GLP-1介导PI3K/Akt通路拮抗AOPP诱导HTR-8/SVneo细胞凋亡的作用  

Glucagon like peptide-1 mediated PI3K/Akt pathway antagonizes advanced oxidized protein products induced apoptosis of HTR-8/SVneo cells

作　　者：王硕石 钟梅[2] Wang Shuoshi;Zhong Mei(Department of Obstetrics and Gynecology,Shenzhen People′s Hospital(The Second Clinical Medical College of Jinan University,the First Affiliated Hospital of Southern University of Science and Technology),Shenzhen 518002,China;Department of Obstetrics and Gynecology,Nanfang Hospital of Southern Medical University,Guangzhou 510515,China)

机构地区：[1]深圳市人民医院(暨南大学第二临床医学院,南方科技大学第一附属医院)妇产科,深圳518002 [2]南方医科大学南方医院妇产科,广州510515

出　　处：《中国医师杂志》2022年第4期522-526,共5页Journal of Chinese Physician

基　　金：国家自然科学基金(81971413);广东省自然科学基金(2020A1515110024)。

摘　　要：目的探究磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)通路在胰高血糖素样肽链-1(GLP-1)拮抗晚期氧化蛋白产物(AOPP)诱导妊娠滋养细胞凋亡的保护作用机制。方法体外培养妊娠滋养细胞HTR-8/SVneo,将细胞分为对照组、AOPP组、GLP-1组、AOPP+GLP-1组、AOPP+GLP-1+LY294002组。对照组采用1640培养基培养;AOPP组采用200μg/ml AOPP刺激;GLP-1组采用50-100 nmol/L GLP-1刺激1 h后进行实验;AOPP+GLP-1组采用200μg/ml AOPP刺激48 h后再加入GLP-1(50-100 nmol/L)1 h后进行实验;AOPP+GLP-1+LY294002组在AOPP+GLP-1组干预基础上再加入PI3K抑制剂LY294002。蛋白免疫印迹(Western blot)法检测PI3K/Akt通路相关蛋白磷酸化蛋白激素B(p-Akt)的表达。CCK-8比色法检测细胞活力。酶联免疫吸附法(ELISA)检测凋亡启动蛋白半胱氨酸天冬氨酸蛋白酶9(caspase-9),caspase-3的含量,及凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤相关X蛋白(Bax)、细胞色素C(Cyto-C)的含量。结果AOPP刺激后,AOPP组的p-AKT表达低于对照组(P<0.05);经50、100 nmol/L两个浓度GLP-1干预后,AOPP+GLP-1组p-AKT表达显著高于AOPP组(均P<0.05)。经100 nmol/L GLP-1干预24、48 h后,AOPP+GLP-1组p-AKT表达显著高于AOPP组(均P<0.05)。AOPP刺激后,AOPP组的细胞活力低于对照组(P<0.05);经GLP-1干预后,AOPP+GLP-1组细胞活力显著高于AOPP组(P<0.05),加入PI3K抑制剂LY294002处理后,AOPP+GLP-1+LY294002组的细胞活力显著低于AOPP+GLP-1组(P<0.05)。ELISA结果发现,AOPP刺激后,AOPP组的凋亡启动蛋白caspase-3、caspase-9,凋亡蛋白Bax及Cyto-c含量高于对照组(均P<0.05),抗凋亡蛋白Bcl-2含量低于对照组(均P<0.05);经GLP-1干预后,AOPP+GLP-1组caspase-3、caspase-9、Bax及Cyto-c含量显著低于AOPP组(均P<0.05),Bcl-2含量高于AOPP组(P<0.05),加入PI3K抑制剂LY294002处理后,AOPP+GLP-1+LY294002组的Bcl-2含量低于AOPP+GLP-1组,Bax及Cyto-c含量高于AOPP+GLP-1组(均P<0.05)。结论GLP-1可能介导PI3K/Akt通路拮抗AOPP诱导妊�Objective To explore the protective mechanism of phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt)pathway in glucagon like peptide-1(GLP-1)antagonizing the apoptosis of gestational trophoblasts(HTR-8/SVneo)induced by advanced oxidized protein products(AOPP).Methods Pregnant trophoblast HTR-8/SVneo were cultured in vitro.The cells were divided into control group,AOPP group,GLP-1 group,AOPP+GLP-1 group and AOPP+GLP-1+LY294002 group.The control group was cultured in 1640 medium;AOPP group was stimulated with 200μg/ml AOPP;GLP-1 group was stimulated with 50-100 nmol/L GLP-1 for 1 h;AOPP+GLP-1 group was stimulated with 200μg/ml AOPP for 48 hours,and then GLP-1(50-100 nmol/L)was added for 1 hour;In AOPP+GLP-1+LY294002 group,PI3K inhibitor LY294002 was added on the basis of the intervention of AOPP+GLP-1 group.The expression of PI3K/Akt pathway related protein p-Akt was detected by Western blot.Cell viability was detected by cell counting kit(CCK-8).Enzyme linked immunosorbent assay(ELISA)was used to detect the contents of apoptosis promoter protease caspase-9 and caspase-3,and the contents of apoptosis related proteins Bcl-2,Bax and Cyto-c.Results After AOPP stimulation,the expression of p-Akt in AOPP group was lower than that in control group(P<0.05);After 50 and 100 nmol/L GLP-1 intervention,the expression of p-Akt in AOPP+GLP-1 group was significantly higher than that in AOPP group(all P<0.05).After 24 and 48 hours of 100 nmol/L GLP-1 intervention,the expression of p-Akt in AOPP+GLP-1 group was significantly higher than that in AOPP group(all P<0.05).After AOPP stimulation,the cell viability of AOPP group was lower than that of control group(P<0.05);After GLP-1 intervention,the cell viability of AOPP+GLP-1 group was significantly higher than that of AOPP group(P<0.05).After adding PI3K inhibitor LY294002,the cell viability of AOPP+GLP-1+LY294002 group was significantly lower than that of AOPP+GLP-1 group(P<0.05).The results of ELISA showed that the contents of apoptosis promoter protein caspase-3,caspase-9,

关 键 词：磷酸肌醇3-激酶 原癌基因蛋白质c-akt 胰高血糖素样肽1 晚期氧化蛋白产物 妊娠滋养细胞 细胞凋亡 

分 类 号：R714[医药卫生—妇产科学]