| Objective To investigate the effect of Zuo-Gui-Wan (ZGW) on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) based on demethylase ALKBH5. Methods Mouse BMSCs were isolated and cultured. The 3-5 generations of cells were randomly divided into: (1) osteogenesis induction 0, 3, 7 d; (2) the blank group and ZGW group (1000 μg/mL); and (3) small interfering RNA (siRNA) was used to transfect mouse BMSCs to construct ALKBH5 knockdown mouse BMSCs. Real-time PCR (RT-qPCR) was used to detect the knockdown efficiency. Then cells were divided into control group (si-NC), ZGW group (si-NC+ZGW), model group (si-ALKBH5), and model+ZGW group (si-ALKBH5+ZGW). The osteogenic differentiation level and mineralization ability of BMSCs in each group were detected using alkaline phosphatase staining (ALP) and Alizarin red (ARS) staining. mRNA expression levels of ALKBH5 and markers of osteogenic differentiation like 1BMP4, COL1A1, and Osx were detected with real-time PCR. The protein expression levels of Runx2, BMP4, and ALKBH5 in BMSCs in each group were detected with Western blotting. Results During osteogenic differentiation of mouse BMSCs, the expressions of ALKBH5 mRNA and protein were up-regulated. ZGW up-regulated the protein expression levels of ALKBH5, Runx2, BMP4, and BMP2, and promoted the osteogenic differentiation of BMSCs in mice. After using siRNA to knockdown ALKBH5, the mRNA and protein expressions of Runx2 and Osx were down-regulated, The protein expression of ALKBH5 was reversed with ZGW intervention in knockdown model cells. The expressions of Runx2 and BMP2 were up-regulated accordingly, and the osteogenic differentiation ability was inhibited. Conclusion The expression level of ALKBH5 demethylase is up-regulated during osteogenic differentiation of mouse BMSCs. ZGW up-regulates ALKBH5 to promote osteogenic differentiation of BMSCs in mice. |