基于溶酶体钙离子通道TRPML1探讨罗哌卡因在诱导心肌细胞自噬性凋亡中的作用机制  

Investigate the mechanism of local anesthetic ropivacaine in inducing autophagic apoptosis of cardiomyocytes based on lysosomal calcium channel TRPML1

作　　者：黄光强 熊槐 欧阳运琼 HUANG Guang-qiang;XIONG Huai;OUYANG Yun-qiong(Department of Anesthesiology,Anzhou District People s Hospital of Mianyang City,Mianyang 622651,Sichuan,China)

机构地区：[1]绵阳市安州区人民医院麻醉科,四川绵阳622651

出　　处：《川北医学院学报》2024年第6期726-731,共6页Journal of North Sichuan Medical College

摘　　要：目的:基于溶酶体钙离子通道瞬时受体电位粘脂素亚家族1(TRPML1)探讨罗哌卡因在诱导心肌细胞自噬性凋亡中的作用机制。方法:将H9c2心肌细胞分为对照组、ROP诱导组、罗哌卡因+TRPML1抑制剂(1.25μmol/L ML-SI1)、罗哌卡因+活性氧(ROS)清除剂(100μmol/L NAC)组。CCK-8检测各组细胞增殖活性;Annexin V-APC/PI流式细胞术检测细胞凋亡;流式细胞术检测ROS含量和钙离子水平;免疫荧光双染LC3与LAMP-1,观察自噬小体与溶酶体膜蛋白的共定位情况;Western blot检测凋亡相关(Caspase-3、cleaved Caspase-3)、自噬相关(LC3-Ⅱ/Ⅰ、P62)蛋白表达情况。结果:与对照组相比,罗哌卡因诱导组细胞增殖活性降低,细胞内ROS和钙离子含量升高(P<0.05);当TRPML1表达被抑制时,能够降低细胞内ROS和Ca^(2+)水平,同时减少自噬小体的蓄积,改善细胞凋亡。结论:局麻药罗哌卡因通过激活ROS/TRPML1途径致自噬流受阻,从而引起ROS、Ca^(2+)和自噬小体的过度累积,导致心肌细胞出现凋亡。Objective:To investigate the mechanism of local anesthetic ropivacaine(ROP)in inducing autophagic apoptosis of cardiomyocytes based on lysosomal calcium channel TRPML1.Methods:H9c2 cells were divided into a control group,a ropivacine-induced group,a ropivacine-treated group with TRPML1 inhibitor(1.25μmol/L ML-SI1),and a ropivacine-treated group with reactive oxygen species(ROS)scavenger(100μmol/L NAC).CCK8 was used to detect the cell proliferation activity.Annexin V-APC/PI flow cytometry was used to detect cell apoptosis.Flow cytometry was used to detect ROS content and calcium ion level.Immunofluorescence double staining of LC3 and LAMP-1 was used to observe the co-localization of autophagosomes and lysosomal membrane proteins.The protein expressions of apoptosis-related(Caspase-3,cleaved Caspase-3)and autophagy markers(LC3-Ⅱ/Ⅰ,P62)were detected by Western blot.Results:Compared with the control group,the ropivacaine-induced group reduced the proliferation activity of the cells,increased the content of intracellular ROS and calcium ions(P<0.05).When TRPML1 expression was inhibited,it could reduce the intracellular ROS and Ca^(2+)levels,and reduce the accumulation of autophagosomes,thereby improving cell apoptosis.Conclusion:Ropivacaine causes the blockage of autophagic flow by activating the ROS/TRPML1 pathway,which leads to the excessive accumulation of ROS,Ca^(2+)and autophagosomes,and then leads to the apoptosis of cardiomyocytes.

关 键 词：罗哌卡因 TRPML1 自噬 凋亡 

分 类 号：R915[医药卫生—微生物与生化药学]