硫酸锌对缺氧/复氧损伤H9c2心肌细胞自噬及氧化应激的影响

Influences of Zinc Sulfate on Autophagy and Oxidative Stress of H9c2 Cardiomyocytes Injured by Hypoxia and Reoxygenation

目的探讨硫酸锌对缺氧/复氧(H/R)H9c2心肌细胞自噬、氧化应激的影响。方法制备H9c2心肌细胞H/R模型,分为对照组、H/R组、低剂量硫酸锌组(LD-Zn组)和高剂量硫酸锌组(HD-Zn组)。流式细胞仪检测心肌H9c2细胞凋亡,硫代巴比妥酸比色法检测丙二醛(MDA)水平,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)水平,蛋白免疫印迹法(Western Blot)检测H9c2心肌细胞自噬体膜型(LC3Ⅱ)蛋白表达水平。结果对照组、H/R组、LD-Zn组和HD-Zn组H9c2心肌细胞凋亡率分别为(0.82±0.15)%、(18.46±2.24)%、(10.07±1.34)%、(6.12±1.05)%,4组比较差异有统计学意义(P<0.01),其中LD-Zn组和HD-Zn组细胞凋亡率明显低于H/R组(P<0.01),HD-Zn组细胞凋亡率明显低于LD-Zn组(P<0.01)。LD-Zn组和HD-Zn组MDA水平明显低于H/R组(P<0.01),HD-Zn组MDA水平明显低于LD-Zn组(P<0.01);LD-Zn组和HD-Zn组SOD水平明显高于H/R组(P<0.01),HD-Zn组SOD水平明显高于LD-Zn组(P<0.01)。对照组、H/R组、LD-Zn组和HD-Zn组H9c2心肌细胞自噬蛋白LC3Ⅱ表达的相对灰度值分别为0.46±0.07、0.07±0.01、0.16±0.03和0.28±0.05,4组比较差异有统计学意义(P<0.01),LD-Zn组和HD-Zn组LC3Ⅱ蛋白相对灰度值明显高于H/R组(P<0.01),HD-Zn组LC3Ⅱ蛋白相对灰度值明显高于LD-Zn组(P<0.01)。结论硫酸锌可以增强H/R损伤H9c2心肌细胞的自噬,抑制氧化应激损伤和心肌细胞凋亡。

Objective To observe the effects of zinc sulfate on autophagy and oxidative stress in cardiomyocytes with hypoxia and reoxygenation(H/R).Methods The cells were divided into control group,H/R group low dose zinc sulfate(LD-Zn)group,and high dose zinc sulfate(HD-Zn)group.The apoptosis of H9c2 cells was detected by flow cytometry.The thiobarbituric acid colorimetry was used to detect malonaldehyde(MDA).The xanthine oxidase method was used to detect superoxide dismutase(SOD).Western Blot was used to detect LC3Ⅱprotein expression in H9c2 cardiomyocytes.Results The apoptosis rate in control group,H/R group,LD-Zn group,and HD-Zn group was(0.82±0.15)%,(18.46±2.24)%,(10.07±1.34)%,and(6.12±1.05)%respctirely.There were significant differences between four groups(P<0.01).The apoptosis rate in LD-Zn and HD-Zn group was lower than that in H/R group(P<0.01),and the apoptosis rate in HD-Zn group was lower than that in LD-Zn group(P<0.01).The MDA levels in LD-Zn group and HD-Zn group were lower than that in H/R group(P<0.01),and the MDA level in HD-Zn group was lower than that in LD-Zn group(P<0.01).SOD levels in LD-Zn group and HD-Zn group were higher than that in H/R group(P<0.01),and SOD level in HD-Zn group was higher than that in LD-Zn group(P<0.01).The relative gray value of LC3Ⅱin control group,H/R group,LD-Zn group,and HD-Zn group was 0.46±0.07,0.07±0.01,0.16±0.03,and 0.28±0.05 respctirely,there were significant differences between four groups(P<0.01).The relative gray value of LC3Ⅱin LD-Zn and HD-Zn group was higher than that in H/R group(P<0.01),and the relative gray value of LC3Ⅱin HD-Zn group was higher than that in LD-Zn group(P<0.01).Conclusion Zinc sulfate can enhance autophagy of cardiomyocytes to inhibit oxidative stress injury and myocardial cell apoptosis induced by H/R injury.