大麻二酚通过BRD4/AMPK/mTOR信号通路拮抗双酚A诱导的猪肠上皮细胞凋亡和自噬  

Cannabidiol Antagonizes BPA-induced Apoptosis and Autophagy in Porcine Intestinal Epithelial Cells through the BRD4/AMPK/mTOR Signaling Pathway

作　　者：侯宛辰 徐童 HOU Wanchen;XU Tong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030

出　　处：《畜牧兽医学报》2025年第4期1919-1933,共15页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：东北农业大学“青年才俊”项目;2023年国家级大学生创新创业训练计划项目(SIPT项目编号:202310224097)。

摘　　要：本研究旨在探究大麻二酚(cannabidiol, CBD)通过BRD4/AMPK/mTOR信号通路缓解双酚A(bisphenol A,BPA)导致的猪肠上皮细胞(pig intestinal epithelial cells, IPEC-J2)的凋亡和自噬的作用机制。首先通过CCK-8法测定细胞活力,筛选出BPA对IPEC-J2细胞的半数抑制浓度和CBD对BPA的最适拮抗剂量。用分组比较方法,将试验分为空白对照组、BPA组(150μmol·L^(-1)BPA)、BPA+CBD组(150μmol·L^(-1)BPA+10μmol·L^(-1)CBD)、BPA+CBD+CQ组(150μmol·L^(-1)BPA+10μmol·L^(-1)CBD+20μmol·L^(-1)CQ)和CBD(10μmol·L^(-1)CBD)组。通过AO/EB染色法以及流式细胞术检测IPEC-J2细胞的凋亡率;通过DCFH-DA法检测细胞中ROS水平;通过氧化应激试剂盒检测氧化应激水平;通过免疫荧光技术检测LC3-Ⅱ蛋白表达水平;采用实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)来检测细胞凋亡、自噬、肠紧密连接蛋白和BRD4/AMPK/mTOR信号通路相关基因的表达水平。结果表明,经150μmol·L^(-1)BPA处理,IPEC-J2细胞凋亡和自噬水平均升高,经10μmol·L^(-1)CBD处理后,细胞凋亡和自噬水平均降低;CBD可以显著下调由BPA引起的ROS和MDA水平升高(P<0.05),上调BPA引起的GSH-Px活性降低(P<0.05);CBD可以显著下调由BPA引起的细胞凋亡相关基因(Bax和caspase-3)、细胞自噬相关基因(P62、LC3-Ⅱ/LC3-Ⅰ和ATG5)和BRD4/AMPK/mTOR通路(AMPK)相关基因的表达水平升高(P<0.05),上调BPA引起的LAMP1、Bcl-2、BRD4、mTOR、ZO-1和Claudin-1的表达水平降低(P<0.05);免疫荧光结果表明,CBD可以显著降低由BPA所诱导的IPEC-J2细胞LC3-Ⅱ的表达与分布(P<0.05)。综上所述,CBD可以缓解BPA导致的IPEC-J2细胞氧化应激,进而通过BRD4/AMPK/mTOR信号通路拮抗BPA诱导的IPEC-J2细胞凋亡和自噬。This study aimed to explore the mechanism of cannabidiol(CBD)in alleviating the apoptosis and autophagy of pig intestinal epithelial cells(IPEC-J2)induced by bisphenol A(BPA)through BRD4/AMPK/mTOR signaling pathway.In this study,the cell viability was measured by CCK-8 method,and the half inhibitory concentration of BPA on IPEC-J2 cells and the optimal dose of CBD antagonist for BPA were screened.Using the group comparison method,The experiment was divided into blank control group,BPA group(150μmol·L^(-1) BPA),BPA+CBD group(150μmol·L^(-1) BPA+10μmol·L^(-1) CBD),BPA+CBD+CQ group(150μmol·L^(-1) BPA+10μmol·L^(-1) CBD+20μmol·L^(-1) CQ)and CBD group(10μmol·L^(-1) CBD).The apop-tosis rate of IPEC-J2 cells was detected by AO/EB staining and flow cytometry.The ROS levels in cells were detected by DCFH-DA method.Oxidative stress was detected by oxidative stress kit.The expression of LC3-Ⅱprotein was detected by immunofluorescence technique.Quantita-tive Real-time PCR(qRT-PCR)and Western blot were used to detect the expression of genes re-lated to apoptosis,autophagy,intestinal tight-junction protein and BRD4/AMPK/mTOR signa-ling pathway.The results showed that apoptosis and autophagy levels of IPEC-J2 cells were in-creased after 150μmol·L^(-1) BPA treatment,and decreased after 10μmol·L^(-1) CBD treatment.CBD could significantly down-regulate the increase of ROS and MDA levels caused by BPA(P<0.05),and up-regulate the decrease of GSH-Px activity(P<0.05);CBD significantly down-reg-ulated the increase of expression levels of apoptosis related genes(Bax and caspase-3),autophagy related genes(P62,LC3-Ⅱ/LC3-Ⅰand ATG5)and BRD4/AMPK/mTOR path-related genes(AMPK)induced by BPA(P<0.05).The expression levels of LAMP1,Bcl-2,BRD4,mTOR,ZO-1 and Claudin-1 were increased(P<0.05);Immunofluorescence results showed that CBD could significantly reduce the expression and distribution of LC3-Ⅱin IPEC-J2 cells induced by BPA(P<0.05).In conclusion,CBD could antagonize BPA-induced apoptosis and autophagy of IPEC-J2 cells

关 键 词：双酚A 大麻二酚 猪肠上皮细胞 细胞凋亡 细胞自噬 BRD4/AMPK/mTOR 

分 类 号：S859.7[农业科学—临床兽医学]