Resveratrol promotes apoptosis and G2/M cell cycle arrest of fibroblast-like synoviocytes in rheumatoid arthritis through regulation of autophagy and the serine-threonine kinase-p53 axis.

Introduction: Resveratrol, a polyphenol extracted from many plant species, has emerged as a promising pro-apoptotic agent in various cancer cells. However, the role of resveratrol in cell proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis (RA-FLS) is not fully understood. The study was aimed at elucidating the role of resveratrol in cell proliferation and apoptosis of RA-FLS and the underlying molecular mechanism. Material and methods: Cultured RA-FLSs were subjected to tumour necrosis factor α (TNF-α). The cell proliferation was measured by Cell Counting Kit-8 assay. Cell apoptosis and cell cycle of RA-FLSs were determined by flow cytometry. The levels of apoptosis or autophagy or cell cycle-related protein were detected by immunoblot analysis. Results: In our study, we confirmed that resveratrol reversed TNF-α mediated cell proliferation in RA-FLS. Meanwhile, resveratrol blocked cells at the G2/M stage and reduced the ratio of S phase cells through upregulation of p53 and consequently led to apoptotic cell death. Quite interestingly, we found that resveratrol reversed TNF-α-induced autophagy. Inhibition of autophagy by resveratrol or autophagy inhibitor or Beclin-1 siRNA suppressed TNF-α mediated cell survival and promoted cell apoptosis. However, the autophagy inducer rapamycin (RAPA) reversed the effect of resveratrol on autophagy and cell proliferation. Mechanistic studies revealed that resveratrol inhibited the activation of the phosphoinositide 3-kinases/serine-threonine kinase (PI3K/AKT) pathway. Inhibition of PI3K/AKT pathway by inhibitor LY294002 or resveratrol increased the expression of p53 and decreased the expression of cycle protein (cyclin B1), which further led to block cells in the G2/M arrest. Conclusions: Our preliminary study indicated that resveratrol may suppress RA-FLS cell survival and promote apoptosis at least partly through regulation of autophagy and the AKT-p53 axis.

Human umbilical cord mesenchymal stem cells improve disease characterization of Sjogren's syndrome in NOD mice through regulation of gut microbiota and Treg/Th17 cellular immunity.

BACKGROUND: For the unclear pathogenesis of Sjogren's syndrome (SS), further exploration is necessary. Mesenchymal stem cells (MSCs) and derived exosomes (MSCs-exo) have exhibited promising results in treating SS. OBJECT: This study aimed to investigate the effect and mechanism of human umbilical cord MSCs (UC-MSCs) on SS. METHODS: Nonobese Diabetic (NOD) mouse splenic T cells were co-cultured with UC-MSCs and UC-MSCs-exo, and interferon-gamma (IFN-γ), interleukin (IL)-6, IL-10, prostaglandin E2 (PGE2), and transforming growth factor-ß1 (TGF-ß1) levels in the supernatant were assessed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Co-cultured T cells were injected into NOD mice via the tail vein. The inflammatory cell infiltration in the intestine and the submandibular gland was characterized by hematoxylin-eosin staining. Treg/Th17 homeostasis within the spleen was determined by flow cytometry. Gut microbiota was detected by 16S rRNA sequencing, and the relationship between differential microbiota and Treg/Th17 cytokines was analyzed by the Pearson correlation coefficient. RESULTS: UC-MSCs, UC-MSCs-exo, and NOD mouse splenic T cells were successfully cultured and identified. After T cells were co-cultured with UC-MSCs and UC-MSCs-exo, both IFN-γ and IL-6 were decreased while IL-10, PGE2, and TGF-ß1 were increased in transcriptional and translational levels. UC-MSCs and UC-MSCs-exo partially restored salivary secretion function, reduced Ro/SSA antibody and α-Fodrin immunoglobulin A levels, reduced inflammatory cell infiltration in the intestine and submandibular gland, raised proportion of Treg cells, decreased IFN-γ, IL-6, IL-2, IL-17, lipopolysaccharide, and tumor necrosis factor-alpha levels, and raised IL-10, Foxp3, and TGF-ß1 levels by affecting co-cultured T cells. The intervention of UC-MSCs and UC-MSCs-exo improved intestinal homeostasis in NOD mice by increasing microbiota diversity and richness. Additionally, differential microbiota was significantly associated with Treg/Th17 cytokine levels. CONCLUSION: Human UC-MSCs and UC-MSCs-exo improved disease characterization of SS in NOD mice through regulation of gut microbiota and Treg/Th17 cellular immunity.

