淫羊藿苷对去卵巢大鼠骨髓间充质干细胞分化的影响

Effect of icariin on the differentiation of bone marrow mesenchymal stem cells in ovariectomized rats

目的探讨不同浓度的淫羊藿苷对去卵巢大鼠骨髓间充质干细胞(BMSCs)分化指标的影响。方法选取8周龄SPF级健康雌性大鼠70只,随机分为正常组、假手术组、骨质疏松组、淫羊藿苷低浓度组、淫羊藿苷中浓度组、淫羊藿苷高浓度组、雌二醇组,每组10只。除正常组和假手术组外,其余组大鼠手术摘除双侧卵巢建立骨质疏松模型。造模成功后,淫羊藿苷低、中、高浓度组分别给予含0.4 g/100 mL、0.8 g/100 mL、1.2 g/100 mL淫羊藿苷的混悬液灌胃,雌二醇组给予含0.8 g/mL 17β-雌二醇的混悬液灌胃,均1次/d,连续灌胃12周。分离培养各组小鼠BMSCs,取传代第3代细胞进行鉴定,并绘制生长曲线;ELISA法检测BMSCs相关碱性磷酸酶(ALP)活性和骨钙素(OCN)水平,Western blot法检测BMSCs中Runx-2蛋白表达情况,茜素红染色观察BMSCs成骨诱导后矿化结节形成情况。结果第3代细胞表面抗原中CD90、CD44、CD29、CD34、CD45、CD11阳性率分别为98.2%,93.3%,99.5%,2.6%,1.7%,4.4%,确认鉴定为BMSCs;不同浓度的淫羊藿苷均能够促进BMSCs的增殖,中浓度淫羊藿苷促增殖作用最强。骨质疏松组ALP活性、OCN水平、Runx-2蛋白相对表达量均明显低于假手术组(P均<0.05);淫羊藿苷中、高浓度组和雌二醇组ALP活性、OCN水平、Runx-2蛋白相对表达量均明显高于骨质疏松组(P均<0.05);淫羊藿苷中浓度组ALP活性、Runx-2蛋白相对表达量和淫羊藿苷高浓度组OCN水平与雌二醇组最为接近。骨质疏松组没有明显的钙化结节;淫羊藿苷各浓度组与雌二醇组出现明显的钙化结节,吸光度值均明显高于骨质疏松组(P均<0.05),其中淫羊藿苷中浓度组钙化结节数量最多。结论淫羊藿苷能够促进去卵巢大鼠BMSCs的增殖和成骨细胞分化,具有防治骨质疏松的作用。

Objective It is to investigate the effects of different concentrations of icariin on the expression of differentiation indicators of bone marrow mesenchymal stem cells(BMSCs).Methods A total of 70 SPF healthy female rats aged 8 weeks were randomly divided into normal control group,sham operation group,osteoporosis group,icariin low concentration group,icariin medium concentration group,icariin high concentration group and estradiol group,with 10 rats in each group.Except for the normal group and the sham operation group,the bilateral ovaries of rats in the remaining groups were surgically removed to establish osteoporosis models.After successful modeling,the icariin low concentration group,icariin medium concentration group,icariin high concentration group were given 0.4 g/100 mL,0.8 g/100 mL and 1.2 g/100 mL icariin suspension by gavage,respectively,and the estradiol group was given 0.8 g/mL 17β-estradiol suspension by gavage,all once daily,continuously treated for 12 weeks.The BMSCs of mice in each group were isolated and cultured,and the cells of the 3rd generation were taken for identification and the growth curves were plotted;the activity of alkaline phosphatase(ALP)and the level of osteocalcin(OCN)associated with BMSCs were detected by ELISA,the expression of Runx-2 protein in BMSCs was detected by Western blot,and the formation of mineralized nodes in BMSCs after osteogenic induction was observed by alizarin red staining.Results The positive rates of CD90,CD44,CD29,CD34,CD45,and CD11 in the cell surface antigens of the 3rd generation were 98.2%,93.3%,99.5%,2.6%,1.7%,and 4.4%,respectively,which were confirmed to be bone marrow stem cells.Different concentrations of icariin were able to promote the proliferation of BMSCs,and the middle concentration of Icariin had the strongest proliferative effect.The activity of ALP,level of OCN,and relative expression of Runx-2 protein in the osteoporosis group were significantly lower than those in the sham operation group(all P<0.05);the activity of ALP,level of OCN,and relative expression of Runx-2 protein in the medium-and high-concentration groups of icariin and in the estradiol group were significantly higher than those in the osteoporosis group(all P<0.05);the activity of ALP,relative expression of Runx-2 protein in the icariin medium-concentration group and the level of OCN in the high concentration group were closest to those in the estradiol group.There were no obvious calcified nodules in the osteoporosis group;the calcified nodules were obvious in each icariin group and estradiol group,in which the absorbance values were significantly higher than those in the osteoporosis group(all P<0.05),the number of calcified nodules in the icariin medium concentration group were the most.Conclusion Icariin can promote the proliferation and osteoblast differentiation of BMSCs in ovariectomized rats,thus to prevent and treat osteoporosis.