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目的:观察黄芪散含药血清对高糖条件下成骨细胞功能的影响及与糖尿病性骨质疏松发病机制相关基因的影响,探讨黄芪散(HQS)对糖尿病性骨质疏松症的疗效机制。方法:选用健康清洁级5周龄Wistar大鼠10只,随机分为正常组和药物组。制备黄芪散含药血清和空白血清,选用健康清洁级新生大鼠乳鼠4只,体外以酶消化法分离培养新生乳鼠颅骨成骨细胞(Ob),行细胞计数(CCK-8)法测定高糖条件下(25.5 mmol·L~(-1))5%,10%,20%,30%含药血清对细胞增殖的影响,选取增殖活性最佳血清浓度,采用实时荧光定量PCR(Real-time PCR)和蛋白免疫印迹(Western blot)法检测黄芪散对成骨功能相关基因骨桥蛋白(OPN),骨钙素(OC),碱性磷酸酶(ALP)和胰腺-骨骼相关基因骨保护素(OPG),叉头转录因子1(Fox O1)表达的影响。结果:10%和30%血清浓度组细胞增殖较空白组有明显升高(P<0.05);20%血清浓度组较空白组有显著升高(P<0.01)。与空白组比较,5%浓度条件下,OPN,OPG mRNA表达明显下调(P<0.05);10%浓度条件下,OC,ALP,OPN mRNA表达明显上调(P<0.05,P<0.01);OPG,Fox O1 mRNA较空白组表达显著下调(P<0.01)。20%浓度和10%浓度条件差异性一致,OPN,OPG,Fox O1 mRNA影响趋势较10%浓度药物血清组更明显。20%浓度血清条件下,与空白组比较,OC和OPN蛋白表达显著上调(P<0.01),OPG蛋白表达显著下调(P<0.01)。结论:黄芪散对糖尿病性骨质疏松的疗效机制可能与其促进高糖作用下成骨细胞增殖作用有关,与下调胰腺-骨骼相关基因OPG,Fox O1表达有关。
Objective: To observe the effect of Astragalus San containing serum on the function of osteoblasts under high glucose condition and its correlation with pathogenesis of diabetic osteoporosis, and to explore the therapeutic mechanism of Astragalus Sangen (HQS) on diabetic osteoporosis. Methods: Ten healthy Wistar rats, 5 weeks old, were randomly divided into normal group and drug group. The astragalus powder-containing serum and blank serum were prepared. Four healthy neonatal rat neonatal rats were selected, and the osteoblasts of newborn rats were isolated and cultured by enzymatic digestion method. The cell counting (CCK-8) The effects of 5%, 10%, 20% and 30% serum containing 25.5 mmol·L -1 high glucose on cell proliferation were studied. The optimal serum concentration of proliferative activity was selected. Real-time fluorescence quantitative PCR (OPG), osteocalcin (OC), alkaline phosphatase (ALP) and pancreatic-skeletal related gene bone (OPG) and FoxO1 expression. Results: The proliferation of cells in 10% and 30% serum was significantly higher than that in blank group (P <0.05), while 20% serum was significantly higher than that in blank group (P <0.01). Compared with the blank group, OPN and OPG mRNA expressions were significantly down-regulated at 5% concentration (P <0.05); OC, ALP and OPN mRNA expressions were significantly increased at 10% concentration (P <0.05, P <0.01) , FoxO1 mRNA expression was significantly lower than the blank group (P <0.01). 20% and 10% concentrations of the same conditions, OPN, OPG, FoxO1 mRNA trends than the 10% concentration of drug serum group more obvious. Compared with the blank group, OC and OPN protein expression was significantly up-regulated (P <0.01) and OPG protein expression was significantly down-regulated (P <0.01) at 20% serum concentration. Conclusion: The therapeutic mechanism of Astragalus Granules on diabetic osteoporosis may be related to its role in promoting proliferation of osteoblasts under high glucose. It may be related to the down-regulation of the expression of OPG and Fox O1 in pancreas-bone.