Plastrum testudinis extracts promote BMSC proliferation and osteogenic differentiation by regulating Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway

Abstract

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, β-catenin, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, β-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.