study, we aim to verify if this mechanism is conserved in patients with ET harboring CALR mutations. To do so, we evaluated the expression of potential orthologs of nhr-2 in human cell lines of interest for the study, as well as in bone marrow (BM) or peripheral blood (PB) mononuclear cells from patients with CALR or JAK2 mutations. The results revealed that this mechanism is conserved in CALR-mutated ET patients, since CALR, but not JAK2 mutations, were associated with an overexpression of RXRA in patients with ET. The use of drugs targeting the activation or blockade of this target in the analyzed cell lines did not result in changes in cell viability. However, RXRA might be relevant in the disease, pointing to the need for future research testing retinoids and other drugs targeting RXRα for the treatment of ET patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Radium-223 is used for the management of metastatic castration-resistant prostate cancer. However, the response to this treatment is poor. Targeting the DNA damage response pathway through pharmacological inhibition has the ability to enhance the radiation response. In this study, we investigated the radiosensitising effects of three different DNA damage response inhibitors targeting ATM, ATR, and PARP in combination with X-rays and radium-223 on human prostate cancer cell lines. Their effects on cell survival, DNA double-strand break repair, cell cycle distribution, and apoptosis were evaluated. We determined that these inhibitors increase the ability of X-rays and radium-223 to kill prostate cells to varying degrees. Moreover, inhibition of the DNA damage response pathways impeded the repair of radiation-induced DNA double-strand breaks and induced changes in cell cycle distribution. Altogether, our study determined DDR inhibition as a promising strategy to increase the effectiveness of treatments utilising X-rays and/or 223Ra for localised and metastatic castration-resistant prostate cancer. Radium-223 (223Ra) and Lutetium-177-labelled-PSMA-617 (177Lu-PSMA) are currently the only radiopharmaceutical treatments to prolong survival for patients with metastatic-castration-resistant prostate cancer (mCRPC); however, mCRPC remains an aggressive disease. Recent clinical evidence suggests patients with mutations in DNA repair genes associated with homologous recombination have a greater clinical benefit from 223Ra. In this study, we aimed to determine the utility of combining DNA damage response (DDR) inhibitors to increase the therapeutic efficacy of X-rays, or 223Ra. Radiobiological responses were characterised by in vitro assessment of clonogenic survival, repair of double strand breaks, cell cycle distribution, and apoptosis via PARP-1 cleavage. Here, we show that DDR inhibitors increase the therapeutic efficacy of both radiation qualities examined, which is associated with greater levels of residual DNA damage. Co-treatment of ATM or PARP inhibition with 223Ra increased cell cycle arrest in the G2/M phase. In comparison, combined ATR inhibition and radiation qualities caused G2/M checkpoint abrogation. Additionally, greater levels of apoptosis were observed after the combination of DDR inhibitors with 223Ra. This study identified the ATR inhibitor as the most synergistic inhibitor for both radiation qualities, supporting further pre-clinical evaluation of DDR inhibitors in combination with 223Ra for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 μg/mL of LPS, with or without 2 μM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 μg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 μg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation. [ABSTRACT FROM AUTHOR]

