补肾活血方冻干粉对低氧诱导HTR-8/SVneo滋养细胞HIF-1α及VEGFA表达的影响

Effect of Bushen Huoxue Recipe Lyophilized Powder(补肾活血方冻干粉)on the Expression of HIF-1αand VEGFA in HTR-8/SVneo Trophoblast Cells Induced by Hypoxia

目的:探讨补肾活血方冻干粉对HTR-8/SVneo滋养细胞小管形成、缺氧诱导因子-1α(HIF-1α)和血管内皮生长因子A(VEGFA)表达的影响及其作用机制。方法:体外常规培养HTR-8/SVneo滋养细胞,采用CoCl2法诱导低氧环境,实验分为空白组、低氧模型组和补肾活血方冻干粉低、中、高剂量组。Tube formation法检测各组细胞小管形成能力,实时定量PCR及Western blotting检测各组细胞HIF-1αmRNA、VEGFA mRNA及其蛋白的表达。结果:小管形成实验结果显示,空白组、低氧模型组和补肾活血方冻干粉低、中、高剂量组形成的小管分支点数分别为12.35±1.25、27.76±1.79、31.61±0.82、33.24±1.22和38.84±1.68。Real-time PCR和Western blotting结果显示,与空白组比较,低氧模型组和补肾活血方冻干粉低、中、高剂量组VEGFA mRNA,以及补肾活血方冻干粉高剂量组HIF-1αmRNA表达均升高(P<0.05);与低氧模型组比较,补肾活血方冻干粉中、高剂量组VEGFA mRNA和补肾活血方冻干粉高剂量组HIF-1αmRNA表达均升高(P<0.05)。与空白组、低氧模型组比较,补肾活血方冻干粉低、中、高剂量组的HIF-1α、VEGFA蛋白表达均升高(P<0.05);与补肾活血方冻干粉低剂量组比较,补肾活血方冻干粉高剂量组HIF-1αmRNA、VEGFA mRNA和蛋白表达均升高(P<0.05);与补肾活血方冻干粉中剂量组比较,补肾活血方冻干粉高剂量组HIF-1α蛋白表达升高(P<0.05)。结论:补肾活血方冻干粉可以提高滋养细胞HTR-8/SVneo小管形成能力,其治疗复发性流产的机制可能与上调HIF-1αmRNA、VEGFA mRNA和蛋白表达相关。

Objective:To investigate the effect of Bushen Huoxue Recipe Lyophilized Powder on the formation of HTR-8/SVneo trophoblast tubules,the expression of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor A(VEGFA)and its mechanism.Methods:HTR-8/SVneo trophoblast cells were routinely cultured in vitro,and hypoxia environment was induced by the CoCl2 method.The experiment was divided into blank group,hypoxia model group and Bushen Huoxue Recipe Lyophilized Powder low,medium and high-dose groups.The ability of tubule formation was detected by tube formation method,and the expressions of HIF-1αmRNA,VEGFA mRNA and their protein were detected by Real-time PCR and Western blotting.Results:The results of the tubule formation experiment showed that the number of tubule branch points formed in the blank group,hypoxia model group,and Bushen Huoxue Recipe Lyophilized Powder low,medium,and high-dose groups were 12.35±1.25,27.76±1.79,31.61±0.82,33.24±1.22 and 38.84±1.68,respectively.The results of Real-time PCR and Western blotting showed that compared with the blank group,the expression of VEGFA m RNA in hypoxia model group and Bushen Huoxue Recipe Lyophilized Powder low,medium and high-dose group and the expression of HIF-1αm RNA in Bushen Huoxue Recipe Lyophilized Powder high-dose group were increase(P<0.05).Compared with hypoxia model group,the expression of VEGFA m RNA and HIF-1αm RNA in Bushen Huoxue Recipe Lyophilized Powder medium and high-dose group were significantly increased(P<0.05).Compared with the blank group and hypoxia model group,the expressions of HIF-1αand VEGFA protein in Bushen Huoxue Recipe Lyophilized Powder low,medium and high-dose group were increased(P<0.05).Compared with Bushen Huoxue Recipe Lyophilized Powder low-dose group,the expressions of HIF-1αm RNA,VEGFA m RNA and their protein in Bushen Huoxue Recipe Lyophilized Powder high-dose group were significantly increased(P<0.05).Compared with Bushen Huoxue Recipe Lyophilized Powder medium-dose group,the expression of HIF-1αprotein in Bushen Huoxue Recipe Lyophilized Powder high-dose group were significantly increased(P<0.05).Conclusion:The Bushen Huoxue Recipe Lyophilized Powder can improve the ability of trophoblast HTR-8/SVneo tubule formation,and its mechanism in the treatment of recurrent spontaneous abortion may be related to the up-regulation of HIF-1αm RNA,VEGFA m RNA and their protein expression.