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2022, Vol. 26 ›› Issue (13): 2020-2026Guilu Erxian Gum medicated serum mediated osteogenic differentiation of rat bone marrow mesenchymal stem cellsChen Jincheng1, 2, Zhu Guotao1, 2, Qin Xiaofei1, 2, Chen Yancheng2, Luo Jun2, Liu Hongwen1, 2, Wu Yijing2, Xu Jie1, 2
Abstract: BACKGROUND: On the basis of studying the proliferation of bone marrow mesenchymal stem cells, revealing their differentiation mechanism is one of the hot topics in the study of prevention and treatment of osteoporosis. OBJECTIVE: To explore the relationship between the molecular mechanism of Guilu Erxian Gum medicated serum that induces osteogenic differentiation of bone marrow mesenchymal stem cells to prevent osteoporosis and the activation of extracellular signal-regulated kinase 1/2/E26 transcription factor 1 signaling pathway factors. METHODS: Passage 3 commercial SD rat bone marrow mesenchymal stem cells were purchased, and divided into fetal bovine serum group, blank serum group, Guilu Erxian Gum medicated serum group, classical induction group, pathway inhibitor group, and Guilu Erxian Gum+pathway inhibitor group. Osteogenic differentiation was conducted according to group conditions, and the fluid was altered every 3 days. At 7 days after intervention, alkaline phosphatase activity detection kit was used to determine the osteogenic differentiation level of bone marrow mesenchymal stem cells in the first four groups. At 14 days after intervention, alizarin red staining was used to verify the level of bone formation and mineralization of bone marrow mesenchymal stem cells in the first four groups. Real-time fluorescent quantitative PCR was used to detect the gene expression levels of alkaline phosphatase, core binding protein 2, peroxisome proliferator activated receptor γ, and E26 transcription factor 1 in bone marrow mesenchymal stem cells of the first four groups. Western blot assay was utilized to determine protein expression of type I collagen, alkaline phosphatase, core binding protein 2, peroxisome proliferator activated receptor γ, E26 transcription factor 1, extracellular signal-regulated kinase 1/2 and phosphorylated extracellular signal-regulated kinase 1/2 in bone marrow mesenchymal stem cells of the six groups. RESULTS AND CONCLUSION: (1) Compared with the blank serum group, the mRNA expression levels of alkaline phosphatase, core binding protein 2, and E26 transcription factor 1 were significantly higher in the Guilu Erxian Gum medicated serum group, while the mRNA expression levels of the adipogenic differentiation specific index peroxisome proliferator activated receptor γ were inhibited. (2) Compared with the fetal calf serum group and the blank serum group, the expression levels of osteogenic differentiation-related proteins, such as type I collagen, alkaline phosphatase, core binding protein 2, E26 transcription factor 1, and phosphorylated extracellular signal-regulated kinase 1/2 were significantly increased; and the expression level of peroxisome proliferator activated receptor γ protein was also suppressed. The use of MAPK signaling pathway inhibitor PD98059 could down-regulate the expression levels of osteogenic differentiation-related proteins and up-regulate the expression levels of peroxisome proliferator activated receptor γ protein. (3) Compared with the fetal bovine serum group and the blank serum group, the alkaline phosphatase activity (P < 0.05) and calcification capacity (P < 0.05) of bone marrow mesenchymal stem cells were significantly improved in the Guilu Erxian Gum medicated serum group. (4) The results show that Guilu Erxian Gum medicated serum may mediate the osteogenic differentiation of bone marrow mesenchymal stem cells through extracellular signal-regulated kinase 1/2/E26 transcription factor 1 signaling pathway factors, which may be one of its important mechanisms for preventing and treating osteoporosis. Key words:stem cells, bone marrow mesenchymal stem cells, Guilu Erxian Gum, osteoporosis, osteogenic differentiation, extracellular signal regulated kinase, mechanism |