基于OPG/RANK/RANKL信号通路探究益肾健骨颗粒对绝经后骨质疏松大鼠BMSCs成骨分化的影响

Effects of Yishen Jiangu Granules on the osteogenic differentiation of BMSCs in postmenopausal osteoporosis rats based on OPG/RANK/RANKL signaling pathway

目的探讨益肾健骨颗粒对绝经后骨质疏松大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的影响及其作用机制。方法给予大鼠分别灌胃益肾健骨颗粒、生理盐水1周,制备含药血清及正常血清。采用背侧双切口切除双侧卵巢建立绝经后骨质疏松模型,分离并培养大鼠BMSCs,流式细胞术鉴定BMSCs,将BMSCs细胞随机分为对照组(N组)、含药血清组(J组)、含药血清+si-NC组(J+C组)、含药血清+si-OPG组(J+O组),按组进行相应处理,对各组BMSCs进行诱导成骨分化,碱性磷酸酶(alkaline phosphatase,ALP)染色与茜素红S染色法检测ALP活性及矿化结节数,实时荧光定量PCR(real-time PCR,RT-qPCR)检测ALP、Runt相关转录因子2(Runt-associated transcription factor 2,Runx2)、骨钙素(osteocalcin,OCN)mRNA表达,Western blot检测骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子(receptor activator of nuclear factor kappa B,RANK)、RANK配体(RANK ligand,RANKL)蛋白表达。结果成骨分离绝经后骨质疏松大鼠BMSCs,并通过形态及CD29、CD44、CD45、CD34表达鉴定;与N组比较,J组与J+C组细胞染色加深,ALP活性、矿化结节数、ALP、Runx2、OCN mRNA表达水平及OPG蛋白表达水平显著升高(P<0.05),RANK、RANKL蛋白表达水平显著降低(P<0.05);与J组、J+C组比较,J+O组细胞ALP染色变浅,ALP活性、矿化结节数、ALP、Runx2、OCN mRNA表达水平及OPG蛋白表达水平显著降低(P<0.05),RANK、RANKL蛋白表达水平显著升高(P<0.05)。结论益肾健骨颗粒通过调节OPG/RANK/RANKL信号通路,促进绝经后骨质疏松大鼠BMSCs成骨分化。

Objective To investigate the the effect and mechanism of Yishen Jiangu Granules on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in postmenopausal osteoporosis rats.Methods Rats were given Yishen Jiangu Granules and normal saline for 1 week to prepare drug-containing serum and normal serum.A rat model of postmenopausal osteoporosis was established by bilateral ovaries resection through dorsal double incision,BMSCs were isolated and cultured,and the BMSCs were identified by flow cytometry.The BMSCs were grouped into control group(N group),medicated serum group(J group),medicated serum+si-NC group(J+C group),and medicated serum+si-OPG group(J+O group),after treated according to the groups,the BMSCs in each group were induced to differentiate into osteogenic cells,alkaline phosphatase(ALP)staining and alizarin red S staining were performed to measure ALP activity and the number of mineralized nodules,respectively,real-time PCR(RT-qPCR)was performed to measure the expressions of ALP,Runt-associated transcription factor 2(Runx2),and osteocalcin(OCN)mRNA,Western blot was performed to measure the protein expression of osteoprotegerin(OPG),receptor activator of nuclear factor kappa B(RANK)and RANK ligand(RANKL).Results BMSCs from postmenopausal osteoporotic rats were isolated,and the morphology and expression of CD29,CD44,CD45 and CD34 were identified.Compared with the N group,the staining of cells in the J group and J+C group was deepened,and the ALP activity,the number of mineralized nodules,ALP,Runx2,OCN mRNA expression levels and OPG protein expression levels were significantly increased(P<0.05),the protein expression levels of RANK and RANKL were significantly decreased(P<0.05);compared with the J group,J+C group,the ALP staining of the J+O group cells became lighter,ALP activity,and the number of mineralized nodules,ALP,Runx2,OCN mRNA expression levels and OPG protein expression levels were significantly decreased(P<0.05),while RANK and RANKL protein expression levels were significantly increased(P<0.05).Conclusion Yishen Jiangu Granules can promote the osteogenic differentiation of BMSCs in postmenopausal osteoporosis rats by regulating the OPG/RANK/RANKL signaling pathway.