Iguratimod inhibits skin fibrosis by regulating TGF-ß1/Smad signalling pathway in systemic sclerosis.

BACKGROUND: Iguratimod (T-614), exerting a powerful anti-inflammatory ability, has therapeutic efficacy in multiple autoimmune diseases. However, the effect of T-614 on systemic sclerosis (SSc) is unclear. Here, we investigate the effect and molecular mechanism of T-614 in experimental SSc models. METHODS: In vitro, cultured dermal fibroblasts from four SSc patients were subjected to different doses of T-614 in the presence or absence of TGF-ß1 stimulation. Cell proliferation, apoptosis and migration were determined by CCK-8, flow cytometry and transwell assay, respectively. Fibrosis markers and smad signalling pathway-related proteins were detected by immunoblotting and immunofluorescence. In vivo, a bleomycin-induced SSc mouse model was used to evaluate the effect of T-614 on skin fibrosis. Pathological changes in skin tissues were evaluated by HE, Masson staining and immunohistochemistry. RESULTS: In the study, we found T-614 inhibited TGF-ß1-induced cell proliferation, migration and promoted apoptosis in a dose-dependent manner (all p < 0.01). T-614 partially reversed TGF-ß1-induced upregulation of fibrosis markers and phosphorylation of smad2 and smad3 and blocked p-Smad3 nuclear translocation (all p < 0.05), suggesting T-614 may inhibit dermal fibroblasts activation by regulating TGF-ß1/smad pathway. In vivo experiments, T-614 alleviated skin thickness in bleomycin-induced SSc mice (all p < 0.05). The expression of fibrosis markers and the infiltration of macrophages in skin tissue were significantly decreased after T-614 treatment (all p < 0.05). CONCLUSION: Our preliminary data indicated T-614 inhibited dermal fibroblasts activation and skin fibrosis at least partly by regulating TGF-ß1/smad pathway in experimental SSc models and may be a promising therapeutic agent for SSc.

Autophagy inhibitor regulates apoptosis and proliferation of synovial fibroblasts through the inhibition of PI3K/AKT pathway in collagen-induced arthritis rat model.

BACKGROUND AND OBJECTIVE: Mounting studies have illustrated an important role of autophagy in various diseases, but few studies have reported its contribution to rheumatoid arthritis (RA) and the underlying mechanism was largely unknown. This study aimed to investigate whether autophagy inhibitors could regulate apoptosis and proliferation through PI3K/AKT pathway in RA. METHODS: RA animal model was established by collagen induction. General observations and degree of joint swelling were observed. Inflammatory response, cell survival related factors and apoptosis were also detected in synovial fibroblasts. In addition, cultured rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were subjected to TNF-α treatment in vitro, and TNF-α induced cell autophagy, synovial cell proliferation and apoptosis were detected. Moreover, cell cycle and cytokine secretion protein, along with the above parameters, were analyzed. RESULTS: Results from the animal model showed that autophagy inhibitors attenuated inflammatory reaction and synovial hyperplasia, while promoted synovial fibroblasts apoptosis. Meanwhile, inhibition of autophagy promoted cell apoptosis and reversed cell proliferation in vitro, also blocked cell in the G2/M arrest and reduced the S phase cells. Furthermore, we observed that inhibition of PI3K/AKT pathway reversed TNF-α mediated autophagy and cytokine secretion. CONCLUSION: autophagy inhibitors could mitigate inflammation response, inhibiting RA-FLS cell proliferation while promoting cell apoptosis by the PI3K/AKT pathway.