Tanshinone IIA regulates fibroblast proliferation and migration and post-surgery arthrofibrosis through the autophagy-mediated PI3K and AMPK-mTOR signaling pathway

Post-surgery arthrofibrosis is one of the most restrictive factors in the development of intra-articular surgery and has presented tremendous obstacles for most orthopaedic surgeons. Tanshinone IIA (Tan IIA), a key active ingredient of Den-shen, has been used to treat fibrosis-related diseases, such as pulmonary, hepatic and myocardial fibrosis. In the present study, we aimed to investigate the effects of Tan IIA on post-surgery arthrofibrosis in vivo and in vitro. Histological analysis indicated that topical application of Tan IIA (10 mg/mL) could significantly alleviate postsurgery arthrofibrosis in rabbits. Immunohistochemistry results showed that proliferating cell nuclear antigen (PCNA) and tubulin protein expression was inhibited, whereas LC3 was activated in vivo. In vitro, EdU and flow cytometry assays demonstrated that Tan IIA could inhibit fibroblast proliferation by arresting cells in G2 phase. Scratch, Transwell and Cytoskeleton protein immunofluorescence assays revealed that fibroblast migration was attenuated. Interestingly, LC3 immunofluorescence staining and transmission electron microscopy indicated that Autophagy flux could be induced in fibroblasts by Tan IIA. However, the inhibitory effects of Tan IIA against fibroblast proliferation and migration were partially restored when fibroblast Autophagy was suppressed after combined treatment with the Autophagy Inhibitor 3-methyladenine (3-MA). Finally, the expression of p-mTOR was suppressed in a dose-dependent manner after Tan IIA treatment but partially restored when Tan IIA treatment was combined with 3-MA intervention. The inhibitory effect of Tan IIA against fibroblast proliferation and migration may be related to Autophagy induction mediated by the PI3K and AMPK-mTOR signaling pathway.