Background: Splicing factor B subunit 4 (SF3B4) has been confirmed to participate in the progression of many cancers and is considered to be a potential target for non‐small cell lung cancer (NSCLC). Thus, the role and molecular mechanism of SF3B4 in NSCLC progression deserves further study. Methods: Quantitative real‐time PCR and western blot were employed to detect the mRNA and protein levels of SF3B4, Sm‐like protein 4 (LSM4) and methyltransferase‐like 3 (METTL3). Cell proliferation, apoptosis, invasion, migration and stemness were tested by cell counting kit‐8, colony formation, flow cytometry, transwell, wound healing, and sphere formation assays. The interaction between SF3B4 and METTL3 or LSM4 was confirmed by MeRIP, RIP and Co‐IP assays. Mice xenograft models were constructed to assess the effects of METTL3 and SF3B4 on NSCLC tumorigenesis. Results: SF3B4 had high expression in NSCLC tissues and was associated with the shorter overall survival of NSCLC patients. Knockdown of SF3B4 suppressed NSCLC cell proliferation, invasion, migration and stemness, while inducing apoptosis. METTL3 promoted SF3B4 mRNA stability by m6A modification, and its knockdown inhibited NSCLC cell growth, metastasis and stemness by downregulating SF3B4. SF3B4 could interact with LSM4, and sh‐SF3B4‐mediated the inhibition on NSCLC cell functions could be reversed by LSM4 overexpression. In addition, reduced METTL3 expression restrained NSCLC tumor growth, and this effect was reversed by SF3B4 overexpression. Conclusion: METTL3‐stablized SF3B4 promoted NSCLC cell growth, metastasis and stemness via positively regulating LSM4. [ABSTRACT FROM AUTHOR]

Background: This study aims to explore how miR-98-5p affects osteoarthritis, focusing on its role in chondrocyte inflammation, apoptosis, and extracellular matrix (ECM) degradation. Methods: Quantitative real-time PCR was used to measure miR-98-5p and CASP3 mRNA levels in OA cartilage tissues and IL-1β-treated CHON-001 cells. We predicted miR-98-5p and CASP3 binding sites using TargetScan and confirmed them via luciferase reporter assays. Chondrocyte viability was analyzed using CCK-8 assays, while pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were quantified via ELISA. Caspase-3 activity was examined to assess apoptosis, and Western blotting was conducted for protein marker quantification. Results: Our results showed lower miR-98-5p levels in both OA cartilage and IL-1β-stimulated cells. Increasing miR-98-5p resulted in reduced pro-inflammatory cytokines, decreased caspase-3 activity, and improved cell viability. Furthermore, miR-98-5p overexpression hindered IL-1β-induced ECM degradation, evident from the decline in MMP-13 and β-catenin levels, and an increase in COL2A1 expression. MiR-98-5p's impact on CASP3 mRNA directly influenced its expression. Mimicking miR-98-5p's effects, CASP3 knockdown also inhibited IL-1β-induced inflammation, apoptosis, and ECM degradation. In contrast, CASP3 overexpression negated the suppressive effects of miR-98-5p. Conclusions: In conclusion, our data collectively suggest that miR-98-5p plays a protective role against IL-1β-induced damage in chondrocytes by targeting CASP3, highlighting its potential as a therapeutic target for OA. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) lacks established biomarkers or molecular targets for predicting or enhancing radiation response. Phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 2 (PREX2) exhibits intricate implications in tumorigenesis and progression. Nevertheless, the precise role and underlying mechanisms of PREX2 in CRC radioresistance remain unclear. Methods: RNA-seq was employed to identify differentially expressed genes between radioresistant CRC cell lines and their parental counterparts. PREX2 expression was scrutinized using Western blotting, real-time PCR, and immunohistochemistry. The radioresistant role of PREX2 was assessed through in vitro colony formation assay, apoptosis assay, comet assay, and in vivo xenograft tumor models. The mechanism of PREX2 was elucidated using RNA-seq and Western blotting. Finally, a PREX2 small-molecule inhibitor, designated PREX-in1, was utilized to enhance the efficacy of ionizing radiation (IR) therapy in CRC mouse models. Results: PREX2 emerged as the most significantly upregulated gene in radioresistant CRC cells. It augmented the radioresistant capacity of CRC cells and demonstrated potential as a marker for predicting radioresistance efficacy. Mechanistically, PREX2 facilitated DNA repair by upregulating DNA-PKcs, suppressing radiation-induced immunogenic cell death, and impeding CD8+ T cell infiltration through the cGAS/STING/IFNs pathway. In vivo, the blockade of PREX2 heightened the efficacy of IR therapy. Conclusions: PREX2 assumes a pivotal role in CRC radiation resistance by inhibiting the cGAS/STING/IFNs pathway, presenting itself as a potential radioresistant biomarker and therapeutic target for effectively overcoming radioresistance in CRC. [ABSTRACT FROM AUTHOR]

