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An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (m6A) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms’ tumor 1-associated protein (WTAP), a member of the m6A functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.

Objective To study the curative effect of bionic tiger‐bone powder on osteoporosis in ovariectomized rats and investigate its mechanism. Methods Overall, a 120 female Wistar rats were randomly divided into Sham (sham‐operated group), ovariectomy (OVX, ovariectomized group), TB (bionic tiger‐bone powder treatment group after ovariectomy) and TB + VD groups (bionic tiger‐bone powder + vitamin D treatment group after ovariectomy). The osteoporotic rat model was established 3 months after ovariectomy, and rats were intragastrically administrated with the corresponding drugs. Serum and bone tissue samples were collected from 10 rats in each group at weeks 4, 12 and 24 after intragastric administration. The bone microstructure of L6 vertebrae was analyzed by MicroCT, the biomechanical strength of left femurs was measured by the three‐point bending test, and serum bone metabolism markers (P1NP and CTX) were detected by ELISA. Changes in bone collagen were analyzed by Masson's trichrome staining and hydroxyproline detection, and members of the BMP2/SMAD/RUNX2 and OPG/RANKL/RANK signal pathways were detected by immunoblotting. Results Compared with the OVX group, the serum level of P1NP in the TB and TB + VD groups was higher (P < 0.05), while the CTX level was lower (P < 0.05). Bone collagen fiber structures in the TB and TB + VD groups were repaired, and the collagen content was significantly higher than that in the OVX group (P < 0.05). In the TB group, BMP‐2, P‐SMAD1/5, RUNX2 and OPG levels were increased in bone tissue (P < 0.01), RANKL levels were decreased (P < 0.01), and the bone microstructure and biomechanical strength were improved. Conclusion Bionic tiger‐bone powder promotes osteogenesis by activating the BMP2/SMAD/RUNX2 signaling pathway, suppresses osteoclasts by downregulating the OPG/RANK/RANKL signaling pathway, increases bone collagen content, and improves bone microstructure and bone biomechanical strength.