水解南珠液通过Wnt/β-catenin通路调节细胞自噬对人微血管内皮细胞氧化应激损伤的影响

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中文摘要：目的:以人微血管内皮细胞（HMEC-1）为研究对象，采用H2O2诱导人微血管内皮细胞氧化应激损伤，探讨水解南珠液调节细胞自噬从而抑制氧化应激损伤的机制。方法:采用细胞计数试剂盒-8（CCK-8）法和活性氧荧光探针（DCFH-DA）法建立人微血管内皮细胞氧化应激损伤模型。采用Western blot法检测氧化应激损伤模型细胞自噬情况和Wnt/β-catenin通路变化情况。水解南珠液作用后，采用逆转录聚合酶链反应（RT-PCR）法检测Wnt信号通路关键蛋白Wnt3a和β-catenin基因的mRNA水平和透射电镜观察细胞自噬小体含量。通过Western blot法检测不同时间点Wnt3a、β-catenin和LC3-Ⅱ蛋白表达的时序性改变。采用WST-1法检测细胞培养液中超氧化物歧化酶活性；采用硝酸还原酶法测定细胞NO分泌量。最后采用TUNEL法检测水解南珠液对氧化应激损伤细胞凋亡情况的影响。结果:使用200 μmol/L浓度的H2O2作用HMEC-1 6 h，成功建立了内皮细胞氧化应激损伤模型。与对照组相比，氧化应激损伤组细胞LC3-Ⅱ/LC3-Ⅰ表达上扬，但Wnt3a和β-catenin蛋白表达却下降（均P＜0.05）。经透射电镜观察，氧化应激损伤组细胞内还可见较多明显的双层膜结构的自噬小体。水解南珠液处理后，随着作用时间延长，细胞内自噬小体的含量越来越少且Wnt3a和β-catenin mRNA表达水平越来越高（均P＜0.05）。且在Wnt3a和β-catenin呈时序性表达上扬的同时，LC3-Ⅱ蛋白表达则呈相对应的时序性减少。经水解南珠液处理后，细胞SOD活性与氧化应激损伤组相比，呈显著增加趋势。水解南珠液组细胞NO分泌量为（121.78±0.17）μmol/L，明显高于氧化应激损伤组的（70.43±0.74）μmol/L（P＜0.05）。氧化应激损伤组的TUNEL阳性细胞为20.3±2.8，水解南珠液作用组则减少为10.4±2.3（P＜0.05）。结论:水解南珠液能促进氧化损伤HMEC-1细胞的SOD活性、NO分泌和减少氧化损伤引起的细胞凋亡率，从而抑制氧化应激损伤程度，这些作用与水解南珠液能激活Wnt/β-catenin信号通路从而减轻细胞自噬有关。

外文标题：Hydrolyzed Nanzhu solution regulates the effect of autophagy on oxidative stress damage in human microvascular endothelial cells through the Wnt/β-catenin pathway

外文摘要：Objective:To explore the protective effect and mechanism of action of Nanzhu hydrolysate on human microvascular endothelial cell-1 (HMEC-1) with regard to oxidative stress damage by using H2O2 to induce oxidative stress in endothelial cells.Methods:An endothelial cell oxidative stress injury model was generated by the cell counting kit-8 (CCK-8) method and dichlorodihydrofluorescein diacetate (DCFH-DA) method. The changes of autophagy and Wnt/β-catenin pathway proteins in oxidative stress injury model cells were detected by Western blot. The mRNA levels of Wnt3a and β-catenin were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the content of autophagosomes in cells was observed by transmission electron microscopy after treatment with Nanzhu hydrolysate. The changes in the expression of Wnt3a, β-catenin, and LC3-Ⅱ proteins at different time points were determined by Western blot. Superoxide dismutase activity in cell culture medium was determined by WST-1 method. Cellular NO secretion was detected by the nitrate reductase method. The effect of hydrolyzed Nanzhu liquid on apoptosis of oxidative stress-injured cells was assessed by TUNEL assay.Results:An endothelial cell oxidative stress damage model was successfully established using H2O2 at a concentration of 200 μmol/L to act on HMEC-1 cells for 6 h. Compared with the control group, the expression of LC3-Ⅱ/LC3-Ⅰ in the oxidative stress injury group increased while the expression of Wnt3a and β-catenin proteins decreased (P＜0.05 for all). More autophagosomes with obvious bilayer membrane structures could be seen in the cells of the oxidative stress injury group by transmission electron microscopy. The content of autophagosomes in cells decreased and the expression levels of Wnt3a and β-catenin mRNA increased as the time of hydrolyzed Nanzhu solution treatment increased (P＜0.05 for all). And the expression of LC3-Ⅱ protein decreased correspondingly while the timing expression of Wnt3a and β-catenin increased. After treatment with hydrolyzed Nanzhu solution, the superoxide dismutase (SOD) activity of cells increased significantly when compared with that in the oxidative stress injury group. The nitrogen monoxide (NO) secretion in the hydrolyzed Nanzhu group was (121.78±0.17) μmol/L, which was significantly higher than that in the oxidative stress injury group [(70.43±0.74) μmol/L, P＜0.05]. The number of TUNEL-positive cells was 20.3±2.8 in the oxidative stress injury group, and it was 10.4±2.3 in the hydrolyzed Nanzhu solution group (P＜0.05).Conclusion:Hydrolyzed Nanhong solution promotes SOD activity, increases NO secretion, and reduces the rate of apoptosis caused by oxidative damage in HMEC-1 cells, thereby inhibiting the degree of oxidative damage, which is related to the activation of the Wnt/β-catenin signaling pathway to reduce autophagy.

作者：

岑妍慧、高月、林江、刘鹏、贾微、杨瑞、黄威、刘鑫、黄泽萍、宁志莹

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作者单位：

530200　南宁，广西中医药大学组织学与胚胎学教研室

关键词：

水解南珠液 Wnt/β-catenin 细胞自噬人微血管内皮细胞氧化应激

基金：

国家自然科学基金面上项目广西自然科学基金面上项目广西一流学科建设开放课题广西高校中青年教师科研基础能力提升项目广西中医药大学重点硕士研究生科研创新项目大学生创新创业训练计划项目广西中医药大学校级科研项目广西一流学科建设项目重点课题广西中医药大学“桂派杏林拔尖人才”项目

项目编号：

822608612020GXNSFAA2590112019XK1372021KY0291YCSZ20220032020106000182019MS0012018XK023

出版年：

2023

DOI：

10.3877/cma.j.issn.1674-0785.2023.01.013

中华临床医师杂志(电子版)

中华医学会

中华临床医师杂志(电子版)

CSTPCD

影响因子：0.99

ISSN：1674-0785

年,卷(期)：2023.17(1)

参考文献量15