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Objective: Protein kinase C-delta (PKC-δ) is involved in apoptosis. This study aimed to establish whether PKC-δ can further promote IL-1β-induced chondrocyte apoptosis by mediating the phosphorylation of the JNK and p38 mitogen-activated protein kinase (MAPK) signaling pathways In osteoarthritis (OA)., Methods: We employed chondrocyte staining to determine the extent of cartilage degeneration. PKC-δ and p38 signal expressions were used in the immunohistochemical (IHC) test and apoptosis was assayed at the TUNEL test in human osteoarthritic and controls. We stimulated rat cartilage cells using IL-1β (10 ng/ml)/rottlerin (10 μM) or lentivirus. To determine the apoptosis rate, we employed flow cytometry. The mRNA of both BCL2-related X (BAX) and cysteine aspartate protease 3 (caspase-3) could be measured via qRT-PCR. Western blot measured the protein levels of BAX, caspase-3, PKC-δ, p-JNK/JNK and p-p38/p38., Results: The positive rate of PKC-δ and the apoptotic rate of chondrocytes in OA were higher than controls. The manifestation of PKC-δ was positively related to the degree of cartilage degeneration, p38 protein expression, and apoptosis rate. IL-1β exposure upregulated PKC-δ expression in chondrocytes in a dose-dependent manner. Decreasing PKC-δ expression and its phosphorylation in OA can inhibit MAPK signaling pathway activation (phosphorylation) by downregulating JNK and p38 protein phosphorylation and expression. This inhibition decreases caspase-3 and BAX levels, consequently lowering the apoptosis rate in chondrocytes., Conclusion: PKC-δ activation by IL-1β in OA promotes chondrocyte apoptosis via activation of the JNK and p38 MAPK signal pathways, thereby promoting the OA progression., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Microglia (μG), the resident immune cells in the central nervous system, surveil the parenchyma to maintain the structural and functional homeostasis of neurons. Besides, they influence neurogenesis and synaptogenesis through complement-mediated phagocytosis. Emerging evidence suggests that μG may also influence development through proinflammatory cytokines. Here, we examined the premise that tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), the two most prominent components of the μG secretome, influence retinal development, specifically the morphological and functional differentiation of human retinal ganglion cells (hRGCs). Using controlled generation of hRGCs and human μG (hμG) from pluripotent stem cells, we demonstrate that TNF-α and IL-1β secreted by unchallenged hμG did not influence hRGC generation. However, their presence significantly facilitated neuritogenesis along with the basal function of hRGCs, which involved the recruitment of the AKT/mTOR pathway. We present ex vivo evidence that proinflammatory cytokines may play an important role in the morphological and physiological maturation of hRGCs, which may be recapitulated for regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1β), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (-73% and -32%, respectively), while IL-1β showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (-65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders.

Objective: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), in part due to activation of the proinflammatory transcription factor NF-κB. Neddylation is modulated by the negative regulator of ubiquitin-like protein (NUB) 1. We determined whether NUB1 and neddylation are aberrant in the models with RA FLS, thereby contributing to their aggressive phenotype., Methods: Models with RA or osteoarthritis (OA) FLS were obtained from arthroplasty synovia. Real-time quantitative polymerase chain reaction and Western blot analysis assessed gene and protein expression, respectively. NUB1 was overexpressed using an expression vector. NF-κB activation was assessed by stimulating FLS with interleukin (IL)-1β. Neddylation inhibitor (MLN4924) and proteasome inhibitor were used in migration and gene expression assays. MLN4924 was used in the model with K/BxN serum-transfer arthritis., Results: Enhanced H3K27ac and H3K27me3 peaks were observed in the NUB1 promoter in the OA FLS compared with the RA FLS. NUB1 was constitutively expressed by FLS, but induction by IL-1β was significantly greater in the OA FLS. The ratio of neddylated cullin (CUL) 1 to nonneddylated CUL1 was lower in the OA FLS than the RA FLS. NUB1 overexpression decreased NF-κB nuclear translocation and IL-6 messenger RNA (mRNA) in IL-1β-stimulated the RA FLS. MLN4924 decreased CUL1 neddylation, NF-κB nuclear translocation, and IL-6 mRNA in IL-1β-stimulated the RA FLS. MLN4924 significantly decreased arthritis severity in the model with K/BxN serum-transfer arthritis., Conclusion: CUL1 neddylation and NUB1 induction is dysregulated in the models with RA, which increases FLS activation. Inhibition of neddylation is an effective therapy in an animal model of arthritis. These data suggest that the neddylation system contributes to the pathogenesis of RA and that regulation of neddylation could be a novel therapeutic approach., (© 2024 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1β and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1β- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1β- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1β may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In this study, we aim to investigate the role of enolase 1 (ENO1) in osteoarthritis (OA) pathogenic process and to uncover the underlying mechanism. To this end, we used IL-1β to induce an in vitro OA‑like chondrocyte model in human immortalized chondrocyte C-28/I2 cells. We manipulated the expression of ENO1 and cytokine receptor-like factor 1 (CRLF1) in IL-1β-induced C-28/I2 cells using siRNA and/or overexpression and tested their effects on IL-1β-induced pathologies including cell viability, apoptosis and inflammatory cytokine levels (IL-6 and TNF-α), and the expression of extracellular matrix-related enzymes and major mediators in the NF-κB signaling pathway (p-p65, p65, p-IκBα and IκBα). We used co-immunoprecipitation and immunofluorescence imaging to study a possible binding between ENO1 and CRLF1. Our data showed that IL-1β induction elevated ENO1 and CRLF1 expression in C-28/I2 cells. Silencing ENO1 or CRLF1 inhibited the IL-1β-induced cell viability damage, apoptosis, inflammation, and extracellular matrix degradation. The inhibitory effect of silencing ENO1 was reversed by CRLF1 overexpression, suggesting a functional connection between ENO1 and CRLF1, which could be attributed to a binding between these two partners. Our study could help validate the role of ENO1 in OA pathogenies and identify novel therapeutic targets for OA treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ca 2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1β binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca 2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca 2+ , generates Ca 2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca 2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1β showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca 2+ ] i . Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca 2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca 2+ -conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca 2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca 2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca 2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca 2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The cytokine profile of primary coronary artery endothelial cells cultivated in the presence of doxorubicin (2 μg/ml and 6 μg/ml) was evaluated using enzyme-linked immunosorbent assay and qPCR. Cultivation of cells in the presence of these concentrations of doxorubicin for 24 h, upregulated expression of the following genes: IL6 (by 2.30 and 2.66 times, respectively), IL1B (by 1.25 and 3.44 times), and CXCL8 (by 6.47 times and 6.42 times), MIF (2.34 and 2.28 times), CCL2 (4.22 and 3.98 times). Under these conditions the following genes were downregulated: IL10, IL1R2, TNF. Cultivation of cells in the presence of doxorubicin (2 μg/ml and 6 μg/ml) for 24 h also increased the secretion of IL-6.

