原代成纤维细胞专用培养基

Background: The role of docosahexaenoic acid (DHA) in fibrosis of other organs has been studied, but its function in oral submucous fibrosis (OSF) has not been reported. This study aimed to investigate the role and mechanism of DHA in OSF. Methods: OSF rat and cell models were established induced by arecoline. Through a series of in vivo and in vitro experiments, the function of DHA in OSF was investigated. Mechanistically, the interaction of TGM-2 with HSP70A1A and p62 proteins was validated using co-immunoprecipitation. Additionally, in cells transfected with overexpression vectors of HSP70A1A or TGM-2 and treated with DHA and arecoline or co-treated with a p62 inhibitor XRK3F2 along with DHA and arecoline, the function of the DHA/HSP70A1A/TGM-2/p62 axis in OSF was explored. Results: In vivo, arecoline caused severe pathological damage and fibrosis in rat oral mucosal tissues and induced overexpression of HSP70A1A. Arecoline treatment also elevated tissue ROS levels and the expression of α-SMA, Collagen I, TGM-2, and LC3 II/I, while decreasing tissue p62 protein expression and serum GSH levels. Treatment with DHA reversed these changes and improved the pathological damage and fibrosis in OSF rats. In vitro, arecoline induced the expression of HSP70A1A in a concentration-dependent manner, and DHA inhibited its expression by directly binding to HSP70A1A and reducing the expression of α-SMA, Collagen I, TGM-2, LC3 II/I, and ROS levels induced by arecoline in cells, while increasing p62 protein expression and GSH levels in cell supernatants. Furthermore, arecoline induced TGM-2 expression, and overexpression of HSP70A1A counteracted the protective effect of DHA on cells and the suppression of TGM-2 expression. TGM-2 interacted with HSP70A1A and p62 proteins. Overexpression of TGM-2 or treatment with XRK3F2 activated autophagy and abolished the protective effect of DHA on cells. Conclusion: DHA inhibits p62-dependent autophagy through targeting the HSP70A1A/TGM-2 axis, thereby alleviating arecoline-induced OSF. These results suggest that DHA and its mediated autophagy regulation mechanism can be a therapeutic target for OSF.

Oral submucous fibrosis (OSF) is an oral condition characterized by chronic progression, which may lead to the development of malignancy. Currently, available treatments for OSF only provide temporary relief of symptoms, and there is a limited availability of effective interventions that can effectively cure this condition. In this study, we aimed to investigate whether adiponectin (APN) could ameliorate OSF and the mechanisms involved in it. First, human oral mucosal fibroblasts (HOMFs) were cultured, an OSF model was established using arecoline, and APN and Imiquimod treatment were administered. Then we overexpressed NLRP3 and knocked down FOXO3A. FOXO3A, fibrosis-related factors (ɑ-SMA, COL1A, CTGF), TGF-β1/Smad3 signaling-related factors (TGF-β1, p-Smad3, Smad3), NLRP3 inflammasome-related factors (NLRP3, Caspase-1, IL-1β), and ROS levels were evaluated. Finally, we explored the effect of APN on OSF in mice by in vivo experiments. We found that arecoline significantly increased ɑ-SMA, COL1A, CTGF, and TGF-β1 expressions and promoted Smad3 phosphorylation, while APN significantly inhibited the elevation of these fibrosis-related factors. ROS production was significantly elevated in HOMFs after arecoline treatment, while APN treatment inhibited ROS production. However, the addition of Imiquimod and overexpression of NLRP3 exhibited a trend of elevated ROS, resisting the inhibitory effect of APN. Furthermore, adding Imiquimod and overexpression of NLRP3 elevated ɑ-SMA, COL1A and CTGF and activated TGF-β1/Smad3 signaling pathway. Additionally, knockdown of FOXO3A enhanced APN-inhibited ɑ-SMA and COL1A. In vivo experiments further confirmed that APN ameliorated OSF in mice by inhibiting ROS/NLRP3 inflammatory pathway. In conclusion, APN ameliorated arecoline-induced OSF by promoting FOXO3A expression and downregulating the ROS/NLRP3 pathway.

Oral submucous fibrosis (OSF) is a precancerous condition characterized by oral mucosal atrophy with fibrosis of the submucosal tissue. OSF has a high prevalence, and treatment requires improvement. Our study aims to investigate the role and mechanism of secreted frizzled-related protein 1 (SFRP1) in OSF. We constructed an arecoline-induced OSF mice model. Through Pearson’s correlation analysis, we investigated the association between SFRP1 levels and expressions of proteins related to the Wnt/β-catenin signaling pathway, as well as the correlation between SFRP1 levels and the degree of neutrophil infiltration. Moreover, neutrophil infiltration intensity, tissue fibrosis degree, and levels of β-catenin, Cyclin D1, and c-myc were evaluated after SFRP1 overexpression treatment through immunohistochemical and biochemical assays. A Wnt/β-catenin pathway activator was used to investigate the molecular mechanism of SFRP1 in the arecoline-induced OSF cell model. Compared with the control group, mice in the OSF group exhibited increased collagen deposition and more severe fibrosis in the oral mucosal tissue (OMT). In the OMT of OSF mice, the levels of SFRP1 were decreased. The levels of SFRP1 exhibited a negative correlation with the levels of Wnt/β-catenin proteins and neutrophil infiltration in the OMT. Upon SFRP1 overexpression, there was a reduction in neutrophil infiltration and fibrosis in the OMT, as well as inhibition of Wnt/β-catenin-related proteins. In vitro, the Wnt/β-catenin pathway activator further reversed the effect of SFRP1 overexpression on OSF. SFRP1 attenuates OSF by reducing neutrophil infiltration and inhibiting the Wnt/β-catenin pathway.