Background: Shenma Jingfu Granule, a traditional Chinese medicine formula, has been used clinically for the treatment of cerebral circulation insufficiency. However, the mechanism involved in alleviating cerebral ischemia has not yet been fully elucidated. Methods: An integrated approach involving network pharmacology and transcriptomics was utilized to clarify the potential mechanisms of SMJF Granule. Molecular docking and surface plasmon resonance (SPR) were employed to identify potential targets and ingredients of SMJF Granule. The anti-CI effect of SMJF Granule was determined on the middle cerebral artery occlusion (MCAO) model by using hematoxylin–eosin (H&E) and Nisslʼs staining, as well as triphenyl tetrazolium chloride (TTC) staining, and the potential targets involved in the mechanisms were validated by RT-qPCR and western blotting. Results: Integrated analysis revealed the mechanism of SMJF Granule intervening in CI injury might be related to the HIF-1 signaling pathway and angiogenesis. Molecular docking and SPR assays demonstrated robust binding interactions between key compounds like salvianolic acid A and naringenin with the core target HIF-1α protein. The experiment confirmed that SMJF Granule lowered neurological scores, diminished infarct volume, and alleviated histopathological changes in vivo. The possible mechanism of SMJF Granule was due to regulating HIF-1 pathway, which contributed to up-regulating expression of VEGF and vWF in the penumbral region, showing a significant promotion of angiogenesis. Conclusion: SMJF Granule promoted angiogenesis through HIF-1α pathway, thereby alleviating cerebral ischemia injury. In addition, our findings provide some evidence that SMJF Granule is a candidate compound for further investigation in treating CI in the clinical. [ABSTRACT FROM AUTHOR]

Cervical cancer significantly impacts women's health due to its high mortality rate and increasing prevalence among younger individuals, thereby posing a severe threat. Echinatin is the primary active component of licorice in traditional Chinese medicine. However, studies on its use in cervical cancer treatment are limited. In our study, 198 targets of Echinatin were identified by some databases. Among these, 40 core targets related to cervical cancer were selected. Enrichment analyses revealed that Echinatin operated through genes associated with cell cycle, apoptosis, senescence, and various cancer-related signaling pathways. Differential expression of intersecting targets was verified in the GEO database, and molecular docking also indicated a strong binding capacity between active compounds and identified targets. Moreover, the results of western blot provided further evidence at the protein level. Echinatin hindered the proliferation, migration, and invasion of HeLa and SiHa cells while increasing their apoptosis. This study predicted the potential targets and beneficial effects of Echinatin in cervical cancer treatment, which provides a new avenue for further research into the molecular mechanisms underlying the role of Echinatin in cervical cancer treatment. [ABSTRACT FROM AUTHOR]