Objective: Reno-renal reflexes are disturbed in cardiovascular and hypertensive conditions when elevated levels of pro-inflammatory mediators/cytokines are present within the kidney. We hypothesised that exogenously administered inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin (IL)-1β modulate the renal sympatho-excitatory response to chemical stimulation of renal pelvic sensory nerves., Methods: In anaesthetised rats, intrarenal pelvic infusions of vehicle [0.9% sodium chloride (NaCl)], TNF-α (500 and 1000 ng/kg) and IL-1β (1000 ng/kg) were maintained for 30 min before chemical activation of renal pelvic sensory receptors was performed using randomized intrarenal pelvic infusions of hypertonic NaCl, potassium chloride (KCl), bradykinin, adenosine and capsaicin., Results: The increase in renal sympathetic nerve activity (RSNA) in response to intrarenal pelvic hypertonic NaCl was enhanced during intrapelvic TNF-α (1000 ng/kg) and IL-1β infusions by almost 800% above vehicle with minimal changes in mean arterial pressure (MAP) and heart rate (HR). Similarly, the RSNA response to intrarenal pelvic adenosine in the presence of TNF-α (500 ng/kg), but not IL-1β, was almost 200% above vehicle but neither MAP nor HR were changed. There was a blunted sympatho-excitatory response to intrapelvic bradykinin in the presence of TNF-α (1000 ng/kg), but not IL-1β, by almost 80% below vehicle, again without effect on either MAP or HR., Conclusion: The renal sympatho-excitatory response to renal pelvic chemoreceptor stimulation is modulated by exogenous TNF-α and IL-1β. This suggests that inflammatory mediators within the kidney can play a significant role in modulating the renal afferent nerve-mediated sympatho-excitatory response., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Osteoarthritis (OA) is one of the most important causes of global disability, and dysfunction of chondrocytes is an important risk factor. The treatment of OA is still a challenge. Orexin-A is a hypothalamic peptide, and its effects in OA are unknown. In this study, we found that exposure to interleukin-1β (IL-1β) reduced the expression of orexin-2R, the receptor of orexin-A in TC-28a2 chondrocytes. Importantly, the senescence-associated β-galactosidase (SA-β-gal) staining assay demonstrated that orexin-A treatment ameliorates IL-1β-induced cellular senescence. Importantly, the presence of IL-1β significantly reduced the telomerase activity of TC-28a2 chondrocytes, which was rescued by orexin-A. We also found that orexin-A prevented IL-1β-induced increase in the levels of Acetyl-p53 and the expression of p21. It is shown that orexin-A mitigates IL-1β-induced reduction of sirtuin 3 (SIRT3). Silencing of SIRT3 abolished the protective effects of orexin-A against IL-1β-induced cellular senescence. These results imply that orexin-A might serve as a promising therapeutic agent for OA.

We investigated the influence of 17β-estradiol (17β-E2) on cartilage extracellular matrix (ECM) homeostasis in postmenopausal women. We focused on the roles of estrogen receptors (ESR) and SOX6 in 17β-E2-mediated stimulation of ECM metabolism during chondrocyte (CH) degeneration. We compared the expression of anabolic genes (collagen II and aggrecan) and catabolic genes (MMPs and TIMPs) in IL-1β-induced CH degeneration in vitro, with and without 17β-E2 supplementation. We separately silenced the SOX6, ESR1, and ESR2 genes in CHs to determine their impact on 17β-E2 treatment. Additionally, we used Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and luciferase assays to investigate protein-DNA interactions within ESR2 and SOX6-promoter complexes. After three days of IL-1β treatment, ESR1/2, SOX6, collagen II, aggrecan, and TIMP1/3 were decreased, while MMP3/9/13 were increased. The addition of 17β-E2 partially reversed these effects, but silencing SOX6, ESR1, or ESR2 weakened the protective effects of 17β-E2. Silencing ESR2, but not ESR1, abolished the upregulation of SOX6 induced by 17β-E2. ESR2 was found to bind the SOX6 promoter and regulate SOX6 expression. 17β-E2 upregulates SOX6 through ESR2 mediation, and the synergistic effect of 17β-E2 and ESR2 on SOX6 balances ECM metabolism in CHs.

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF- K B) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF- K B inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF- K B-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF- K B-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF- K B signaling. Administering direct pharmacological inhibitors of NF- K B did reduce NF- K B-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF- K B, signalling., (© 2023. The Author(s).)

Objective: This study explored the pharmacological mechanism of Tanshinone IIA (TAN IIA) in the treatment of Osteoarthritis (OA), which provided a certain reference for further research and clinical application of Tan IIA in OA., Methods: CHON-001 cells were stimulated with 10 μg/mL IL-1β for 48 h and treated with 10 μM TAN IIA for 48 h. Cellular viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, and Cleaved caspase-3 was measured by Immunoblot assay and RT-qPCR. TNF-α, IL-6, and iNOS in CHON-001 cells were determined by RT-qPCR and ELISA. To further verify the effect of TAN IIA on OA, a rat model of OA in vivo was established by right anterior cruciate ligament transection. TAN IIA was administered at 50 mg/kg or 150 mg/kg for 7 weeks. The degree of cartilage destruction in OA rats was observed by TUNEL and HE staining. Cleaved caspase-3 and FBXO11 were measured by immunohistochemical staining, RT-qPCR, and Immunoblot. TNF-α, IL-6, and iNOS in chondrocytes of OA rats were detected by ELISA., Results: IL-1β stimulated CHON-001 cell apoptosis and inflammation, and TAN IIA had anti-apoptosis and anti-inflammatory effects on IL-1β-regulated CHON-001 cells. TAN IIA down-regulated FBXO11 and inhibited PI3K/AKT and NF-κB pathways, thereby alleviating apoptotic and inflammatory reactions in CHON-001 cells under IL-1β treatment. Moreover, TAN IIA treatment improved chondrocyte apoptosis and inflammations in OA rats., Conclusion: TAN IIA inhibits PI3K/Akt and NF-κB pathways by down-regulating FBXO11 expression, alleviates chondrocyte apoptosis and inflammation, and delays the progression of OA., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Osteoarthritis (OA) is a degenerative disease that affects millions of individuals worldwide. Despite its prevalence, the exact causes and mechanisms behind OA are still not fully understood, resulting in a lack of effective treatments to slow down or halt disease progression. Recent research has discovered that extracellular vesicles (EVs) present in the circulation of young mice have a remarkable ability to activate musculoskeletal stem cells in elderly mice. Conversely, EVs derived from elderly mice do not exhibit the same potential, indicating that EVs obtained from young individuals may hold promise to activate aging cells in degenerative tissue. However, it remains unknown whether EVs derived from young individuals can also address cartilage degeneration caused by aging. In this study, we first evaluated EVs derived from young human plasma (YEVs) and EVs derived from old human plasma (OEVs) in an in vitro experiment using chondrocytes. The results revealed that YEVs effectively stimulated chondrocyte proliferation and migration, while OEVs from old plasma did not exhibit a similar effect. Given that OA represents a more complex inflammatory microenvironment, we further determine whether the benefits of YEVs on chondrocytes can be maintained in this context. Our findings indicate that YEVs have the ability to positively regulate chondrocyte function and protect them against apoptosis induced by IL-1β and TNF-α in an in vitro OA model. Furthermore, we discovered that lyophilized EVs could be stored under mild conditions without any alterations in their physical characteristics. Considering the exceptional therapeutic effects and the wide availability of EVs from young plasma, they hold significant promise as a potential approach to activate chondrocytes and promote cartilage regeneration in early-stage OA., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

