妊娠期肝内胆汁淤积症中Beclin2通过介导自噬对滋养细胞凋亡的影响

Effects of Beclin2 on trophoblast apoptosis by mediating autophagy in intrahepatic cholestasis of pregnancy

目的探讨Beclin2在妊娠期肝内胆汁淤积症(ICP)产妇胎盘中的表达及其对滋养细胞的作用机制。方法选取2018年3月至2019年3月于株洲市中心医院产科住院分娩的ICP产妇30例作为研究对象,ICP轻度产妇和ICP重度产妇的胎盘组织各15例,另取同时期住院分娩的15例正常孕产妇作为对照组。免疫组化检测胎盘组织Beclin2蛋白表达。采用不同浓度(0、2、20、100μmol/L)的牛磺胆酸(TCA)处理滋养细胞,以建立ICP体外细胞模型。后期实验另设空白对照组(细胞未做任何处理)、si-NC组(转染si-NC)和si-Beclin2组(转染si-Beclin2)、TCA组(添加100μmol/L TCA)、TCA+si-NC组(添加100μmol/L TCA并转染si-NC)和TCA+si-Beclin2组(添加100μmol/L TCA并转染si-Beclin2);CCK-8检测滋养细胞增殖;实时荧光定量PCR检测Beclin2 mRNA表达;流式细胞术检测滋养细胞凋亡;Western blot检测滋养细胞Beclin2、LC3和p62蛋白表达。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验;胎盘中Beclin2基因的3组比较采用x^(2)检验,组间两两比较用Bonferroni方法。结果与健康产妇相比,ICP轻度产妇和ICP重度产妇胎盘组织中Beclin2蛋白阳性率和强阳性率(33.33﹪比86.67﹪、93.33﹪;13.33﹪比46.67﹪、66.67﹪)升高(P均<0.05/3)。与0、2μmol/L组比较,20、100μmol/L组细胞增殖能力24 h(0.53±0.04、0.56±0.04比0.42±0.05、0.38±0.06)、48 h(0.78±0.06、0.81±0.06比0.60±0.05、0.55±0.06)和72 h(0.94±0.08、1.02±0.09比0.73±0.07、0.68±0.08)及p62蛋白表达(1.17±0.12、0.98±0.08比0.85±0.07、0.38±0.03)均降低,Beclin2蛋白表达(0.15±0.01、0.23±0.01比0.46±0.05、0.58±0.04)、LC3Ⅱ/LC3Ⅰ蛋白表达(0.76±0.08、1.26±0.11比1.87±0.15、2.24±0.19)和细胞凋亡率[(3.87±0.25)﹪、(4.35±0.32)﹪比(8.67±0.59)﹪、(26.19±1.27)﹪]均升高(P均<0.05),且呈剂量依赖性。与空白对照组相比,TCA组滋养细胞Beclin2蛋白表达(0.25±0.03比1.28±0.11)和LC3Ⅱ/LC3Ⅰ蛋白表达(1.41±0.12比6.39±0.51)、细胞凋亡率[(2.53±0.17)﹪比(28.67±2.08)﹪]均升高,p62蛋白表达(0.97±0.08比0.53±0.04)降低(P均<0.05);与TCA+si-NC组相比,TCA+si-Beclin2组滋养细胞Beclin2蛋白表达(1.16±0.13比0.37±0.04)和LC3Ⅱ/LC3Ⅰ蛋白表达(5.88±0.47比2.35±0.24)、细胞凋亡率[(27.18±2.14)﹪比(16.33±0.79)﹪]均降低,p62蛋白(0.48±0.04比0.95±0.09)升高(P均<0.05)。结论Beclin2在ICP产妇胎盘组织中高表达;Beclin2通过上调自噬水平促进TCA处理后滋养细胞凋亡。

