Exogenous hydrogen sulfide promotes osteogenic differentiation of senescent BMSCs through Wnt/β-catenin signaling pathway
FAN Qinchen 1， HE Dawei 2， LI Chong 3
(1. School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013; 2. Clinical Research & Lab Center, Kunshan Hospital Affiliated to Jiangsu University, Suzhou Jiangsu 215300; 3. Department of Spinal Surgery, Kunshan Hospital Affiliated to Jiangsu University,Suzhou Jiangsu 215300, China)
Abstract：Objective： To investigate the effect of exogenous hydrogen sulfide donor GYY4137 on the osteogenic ability of D-galactose (D-Gal)induced senescent bone marrow mesenchymal stem cells (BMSCs) and its potential mechanism. Methods： BMSCs senescence was induced by different concentrations of D-Gal. The cell activity was detected by CCK8 method and the degree of cellular senescence was evaluated by β-galactosidase staining to choose the optimal concentration of D-Gal. After treatment with various concentrations of GYY4137, the effects of GYY4137 on the activity of normal BMSCs and senescent BMSCs were detected, and the optimal concentration of GYY4137 was determined. CON group, GYY4137 group, D-Gal group and D-Gal+GYY4137 group were set up, and β-galactosidase staining was performed on BMSCs in each group after corresponding treatment. The mRNA expression of cellular senescence-related genes (P16, P21, P53) and cellular senescencerelated inflammatory factors (IL-6, IL-8, TNF-α) was detected by qRT-PCR. Osteoblastic induction differentiation was performed on cells in each group. mRNA expressions of osteogenic differentiation-related factors alkaline phosphatase (ALP), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and human bone morphogenetic protein 2 (BMP2) were detected by qRT-PCR. The number of calcium nodules formed was detected by alizarin red staining. ALP was detected by ALP staining. The differentially expressed genes of D-Gal group and D-Gal+GYY4137 group were detected by transcriptome sequencing. According to the sequencing results, the differentially expressed genes were verified by qRT-PCR. After the expression of Wnt10b was inhibited by DKK1, the osteogenic differentiation ability of senescent BMSCs in each group was detected. Results： The optimal induction concentration of D-Gal was 30 g/L, and the optimal intervention concentration of GYY4137 was 10 μmol/L. The β-galactosidase content in D-Gal group was significantly increased. The mRNA expression levels of senescence related genes (P16, P21, P53) and senescence related inflammatory factors (IL-6, IL-8, TNF-α) were significantly increased (P<0.05 or P<0.01), and the above cell senescence related indicators were significantly decreased after GYY4137 intervention (P<0.05 or P<0.01). During osteogenic differentiation, the mRNA expressions of ALP, OCN, RUNX2 and BMP2 in D-Gal+GYY4137 group were significantly higher than those in D-Gal group (P<0.05 or P<0.01). The sequencing results showed that there were significant differences in Wnt10b expression in Wnt/βcatenin signaling pathway, and qRT-PCR results showed that GYY4137 increased the expression of Wnt10b and its downstream gene β-catenin in senescent cells (P<0.05 or P<0.01), which was consistent with the sequencing results. The expression of Wnt10b was inhibited by 100 μg/L DKK1, and the mRNA expressions of ALP, OCN, RUNX2 and BMP2 related to osteogenic differentiation of senescent BMSCs treated with GYY4137 were significantly decreased (P<0.05 or P<0.01). Conclusion： GYY4137 can alleviate the D-Gal-induced senescent process of BMSCs, and may promote osteogenic differentiation of senescent BMSCs through Wnt/β-cantenin signaling pathway. ［Key words］bone marrow mesenchymal stem cells; cellular senescence; hydrogen sulfide donor; senile osteoporosis; Wnt/β-catenin signaling pathway