Osteoarthritis (OA) is a chronic degenerative joint disease that affects worldwide. Oxidative stress plays a critical role in the chronic inflammation and OA progression. Scavenging overproduced reactive oxygen species (ROS) could be rational strategy for OA treatment. Bilirubin (BR) is a potent endogenous antioxidant that can scavenge various ROS and also exhibit anti-inflammatory effects. However, whether BR could exert protection on chondrocytes for OA treatment has not yet been elucidated. Here, chondrocytes were exposed to hydrogen peroxide with or without BR treatment. The cell viability was assessed, and the intracellular ROS, inflammation cytokines were monitored to indicate the state of chondrocytes. In addition, BR was also tested on LPS-treated Raw264.7 cells to test the anti-inflammation property. An in vitro bimimic OA microenvironment was constructed by LPS-treated Raw264.7 and chondrocytes, and BR also exert certain protection for chondrocytes by activating Nrf2/HO-1 pathway and suppressing NF-κB signalling. An ACLT-induced OA model was constructed to test the in vivo therapeutic efficacy of BR. Compared to the clinical used HA, BR significantly reduced cartilage degeneration and delayed OA progression. Overall, our data shows that BR has a protective effect on chondrocytes and can delay OA progression caused by oxidative stress., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the β-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1β) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1β. Indeed, IL-18 and IL-1β treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1β signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1β may be promising therapeutic targets., (© 2024 Wiley Periodicals LLC.)

Objective: Studies have shown that the levels of pleiotrophin (PTN) are greatly elevated in the synovial fluid and cartilage in osteoarthritis. Therefore, the purpose of this study was to investigate the effect and mechanism of PTN on the chondrogenic differentiation of DPSCs in inflammatory and normal microenvironments., Materials and Methods: A lentiviral vector was used to deplete or overexpress PTN in DPSCs. The inflammatory microenvironment was simulated in vitro by the addition of IL-1β to the culture medium. The chondrogenic differentiation potential was assessed using Alcian Blue staining and the main chondrogenic markers. A dual-luciferase reporter assay was used to explore the relationship between miR-137 and PTN., Results: The results showed that 0.1 ng/mL IL-1β treatment during chondrogenic induction greatly impaired the chondrogenic differentiation of DPSCs. Supplementation with PTN and PTN overexpression inhibited chondrogenic differentiation of DPSCs, while PTN depletion promoted chondrogenic differentiation. MiR-137 negatively regulated the expression of PTN by binding to the 3'UTR of its mRNA. Moreover, miR-137 promoted chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments., Conclusion: Our results suggest that PTN may play an inhibitory role in the chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments, which is regulated by miR-137., (© 2023 Wiley Periodicals LLC.)

Objective: To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA)., Methods: Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay., Results: After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G 2 phase decreased significantly, and the proportion of cells at G 1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes., Conclusion: miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

Objective To investigate the regulation of IL-1β on the expression of CD200 in human umbilical cord mesenchymal stem cells (hUC-MSCs), its role in macrophage polarization and the underlying mechanism. Methods hUC-MSCs were isolated and cultured in serum-free medium. Morphological observation and the expressions of CD73, CD90, CD105, CD14, CD34, CD45 and HLA-DR were detected by flow cytometry to confirm the properties of mesenchymal stem cells. hUC-MSCs were treated with IL-1β at the final concentration of 20 ng/mL for 24 hours. The proportion of CD200 positive cells was measured by flow cytometry. Real-time quantitative PCR and Western blot analysis were used to detect CD200 mRNA and protein expression levels. hUC-MSCs infected with CD200 overexpression (OE-CD200) and its negative control (OE-NC) lectin virus were treated with IL-1β and co-cultured with PMA-activated THP-1 macrophages. The proportion of CD11c and CD206 positive cells was measured by flow cytometry. hUC-MSCs were treated with IL-1β in combination with PD98059, and the expression of MAPK signaling pathway-related proteins and its effect on CD200 expression were detected by Western blot analysis. Results IL-1β significantly down-regulated the expression of CD200 protein and the proportion of CD200 positive cells. Overexpression of CD200 significantly up-regulated the expression of CD200 in hUC-MSCs, and increased the proportion of CD206-positive macrophages. IL-1β activated the ERK1/2 signaling pathway in hUC-MSCs, and PD98059 up-regulated the expression of CD200 protein in hUC-MSCs treated with IL-1β. Conclusion IL-1β inhibits the expression of CD200 by activating ERK1/2 signaling pathway, and reduces the immunosuppressive effect of hUC-MSCs on regulating the M2-type polarization of macrophages.

Background: Osteoarthritis (OA) is a common disease that causes pain to many older adults. Because the pathogenesis is not fully elucidated, effective drug therapies are currently lacking. This study aimed to determine how salidroside (Sal)-mediated reduction of osteoarthritis development in mice worked and to identify the underlying mechanism., Methods: Using in vitro experiments, ATDC5 cells were treated with various concentrations of Sal and interleukin (IL)-1β for 24 hours to mimic OA. An enzyme-linked immunosorbent assay (ELISA) was conducted to detect the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Western blotting was performed to observe the nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. In in vivo experiments, pathological examination was used to assess the effects of Sal on alleviating OA progression in mice. Nrf2 signaling and its downstream proteins were further tested by immunofluorescence analysis., Results: The results showed that both pro-inflammatory cytokines and ROS were significantly reduced following Sal treatment in a concentration-dependent manner. Western blotting revealed that Sal could inhibit the expression of the NF-κB/hypoxia-inducible factor-2α pathway and activate the Nrf2/heme oxygenase-1 pathway. In vivo experiments showed that the cartilage surface in the saline-treated group eroded to a greater extent than the Sal-treated groups ( p < 0.001). Immunohistochemistry analysis revealed that matrix metallopeptidase (MMP) 9, MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) decreased expression level. In contrast, collagen-II and aggrecan increased in the Sal-treated groups compared to the saline-treated group., Conclusions: Our findings indicate that Sal can alleviate OA progression by promoting anti-oxidant expression and inhibiting degradation enzyme expression. These findings suggest that Sal inhibits the NF-κB pathway and its downstream targets through up-regulating the Nrf2 pathway.

