黄芪桂枝五物汤对H_(2)O_(2)诱导人脐静脉细胞损伤的保护作用及机制

Protective effects and mechanism of Huangqi Guizhi Wuwu Decoction on H_(2)O_(2)-induced injury of human umbilical vein endothelial cells

目的探讨黄芪桂枝五物汤对H_(2)O_(2)诱导人脐静脉细胞(HUVECs)损伤的保护作用及机制。方法HUVECs分为正常组,模型组,阳性药组(卡维地洛),黄芪桂枝五物汤高、中、低剂量组,各组水提醇沉液培养24 h后加入H_(2)O_(2)溶液使终浓度达到400μmol/L)。MTT法检测细胞存活率;检测细胞上清LDH(乳酸脱氢酶)活性,细胞内T-SOD(总超氧化物歧化酶)、NO、GSH-Px(谷胱甘肽过氧化物酶)、ET-1(内皮素-1)水平;Real-time PCR检测Bax、Bcl-2 mRNA表达;Western blot法检测Bax、Bcl-2蛋白表达。结果与正常组比较,模型组细胞存活率减低(P<0.01);与模型组比较,黄芪桂枝五物汤各组存活率升高(P<0.01);LDH活性降低、NO水平升高、T-SOD活力增强、GSH-Px水平升高、ET-1水平降低(P<0.05)。Bax mRNA表达降低,Bcl-2 mRNA表达增加(P<0.05);Bax蛋白表达降低,Bcl-2蛋白表达增加(P<0.05)。结论黄芪桂枝五物汤对H_(2)O_(2)损伤HUVECs有保护作用,其机制可能与提高内皮细胞T-SOD活性、促进细胞NO分泌、提高细胞内GSH-Px水平,降低细胞内ET-1水平以拮抗氧化应激造成的细胞损伤,调控凋亡相关基因及蛋白Bax、Bcl-2,抑制线粒体凋亡途径有关。

AIM To explore the protective effect and mechanism of Huangqi Guizhi Wuwu Decoction on human umbilical vein endothelial cells(HUVECs)injured by H_(2)O_(2).METHODS Cells divided into normal group,model group,positive drug group(carvedilol),and high-dose,medium-dose and low-dose Huangqi Guizhi Wuwu Decoction groups were given H_(2)O_(2) solution after 24 hours exposure to the aqueous extraction and alcohol precipitation to achieve a final concentration of 400μmol/L.HUVECs were subjected to the detection of cell survival rate by MTT method;the measurement of LDH,T-SOD,NO,GSH-Px,ET-1 level;the determination of Bax,Bcl-2 mRNA expression by real-time PCR;the detection of Bax,Bcl-2 protein expression by Western blot.RESULTS The model group shared lower cell survival rate than the blank group(P<0.01).Compared with the model group,Huangqi Guizhi Wuwu Decoction groups had higher cell survival rate(P<0.01),decreased LDH activity and ET-1 level,increased NO concentration,and T-SOD activity,GSH-Px level(P<0.05),decreased Bax mRNA expression,and increased relative Bcl-2 mRNA expression(P<0.05);as well as decreased relative expression of Bax protein and increased expression of Bcl-2 protein(P<0.05).CONCLUSION Huangqi Guizhi Wuwu Decoction can improve the survival rate of H_(2)O_(2)-injured HUVECs and reduce the LDH leakage,revealing its protective effect on HUVECs.The mechanism may contribute to the improvement in T-SOD activity in endothelial cells,NO and GSH-Px production,and lower ET-1 level to antagonize the cell damage caused by oxidative stress,regulate the apoptosis-related genes and proteins Bax and Bcl-2,and inhibit the mitochondrial apoptosis pathway.