2022, Vol. 26 ›› Issue (31): 4954-4961

Expression of autophagy in rotator cuff tendon stem cells induced by oxidative stress

Wei Hewei1, Zheng Weipeng1, Liu Zhijun1, Zhao Guoyuan2, Fang Weihua2, Chen Sheng1, Liao Zhihao1, Wan Lei1   

Abstract: BACKGROUND: The mechanism of rotator cuff tendon-bone healing has not yet been fully elucidated. Studies have shown that oxidative stress can lead to tissue cell damage, which is not conducive to wound healing. It is clinically found that the tendon-bone insertion point of rotator cuff injury often has a poor healing effect, and it is speculated that this may be related to the abnormal cell apoptosis in the wound under ischemia and hypoxia stress.
OBJECTIVE: To induce autophagy of rotator cuff tendon stem cells to promote cell survival as a breakthrough point by oxidative stress stimulation, it is envisaged that autophagy can alleviate cellular oxidative stress damage.
METHODS: The rotator cuff tendon stem cells were cultured in vitro and treated with 0, 50, 100, 200, 400 μmol/L H2O2 respectively for 24, 48 and 72 hours, followed by CCK-8 assay to detect the cell viability. The oxidative stress injury model of rotator cuff tendon stem cells in vitro was established with an appropriate concentration of H2O2. The cultured rotator cuff tendon stem cells at passage 3 were divided into three groups: blank group, H2O2 (50 μmol/L) intervention group, and 3-methyladenine pretreatment (5 mmol/L, 30 minutes)+H2O2 group. At 48 hours after intervention, autophagy was detected by MDC and Lyso-Tracker Red staining. Autophagy related genes were detected by gene expression microarray. Reactive oxygen species level was detected by DCFH-DA. Apoptosis was detected by Annexin V/PI. Protein expression levels of Beclin-1, p-mTOR, mTOR, LC3A/B and Caspase-3 were detected by western blot assay.
RESULTS AND CONCLUSION: (1) With the increase of H2O2 concentration, the proliferation activity of rotator cuff tendon stem cells significantly decreased (P < 0.05). When the concentration of H2O2 was 50 μmol/L, the proliferation activity of rotator cuff tendon stem cells was the strongest. (2) Compared with the blank group and 3-methyladenine+H2O2 group, the autophagy level of H2O2 group was significantly increased (P < 0.05). (3) Compared with the blank group and 3-methyladenine+H2O2 group, the level of reactive oxygen species was significantly increased (P < 0.05); the expression of Beclin-1 protein was up-regulated (P < 0.05); the expression of mTOR protein was down-regulated (P < 0.05); the expression of LC3A/B protein was up-regulated (P < 0.05); and the expression of Caspase-3 protein was up-regulated (P < 0.05) in the H2O2 group. (4) These results indicate that the low concentration of H2O2 can promote the proliferation of human tendon stem cells, and the degree of apoptosis and reactive oxygen species level also increase slightly in a certain range, which is a state of high cell transformation. Autophagy may play a cytoprotective role and promote this high transformation state, but the specific mechanism needs to be further studied.
Key words: rotator cuff, tendon stem cells, oxidative stress, autophagy, apoptosis, tendon bone healing