This study aimed to evaluate the effects of general control non-derepressible 2 (GCN2) on osteoarthritis (OA) in vivo and in vitro. First, anterior cruciate ligament transection (ACLT)-induced rat model and interleukin (IL)-1β-induced ATDC5 chondrocyte were established. Hematoxylin and eosin staining and safranin O/fast green staining were employed for analyzing the histological changes in the rat cartilage. In addition, immunohistochemistry, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and immunofluorescence staining were employed for examining cartilage degeneration-, inflammation-, autophagy-, and NLR family pyrin domain containing 3 (NLRP3) inflammasome-associated genes expression. Moreover, 2,7-dichlorodihydrofluorescein acetoacetic acid probe was utilized for examining the intracellular reactive oxygen species. In addition, 5-ethynyl-2'-deoxyuridine assay and flow cytometry were applied for detecting chondrocyte proliferation and apoptosis IL-1β-treated ATDC5 chondrocytes. GCN2 overexpression ameliorated articular cartilage degeneration and inflammation but promoted chondrocyte autophagy in ACLT-induced OA rats. Similarly, we demonstrated that the upregulation of GCN2 could promote chondrocyte proliferation, suppress chondrocyte apoptosis, attenuate chondrocyte inflammation and extracellular matrix degradation, and promote chondrocyte autophagy. Moreover, GCN2 overexpression could inhibit the activation of NLRP3 inflammasome in IL-1β-induced ATDC5 chondrocyte. Furthermore, 3-methyladenine neutralized the protective and autophagy-promoting effects of GCN2 overexpression on ATDC5 chondrocytes. GCN2 could attenuate inflammation and cartilage degeneration, promote chondrocyte autophagy, and inhibit NLRP3 inflammasome activation in OA., Competing Interests: Competing InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Immune responses play a significant role in hypertension, though the importance of key inflammatory mediators remains to be defined. We used a systematic literature review and meta-analysis to study the associations between key cytokines and incident hypertension., Methods: We performed a systematic search of Pubmed/Medline, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL), for peer-reviewed studies published up to August 2022. Incident hypertension was defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg and/or the use of antihypertensive medications. Random effects meta-analyses were used to calculate pooled hazard ratios (HRs)/risk ratios (RRs) and 95% confidence intervals by cytokine levels (highest vs. lowest quartile)., Results: Only IL-6 and IL-1β levels have evidence allowing for quantitative evaluation concerning the onset of hypertension. Six studies (10406 participants, 2932 incident cases) examined the association of IL-6 with incident hypertension. The highest versus lowest quartile of circulating IL-6 was associated with a significant HR/RR of hypertension (1.61, 95% CI: 1.00 to 2.60; I 2 =87%). After adjusting for potential confounders, including body mass index (BMI), HR/RR was no longer significant (HR/RR: 1.24; 95% CI, 0.96 to 1.61; I 2 = 56%). About IL-1β, neither the crude (HR/RR: 1.03; 95% CI, 0.60 to 1.76; n = 2) nor multivariate analysis (HR/RR: 0.97, 95% CI, 0.60 to 1.56; n = 2) suggested a significant association with the risk of developing hypertension., Conclusions: A limited number of studies suggest that higher IL-6, but not IL-1β, might be associated with the development of hypertension., Competing Interests: Declaration of Competing Interest Tomasz Jan Guzik reports consulting fees for Moderna. All the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule (XFC) on interleukin (IL)-1β-induced impairment of chondrocytes., Methods: XFC-medicated serum was collected from SD rats with XFC gavage, and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry. Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2. In cultured human chondrocytes induced with IL-1β, the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum, alone or in combination, on expression levels of IL-1β, tumor necrosis factor- α (TNF- α ), IL-4, and IL-10 were examined with ELISA, and the changes in the expressions of collagen type Ⅱ alpha 1 (COL2A1), matrix metalloproteinase 13 (MMP13), adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR, Western blotting, and immunofluorescence assay., Results: In cultured human chondrocytes, treatment with IL-1β significantly decreased the cell viability, increased cell apoptosis rate, lowered miR-502-5p, IL-4, IL-10, and COL2A1 expressions, and enhanced IL-1β, TNF- α , ADAMTS5, MMP13, TRAF2, and NF-κB p65 expressions ( P < 0.05), and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20% ( P < 0.05). Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1β, TNF- α , ADAMTS5, MMP13, TRAF2, and NF-κB p65 and lowered expressions of miR-502-5p, IL-4, IL-10, and COL2A1, and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor., Conclusion: XFC can inhibit IL-1β-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Background: Osteoarthritis (OA) is a common degenerative disease involving articular cartilage, in which ferroptosis of chondrocytes plays an important role. Baicalin (BAI) exerts regulatory effects in a wide range of orthopedic diseases including OA, but its effect on ferroptosis of chondrocytes (CHs) is still unclear. The purpose of this study was to determine the effect of BAI on ferroptosis in human OA chondrocytes (OACs), and to explore its possible mechanism., Methods: CHs were treated with IL-1β (10 ng/mL) to simulate inflammation in vitro. Immunofluorescence, quantitative RT-PCR, Western blotting and cell viability assay were performed to evaluate the impacts of BAI on Fe 2+ level, mitochondrial dysfunction, ferroptosis-related proteins, oxidative stress and cytotoxicity in CHs. Additionally, siRNA was made use of to knock out nuclear factor E2-related factor 2 (Nrf2) to analyze the role played by Nrf2 in BAI-induced CH ferroptosis., Results: BAI eliminated IL-1β-induced Fe 2+ accumulation, changes in mitochondrial membrane potential and ferroptosis-related protein GPX4, SLC7A11, P53 and ACSL4 levels, as well as reactive oxygen species (ROS), lipid peroxidation (LPO) and malondialdehyde (MDA) accumulation in CHs. Besides, BAI reversed IL-1β-induced decrease of Collagen II and increase of MMP13 in CHs. Meanwhile, BAI attenuated IL-1β-induced CH toxicity and promoted Nrf2 antioxidant system activation. When Nrf2 was knocked down by siRNA, the effects of BAI on IL-1β-induced ferroptosis-related proteins and antioxidant stress in CHs were significantly weakened., Conclusions: This study demonstrates that IL-1β can induce CH ferroptosis. BAI is able to inhibit IL-1β-induced CH ferroptosis and ECM degradation, and the specific mechanism may be that it can inhibit IL-1β-induced CH ferroptosis by activating Nrf2 antioxidant system to attenuate the accumulation of intracellular ROS and lipid ROS., (© 2023. The Author(s).)

The aim of this study was to explore the effect and mechanism of Sirt6 on DNA damage repair in OA chondrocytes. Cartilage tissues were collected from OA patients with knee arthroplasty and traumatic amputation patients without OA. Besides, 7-week-old male C57BL/6 mice were randomly divided into Control and OA groups; CHON-001 cells of corresponding groups were treated with 10 ng/ml interleukin (IL)-1β, respectively. Subsequently, Sirt6 or siNrf2 was over-expressed in CHON-001 cells to observe the effect of Sirt6 on DNA damage and senescence of chondrocytes by IL-1β through the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. The expression level of Sirt6 in human and mouse OA cartilage tissues was significantly decreased. However, 24 h of treatment with IL-1β significantly decreased the expression of Sirt6 in chondrocytes, induced DNA damage, and promoted cellular senescence. In addition, over-expression of Sirt6 promoted DNA damage repair and inhibited cellular senescence in IL-1β-induced chondrocytes. Moreover, the overexpression of Sirt6 activated the Keap1/Nrf2/HO-1 signaling pathway in chondrocytes, while knockdown of Nrf2 expression inhibited the DNA damage repair and anti-senescence effects of Sirt6 on IL-1β-treated chondrocytes. Sirt6 may reduce DNA damage and cellular senescence in OA chondrocytes induced by IL-1β through activating the Keap1/Nrf2/HO-1 signaling pathway.

