Abstract
BACKGROUND CONTEXT The osteoprotective effect on glucocorticoid-induced osteoporosis in vivo and osteogenesis effectin vitro of the extracts from plastrum testudinis (PTE) have been demonstrated in previous studies. However, its effects and mechanisms of action on promoting bone mesenchymal stem cells (BMSCs) osteogenic differentiation remain to be determined. PURPOSE The present study aimed to determine the effect of PTE on BMSCs proliferation and osteogenic differentiation, and to elucidate the possible mechanism of their action. STUDY DESIGN/SETTING Bone mesenchymal stem cell proliferation was examined with a cell proliferation assay Cell Counting Kit-8 (CCK-8), ALP staining was used to visualize early BMSCs differentiation, and Alizarin red staining was performed to determine the BMSCs osteogenic mineralization ability. The mRNA expression level of let-7f-5p, TNFR2, TRAF2, PI3K, Akt, β-catenin, and GSK3β were measured by quantitative real time RT-PCR. The protein expressions of TNFR2, TRAF2, p-PI3K, p-Akt, p-β-catenin, and p-GSK3β were measured by Western-blot assay. The functional targeting relationship of let-7f-5p and TNFR2 was determined by luciferase reporter assays. OUTCOME MEASURES Our results show that a direct targeted relationship between let-7f-5p and TNFR2, suggesting that PTE may promote BMSCs proliferation and osteogenic differentiation via let-7f-5p targeting TNFR2/PI3K/Akt signaling pathway. METHODS Bone mesenchymal stem cells proliferation was examined with a cell proliferation assay Cell Counting Kit-8 (CCK-8), ALP staining was used to visualize early BMSCs differentiation, and Alizarin red staining was performed to determine the BMSCs osteogenic mineralization ability. The mRNA expression level of let-7f-5p, TNFR2, TRAF2, PI3K, Akt, β-catenin, and GSK3β were measured by quantitative real time RT-PCR. The protein expressions of TNFR2, TRAF2, p-PI3K, p-Akt, p-β-catenin, and p-GSK3β were measured by Western-blot assay. The functional targeting relationship of let-7f-5p and TNFR2 was determined by luciferase reporter assays. RESULTS We found that 30 μg/ml was the optimum concentration for PTE intervention. PTE significantly promoted BMSCs osteogenic differentiation and mineralization after 7days culture and 14days culture, respectively. And the combination of PTE and osteogenic induction exhibited significant synergies. Moreover, PTE up-regulated let-7f-5p and β-catenin mRNA expression, and down-regulated TNFR2, TRAF2, PI3K, Akt, and GSK3β mRNA expression. Plastrum testudinis inhibited TNFR2, TRAF2, and β-catenin protein expression, and promoted p-PI3K, p-Akt, and p-GSK3β protein expression. We also demonstrated that TNFR2 is a functional target of let-7f-5p. CONCLUSIONS Together, these results showed that PTE may promote BMSCs proliferation and osteogenic differentiation via let-7f-5p targeting TNFR2/PI3K/Akt signaling pathway.
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