2021 年第 5 期 第 16 卷

右美托咪定对缺氧复氧诱导心肌细胞凋亡的防护作用及机制

Protective effect and mechanism of dexmedetomidine of hypoxia/reoxygenation-mediated cardiomyocyte apoptosis

作者：车昊马骏

英文作者：Che Hao Ma Jun

单位：首都医科大学附属北京安贞医院麻醉中心100029

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

• 摘要：

• 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.