Uveitis, or intraocular inflammation, is a potentially blinding condition that mostly affects the working-age population. The cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play a role in the pathogenesis of non-infectious uveitis and have been linked to the breakdown of the inner blood-retinal barrier, composed mainly of retinal endothelial cells, leading to macular oedema and vascular leakage. However, the effects of TNF-α and IL-1β on human retinal endothelial function are not fully understood. In this work, we investigated the impact of TNF-α and IL-1β on several aspects of human retinal endothelial cell biology. Through a real-time biosensor, the impact of TNF-α and IL-1β on formation of a retinal endothelial cell barrier was analyzed. Changes in junctional components were assessed via RT-qPCR and immunolabelling. Cell survival, necrosis and apoptosis were appraised via cell proliferation and flow cytometric studies. Tumor necrosis factor-α and IL-1β impaired the electrical resistance of the retinal endothelial cell barrier, while the addition of a potentially barrier-impairing cytokine, IL-6, did not enhance the effect of TNF-α and IL-1β. Level of the gene transcript encoding zonula occludens (ZO)-1 was diminished, while ZO-1 protein configuration was changed by TNF-α and IL-1β. Both cytokines affected human retinal endothelial cell proliferation and viability, while only TNF-α increased rates of necrosis. These results indicate that TNF-α and IL-1β are important drivers of retinal endothelial dysfunction in non-infectious uveitis, suggesting that targeting these cytokines is critical when treating complications of uveitis, such as macular oedema and vascular leakage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Inflammation is characterized by a biphasic cycle consisting initially of a proinflammatory phase that is subsequently resolved by anti-inflammatory processes. Interleukin-1β (IL-1β) is a master regulator of proinflammation and is encoded within the same topologically associating domain (TAD) as IL-37, which is an anti-inflammatory cytokine that opposes the function of IL-1β. Within this TAD, we identified a long noncoding RNA called AMANZI, which negatively regulates IL-1β expression and trained immunity through the induction of IL37 transcription. We found that the activation of IL37 occurs through the formation of a dynamic long-range chromatin contact that leads to the temporal delay of anti-inflammatory responses. The common variant rs16944 present in AMANZI augments this regulatory circuit, predisposing individuals to enhanced proinflammation or immunosuppression. Our work illuminates a chromatin-mediated biphasic circuit coordinating expression of IL-1β and IL-37, thereby regulating two functionally opposed states of inflammation from within a single TAD., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1β (IL-1β)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1β was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1β-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.

Osteoarthritis (OA) is a common musculoskeletal disorder that impairs function and reduces the quality of life. Extracellular matrix (ECM) degradation and inflammatory mechanisms are crucial to the progression of OA. In this study, we aimed to investigate the anti-inflammatory activity, anti-ECM degradation property, and glucose transport capacity of quercitrin (QCT) on IL-1β-treated rat primary chondrocytes. Rat primary chondrocytes were treated with IL-1β to simulate inflammatory environmental conditions and OA in vitro. We examined the effects of QCT at concentrations ranging from 0 to 200 μM on the viability of rat chondrocytes and selected 5 μM for further study. Using qRT-PCR, immunofluorescent, immunocytochemistry, and western blotting techniques, we identified the potential molecular mechanisms and signaling pathways that are responsible for these effects. We established an OA rat model through anterior cruciate ligament transection (ACLT). The animals were then periodically injected with QCT into the knee articular cavity. Our in vivo and in vitro study showed that QCT could inhibit IL-1β-activated inflammation and ECM degradation in chondrocyte. Furthermore, QCT could inhibit the NF-κB signal pathway and enhance glucose transport capacity in the IL-1β-stimulated chondrocytes. In vivo study proved that QCT attenuates OA progression in rats. Overall, QCT inhibited the activation of NF-κB and enhanced glucose transport capacity to alleviate the progression of OA., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Prostate cancer is one of the main global health threats for men which is in close association with chronic inflammation. Neuropeptide substance P (SP), acting through neurokinin receptor (NK-1R), induces various pro-inflammatory responses which are strongly involved in the pathogenesis of several diseases as well as cancer. Therefore, we aimed to investigate the pro-inflammatory functions of the SP/NK1R complex in prostate cancer and the therapeutic effects of its inhibition by NK-1R antagonist, aprepitant, in vitro. MTT assay was conducted for the cytotoxicity assessment of aprepitant in prostate cancer cells. The protein expression levels were evaluated by Western blot assay. Quantitative real-time PCR (qRT-PCR) was applied to measure mRNA expression levels of pro-inflammatory cytokines. Concurrently, the protein concentrations of pro-inflammatory cytokines were also analyzed by enzyme-linked immunosorbent assay. We observed that SP increased the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), while treatment with aprepitant reduced the effects of SP. We also indicated that SP increased the protein levels of nuclear factor-kappa B (NF-κB), as the main regulator of inflammatory processes, and also an NF-κB target gene, cyclooxygenase 2 (COX-2) in prostate cancer cells, while treatment with aprepitant reversed these effects. Taken together, our findings highlight the importance of the SP/NK1R system in the modulation of pro-inflammatory responses in prostate cancer cells and suggest that aprepitant may be developed as a novel anti-inflammatory agent for the management of cancer-associated inflammation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To explore the effect of baicalin on human nucleus pulposus cells (NPCs) in response to interleukin (IL)-1β stimulation., Methods: Viability of NPCs was measured by cell counting kit-8 (CCK-8) assay. TUNEL staining assay and flow cytometry were performed to detect apoptotic cell death of NPCs. Western blot analysis was conducted to detect the expression levels of proteins. Enzyme-linked immunosorbent assay (ELISA) was applied for the determination of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and IL-6. Oxidative stress indicators including reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity were measured., Results: Baicalin attenuated IL-1β-caused cell viability reduction and apoptosis in NPCs. IL-1β-induced increase in Bax expression and decrease in Bcl-2 expression were attenuated by baicalin treatment. IL-1β-induced production of iNOS, COX-2, IL-6, and TNF-α in NPCs was inhibited by baicalin treatment. Baicalin treatment reversed IL-1β-induced increase in ROS production and MDA level, as well as decrease in SOD activity. Furthermore, baicalin treatment elevated the expression levels of Col II and Aggrecan and downregulated the expression levels of MMP3, MMP13, and ADAMTS5 in IL-1β-induced NPCs. A total of 402 related targets of baicalin and 134 related targets of intervertebral disk degeneration were found, and nine intersection targets were screened out. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mitogen-activated protein kinase (MAPK) pathway was found to be involved in the effects of baicalin., Conclusions: Baicalin exhibited protective effects on IL-1β-caused cell viability reduction, apoptosis, oxidative stress, inflammation, and extracellular matrix degradation in NPCs. In addition, we found c-Jun N-terminal kinase (JNK) and p38 MAPK pathways as targets of baicalin through bioinformatic analysis.

Background: Recent studies have shown that circRNAs are involved in the pathogenesis of osteoarthritis (OA) by affecting various fundamental cellular characteristics of chondrocytes. The purpose of this paper is to investigate the role and regulatory mechanism of hsa&#95;circ&#95;0020014 (circ&#95;0020014) in chondrocytes of OA., Methods: The inflammatory cytokine interleukin 1 beta (IL-1β) was used to stimulate human chondrocytes. Cell viability, proliferation, and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-Ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Several protein levels were determined by western blotting (WB). Levels of inflammatory cytokines and malondialdehyde (MDA) were determined by enzyme-linked immunosorbent assay (ELISA). Relative expression of circ&#95;0020014 was estimated by real-time polymerase quantitative chain reaction (RT-qPCR). Bioinformatics prediction combined with dual-luciferase reporter, RIP and RNA pull-down assays were done to probe into the regulatory mechanism of circ&#95;0020014., Results: Circ&#95;0020014 was overexpressed in OA patient-derived articular cartilages and IL-1β-stimulated chondrocytes. Silencing of circ&#95;0020014 lighted IL-1β-prompted chondrocyte proliferation repression, apoptosis, inflammation, and oxidative stress. Mechanically, circ&#95;0020014 functioned as a miR-24-3p molecular sponge to regulate cathepsin B (CTSB) expression. Furthermore, miR-24-3p inhibition alleviated circ&#95;0020014 knockdown-mediation repression of IL-1β-urged chondrocyte injury. In addition, CTSB overexpression whittled miR-24-3p upregulation-mediated suppression of IL-1β-urged chondrocyte injury., Conclusion: Our findings demonstrated that the circ&#95;0020014/miR-24-3p/CTSB axis was associated with IL-1β-prompted chondrocyte injury, supporting the involvement of circ&#95;0020014 in the OA pathogenesis., (© 2023. The Author(s).)

