Docosahexaenoic Acid Inhibits p62-Dependent Autophagy by Targeting HSP70A1A/TGM-2 Axis to Alleviate Arecoline-Induced Oral Submucosal Fibrosis

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.