Background: Myocardial ischemia/reperfusion injury (MIRI) is an important complication of reperfusion therapy, and has a lack of effective prevention and treatment methods. Oleuropein (OP) is a natural strong antioxidant with many protective effects on cardiovascular diseases, but its protective effect on MIRI has not yet been studied in depth. Methods: Tert-Butyl hydroperoxide (tBHP) was used to establish an in vitro oxidative stress model. Cell viability was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and lactate dehydrogenase (LDH). Flow cytometry and fluorescence assays were performed for evaluating the ROS levels and mitochondrial membrane potential (MMP). Immunofluorescence analysis detected the NRF2 nuclear translocation and autophagy indicators. Further, Western blotting and quantitative real-time PCR were performed to evaluate the expression levels of proteins and mRNAs. Molecular docking, CETSA, and molecular interaction analysis explored the binding between OP and TLR4. The protective effects of OP in vivo were determined using a preclinical MIRI rat model. Results: OP protected against tBHP-treated injury, reduced ROS levels and reversed the damaged MMP. Mechanistically, OP activated NRF2-related antioxidant pathways, inhibited autophagy and attenuated the TLR4/MAPK signaling pathway in tBHP-treated H9C2 cells with a high binding affinity to TLR4 (KD = 37.5 µM). The TLR4 inhibitor TAK242 showed a similar effect as OP. In vivo, OP could alleviate cardiac ischemia/reperfusion injury and it ameliorated adverse cardiac remodeling. Consistent with in vitro studies, OP inhibited TLR4/MAPK and autophagy pathway and activated NRF2-dependent antioxidant pathways in vivo. Conclusion: This study shows that OP binds to TLR4 to regulate oxidative stress and autophagy for protecting damaged cardiomyocytes, supporting that OP can be a potential therapeutic agent for MIRI. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) can result in structural and functional damage to the spinal cord, which may lead to loss of limb movement and sensation, loss of bowel and bladder control, and other complications. Previous studies have revealed the critical influence of trans-acting transcription factor 1 (SP1) in neurological pathologies, however, its role and mechanism in SCI have not been fully studied. Methods: The study was performed using mouse microglia BV2 stimulated using lipopolysaccharide (LPS) and male adult mice subjected to spinal hitting. Western blotting was performed to detect protein expression of SP1, 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), BCL2-associated x protein (Bax), B-cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS), clusters of differentiation 86 (CD86), Arginase 1 (Arg-1) and clusters of differentiation 206 (CD206). Cell viability and apoptosis were analyzed by MTT assay and TUNEL assay. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4) and tumor necrosis factor-β (TNF-β) were quantified by quantitative real-time polymerase chain reaction. The association of SP1 and HTR2B was identified by chromatin immunoprecipitation assay and dual-luciferase reporter assay. HE staining assay was performed to analyze the pathological conditions of spinal cord tissues. Results: LPS treatment induced cell apoptosis and inhibited microglia polarization from M1 to M2 phenotype, accompanied by an increase of Bax protein expression and a decrease of Bcl-2 protein expression, however, these effects were relieved after SP1 silencing. Mechanism assays revealed that SP1 transcriptionally activated HTR2B in BV2 cells, and HTR2B knockdown rescued LPS-induced effects on BV2 cell apoptosis and microglial M1/M2 polarization. Moreover, SP1 absence inhibited BV2 cell apoptosis and promoted microglia polarization from M1 to M2 phenotype by decreasing HTR2B expression. SCI mouse model assay further showed that SP1 downregulation could attenuate spinal hitting-induced promoting effects on cell apoptosis of spinal cord tissues and microglial M1 polarization. Conclusion: SP1 transcriptionally activated HTR2B to aggravate traumatic SCI by shifting microglial M1/M2 polarization. [ABSTRACT FROM AUTHOR]

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'. [ABSTRACT FROM AUTHOR]

