主管单位:中华人民共和国
国家卫生健康委员会
主办单位:
总编辑:杨秋
编辑部主任:吴翔宇
邮发代号:80-528
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全年:336.00元
Email:zgyy8888@163.com
电话(传真):010-64428528;
010-64456116(总编室)
英文单位:Anesthesia Center Beijing Anzhen Hospital Capital Medical University Beijing 100029 China
关键词:右美托咪定;缺氧复氧损伤;心肌细胞凋亡;微小RNA-340-5p
英文关键词:Dexmedetomidine;Hypoxia/reoxygenationinjury;Myocardialapoptosis;MicroRNA-340-5p
目的 探讨右美托咪定(Dex)对缺氧复氧诱导心肌细胞凋亡的防护作用及机制。方法 选取大鼠心肌细胞H9c2,根据微小RNA(miR)-340-5p抑制剂及其阴性对照抑制剂转染情况,分为对照组(未处理)、阴性对照抑制剂组和miR-340-5p抑制剂组;根据miR-340-5p抑制剂及其阴性对照抑制剂转染、Dex以及缺氧复氧干预,分为对照组(未处理)、缺氧复氧组、缺氧复氧+Dex组、缺氧复氧+阴性对照抑制剂+Dex组、缺氧复氧+miR-340-5p抑制剂+Dex组;根据miR-340-5p模拟物及其阴性对照模拟物转染、高迁移率族蛋白B1(HMGB1)碱基突变情况,分为HMGB1-wtUTR+阴性对照模拟物组、HMGB1-mutUTR+阴性对照模拟物组、HMGB1-wtUTR+miR-340-5p模拟物组、HMGB1-mutUTR+miR-340-5p模拟物组。采用细胞计数试剂盒8检测细胞增殖水平。采用实时荧光定量聚合酶链反应法检测HMGB1 mRNA、miR-340-5p表达量。采用蛋白质印迹法检测HMGB1、B细胞淋巴瘤2家族蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)表达量。采用流式细胞术检测细胞凋亡情况。采用双荧光素酶试验验证miR-340-5p与HMGB1的靶向结合。结果 缺氧复氧+Dex组miR-340-5p表达量高于缺氧复氧组[(0.80±0.04)比(0.25±0.01)],HMGB1 mRNA及其蛋白表达量均低于缺氧复氧组[(1.47±0.13)比(5.02±0.52)、(2.09±0.08)比(4.87±0.16)],差异均有统计学意义(均P<0.05)。缺氧复氧+Dex组细胞增殖水平低于对照组,但高于缺氧复氧组;缺氧复氧+miR-340-5p抑制剂+Dex组细胞增殖水平低于缺氧复氧+阴性对照抑制剂+Dex组;差异均有统计学意义(均P<0.05)。缺氧复氧+miR-340-5p抑制剂+Dex组Bcl-2表达量低于缺氧复氧+阴性对照抑制剂+Dex组,Bax表达量、凋亡率均高于缺氧复氧+阴性对照抑制剂+Dex组,差异均有统计学意义(均P<0.05)。双荧光素酶试验结果 显示,HMGB1-wtUTR+阴性对照模拟物组、HMGB1-mutUTR+阴性对照模拟物组、HMGB1-wtUTR +miR-340-5p模拟物组及HMGB1-mutUTR+miR-340-5p模拟物组相对荧光比值比较,差异有统计学意义(F=22.43,P<0.01),且HMGB1-wtUTR+miR-340-5p模拟物组相对荧光比值低于其他3组,差异均有统计学意义(均P<0.05)。结论 Dex可通过上调miR-340-5p表达,从而靶向抑制HMGB1的表达,改善细胞凋亡情况,进而发挥心肌保护作用。
Objective To explore the protective effect and mechanism of dexmedetomidine (Dex) on hypoxia/reoxygenation(H/R)-mediated cardiomyocyte apoptosis. Methods Rat cardiomyocytes H9c2 were selected and divided into control group(no intervention), negative-control(NC) inhibitor group and microRNA(miR)-340-5p inhibitor group, according to transfection by miR-340-5p inhibitor or its NC inhibitor. According to miR-340-5p inhibitor or its NC inhibitor transfection, Dex and H/R intervention, H9c2 were divided into control group(no intervention), H/R group, H/R+Dex group, H/R+NC inhibitor+Dex group, H/R+miR-340-5p inhibitor+Dex group. According to miR-340-5p mimic and its NC mimic transfection and high mobility group protein B1(HMGB1) base mutation, H9c2 were divided into HMGB1-wtUTR+NC mimic group, HMGB1-mutUTR+NC mimic group, HMGB1-wtUTR+miR-340-5p mimic group and HMGB1-mutUTR+miR-340-5p mimic group. Cell counting kit-8 was used to detect cells proliferation level, real-time fluorescence quantitative polymerase chain reaction was used to detect expressions of HMGB1 mRNA and miR-340-5p, and western blotting was used to detect the expressions of HMGB1, B cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax). Flow cytometry was used to detect cell apoptosis. Dual luciferase experiment was used to confirm the direct binding of miR-340-5p to HMGB1. Results The miR-340-5p expression in H/R+Dex group was higher than that in H/R group[(0.80±0.04) vs (0.25±0.01)], and expressions of HMGB1 mRNA and HMGB1 in H/R+Dex group were lower than those in H/R group[(1.47±0.13) vs (5.02±0.52),(2.09±0.08) vs (4.87±0.16)](all P<0.05). Cells proliferation level in H/R+Dex group was lower than that in control group but higher than that in H/R group; that in H/R+miR-340-5p inhibitor+Dex group was lower than that in H/R+NC inhibitor+Dex group(all P<0.05). Bcl-2 expression in H/R+miR-340-5p inhibitor+Dex group was lower than that in H/R+NC inhibitor+Dex group, and Bax expression and apoptosis rate in H/R+miR-340-5p inhibitor+Dex group were higher than those in H/R+NC inhibitor+Dex group(all P<0.05). Dual luciferase experiment showed that there was difference in relative fluorescence value among HMGB1-wtUTR+NC mimic group, HMGB1-mutUTR+NC mimic group, HMGB1-wtUTR+miR-340-5p mimic group and HMGB1-mutUTR+miR-340-5p mimic group(F=22.43,P<0.01), and that in HMGB1-wtUTR+miR-3405p mimic group was lower than that in other three groups (all P<0.05). Conclusion Dex can target to inhibit the expression of HMGB1 by up-regulating the expression of miR-340-5p, improve cell apoptosis, and play a cardioprotective effect.
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