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12 myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1β (IL-1β) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1β treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12 myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1β (IL-1β) expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress., (© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.)

Previous studies have reported that circular RNA hsa&#95;circ&#95;0010024 (circDHRS3), microRNA (miR)-193a-3p, and Methyl CpG binding protein 2 (MECP2) are unconventionally expressed in osteoarthritis (OA) cartilage samples. However, the regulatory mechanisms among circDHRS3, miR-193a-3p, and MECP2 in OA pathogenesis are unclear. Changes of circDHRS3, miR-193a-3p, and MECP2 mRNA were detected by qRT-PCR. Several protein levels were evaluated using western blotting. Cell proliferation was analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) and cell counting assays. Cell apoptosis was determined by flow cytometry assay. Detection of pro-inflammatory cytokines was conducted using ELISA. The relationship between circDHRS3 or MECP2 and miR-193a-3p was validated by dual-luciferase reporter assay. We verified that circDHRS3 and MECP2 were overexpressed in OA cartilage samples, whereas miR-193a-3p was downregulated. CircDHRS3 silencing weakened IL-1β-induced chondrocyte cartilage extracellular matrix (ECM) degradation, apoptosis, and inflammatory response. CircDHRS3 adsorbed miR-193a-3p to modulate MECP2 expression. Also, silencing of miR-193a-3p impaired circDHRS3 silencing-mediated suppression on IL-1β-induced chondrocyte injury. Also, MECP2 overexpression alleviated miR-193a-3p mimic-mediated inhibition on IL-1β-prompted chondrocyte injury. CircDHRS3 silencing reduced MECP2 expression via sponging miR-193a-3p, thereby weakening IL-1β-induced chondrocyte ECM degradation, apoptosis, and inflammatory response., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The association between interleukin-1β (IL-1β) concentrations during ex vivo lung perfusion (EVLP) with donor organ quality and post-lung transplant outcome has been demonstrated in several studies. The mechanism underlying IL-1β-mediated donor lung injury was investigated using a paired single-lung EVLP model., Methods: Human lung pairs were dissected into individual lungs and perfused on identical separate EVLP circuits, with one lung from each pair receiving a bolus of IL-1β. Fluorescently labeled human neutrophils isolated from a healthy volunteer were infused into both circuits and quantified in perfusate at regular timepoints. Perfusates and tissues were subsequently analyzed, with perfusates also used in functional assays., Results: Neutrophil numbers were significantly lower in perfusate samples collected from the IL-1β-stimulated lungs consistent with increased neutrophil adhesion ( P = 0.042). Stimulated lungs gained significantly more weight than controls ( P = 0.046), which correlated with soluble intercellular adhesion molecule-1 (R 2 = 0.71, P = 0.0043) and von-Willebrand factor (R 2 = 0.39, P = 0.040) in perfusate. RNA expression patterns for inflammatory genes were differentially regulated via IL-1β. Blockade of IL-1β significantly reduced neutrophil adhesion in vitro ( P = 0.025)., Conclusion: These data illustrate the proinflammatory functions of IL-1β in the context of EVLP, suggesting this pathway may be susceptible to therapeutic modulation before transplantation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: To elucidate whether pro-inflammatory cytokines might influence the commitment of intervertebral disc (IVD)- and ligamentum flavum (LF)-derived progenitor cells toward either osteogenesis or adipogenesis, specifically Interleukin-1β (IL-1β), IL-19, and IL-20., Methods: Sixty patients with degenerative spondylolisthesis and lumbar or lumbosacral spinal stenosis were included in the study. Injuries to the spine, infections, and benign or malignant tumors were excluded. From nine patient samples, IVD- and LF-derived cells were isolated after primary culture, and two clinical samples were excluded due to mycoplasma infection. The effects of IL-1β, IL-19, as well as IL-20 in regulating osteogenic and adipogenic differentiation in vitro were investigated., Results: Primary IVD- and LF-derived cells were found to have a similar cell morphology and profile of surface markers (CD44, CD90, and CD105) as placenta-derived mesenchymal stem cells (MSCs). Primary IVD/LF cells have a high capacity to differentiate into osteocytes and adipocytes. IL-19 had a tendency to promote adipogenesis. IL-20 inhibited osteogenesis and promoted adipogenesis; IL-1β promoted osteogenesis but inhibited adipogenesis., Conclusion: IL-1β, IL-19, and IL-20 impact the adipogenic and osteogenic differentiation of IVD-derived and LF-derived cells. Modulating the expression of IL-1β, IL-19, and IL-20 provides a potential avenue for controlling cell differentiation of IVD- and LF-derived cells, which might have beneficial effect for degenerative spondylolisthesis and spinal stenosis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1β secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1β production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1β production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1β, mature IL-1β, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1β, mature IL-1β and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1β and mature IL-1β in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1β production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1β production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1β transcription in macrophage-like J774.1 cells., (© 2023. The Author(s), under exclusive licence to The Japanese Society of Hypertension.)

The role and mechanism of Gremlin-1 in osteoarthritis (OA) were expected to be probed in this study. Firstly, an in vitro OA model was constructed by stimulating human chondrocyte cell line CHON-001 with IL-1β. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were utilized for assessing the effect of IL-1β with different concentrations (5, 10, and 20 ng/mL) on the activity and Gremlin-1 messenger RNA of CHON-001 cells, respectively. Besides, the influence of knocking down/over-expressing Gremlin-1 on the inflammatory factors (IL-6, TNF-α, IL-18 and PGE2), oxidative stress-related substances (malondialdehyde [MDA]; superoxide dismutase [SOD]; lactate dehydrogenase [LDH]), extracellular matrix (ECM) degradation-related proteins, and mitogen-activated protein kinase (MAPK) pathway proteins in IL-1β-stimulated CHON-001 cells were tested by enzyme-linked immunosorbent assay, related kits, qRT-PCR, and western blot, respectively. IL-1β inhibited CHON-001 cell proliferation and upregulated Gremlin-1 expression in a concentration-dependent manner. Overexpression of Gremlin-1 increased the IL-6, TNF-α, IL-18, PGE2, and MDA levels, enhanced the LDH activity, and decreased the SOD activity in IL-1β-induced CHON-001 cells; while the effect of Gremlin-1 knockdown on the above factors was in contrast with that of the overexpression. Furthermore, overexpression of Gremlin-1 upregulated protein expression of matrix metalloproteinase (MMP)-3, MMP-13, and ADAMTS4 while downregulated protein expression of collagen III, aggrecan, and SOX-9 in IL-1β-stimulated CHON-001 cells. Besides, overexpression of Gremlin-1 increased the p-p38/p38 value while decreased the p-JNK/JNK value in L-1β-stimulated CHON-001 cells; however, knockdown of Gremlin-1 reversed the above results. Gremlin-1 may promote IL-1β-stimulated CHON-001 cell inflammation and ECM degradation by activating the MAPK signaling pathway., (© 2023 Wiley Periodicals LLC.)

