水解南珠液抑制细胞自噬减轻人微血管内皮细胞氧化损伤

Nanzhu hydrolysate attenuate oxidative stress damage of human microvascular endothelial cells by inhibiting autophagy

[目的]以人微血管内皮细胞(human microvascular endothelial cell-1,HMEC-1)为研究对象,采用H_(2)O_(2)诱导内皮细胞氧化应激,探讨南珠水解液对HMEC-1细胞氧化损伤的保护作用及机制。[方法]采用生化检验法检测南珠水解液作用后,HMEC-1细胞内超氧化物歧化酶(Superoxide Dismutase,SOD)和谷胱甘肽过氧化物酶(Glutathione Peroxidase,GSH-PX)活性及丙二醛(Malondialdehyde,MDA)含量变化;采用流式细胞仪检测细胞内活性氧(Reactive Oxygen,ROS)含量变化;采用透射电镜观察自噬体;采用免疫荧光实验检测细胞微管相关蛋白轻链3Ⅰ/Ⅱ(Microtubuleassociated protein light chainⅠ/Ⅱ,LC3Ⅰ/Ⅱ)表达变化。[结果]与氧化损伤组相比,不同浓度南珠水解处理组SOD活性显著增加,分别提高8.68%、24.99%和49.43%;GSH-PX活性无显著变化;MDA显著降低,分别降低36.99%、35.82%和60.25%;且给药组细胞内ROS含量相比氧化损伤模型组显著降低,分别降低5.18%、15.55%和32%;透射电镜结果显示对照组细胞内无明显自噬小体,氧化损伤组可见明显的双层膜结构的自噬小体,用不同浓度南珠水解液处理后,随着浓度的增加自噬小体含量显著减少;免疫荧光实验结果显示,与氧化损伤组相比,南珠水解液组LC3-Ⅰ/LC3-Ⅱ蛋白比值显著降低,分别降低19.21%、29.76%和33.71%。[结论]南珠水解液对氧化损伤的HMEC-1细胞有一定的修复作用,这种作用可能是通过抑制细胞自噬,并促进SOD活性水平提升,降低MDA和ROS含量,从而减轻H_(2)O_(2)诱导的HMEC-1细胞氧化损伤。

[Objective]The HMEC-1 cells were used as the research object,the oxidative stress was induced by H2 O2,and then to explore the protective effect and mechanism of Nanzhu hydrolysate on oxidative damage of HMEC-1 cells. [Method]The activities of SOD and GSH-PX and the content of MDA in HMEC-1 cells treated with Nanzhu hydrolysate were detected by routine biochemical assay,the ROS was detected by flow cytometry,and autophagosomes were observed by transmission electron microscope. Immunofluorescence assay was used to detect the expression of LC3 I/II.[Result]Compared with the oxidative damage group,the SOD activity of different concentrations of Nanzhu hydrolysis group increased significantly,increased by8. 68%,24. 99% and 49. 43%,respectively;There was no significant change in GSH-PX activity;MDA significantly decreased by 36. 99%,35. 82% and 60. 25%,respectively;And the ROS content in the treatment group was significantly lower than that in the oxidative damage model group by 5. 18%,15. 55% and 32%,respectively. The results of transmission electron microscope showed that there were no obvious autophagosomes in the control group,but obvious autophagosomes with bilayer membrane structure could be seen in the oxidative damage group. After being treated with different concentrations of Nanzhu hydrolysate,the content of autophagosomes decreased significantly with the increase of concentration. The results of immunofluorescence experiment showed that compared with the oxidative damage group,the ratio of LC3-Ⅰ/LC3-Ⅱ protein in Nanzhu hydrolysate group decreased by 19. 21%,29. 76% and 33. 71%,respectively. [Conclusion] Nanzhu hydrolysate has a certain repair effect on oxidative damage of HMEC-1 cells,which may be through inhibiting autophagy,promoting the level of SOD activity,reducing the content of MDA and ROS,thus reducing the oxidative damage of HMEC-1 cells induced by H2 O2.