Effect of The Autophagy Mechanism of TSCs Induced by Oxidative Stress Under Beclin1-mTOR Interaction

Abstract

Background Tendinopathy is a chronic injury disease caused by repeated traction. It is characterized by exercise-related pain, increased local tendon sensitivity, and imaging changes in the tendon. Rotator cuff injury is one of the typical tendinopathy. Tendon-derived stem cells (TDSC) play a vital role in the development of tendinopathy. Our previous studies have found that reactive oxygen species increase after rotator cuff injury and the oxidative stress response is strengthened, but whether oxidative stress induces TDSC autophagy to promote tendon bone healing is not clear. Methods First, we collected the injured and normal tendon tissues of patients with rotator cuff injury, detected the levels of reactive oxygen species (ROS) and superoxide anion (SOD) in the tissues, detected Beclin1, mTOR gene expression by qPCR, and WB (Western blotting). Beclin1, p-mTOR/mTOR protein expression.Then, we extracted human tendon stem cells (TDSC) from tendon tissue, infected TDSC with recombinant lentivirus pLKO.1-shBeclin1, and verified the expression of Beclin1 by qPCR and WB.Finally, H2O2 and 3-MA were used to intervene TSCs. CCK8 was used to detect the proliferation ability of H2O2 on human TSCs; autophagy staining (MDC), autophagy-lysosome staining (Lyso-Tracker Red) and transmission electron microscopy were used to observe autophagy. Immunofluorescence staining detects the expression of autophagy factor LC3A/B; DCFH-DA detects cellular reactive oxygen species ROS level, Annexin V/PI detects cell apoptosis; WB detects Beclin1, mTOR, p-mTOR (Ser2448), LC3A/B, cleaved caspase-3 protein expression. Results In this study, it was found that the expression levels of ROS and Beclin1 in the damaged rotator cuff tissue were higher, while the expression levels of SOD and mTOR were lower. After the recombinant lentivirus pLKO.1-shBeclin1 was infected with TDSC, the expression of Beclin1 decreased. After treating TDSCs with H2O2 and 3-MA, it was found that H2O2 caused an increase in reactive oxygen species ROS content, autophagy levels, and LC3A/B expression in TDSCs, and an increase in Beclin1, mTOR, LC3A/B, and cleaved caspase-3 protein expression. Lead to a decrease in the level of apoptosis. Conclusion Under the mutual regulation of Beclin1-mTOR, oxidative stress induces the occurrence of autophagy in TSCs, and autophagy may protect TSCs from oxidative stress by reducing the accumulation of ROS.