加味宫外孕Ⅱ号方对人滋养叶细胞Caspase-12通路影响的研究

Effect of modified ectopic pregnancy formula Ⅱ on the Caspase-12 pathway in human trophoblasts

目的观察加味宫外孕Ⅱ号方对人滋养叶细胞内质网应激Caspase-12信号通路凋亡相关蛋白Caspase-7、Caspase-9、Caspase-3水平的影响及细胞超微结构变化,探讨其治疗异位妊娠的作用机制。方法将SD大鼠分为阴性对照组(B组)、低、中、高剂量中药组(C组、D组、E组)、甲氨蝶呤组(F组)、中西药结合组(G组),予相应药物处理后取血制备含药血清,将各组含药血清分别作用于HTR-8/SVneo细胞24 h,并设置未用含药血清处理的细胞作为空白对照组(A组)。采用蛋白质免疫印迹法(Western blot)与实时荧光定量PCR(Real-time PCR)检测HTR-8/SVneo细胞中凋亡基因Caspase-7、Caspase-9、Caspase-3蛋白及mRNA的表达水平;采用透射电镜观察细胞超微结构的变化。结果(1)Caspase-7、Caspase-9、Caspase-3蛋白水平及mRNA相对表达量:A组与B组比较无差异(P>0.05);与B组比较,C组Caspase-7 mRNA、Caspase-9蛋白及Caspase-3蛋白与mRNA表达均上调明显(P<0.05),而Caspase-7蛋白、Caspase-9 mRNA表达无差异(P>0.05);D、E、F、G组Caspase-7、Caspase-9、Caspase-3蛋白及mRNA表达量均较B组明显上调(P<0.05);E组较D组上调明显(P<0.05);E组与F组比较,Caspase-7、Caspase-9蛋白与mRNA及Caspase-3蛋白表达上调明显(P<0.05);E组与G组比较,Caspase-9蛋白与mRNA及Caspase-3蛋白表达上调明显(P<0.05);F组与G组比较无差异(P>0.05)。(2)透射电镜结果显示:A组与B组细胞染色质均匀散布于核膜内,细胞内质网、线粒体、高尔基体清晰可见;与B组比较,C、D、E、F、G组细胞微绒毛减少,染色质固缩,异染色质边缘化,部分线粒体肿胀、脊紊乱或断裂、甚至消失,线粒体空泡化等典型的凋亡特征改变。结论加味宫外孕Ⅱ号方可能通过激活内质网应激Caspase-12信号通路,破坏滋养叶细胞的形态学结构,上调Caspase-7、Caspase-9、Caspase-3凋亡相关基因的表达,促进滋养叶细胞凋亡。

Objective To examine the effect of modified ectopic pregnancy formula II on levels of apoptosisrelated proteins Caspase-7,Caspase-9,and Caspase-3 in the endoplasmic reticulum stress Caspase-12 signaling pathway in human trophoblasts,as well as associated changes in cell ultrastructure,to explore its treatment mechanism for ectopic pregnancy.Methods SD rats were divided into a negative control group(group B),low-,medium-,and high-dose Chinese medicine groups(groups C,D,E),a methotrexate group(group F),and a combined Chinese and Western medicine group(group G).After receiving the corresponding drug treatment,we collected blood to prepare a drugcontaining serum.The drug-containing serum from each group was applied to HTR-8/SVneo for 24 h,and cells not treated with drug-containing serum were set as a blank control group(group A).The expression levels of apoptotic genes Caspase-7,Caspase-9,Caspase-3 protein and mRNA in HTR-8/SVneo cells were detected via Western blot and real-time quantitative PCR.Transmission electron microscopy was used to observe changes in cell ultrastructure.Results(1)There was no difference between group A and group B in terms of Caspase-7,Caspase-9,or Caspase-3 protein levels and relative mRNA expression(P>0.05).Compared with group B,expression levels of Caspase-7 mRNA,Caspase-9 protein,and Caspase-3 protein and mRNA were significantly up-regulated in group C(P<0.05),but we found no differences in Caspase-7 protein or Caspase-9 mRNA expression(P>0.05).In terms of Caspase-7,Caspase-9,and Caspase-3 protein and mRNA expression,expression was significantly increased in groups D,E,F,and G compared with group B(P<0.05),and expression in Group E was significantly up-regulated compared with group D(P<0.05).Group E was compared with group F,expression levels of Caspase-7,Caspase-9 protein and mRNA,and Caspase-3 protein were significantly up-regulated(P<0.05).Group E was compared with group G,expression of Caspase-9 protein and mRNA and Caspase-3 protein was significantly increased(P<0.05).There was no difference between group F and group G(P>0.05).(2)Transmission electron microscopy indicated that the cell chromatin in group A and group B was evenly distributed in the nuclear membrane,and the endoplasmic reticulum,mitochondria,and Golgi apparatus were clearly visible.Compared with group B,the cell microvilli in groups C,D,E,F,and G were reduced,chromatin was condensed,heterochromatin was marginalized,some mitochondria were swollen,disordered,broken,or even absent,and mitochondrial vacuolation were typical apoptotic characteristics.Conclusions Modified ectopic pregnancy formula II may activate the Caspase-12 signaling pathway of endoplasmic reticulum stress,destroy the morphological structure of trophoblasts,up-regulate the expression of Caspase-7,Caspase-9,and Caspase-3 apoptosis-related genes,and promote trophoblast apoptosis.