Simvastatin promotes osteogenic differentiation of mesenchymal stem cells in rat model of osteoporosis through BMP-2/Smads signaling pathway

C. Feng, L. Xiao, J.-C. Yu, D.-Y. Li, T.-Y. Tang, W. Liao, Z.-R. Wang, A.-Q. Lu

Department of Orthopedics, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, China. zjgzyyxl@163.com

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• Pharmacology

OBJECTIVE: By establishing osteoporosis (OP) model in rats, the specific regulatory effect of simvastatin on promoting the differentiation of mesenchymal stem cells (MSCs) into osteoblasts through the bone morphogenetic protein 2 (BMP-2)/Smads signaling pathway was investigated.

MATERIALS AND METHODS: A total of 45 Sprague-Dawley rats were selected to establish the OP model by performing ovariectomy. The rats were divided into OP model group (OP group, n=15), 10-7 mmol/L simvastatin treatment group (SIM group, n=15), and normal control group (Control group, n=15). After the experimental period, the enzyme-linked immunosorbent assay (ELISA) was applied to observe the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was adopted to detect the contents of the differentiation-associated genes [runt-related transcription factor 2 (RUNX2) and Osterix (Osx)]. Later, the bone marrow MSCs (BMSCs) were selected and divided into Control group, 10-7 mol/L simvastatin group (SIM group), and osteoinduction medium group (OM group). Cell morphology in each group was observed. The Cell Counting Kit-8 (CCK-8) was performed to determine the proliferation activity of BMSCs. ELISA was performed to measure the level of alkaline phosphatase (ALP). RT-PCR was conducted to examine the levels of key differentiation-associated gene RUNX2 and those in BMP-2/Smads pathway. Moreover, the Western blotting was adopted to analyze the expressions of RUNX2 and genes in BMP-2/Smads pathway.

RESULTS: The serum levels of TNF-α, IL-6, and IL-1 in OP group were remarkably higher than those in the Control group, and their levels in the SIM group were close to those in the Control group. The elevated messenger ribonucleic acid (mRNA) levels of the key differentiation-associated factors RUNX2, osteoprotegerin (OPG), osteopontin (OPN), and Osx were observed in the SIM group. In vitro cell culture revealed that the cells were in a favorable growth status in the SIM group and OM group, mostly manifesting in fusiform or spindle shape, and proliferated rapidly. In addition, the ALP level notably increased in the two groups compared with that in the Control group (p<0.05). Both SIM group and OM group had evidently higher mRNA expression levels of RUNX2, OPG, OPN, and Osx than those in the Control group (p<0.05), consistent with the expression trends of the genes in BMP-2/Smads pathway. The Western blotting indicated that the expression levels of RUNX2 and genes in BMP-2/Smads pathway in the SIM group were significantly higher than those in the Control group.

CONCLUSIONS: Simvastatin can promote the differentiation of MSCs into osteoblasts in the OP rat model through the BMP-2/Smads signaling pathway.

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