化瘀消癥颗粒对低氧状态下滋养细胞增殖、黏附与自噬的影响

Effects of Huayu Xiaozheng Granules(化瘀消癥颗粒)on Proliferation,Adhesion and Autophagy of Tropho⁃blast Cells under Hypoxia

目的探讨化瘀消癥颗粒治疗输卵管妊娠的可能作用机制。方法人源妊娠滋养细胞系HTR-8/SVneo细胞消化计数后接种至96孔板,分别设空白组、低氧组和化瘀消癥颗粒低、中、高剂量组,每组3个复孔。低氧组和化瘀消癥颗粒低、中、高剂量组每孔加入含400μmol/L二氯化钴(CoCl2)0.1 ml作用24 h建立低氧模型。造模后化瘀消癥颗粒低、中、高剂量组在培养液中分别加入20 mg/ml化瘀消癥颗粒母液50、100、150μl分别作用24 h、48 h、72 h后,采用CCK8法筛选后续实验药物干预时间。采用同上分组及干预方法,根据CCK8检测结果确定作用时间,采用细胞划痕实验观察各组细胞迁移情况并计算细胞迁移率,采用Western-blot法检测细胞缺氧诱导因子1α(HIF-1α)、上皮细胞钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)与自噬标志蛋白(Beclin-1)、微管相关蛋白轻链3蛋白Ⅱ(LC3-Ⅱ)表达。结果根据CCK8法检测结果确定作用时间为48 h继续后续实验。与空白组比较,低氧组细胞迁移率升高,HIF-1α、N-cadherin、Beclin-1、LC3-Ⅱ蛋白相对表达量均升高,E-cadherin蛋白相对表达量降低(P<0.05)。与低氧组比较,化瘀消癥颗粒低、中、高剂量组细胞迁移率降低,HIF-1α、N-cadherin、Beclin-1、LC3-Ⅱ蛋白相对表达量降低,且呈浓度依赖性(P<0.05);化瘀消癥颗粒高、低剂量组E-cadherin蛋白相对表达量升高,且化瘀消癥颗粒高剂量组升高更显著(P<0.05)。结论化瘀消癥颗粒可能通过抑制低氧所导致的滋养细胞的增殖、黏附、自噬活性,而发挥治疗输卵管妊娠的作用,以高剂量效果更佳。

Objective To explore the possible mechanism of Huayu Xiaozheng Granules(化瘀消癥颗粒,HXG)in treatment of tubal pregnancy.Methods The human gestational trophoblast cell line HTR-8/SVneo cells were counted after digestion and seeded in 96-well plates,with three duplicate wells in each group of blank group,hypoxia group,HXG low,medium and high dose groups.The hypoxia model was established 24 hours after giving 0.1 ml CoCl2(400μmol/L)into each duplicate well in the hypoxia group,HXG low,medium and high dose groups.After modeling,the mother liquor of HXG(20 mg/ml)at the dose of 50μl,100μl,and 150μl were added into the culture medium of the HXG low,medium and high dose groups correspondingly,and were observed for 24 h,48 h and 72 h,based on which the intervention time of the subsequent experiments was determined through CCK8 method.The scratch assay was used to observe the cell migration of each group,and the cell migration rate was calculated.The expression of cellular hypoxia-inducible factor 1α(HIF-1α),epithelial cell cadherin(E-cadherin),neural cadherin(N-cadherin),the iconic protein of autophagy(Beclin-1)and microtubule-associated protein light chain 3 proteinⅡ(LC3-Ⅱ)were detected through Western blotting.Results The intervention time was determined as 48h for the subsequent experiments through CCK8 method.Compared to the blank group,the hypoxia group had higher cell migration rate,increased expression of HIF-1α,N-cadherin,Beclin-1 and LC3-Ⅱ,as well as decreased expression of E-cadherin(P<0.05).Compared to those of the hypoxia group,the cell migration rate of the HXG low,medium and high dose groups were significantly lower,and the expression of HIF-1α,N-cadherin,Beclin-1 and LC3-Ⅱsignificantly decreased,in a concentration dependent manner(P<0.05);the expression of E-cadherin of the HXG low and high dose groups significantly increased compared to that of the hypoxia group,and more increase was seen in the HXG high dose group than low dose group(P<0.05).Conclusion HXG may act on tubal pregnancy by inhibiting the proliferation,adhesion and autophagy activity under hypoxia,and higher dose is associated with better effect.