柚皮苷在促大鼠骨髓间充质干细胞骨向分化过程中对丝裂原活化蛋白激酶信号通路的影响

Effects of naringin on MAPK signaling pathway in promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells

目的分析补肾活血治则指导下柚皮苷促进骨髓间充质干细胞(MSCs)成骨分化及骨段迁移修复能力。方法对60只6月龄双侧卵巢摘除术后12周的大鼠,行单侧胫骨水平切开术,处死后,在无菌条件下取其双侧股骨和胫骨,进行传代培养。培养的第1、2、3、5、7、9、11 d,采用四甲基偶氮唑盐(MTT)比色法测定并绘制生长曲线,探讨大鼠MSCs增殖能力。采用MTT法检测第三代MSCs(生长状态良好)被柚皮苷(浓度:10^(-8)mol/L、10^(-7)mol/L、10^(-6)mol/L、10^(-5)mol/L、10^(-4)mol/L)干预后的吸光度(A)值,筛选最优促增值浓度,确认柚皮苷诱导MSCs向成骨细胞分化的能力。结果MTT法测定生长曲线成S形,培养第3天后呈对数生长,第9天后进入平台期。不同浓度柚皮苷对MSCs增殖影响及磷性磷酶酶(ALP)染色阳性细胞表达率两两比较,差异均有统计学意义(P<0.05)。结论柚皮苷可促进实验大鼠骨髓基质细胞的分化与增殖,增加骨钙素的表达,改善卵巢切除大鼠的骨质疏松症状。

Objective To explore the impact of naringin on enhancing the osteogenic differentiation and bone segment migration repair capability of bone marrow mesenchymal stem cells(MSCs)following the principles of kidney tonification and blood circulation promotion.Methods A total of 64 six-month-old rats undergoing bi-lateral ovariectomy,unilateral tibial plateau osteotomy was performed 12 weeks postoperatively.After euthanasia,bilateral femurs and tibias of the rats were collected under sterile conditions for MSCs culture.On the 1st,2nd,3rd,5th,7th,9th,and 11th day of cultivation,the viability of MSCs was measured using MTT method and growth curves were plotted to explore the proliferation ability of rat MSCs.The surface labeled antigens CD29,CD44,and CD45 were detected using flow cytometry.The optical density(A)values of the third-generation MSCs(in good growth status)after intervention with naringin(at concentrations of 10^(-8) mmol/L,10^(-7) mmol/L,10^(-6) mmol/L,10^(-5) mmol/L,and 10^(-4) mmol/L)were detected using the MTT assay.The optimal concentration promoting proliferation was screened to confirm the effect of naringin in inducing MSC differentiation into osteoblasts.Results The A value was measured by MTT method,and the growth curve formed an S-shape.MSCs showed logarithmic growth after 3 days of cultivation,and entered the plateau phase after 9 days.The proliferative effects of different concen-trations of naringin on MSCs and the expression rate of ALP-positive cells were compared,and the differences were statistically significant(P<0.05).Conclusion Naringin can promote the differentiation and proliferation of MSCs in experimental rats,increase the expression of osteocalcin,and improve the symptoms of osteoporosis in ovariectomized rats.