PCSK9通过溶酶体稳态-自噬途径调控心肌缺氧再复氧损伤  

PCSK9 regulates myocardial hypoxia⁃reoxygenation injury through lysosomal homeostasis/autophagy pathway

作　　者：许卫攀 徐航 刘滴 钟可文 李柳青 王琪[1,3] XU Weipan;XU Hang;LIU Di;ZHONG Kewen;LI Liuqing;WANG Qi(Postgraduate Joint Training Base,Huangshi Central Hospital,Wuhan University of Science and Technology,Huangshi 435000,China;School of Medicine,Wuhan University of Science and Technology,Wuhan 430081,China;Department of Cardiovascular Medicine,Affiliated Hospital of Hubei Polytechnic University,Huangshi 435000,China)

机构地区：[1]武汉科技大学黄石市中心医院研究生联合培养基地,湖北黄石435000 [2]武汉科技大学医学部医学院,湖北武汉430081 [3]湖北理工学院附属医院心内科,湖北黄石435000

出　　处：《海南医学院学报》2024年第21期1601-1606,共6页Journal of Hainan Medical University

基　　金：湖北省自然科学基金面上项目(2022CFB159);湖北省卫生健康委科研资助(WJ2023F061);湖北理工学院校级科研项目(22xjz6Q);黄石市中心医院科研项目(ZX2023M02)。

摘　　要：目的:研究前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)在心肌缺氧再复氧(hypoxia-reoxygenation,H/R)损伤中的作用及机制。方法:C57BL/6乳鼠原代心肌细胞,随机分为4组:空白对照组(Control组)、缺氧/复氧组(H/R组)、阴性腺相关病毒组(H/R+Scramble组)、AAV9-shPCSK9重组腺相关病毒组(H/R+shPCSK9组)。建立H/R模型(缺氧2 h,复氧4 h),使用CCK-8检测各组细胞活力的变化,LDH试剂盒检测细胞损伤程度,白光显微镜观察心肌细胞形态变化,共聚焦显微镜观察细胞内染色质凝集、自噬小体、溶酶体数量和形态等超微结构变化,western blot检测PCSK9、LC3Ⅱ、P62、LAMP2、Cathepsin B等蛋白表达变化。结果:H/R组和H/R+Scramble组较Control组的心肌细胞活力明显降低,LDH水平显著升高,PCSK9、LC3Ⅱ、P62、Cathepsin B蛋白显著升高,而LAMP2蛋白明显降低(P<0.05),这些变化在H/R+shPCSK9组则被逆转。此外,与H/R+Scramble组相比,白光显微镜下H/R+shPCKS9组的部分心肌细胞形态明显较为正常,部分细胞亦有缩短、肿胀以及成团状,但心肌细胞数量仍较多,同时共聚焦显微镜下H/R+shPCKS9组溶酶体数量及比例明显升高,而自噬小体数量虽增加但所占比例显著降低。结论:PCSK9通过破坏溶酶体稳态引起自噬通路受阻,导致自噬小体聚集以及自噬性死亡,进而加剧了心肌缺氧再复氧损伤,而下调PCSK9可通过维持溶酶体稳态,促进自噬通量,从而减轻H/R所导致的心肌细胞损伤。Objective:To investigate the role and mechanism of proprotein convertase subtilisin/kexin type 9(PCSK9)in myocardial hypoxia-reoxygenation(H/R)injury.Methods:Primary cardiomyocytes from neonatal C57BL/6 mice were randomly divided into 4 groups:Control group,H/R group,H/R+Scramble group,and H/R+shPCSK9 group.CCK-8 was used to detect the changes in cell viability,LDH kit was used to detect the degree of cell damage,white light microscope was used to observe the morphological changes of cardiomyocytes,and confocal microscope was used to observe the ultrastructural changes such as chromatin condensation and the number as well as morphology of autophagosomes and lysosomes.The protein expressions of PCSK9,LC3Ⅱ,P62,LAMP2 and Cathepsin B were detected by Western blot.Results:Compared to the control group,the viability of cardiomyocytes in the H/R group and the H/R+Scramble group was significantly decreased,the LDH level was significantly increased,the protein levels of PCSK9,LC3Ⅱ,P62,and Cathepsin B were significantly increased,and the protein level of LAMP2 was significantly decreased(P<0.05).These changes were reversed in the H/R+shPCSK9 group.In addition,compared to the H/R+Scramble group,the morphology of some cardiomyocytes in the H/R+shPCKS9 group was significantly normal under the white light microscope,and some cells were shortened,swollen and clumps,but the number of cardiomyocytes was still large.At the same time,the number and proportion of lysosomes in the H/R+shPCKS9 group were significantly increased under the confocal microscope.However,the number of autophagosomes increased but the proportion of autophagosomes decreased significantly.Conclusions:PCSK9 causes the block of the autophagy pathway by disrupting the lysosomal homeostasis,leading to autophagosome aggregation and autophagic death to aggravate myocardial hypoxia-reoxygenation injury,while PCSK9 down-regulation can reduce H/R-induced myocardial cell injury by maintaining lysosomal homeostasis and promoting autophagic flux.

关 键 词：心肌缺血再灌注损伤 溶酶体 自噬 PCSK9 

分 类 号：R542.22[医药卫生—心血管疾病]