自噬标志物LC3的节律表达破坏参与β_(1)-AA诱导的H9c2大鼠心肌细胞死亡  

Destruction of autophagy marker LC3 rhythm is involved in β_(1)-AA-induced H9c2 rat cardiomyocyte death

作　　者：贾微微 宁娜 孙聪 李朋佳 陆洁蓓 张晟 原媛 王丽[1,3] 王晓晖[1,3] JIA Wei-wei;NING Na;SUN Cong;LI Peng-jia;LU Jie-bei;ZHANG Sheng;YUAN Yuan;WANG Li;WANG Xiao-hui(Department of Pathology,Shanxi Medical University,Taiyuan 030001,China;Department of Morphology,Shanxi Medical University,Taiyuan 030001,China;Basic Medical Research Center,School of Basic Medicine,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学基础医学院病理教研室,山西太原030001 [2]山西医科大学基础医学院形态学实验室,山西太原030001 [3]山西医科大学基础医学院基础医学研究中心,山西太原030001

出　　处：《中国病理生理杂志》2021年第1期10-17,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31871177);山西省高等学校优秀成果(科学技术)培育项目(No.2020KJ034);山西省研究生教育创新项目(No.2020SY230);山西省“1331工程”重点学科建设计划经费资助。

摘　　要：目的:探讨β_(1)-肾上腺素受体(β_(1)-AR)自身抗体(β_(1)-AA)对大鼠心肌细胞自噬标志物微管相关蛋白1轻链3(LC3)节律表达的影响及其在心肌细胞死亡中的作用。方法:实验材料为Sprague-Dawley(SD)大鼠和H9c2大鼠心肌细胞。将SD大鼠随机分为免疫组(β_(1)-AR组)和对照(control)组,每组6只;将H9c2细胞随机分为control组、β_(1)-AA组、慢病毒(LV)-NC组和LV-shPer2组(n=6);合成β_(1)-AR细胞外第二环抗原肽段,用于主动免疫大鼠,并使用亲和层析法从大鼠血清中提纯β_(1)-AA;β_(1)-AA处理H9c2细胞24 h后使用CCK-8法检测细胞活力;用地塞米松同步化细胞后,再给予β_(1)-AA处理,采用real-time PCR及Western blot法检测LC3的表达情况,采用Western blot法检测生物钟蛋白Per2的表达情况,使用JTK_CYCLE算法分析昼夜节律参数;用LV-shPer2感染H9c2细胞以破坏LC3的节律表达,进而采用CCK-8法检测细胞活力。结果:β_(1)-AR组大鼠血清中β_(1)-AA的A值与control组相比显著升高(P<0.05)。β_(1)-AA组H9c2细胞的活力显著低于control组(P<0.05)。β_(1)-AA可破坏H9c2细胞LC3和Per2的节律表达(JTK_CYCLE P<0.05)。通过LV-shPer2干扰Per2基因而破坏H9c2细胞LC3节律表达(JTK_CYCLE P<0.05)后,细胞活力显著降低(P<0.05)。结论:β_(1)-AA破坏H9c2大鼠心肌细胞自噬标志物LC3的节律表达,从而促进细胞死亡。AIM:To investigate the effect ofβ_(1)-adrenergic receptor autoantibodies(β_(1)-AA)on the rhythm of autophagy marker microtubule-associated protein 1 light chain 3(LC3),and the underlying mechanism of cardiomyocyte death.METHODS:The test materials were Sprague-Dawley(SD)rats and H9c2 rat cardiomyocytes.The SD rats were randomly divided into immunization group and control group with 6 rats in each group.The H9c2 cells were randomly divided into control group,β_(1)-AA group,lentivirus(LV)-NC group,and LV-shPer2 group(n=6).Affinity chromatography was used for purification ofβ_(1)-AA from rat serum.CCK-8 assay was used to observe the viability of cardiomyocytes treated withβ_(1)-AA for 24 h.The cells were synchronized by dexamethasone and then treated withβ_(1)-AA.The mRNA and protein levels of LC3 at different time points were determined by real-time PCR and Western blot,respectively.The Per2 protein level at different time points was also determined by by Western blot.JTK_CYCLE algorithm was used to estimate the circadian rhythm parameters.After destruction of LC3 circadian rhythm via LV-shPer2,CCK-8 assay was used to measure the viability of H9c2 cells.RESULTS:High level ofβ_(1)-AA in rat serum was found after active immunization compared with control group(P<0.05).The viability of H9c2 cells inβ_(1)-AA group was significantly lower than that in control group(P<0.05).The LC3 and Per2 rhythms were both disrupted in H9c2 cells induced byβ_(1)-AA(JTK_CYCLE P<0.05).After LV-shPer2 infection,the LC3 rhythm was disrupted(JTK_CYCLE P<0.05)and the cell viability was reduced(P<0.05).CONCLUSION:β_(1)-AA may induce the destruction of autophagy marker LC3 rhythm in rat cardiomyocytes and then promote cell death.

关 键 词：心肌细胞 β_(1)-肾上腺素受体自身抗体 微管相关蛋白1轻链3 昼夜节律 自噬 

分 类 号：R541[医药卫生—心血管疾病] R363.2[医药卫生—内科学]