Background: Gamma-glutamyltransferase 5 (GGT5), one of the two members in the GGT family (GGT1 and GGT5), plays a crucial role in oxidative regulation, inflammation promotion, and drug metabolism. Particularly in the tumorigenesis of various cancers, its significance has been recognized. Nevertheless, GGT5's role in gastric cancer (GC) remains ambiguous. This study delves into the function and prognostic significance of GGT5 in GC through a series of in vitro experiments. Methods: Employing online bioinformatics analysis tools such as The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan–Meier plotter, and cBioPortal, we explored GGT5 characteristics and functions in GC. This encompassed aberrant expression, prognostic value, genomic alterations and mutations, immune cell infiltration, and associated signaling pathways. Immunohistochemistry was conducted to assess GGT5 expression in GC and adjacent normal tissues. Subsequently, univariate and multivariate logistic regression analyses were applied to investigate the associations between GGT5 and clinical characteristics. CCK8, wound healing, and migration assays were utilized to evaluate the impact of GGT5 on cell viability and migration. Additionally, Gene Set Enrichment Analysis (GSEA) and Western blot analysis were performed to scrutinize the activity of the epithelial–mesenchymal transformation (EMT) signaling pathway under GGT5 regulation. Results: GGT5 exhibits upregulation in gastric cancer, with its overexpression significantly linked to histological differentiation in GC patients (P < 0.05). Multivariate analysis indicates that elevated GGT5 expression is an independent risk factor associated with poorer overall survival in gastric cancer patients (P < 0.05). In vitro experiments reveal that downregulation of GGT5 hampers the proliferation and migration of GC cell lines. Finally, GSEA using TCGA data highlights a significant correlation between GGT5 expression and genes associated with EMT, a finding further confirmed by Western blot analysis. Conclusions: GGT5 emerges as a promising prognostic biomarker and potential therapeutic target for GC. [ABSTRACT FROM AUTHOR]

Background: The objective of this study was to determine the role and regulatory mechanism of miR-380 in cholangiocarcinoma. Methods: The TargetScan database and a dual-luciferase reporter assay system were used to determine if LIS1 was a target gene of miR-380. The Cell Counting Kit 8 assay, flow cytometry, and Transwell assay were used to detect the effects of miR-380 and LIS1 on the proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. Western blotting was used to determine the effect of miR-380 on MMP-2/p-AKT. Immunohistochemistry detected the regulatory effect of miR-380 on the expression of MMP-2/p-AKT/LIS1. Results: Expression of miR-380 in cholangiocarcinoma was decreased but expression of LIS1 was increased. LIS1 was confirmed to be a target gene of miR-380. Transfection with miR-380 mimics inhibited the proliferation, S-phase arrest, and invasion of HCCC-9810/HuCCT1/QBC939 cells, and LIS1 reversed these inhibitory effects. miR-380 inhibitor promoted proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. si-LIS1 salvaged the promotive effect of miR-380 inhibitor. Overexpression of miR-380 inhibited expression of MMP-2/p-AKT/LIS1, but miR-380 inhibitor promoted their expression. Conclusion: An imbalance of miR-380 expression is closely related to cholangiocarcinoma, and overexpression of miR-380 inhibits the expression of MMP-2/p-AKT by directly targeting LIS1. [ABSTRACT FROM AUTHOR]

Bilobalide has shown strong anti-inflammatory activity. Colorectal cancer (CRC) is closely associated with inflammation. However, no studies have reported on the use of bilobalide for treating CRC. This study aims to evaluate the effect of bilobalide on CRC prevention. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (RT-qPCR), Western blotting, and immunofluorescence showed that bilobalide significantly inhibits the M2 polarization of macrophages dependent on phorbol 12-myristate 13-acetate (PMA) and interleukin-4 (IL-4). Analysis of signaling pathways showed that the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT3) was regulated. In particular, human CRC organoids were established. Western blotting, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and analysis of cell viability and morphology further supported the hypothesis that the anti-CRC effects of bilobalide could be explained by its ability to suppress M2 macrophage polarization and promote M1 transformation. C57BL/6 mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) were divided into three groups, i.e., control, AOM/DSS, low (2.5 mg/kg), and high (5 mg/kg). High-dose bilobalide markedly inhibited the progression of CRC, as evidenced by the increased body weight, decrease in disease activity index (DAI) death rate, and alleviation of colon length reduction and tumorigenesis. According to the in vivo results, reduced levels of inflammatory cytokines in the serum included tumor necrosis factor (TNF-α), IL-6, IL-1β, and IL-10. Bilobalide reduced oxidative stress indices, lipid peroxide (LPO), and malondialdehyde (MDA) and increased reduced glutathione (GSH). In addition, the expression of proliferating cell nuclear antigen (PCNA), Ki67, cellular Myc (c-Myc), and CD206 was downregulated in the drug-treated groups, as confirmed by the immunohistochemical staining. Collectively, these results indicated that bilobalide administration improve experimental CRC by inhibiting M2 macrophage polarization and oxidative stress. Thus, bilobalide may prevent CRC and serve as a potential therapeutic target for CRC. [ABSTRACT FROM AUTHOR]