Chondrocyte inflammation and catabolism are two major features in the progression of osteoarthritis (OA). Chelidonine, a principal alkaloid extracted from Chelidonium majus, is suggested to show anti-inflammation, anti-apoptosis, and anti-oxidation activities in various diseases. However, its potential effects on OA cartilage degeneration remains unclear. To evaluate the effect of chelidonine on OA and its underlying mechanism, we incubated chondrocytes with interleukin (IL)-1β and chelidonine at varying concentrations. Then, we performed the CCK-8 assay, fluorescence immunostaining, reverse transcription PCR, ELISA, and western blotting to evaluate cell viability, catabolic/inflammatory factors, levels of extracellular matrix (ECM)-related proteins, and the involved pathways. H&E and Safranin-O staining and ELISA were performed to measure cartilage degradation and synovial inflammation. Chelidonine suppressed the IL-1β-mediated catabolism and inflammation of chondrocytes. Chelidonine suppressed the NF-κB pathway activation. Similarly, our in vivo experiment showed that chelidonine partially attenuated cartilage degradation while inhibiting synovial inflammation. Chelidonine inhibited inflammation and catabolism through modulation of NF-κB pathways in vitro, thereby avoiding rat cartilage degeneration and synovial inflammation within OA.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy. Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Osteoarthritis(OA) is an age-related degenerative disease involving chondrocyte apoptosis and extracellular matrix(ECM) degradation.Brain acid soluble protein 1(BASP1) has been reported to induce apoptosis.Thus, we speculated that BASP1 might regulate OA progression by inducing apoptosis, which is also the purpose of this study.The cartilage of the knee joint was collected from OA patients who received the joint replacement.In OA cartilage tissue,we found BASP1 expression was highly expressed, which inferred that BASP1 might be involved in OA.To validate our hypothesis, destabilization of the medial meniscus (DMM) surgery-induced male C57BL/6mice and interleukin-1β (IL-1β)-treated human chondrocytes were used to mimic the OA environment.BASP1 knockdown in mice and chondrocytes was achieved by adenovirus carried with BASP1-specific shRNA.High expression of BASP1 was observed in OA mice, which was also verified in IL-1β-treated chondrocytes.The potential mechanism of BASP1 in OA was further explored in vitro.BASP1 knockdown alleviated IL-1β-induced apoptosis and ECM degradation, as reflected by the decreased number of apoptotic cells and matrix metalloproteases 13 expression,and the increased collagen II expression.Our findings indicated that BASP1 knockdown alleviated OA progression by inhibiting apoptosis and ECM degradation, suggesting that inhibiting BASP1 may be a potentially applicable method for preventing OA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Osteoarthritis (OA) is a chronic disease of the bones and joints that commonly affects middle-aged and elderly individuals, characterized by the degeneration of articular cartilage and inflammation of the joints. The molecular mechanisms of OA urgently need to be further examined. Our study intended to uncover circ-NFKB1/miR-203a-5p/ERBB4 axis in regulating interleukin-1β (IL-1β) induced chondrocytes apoptosis., Methods: GSE178724, GSE79258 and GSE169077 were downloaded from Gene Expression Omibus (GEO) database and differentially expressed circRNAs, miRNAs and mRNAs were obtained by R software. Annexin V assay was used to determine cell apoptosis rate. ELISA was further performed to identify the inflammation response. Dual-luciferase reporter gene assay was conducted to examine the combination among circ-NFKB1, miR-203a-5p and ERBB4., Results: Our research demonstrated that circ-NFKB1 and ERBB4 were significantly upregulated through bioinformatic analysis. MiR-203a-5p was significantly downregulated through bioinformatic analysis. Silencing of circ-NFKB1 notably inhibited the IL-1β induced chondrocytes apoptosis and upregulated ERBB4 expression. Through prediction on bioinformatics analysis, miR-203a-5p was the target binding circ-NFKB1, and ERBB4 was the potential target of miR-203a-5p. Subsequently, these changes induced by the silencing of circ-NFKB1 were reversed upon addition of pcDNA/ERBB4., Conclusions: Silencing circ-NFKB1 could sponge miR-203a-5p to regulate ERBB4 expression and alleviate OA progression., (© 2023. The Author(s).)

Purpose: Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of the key elements. The phosphorylation of αB-crystallin/CRYAB modulates its molecular dynamics and chaperone activity, and attenuates αB-crystallin secretion via exosomes. In this study, we investigated the effect of phosphorylated αB-crystallin in retinal Müller cells on diabetic mimicking conditions, including interleukin (IL)-1β stimuli., Methods: Human retinal Müller cells (MIO-M1) were used to examine gene and protein expressions with real-time quantitative PCR, enzyme linked immunosorbent assay (ELISA), and immunoblot analyses. Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling staining. Retinal tissues isolated from the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic animal model with obesity, and fibrovascular membranes from patients with PDR were examined by double-staining immunofluorescence., Results: CRYAB mRNA was downregulated in MIO-M1 cells with the addition of 10 ng/mL IL-1β; however, intracellular αB-crystallin protein levels were maintained. The αB-crystallin serine 59 (Ser59) residue was phosphorylated with IL-1β application in MIO-M1 cells. Cell apoptosis in MIO-M1 cells was induced by CRYAB knockdown. Immunoreactivity for Ser59-phosphorylated αB-crystallin and glial fibrillary acidic protein was colocalized in glial cells of SDT fatty rats and fibrovascular membranes., Conclusions: The Ser59 phosphorylation of αB-crystallin was modulated by IL-1β in Müller cells under diabetic mimicking inflammatory conditions, suggesting that αB-crystallin contributes to the pathogenesis of PDR through an anti-apoptotic effect.

Objective: Poly (ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in DNA damage repair, gene transcription, cell growth, death and apoptosis. In our study, we aimed to explore the dynamic role of PARP-1 in chondrocyte (CH) degeneration in vitro., Methods: We used the primary CHs and treated them with interleukin-1 beta for up to 5 days. (IL-1β) to induce degeneration. Meanwhile, we used AG-14361 (AG) to inhibit endogenous PARP-1 expression. Cell survival and collagen II expression were used to define the cell function of CHs. In addition, other metabolic indicators were measured containing the reactive oxygen species (ROS) level, 8-Hydroxy-2'-deoxyguanosine (8-OH-dG), IL-1β, tumor necrosis factor alpha (TNF-α) and caspase 3/9 expression., Results: With IL-1β treatment, the PARP1 expression of CHs was gradually increased from day 1 to day 5, accompanied by a reduction in cell survival and collagen II expression, and an increase in ROS, 8-OH-dG, IL-1β, TNF-α and caspase 3/9 levels. We suppressed PARP1 expression on the first day of IL-1β stimulation and found severe destruction of cell survival and collagen II content with a higher expression of caspase 3/9. However, when we cultured the CHs with AG from day 3 of the 5-day IL-1β stimulation, cell survival and collagen II expression were rescued, and the ROS, 8-OH-dG, IL-1β, TNF-α, and caspase 3/9 were downregulated., Conclusions: On day 1 of degeneration, increased PARP-1 played a protective role in CHs. However, from days 3 to 5 of degeneration, the accumulated PARP-1 presented a more destructive function in CHs.

