【摘要】 目的探讨四物汤通过G蛋白偶联雌激素受体(GPER)介导的表皮生长因子受体(EGFR)/磷脂酰肌醇3-激酶(PI3K)途径影响小鼠胚胎成骨细胞(MC3T3-E1)的增殖效应及其分子机制。方法将40只SD雌性大鼠随机分为5组:正常组,雌二醇组,四物汤高、中、低剂量组,每组8只。以雌二醇药物血清为阳性对照,用高、中、低剂量四物汤药物血清干预MC3T3-E1细胞,以MTT细胞增殖实验检测各药物血清干预24 h对MC3T3-E1细胞增殖的影响;Western blot及免疫荧光技术检测高剂量四物汤对MC3T3-E1细胞EGFR、PI3K、cyclin B1、cyclin D1蛋白表达情况的影响;并应用GPER激动剂G1、拮抗剂G15,EGFR抑制剂AG1478,PI3K抑制剂LY294002进一步检测高剂量四物汤影响MC3T3-E1细胞增殖的作用途径和分子机制。结果与正常血清组比较,高、中、低剂量四物汤药物血清均能促进MC3T3-E1细胞的增殖(P<0.01),加入G1可使高剂量四物汤促增殖作用提高(P<0.01),而加入G15、AG1478、LY294002使高剂量四物汤的促增殖作用减弱(P<0.01)。Western blot和免疫荧光结果均显示,四物汤药物血清增加MC3T3-E1细胞EGFR、PI3K、cyclin B1、cyclin D1的蛋白表达,与正常血清组比较差异有统计学意义(P<0.01),G1同时干预时上述蛋白表达量较高剂量四物汤增加(P<0.01),分别加入G15、AG1478、LY294002后上述蛋白的表达量较高剂量四物汤组降低(P<0.01或P<0.05)。结论四物汤对小鼠MC3T3-E1细胞增殖有明显的促进作用,其作用机制可能为GPER介导的EGFR/PI3K信号通路的传导。更多还原

【Abstract】 Objective To investigate the influence of Siwu Tang(Four Ingredients Decoction) on proliferation efficacy of mouse embryonic osteoblasts(MC3 T3-E1) based on G-protein-coupled estrogen receptor(GPER)-mediated pathway of epidermal growth factor receptor(EGFR)/phosphatidylinositol 3-kinase(PI3 K), and study the molecular mechanism. Methods Female SD rats(n=40) were randomly divided into normal group, estradiol group, and high-dose, mid-dose and low-dose Siwu Tang groups(each n=8). At the same time, the high-dose Siwu Tang group was divided into high-dose Siwu Tang+G1 group, high-dose Siwu Tang+G15 group, high-dose Siwu Tang+AG1478 group and high-dose Siwu Tang+LY294002 group. Taken E2 medicated serum as positive control, MC3 T3-E1 was intervened with high-dose, mid-dose and low-dose Siwu Tang medicated serum. The influence of 24-h intervention with medicated serum on MC3 T3-E1 proliferation was detected by using methyl thiazolyl tetrazolium test(MTT) in all groups. The influence of high-dose Siwu Tang in high dose on protein expressions of EGFR, PI3 K, cyclin B1 and cyclin D1 in MC3 T3-E1 were detected by suing Western blot assay and immunofluorescence technique(IF). The effective pathway and molecular mechanism of Siwu Tang in high dose influencing MC3 T3-E1 proliferation were detected by using GPER agonist(G1), GPER antagonist(G15), EGFR inhibitor(AG1478) and PI3 K inhibitor(LY294002). Results The proliferation of MC3 T3-E1 was improved in high-dose, mid-dose and low-dose Siwu Tang groups compared with normal group(P<0.01), and G1 increased significantly the improving effect of Siwu Tang in high dose on proliferation of MC3 T3-E1(P<0.01). G15, AG1478 and LY294002 decreased the improving effect of Siwu Tang in high dose on proliferation of MC3 T3-E1(P<0.01). The results of Western blotting assay and FIA showed that the protein expressions of EGFR, PI3 K, cyclin B1 and cyclin D1 in MC3 T3-E1 increased significantly in all Siwu Tang groups compared with normal group(P<0.01). G1 promoted the above-mentioned expressions(P<0.01), and G15, AG1478 and LY294002 reduced the above-mentioned expressions(P<0.01, P<0.05) compared with Siwu Tang in high dose. Conclusion Siwu Tang has significant improving effect on the proliferation of MC3 T3-E1, and the effect mechanism may be related to the conduction of GPER-mediated EGFR/PI3 K signaling pathway.更多还原