Objective To investigate the expression of Beclin2 in the placenta tissues of pregnant women with intrahepatic cholestasis(ICP)and its effects on trophoblasts.Methods Thirty women with ICP who were hospitalized and delivered in the Department of Obstetrics,Zhuzhou Central Hospital from March 2018 to March 2019 were enrolled.15 placental tissues of the women with mild ICP and 15 women with severe ICP were selected,and the other 15 normal pregnant women were selected as the control group.Immunohistochemistry was used to detect the expression of Beclin2 protein in placental tissues.In vitro cultured trophoblasts were divided into 0μmol/L group(supplemented with saline),2μmol/L group(supplemented with 2μmol/L TCA),20μmol/L group(supplemented with 20μmol/L TCA),and 100μmol/L group(supplemented with 100μmol/L TCA).In the following experiment,a blank control group(cells without any treatment),Si-NC group(cells were transfected with Si-NC)and si-Beclin2 group(cells were transfected with si-Beclin2),TCA group(cells were transfected with 100μmol/L TCA),TCA+Si-NC group(cells were added with 100μmol/L TCA and transfected with Si-NC)and TCA+si-Beclin2 group(cells were transfected with 100μmol/L TCA and transfected with si-Beclin2)were set up,and CCK-8 was used to detect trophoblast proliferation.The mRNA expression of Beclin2 was detected by real-time PCR,trophoblast apoptosis was detected by flow cytometry,and the protein expression of Beclin2,LC3 and p62 in trophoblasts was detected by Western blot.One way ANOVA was used for comparison between multiple groups,and LSD-t test was used for pairwise comparison between groups.Comparisons between control,ICP mild,and ICP severe groups were performed by x^(2) test,and pairwise comparisons between groups were performed by Bonferroni method.Results Compared with healthy pregnant women,the positive rate and strong positive rate of Beclin2 protein in placenta tissue of mild ICP and severe ICP pregnant women(33.33﹪vs 86.67﹪and 93.33﹪,13.33﹪vs 46.67﹪and 66.67﹪)were significantly increased(P<0.05/3).Compared with the 0μmol/L group and the 2μmol/L group,the proliferation ability of cells in the 20μmol/L group and 100μmol/L group at 24 h(0.53±0.04 and 0.56±0.04 vs 0.42±0.05 and 0.38±0.06),48 h(0.78±0.06 and 0.81±0.06 vs 0.60±0.05 and 0.55±0.06)and 72 h(0.94±0.08 and 1.02±0.09 vs 0.73±0.07 and 0.68±0.08)and expression of p62 protein(1.17±0.12 and 0.98±0.08 vs 0.85±0.07 and 0.38±0.03)were all decreased(P<0.05).Expressions of Beclin2(0.15±0.01 and 0.23±0.01 vs 0.46±0.05 and 0.58±0.04),LC3Ⅱ/LC3Ⅰprotein(0.76±0.08 and 1.26±0.11 vs 1.87±0.15 and 2.24±0.19)and apoptosis rate[(3.87±0.25)﹪and(4.35±0.32)﹪ratio(8.67±0.59)﹪and(26.19±1.27)﹪]were increased(P<0.05),and it was dose-dependent.Compared with the blank control group,trophoblast Beclin2 in the TCA group(0.25±0.03 vs 1.28±0.11)and LC3Ⅱ/LC3Ⅰprotein expression(1.41±0.12 vs 6.39±0.51),cell apoptosis rate[(2.53±0.17)﹪vs(28.67±2.08)﹪]were significantly increased,p62 protein expression(0.97±0.08 vs 0.53±0.04)was significantly decreased(P<0.05).Compared with TCA+si-NC group,trophoblast Beclin2 in the TCA+Si-Beclin2(1.16±0.13 vs 0.37±0.04)and LC3Ⅱ/LC3Ⅰprotein expression(5.88±0.47 vs 2.35±0.24)and cell apoptosis rate[(27.18±2.14)﹪vs(16.33±0.79)﹪]were significantly reduced,and p62 protein expression(0.48±0.04 vs 0.95±0.09)was significantly increased(P<0.05).Conclusion Beclin2 is highly expressed in ICP maternal placental tissues,and Beclin2 promotes trophoblast apoptosis after TCA treatment by upregulating the level of autophagy.