背景:骨质疏松症和骨质疏松性骨折的发生率每年都在增加。中药(TCM)可以为骨质疏松症的治疗提供新的思路。本研究旨在探讨芍药苷促进成骨细胞前体细胞(MC3T3-E1)成骨分化的作用及机制。
方法:将MC3T3-E1细胞在成骨诱导培养基(OIM)和OIM联合不同浓度的芍药苷中培养。通过细胞计数试剂盒-8(CCK-8)测定来评估芍药苷的最佳剂量。然后,进行碱性磷酸酶(ALP)和茜素红S(ARS)染色以评估芍药苷的成骨能力。通过RT-PCR和Westernblotting评估成骨基因的转录和成骨蛋白的表达。分别。通过RT-PCR和Westernblotting检测Wnt/β-catenin信号通路基因和蛋白的转录,分别。最后,Dickkopf-1(DKK-1),一种Wnt/β-catenin信号通路抑制剂,用于鉴定Wnt/β-catenin信号通路是否参与芍药苷的成骨分化。通过抗酒石酸酸性磷酸酶(TRAP)染色鉴定RAW264.7细胞中的破骨细胞生成。
结果:在0.1至100μM的浓度范围内,芍药苷对MC3T3-E1细胞无细胞毒性。芍药苷可显著促进MC3T3-E1细胞成骨分化,且呈剂量依赖性。此外,芍药苷显著增加成骨分化基因和蛋白表达。通过生物信息学分析,发现受芍药苷影响的基因参与不同的信号通路,如Wnt/β-catenin信号通路。与对照组相比,芍药苷增强了β-catenin和CyclinD1的表达。DKK-1部分逆转芍药苷促进MC3T3-E1细胞成骨分化的促进作用。此外,芍药苷抑制RAW264.7细胞的破骨细胞生成。
结论:芍药苷通过调节Wnt/β-catenin通路促进MC3T3-E1细胞成骨分化。芍药苷是治疗骨质疏松症的潜在治疗剂。
BACKGROUND: The incidence of osteoporosis and osteoporotic fractures is increasing every year. Traditional Chinese Medicine (TCM) can shed new light on the treatment of osteoporosis. This study aimed to explore the role and mechanism of paeoniflorin in promoting osteogenic differentiation of an osteoblast precursor cell line (MC3T3-E1).
METHODS: MC3T3-E1 cells were cultured in osteogenic induction medium (OIM) and OIM combined with different concentrations of paeoniflorin. The optimal dose of paeoniflorin was assessed by a cell counting kit-8 (CCK-8) assay. Then, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed to assess the osteogenic capacity of paeoniflorin. The transcription of osteogenic genes and the expression of osteogenic proteins were assessed by RT-PCR and Western blotting, respectively. The transcription of Wnt/β-catenin signaling pathway genes and proteins was assessed by RT-PCR and Western blotting, respectively. Finally, Dickkopf-1 (DKK-1), a Wnt/β-catenin signaling pathway inhibitor, was used to identify whether the Wnt/β-catenin signaling pathway was involved in the osteogenic differentiation of paeoniflorin. Osteoclastogenesis in RAW264.7 cells was identified by tartrate-resistant acid phosphatase (TRAP) staining.
RESULTS: At concentrations ranging from 0.1 to 100 μM, paeoniflorin was not cytotoxic to MC3T3-E1 cells. Paeoniflorin significantly increased the osteogenic differentiation of MC3T3-E1 cells in a dose-dependent manner. Moreover, paeoniflorin significantly increased osteogenic differentiation gene and protein expression. Through bioinformatic analysis, paeoniflorin-affected genes were found to be involved in different signaling pathways, such as the Wnt/β-catenin signaling pathway. Paeoniflorin enhanced β-catenin and CyclinD1 expression compared with that of the control groups. DKK-1 partially reversed the promoting effects of paeoniflorin in promoting osteogenic differentiation of MC3T3-E1 cells. Moreover, paeoniflorin inhibited the osteoclastogenesis of RAW264.7 cells.
CONCLUSIONS: Paeoniflorin promotes osteogenic differentiation in MC3T3-E1 cells by regulating the Wnt/β-catenin pathway. Paeoniflorin is a potential therapeutic agent for the treatment of osteoporosis.