Background: Lactate has emerged as a critical regulator within the tumor microenvironment, including glioma. However, the precise mechanisms underlying how lactate influences the communication between tumor cells and tumor-associated macrophages (TAMs), the most abundant immune cells in glioma, remain poorly understood. This study aims to elucidate the impact of tumor-derived lactate on TAMs and investigate the regulatory pathways governing TAM-mediated tumor-promotion in glioma. Methods: Bioinformatic analysis was conducted using datasets from TCGA and CGGA. Single-cell RNA-seq datasets were analyzed by using UCSC Cell Browser and Single Cell Portal. Cell proliferation and mobility were evaluated through CCK8, colony formation, wound healing, and transwell assays. Western blot and immunofluorescence staining were applied to assess protein expression and cell distribution. RT-PCR and ELISA were employed to identify the potential secretory factors. Mechanistic pathways were explored by western blotting, ELISA, shRNA knockdown, and specific inhibitors and activators. The effects of pathway blockades were further assessed using subcutaneous and intracranial xenograft tumor models in vivo. Results: Elevated expressions of LDHA and MCT1 were observed in glioma and exhibited a positive correlation with M2-type TAM infiltration. Lactate derived from glioma cells induced TAMs towards M2-subtype polarization, subsequently promoting glioma cells proliferation, migration, invasion, and mesenchymal transition. GPR65, highly expressed on TAMs, sensed lactate-stimulation in the TME, fueling glioma cells malignant progression through the secretion of HMGB1. GPR65 on TAMs triggered HMGB1 release in response to lactate stimulation via the cAMP/PKA/CREB signaling pathway. Disrupting this feedback loop by GPR65-knockdown or HMGB1 inhibition mitigated glioma progression in vivo. Conclusion: These findings unveil the intricate interplay between TAMs and tumor cells mediated by lactate and HMGB1, driving tumor progression in glioma. GPR65, selectively highly expressed on TAMs in glioma, sensed lactate stimulation and fostered HMGB1 secretion via the cAMP/PKA/CREB signaling pathway. Blocking this feedback loop presents a promising therapeutic strategy for GBM. [ABSTRACT FROM AUTHOR]

Synaptopodin (SP), an actin-associated protein found in telencephalic neurons, affects activity-dependant synaptic plasticity and dynamic changes of dendritic spines. While being required for long-term depression (LTD) mediated by metabotropic glutamate receptor (mGluR-LTD), little is known about its role in other forms of LTD induced by low frequency stimulation (LFS-LTD) or spike-timing dependent plasticity (STDP). Using electrophysiology in ex vivo hippocampal slices from SP-deficient mice (SPKO), we show that absence of SP is associated with a deficit of LTD at Sc-CA1 synapses induced by LFS-LTD and STDP. As LTD is known to require AMPA- receptors internalization and IP3-receptors calcium signaling, we tested by western blotting and immunochemistry if there were changes in their expression which we found to be reduced. While we were not able to induce LTD, long-term potentiation (LTP), albeit diminished in SPKO, can be recovered by using a stronger stimulation protocol. In SPKO we found no differences in NMDAR, which are the primary site of calcium signalling to induce LTP. Our study shows, for the first time, the key role of the requirement of SP to allow induction of activity-dependant LTD at Sc-CA1 synapses. [ABSTRACT FROM AUTHOR]

Background: The protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression. Methods: The modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient's overall survival versus AnxA6 expression level was evaluated by the Kaplan–Meier method. Results: Lys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients. Conclusions: AnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis. [ABSTRACT FROM AUTHOR]