基于ALKBH5调控GSK3β/mTOR通路抑制过度自噬探讨速效救心丸减轻心肌细胞缺氧/复氧损伤的作用机制  

Mechanism of Suxiao Jiuxin Pills on Hypoxia/Reoxygenation Injury in Cardiomyocytes Based on ALKBH5 Regulation of GSK3β/mTOR Pathway to Inhibit Excessive Autophagy

作　　者：李檫 李益萍[1] 王可妍 王丹 王肖龙[1] LI Cha;LI Yiping;WANG Keyan;WANG Dan;WANG Xiaolong(Shuguang Hospital Shanghai University of Traditional Chinese Medicine,Shanghai Baoshan Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院,上海市宝山区中西医结合医院,上海201203

出　　处：《中西医结合心脑血管病杂志》2023年第24期4510-4519,共10页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：国家自然科学基金面上项目(No.82174152,82074222);上海市临床重点专科项目(No.shslczdzk05301)。

摘　　要：目的:基于HL-1细胞自噬水平及相关通路关键蛋白表达探讨速效救心丸经Alk B同源蛋白5(ALKBH5)信号转导调控糖原合成酶激酶3β(GSK3β)/雷帕霉素靶蛋白(m TOR)通路抑制过度自噬而发挥对心肌细胞缺氧/复氧(H/R)损伤保护作用的机制。方法:构建慢病毒干扰载体,筛选最佳干扰慢病毒。将HL-1细胞随机分为3组,并进行空白转染、ALKBH5转染、GSK3转染,各组再随机分为4组分别进行缺氧/复氧(模型组)、缺氧/复氧+速效(速效组)、缺氧/复氧+川芎嗪(川芎嗪组)、缺氧/复氧+冰片(冰片组)处理。通过自噬双标腺病毒(m RFP-e GFP-LC3)双荧光染色检测自噬流,细胞计数法(CCK8)检测细胞增殖,双染色双变量流式细胞分析法(Annexin V/PI)检测细胞凋亡,透射电镜观察自噬小体的数量和形态,蛋白免疫印迹法(Western Blot)检测GSK3β、m TOR含量及自噬相关基因(Atg)5、苄氯素1(Beclin1)、自噬相关蛋白微管相关蛋白轻链3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)、自噬标志物p62蛋白表达。结果:ALKBH5过表达可抑制GSK3β表达,增强m TOR表达(P<0.05)。CCK8法检测m RFP-e GFP-LC3的双荧光染色检测自噬流结果显示,与空白转染比较,ALKBH5转染可促进自噬,GSK3β转染可抑制自噬(P<0.05)。CCK8法检测细胞增殖结果显示,与模型组比较,速效组、川芎嗪组、冰片组均可促进细胞增殖;与空白转染比较,ALKBH5组可抑制细胞增殖,GSK3β可促进细胞增殖(P<0.05)。Annexin V/PI双染色法检测心肌细胞凋亡显示,与模型组比较,速效组、川芎嗪组、冰片组均可抑制细胞凋亡;与空白组比较,ALKBH5转染及GSK3β转染均可增加细胞凋亡(P<0.05)。Western Blot检测LC3Ⅱ/Ⅰ比值、Atg5、Atg7、p62表达显示,与模型组比较,速效组、川芎嗪组、冰片组可抑制LC3Ⅱ/Ⅰ比值、Atg5、Atg7、p62表达,增强m TOR表达(P<0.05)。与空白转染比较,ALKBH5转染可增强LC3Ⅱ/Ⅰ比值、Atg5、Atg7、p62表达,抑制m TOR表达;GSK3β转染可抑�Objective:Based on the level of autophagy in HL-1 cells and the expression of key proteins of related pathways,to explore the mechanism of Suxiao Jiuxin Pills on hypoxia/reoxygenation(H/R)injury through the Alk B homolog 5(ALKBH5)regulation of glycogen synthetase kinase 3beta(GSK3β)/mammalian target of rapamycin(mTOR)pathway to inhibit excessive autophagy.Methods:After construction of lentiviral interference carrier and screening of optimal interfering lentiviruses,the HL-1 cells were randomly divided into 3 groups and blank transfection,ALKBH5 transfection,and GSK3βtransfection.Each group was randomly divided into 4 groups;hypoxia/reoxygenation(model group),hypoxia/reoxygenation+Suxiao Jiuxin Pills treatment(Suxiao group),hypoxia/reoxygenation+ligustrazine treatment(ligustrazine group),hypoxia/reoxygenation+borneol treatment(borneol group).Autophagy flow was detected by double fluorescence staining of autophagy double-labeled adenovirus(mRFP-eGFP-LC3),cell proliferation was detected by cell counting(CCK8),apoptosis was detected by double-stained bivariate flow cytometric analysis(Annexin V/PI),the number and morphology of autophagic vesicles were observed by transmission electron microscopy,and GSK3β,mTOR content,and autophagy-related gene(autophagy-related gene,Atg5),benzyl chloride 1(Beclin1),autophagy-related protein microtubule-associated protein light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),and autophagy marker p62 were detected by Western Blot.Results:ALKBH5 overexpression inhibited GSK3βexpression and enhanced mTOR expression(P<0.05).mRFP-eGFP-LC3 dual fluorescence staining for autophagy flow detection showed that compared with the blank transfected under the condition of H/R,ALKBH5 transfection promoted autophagy and GSK 3βtransfection inhibited autophagy(P<0.05).The results of CCK8 assay showed that compared with the model group,cell proliferation was promoted in the Suxiao group,ligustrazine group,and borneol group.Compared with the blank transfection,cell proliferation was inhibited in the ALKBH5 group,and ce

关 键 词：心肌细胞 缺氧/复氧损伤 速效救心丸 Alk B同源蛋白5 ALKBH5 糖原合成酶激酶3Β GSK3Β 雷帕霉素靶蛋白 mTOR 自噬 实验研究 

分 类 号：R285.5[医药卫生—中药学]