Osteoarthritis (OA) is a form of chronic degenerative disease contributing to elevated disability rate among the elderly. Genkwanin is an active component extracted from Daphne genkwa possessing pharmacologic effects. Here, this study is designed to expound the specific role of genkwanin in OA and elaborate the probable downstream mechanism. First, the viability of chondrocytes in the presence or absence of interleukin-1 beta (IL-1β) treatment was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to assess cell apoptosis. Inflammatory response was estimated through enzyme-linked immunosorbent assay and Western blot. In addition, immunofluorescence staining and Western blot were utilized to measure the expression of extracellular matrix (ECM)-associated proteins. Dual-specificity protein phosphatase-1 (DUSP1) expression was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Following DUSP1 elevation in genkwanin-treated chondrocytes exposed to IL-1β, inflammatory response and ECM-associated factors were evaluated as forementioned. In addition, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining was to assess the mitochondrial membrane potential. Adenosine triphosphate (ATP) level was examined with ATP assay kit, and RT-qPCR was used to test mitochondrial DNA expression. Results indicated that genkwanin administration enhanced the viability while ameliorated the apoptosis, inflammatory response, and ECM degradation in IL-1β-induced chondrocytes. Besides, genkwanin treatment fortified DUSP1 expression in IL-1β-exposed chondrocytes. DUSP1 interference further offsets the impacts of genkwanin on the inflammation, ECM degradation, and mitochondrial dysfunction in IL-1β-challenged chondrocytes. In short, genkwanin enhanced DUSP1 expression to mitigate mitochondrial dysfunction, thus ameliorating IL-1β-elicited inflammation, apoptosis, and degradation of ECM in chondrocytes., Competing Interests: None

Aims: Osteoarthritis (OA) is a chronic inflammatory disease, characterized by degradation of extracellular matrix, apoptosis of chondrocytes and inflammation in articular cartilage. Zinc finger E-box binding homebox 2 (ZEB2), a transcription repressor, has been demonstrated with anti-inflammatory role in some cells. The analysis from GEO data reveals that ZEB2 expression is upregulated in articular cartilage of OA patients and experimental OA rodents. This study aims to confirm the function of ZEB2 in OA process., Material and Methods: The experimental OA was induced by anterior cruciate ligament transaction (ACLT) in rats, and the adenovirus loaded with ZEB2 coding sequence was intra-articularly injected into rats (1 × 10 PFU). The primary articular chondrocytes were stimulated by interleukin-1β (IL-1β) (10 ng/ml) to mimic the osteoarthritic injury, and transfected with the adenovirus carrying ZEB2 coding or silencing sequence. The apoptosis, content of extracellular matrix, inflammation, and the activity of NFκB signaling in chondrocytes and cartilage were determined., Results: ZEB2 was highly expressed in osteoarthritic cartilage tissues and IL-1β-treated chondrocytes. The overexpression of ZEB2 restrained ACLT- or IL-1β administration-induced apoptosis, matrix degradation and inflammation in vivo or in vitro, evidenced by changed levels of cleaved caspase-3/PARP, collagen-II, aggrecan, matrix metalloproteinase 3/13, tumor necrosis factor-α and interleukin-6. Additionally, the phosphorylation of NFκB p65, IκBα and IKKα/β, and the nuclear translocation of p65 was blocked by ZEB2, implying inactivation of this signaling., Conclusions: ZEB2 mitigated osteoarthritic symptoms in rats and chondrocytes, and NFκB signaling might be involved. These findings may provide novel insights for clinical treatment of OA.

Meibomian glands (MGs) are vital for ocular surface health. However, the roles of inflammation in the progression of meibomian gland dysfunction (MGD) are largely unknown. In this study, the roles of the inflammation factor interleukin-1β (IL-1β) via the p38 mitogen-activated protein kinases (MAPK) signaling pathway on rat meibomian gland epithelial cells (RMGECs) were explored. Eyelids from adult rat mice at 2 months and 2 years of age were stained with specific antibodies against IL-1β to identify inflammation levels. RMGECs were exposed to IL-1β and/or SB203580, a specific inhibitor of p38 MAPK signaling pathway, for 3 days. Cell proliferation, keratinization, lipid accumulation, and matrix metalloproteinases 9 (MMP9) expression were evaluated by MTT assay, polymerase chain reaction (PCR), immunofluorescence staining, apoptosis assay, lipid staining, and Western blot analyses. We found that IL-1β was significantly higher in the terminal ducts of MGs in rats with age-related MGD than in young rats. IL-1β inhibited cell proliferation, suppressed lipid accumulation and peroxisome proliferator activator receptor γ (PPARγ) expression, and promoted apoptosis while activating the p38 MAPK signaling pathway. Cytokeratin 1 (CK1), a marker for complete keratinization, and MMP9 in RMGECs were also up-regulated by IL-1β. SB203580 effectively diminished the effects of IL-1β on differentiation, keratinization, and MMP9 expression by blocking IL-1β-induced p38 MAPK activation, although it also inhibited cell proliferation. The inhibition of the p38 MAPK signaling pathway blocked IL-1β-induced differentiation reduction, hyperkeratinization, and MMP9 overexpression of RMGECs, which provides a potential therapy for MGD., Competing Interests: Declaration of competing interest All authors indicate no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Osteoarthritis (OA) is a common, progressive, and chronic disorder of the joints that is characterized by the inflammation and degradation of articular cartilage and is known to significantly impair quality of daily life. Stevioside (SVS) is a natural diterpenoid glycoside that has anti-inflammatory benefits. Hence, in the current research, it was hypothesized that SVS might exert anti-inflammatory effects on articular chondrocytes and alleviate cartilage degradation in mice with OA. The expression of inflammatory cytokines, like inducible nitric oxide synthase (iNOS), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and cyclooxygenase-2 (COX-2) in chondrocytes after interleukin-1β (IL-1β) exposure, was inhibited by the pretreatment of SVS. As well, SVS inhibited the reduction of collagen II and sry-box transcription factor 9 (SOX9) in chondrocytes stimulated by IL-1β and suppressed the expression of MMP3 and MMP13. Further, after treatment with SVS, cell cytometry, autophagy flux, and related protein expression showed diminished cell apoptosis and reduced autophagy impairment. Moreover, SVS blocked the activation of phosphoinositide-3-kinase/Akt/nuclear factor-kappa beta (PI3K/Akt/NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways stimulated by IL-1β. This resulted in decreased cellular inflammation. In vivo experiments with intra-articular injections of SVS in mice with the DMM mouse model demonstrated a decrease in cartilage degradation and an improvement in subchondral bone remodeling. After the integrin αVβ3-related knockdown using siRNA, a reversed effect was observed on the anti-inflammatory, anabolic promoting, catabolic blocking, and NF-κB and MAPK signaling pathway inhibition of SVS on chondrocytes treated with IL-1β. The above findings highlighted that SVS blocked IL-1β, triggered an inflammatory response in mice chondrocytes, and prevented cartilage degradation in vivo through integrin αVβ3. This suggested that SVS might serve as a novel therapeutic option for OA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization., Methods: hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model., Results: IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation., Conclusion: Cxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM., (© 2023. The Author(s).)