基于G蛋白偶联雌激素受体介导的EGFR/PI3K途径探讨四物汤对MC3T3-E1细胞增殖的影响

Influence of Siwu Tang on proliferation of MC3T3-E1 cells based on GPER-mediated EGFR/PI3K pathway

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。

Objective To investigate the influence of Siwu Tang(Four Ingredients Decoction)on proliferation efficacy of mouse embryonic osteoblasts(MC3T3-E1)based on G-protein-coupled estrogen receptor(GPER)-mediated pathway of epidermal growth factor receptor(EGFR)/phosphatidylinositol 3-kinase(PI3K),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,MC3T3-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 MC3T3-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,PI3K,cyclin B1 and cyclin D1 in MC3T3-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 MC3T3-E1 proliferation were detected by using GPER agonist(G1),GPER antagonist(G15),EGFR inhibitor(AG1478)and PI3K inhibitor(LY294002).Results The proliferation of MC3T3-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 MC3T3-E1(P<0.01).G15,AG1478 and LY294002 decreased the improving effect of Siwu Tang in high dose on proliferation of MC3T3-E1(P<0.01).The Results of Western blotting assay and FIA showed that the protein expressions of EGFR,PI3K,cyclin B1 and cyclin D1 in MC3T3-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 MC3T3-E1,and the effect mechanism may be related to the conduction of GPER-mediated EGFR/PI3K signaling pathway.