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目的培养H9c2心肌细胞建立氧化应激模型,同时选择并选用ERK1/2途径作为研究对象,利用ERK1/2通路特异性阻断剂UO126进一步探讨可能的作用机制,观测白藜芦醇对心肌细胞抗氧化应激损伤的保护及机制。方法通过传代方法培养心肌细胞细胞。实验分为以下5组:正常对照组、过氧化氢(H2O2)组、白藜芦醇+H2O2组、白藜芦醇+UO126+H2O2组、UO126+H2O2组,实验终止后,在倒置相差显微镜下观察心肌细胞形态的变化,测定乳酸脱氢酶(LDH)的释放,测定超氧化物歧化酶(SOD)的活性,通过流式细胞术来检测心肌细胞的凋亡。结果H2O2组心肌细胞LDH量较对照组有所增加(998.08±23.07Vs477.11±17.78,p<0.05),SOD活性较对照组有所下降(15.43±4.52vs35.03±2.17,p<0.05);白藜芦醇+H2O2组LDH的量较H2O2组下降(708.51±17.84Vs998.08±23.07,p<0.05),SOD的量较H2O2组下降(29.03±3.18Vs15.43±4.52,p<0.05);而白藜芦醇+UO126+H2O2组与白藜芦醇+H2O2组相比LDH的量有所下降(617.3±11.23Vs708.51±17.84,p<0.05),SOD的量有所下降(25.12±2.23Vs29.03±3.18,p<0.05),UO126+H2O2组与H2O2组比较差异有统计学意义,p<0.05。凋亡率结果显示,空白组细胞生长良好,对照组细胞集中在B3区,在B4区和B2区内无分布或分布甚少,凋亡率极低为6.73±2.38;H2O2组出现大量凋亡细胞,其凋亡率为56.2±4.73,与空白对照组相比差别有统计学意义(p<0.05);白藜芦醇+H2O2组其凋亡率显著降低为48.17±3.08,与H2O2组相比差别有统计学意义(p<0.05);而白藜芦醇+UO126+H2O2组与白藜芦醇+H2O2组相比凋亡率有所下降(48.17±3.08Vs40.9±1.66,p<0.05),差别均有统计学意义。结论H2O2可以诱导心肌细胞的损伤及凋亡;白藜芦醇可以减轻H2O2所致的心肌细胞损伤及凋亡作用,ERK1/2通路可能为白藜芦醇发挥保护作用的途径之一。
Objective To establish oxidative stress model of H9c2 cardiomyocytes and to select and select the ERK1 / 2 pathway as the research object. The possible mechanism of ERK1 / 2 pathway-specific blocker UO126 was further explored to observe the effect of resveratrol on cardiomyocyte Protection and Mechanism of Oxidative Stress Damage. Methods Cardiomyocytes were cultured by passage method. The experiment was divided into the following five groups: normal control group, hydrogen peroxide (H2O2) group, resveratrol + H2O2 group, resveratrol + UO126 + H2O2 group, UO126 + H2O2 group, after the experiment was terminated, inverted phase contrast microscope The morphological changes of cardiomyocytes were observed. The release of lactate dehydrogenase (LDH) was measured. The activity of superoxide dismutase (SOD) was measured. The apoptosis of cardiomyocytes was detected by flow cytometry. Results Compared with control group, the amount of LDH in H2O2 group was increased (998.08 ± 23.07 vs 447.11 ± 17.78, p <0.05), and SOD activity was decreased (15.43 ± 4.52 vs 35.03 ± 2.17, p <0.05) ; The amount of LDH in resveratrol + H2O2 group was lower than that in H2O2 group (708.51 ± 17.84Vs998.08 ± 23.07, p <0.05); the amount of SOD was decreased compared with H2O2 group (29.03 ± 3.18Vs15.43 ± 4.52, p <0.05 ), While the amount of LDH decreased in resveratrol + UO126 + H2O2 group compared with resveratrol + H2O2 group (617.3 ± 11.23 vs708.51 ± 17.84, p <0.05), and the amount of SOD decreased 25.12 ± 2.23Vs29.03 ± 3.18, p <0.05). There was significant difference between UO126 + H2O2 group and H2O2 group, p <0.05. The results of apoptosis showed that the cells in the blank group grew well, and the cells in the control group were concentrated in the B3 area. There was no distribution or distribution in the area B4 and B2, with a very low apoptotic rate of 6.73 ± 2.38; The apoptotic rate was 56.2 ± 4.73, which was significantly different from that of the blank control group (p <0.05). The apoptosis rate of resveratrol + H2O2 group was significantly reduced to 48.17 ± 3.08, (P <0.05). The apoptosis rate of resveratrol + UO126 + H2O2 group was lower than that of resveratrol + H2O2 group (48.17 ± 3.08Vs40.9 ± 1.66, p < 0.05), the difference was statistically significant. Conclusion H2O2 can induce cardiomyocyte injury and apoptosis. Resveratrol can reduce the damage of H2O2-induced cardiomyocytes and apoptosis. ERK1 / 2 pathway may play a protective role in resveratrol.