基于GPER介导的EGFR/MEK/ERK途径探讨四物汤含药血清促进成骨细胞增殖的分子机制

Molecular mechanism of Siwu Decoction containing serum promoting proliferation of osteoblasts based on EGFR/MEK/ERK signaling pathway mediated by GPER

目的:探究四物汤对小鼠胚胎成骨细胞前体细胞MC3T3-E1增殖的影响及其基于G蛋白偶联雌激素受体(GPER)介导的表皮生长因子受体(EGFR)/丝裂原细胞外信号调节激酶(MEK)/细胞外信号调节激酶(ERK)信号通路发挥效应的分子机制。方法:将20只雌性SD大鼠随机分为对照组,雌二醇(0.16 mg·kg^(-1)·d^(-1))组,四物汤高、低剂量(2.08、0.52 g·kg^(-1)·d^(-1))组。灌胃4 d后,经腹主动脉取血,制备含药血清。MTT法检测四物汤含药血清对MC3T3-E1细胞增殖的影响;Western blot法检测四物汤含药血清对MC3T3-E1细胞中EGFR/MEK/ERK信号通路相关蛋白EGFR、MEK、ERK1/2表达的调控作用。为了进一步明确GPER在四物汤影响成骨细胞增殖中的介导作用,在四物汤高剂量组中应用GPER特异性激动剂G1(终浓度1×10^(-8)mol/L)与抑制剂G15(终浓度1×10^(-8)mol/L)进行干预。结果:四物汤含药血清可促进MC3T3-E1细胞增殖(P<0.01)。与对照组比较,四物汤高剂量组可上调MC3T3-E1细胞中EGFR、MEK、ERK1/2蛋白表达水平(P<0.01)。与四物汤高剂量组比较,在加入G1后,上述蛋白表达进一步增高(P<0.01);加入G15干预则会拮抗此效应,即升高的蛋白表达水平将有所下降(P<0.05)。结论:四物汤可以促进MC3T3-E1细胞增殖,该效应可能是通过在GPER的介导下,上调EGFR/MEK/ERK信号通路实现的。

Objective:To explore the effects of Siwu Decoction on the proliferation of osteoblast precursor cells(MC3T3-E1) in mouse embryos and its molecular mechanism based on EGFR/MEK/ERK signaling pathways via GPER.Methods:Twenty female SD rats were randomly grouped:control group,estradiol group (0.16 mg·kg^(-1)·d^(-1)),high-dose Siwu Decoction group(2.08 g·kg^(-1)·d^(-1)) and low-dose Siwu Decoction group (0.52 g·kg^(-1)·d^(-1)).After 4 days of gastric treatment with estradiol and Siwu Decoction,blood was collected from the abdominal aorta to prepare serum containing the drug.MTT assay was applied to detect the effects of Siwu Decoction on the proliferation of MC3T3-E1 cells.Western blot was applied to detect the regulatory effect of the Siwu Decoction on the expression of EGFR/MEK/ERK signaling pathway-related proteins including EGFR,MEK and ERK1/2 in MC3T3-E1 cells.In order to further clarify whether the role of GPER in osteoblast proliferation affected by Siwu,the GPER-specific agonist G1 (1×10^(-8)mol/L) and inhibitor G15 (1×10^(-8)mol/L) were used.Results:The drug serum could promote the proliferation of MC3T3-E1 cells (P<0.01).Compared with control group,high-dose Siwu Decoctio group could increase the protein expression of EGFR,MEK and ERK1/2 in MC3T3-E1 cells (P<0.01).Compared with high-dose Siwu Decoction group,the expression of these of these proteins was further enhanced by G1 (P<0.01) and weakened by G15 (P<0.05).Conclusion:Siwu Decoction could promote MC3T3-E1 cell proliferation based on EGFR/MEK/ERK signaling pathway via GPER.