终末期肾病患者血清对内皮细胞凋亡的影响及黄芪总黄酮的干预作用

Influence of End Stage Renal Disease Serum on Endothelial Cell Apoptosis and Intervention Action of Total Flavonoids of Astragalus

目的:探讨终末期肾病患者血清对内皮细胞凋亡的影响及黄芪总黄酮的干预作用。方法:收集22例健康志愿者和25例终末期肾病规律血液透析患者的血清,在体外模拟终末期肾病患者内环境,以人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)为对象,分为空白对照组(10%胎牛血清),正常对照组(10%健康人血清),终末期肾病组(10%终末期肾病患者血清),低剂量(TFA1),中剂量(TFA2),高剂量(TFA3)组(在终末期肾病组基础上分别加入0.5,1.0,2.0 g·L-1黄芪总黄酮),培养24 h后光镜下观察血管内皮细胞形态,MTT法检测细胞生存率,免疫组化法检测C/EBP同源蛋白(CHOP)的表达,ELISA法检测4-羟基壬烯醛(4-HNE)的含量,TUNEL法检测细胞凋亡。结果:与正常对照组相比,终末期肾病患者组细胞生存率降低(P<0.01),CHOP和4-HNE升高(P<0.05),凋亡细胞数增高(P<0.01)。黄芪总黄酮干预后,各组HUVECs生存率提高(P<0.05);CHOP的表达降低(P<0.05),中剂量剂量组优于低剂量组(P<0.01),但高剂量组与中剂量组差异无统计学意义;4-HNE含量降低(P<0.05),细胞凋亡减少(P<0.05),呈剂量依赖性(P<0.05)。相关性分析显示:CHOP与4-HNE的含量呈正相关(P<0.01)。结论:终末期肾病患者血清可诱导HUNFCs凋亡,其机制部分与内质网应激诱导的细胞凋亡通路相关,黄芪总黄酮可通过减轻内质网应激从而抑制终末期肾病患者血清诱导的内皮细胞凋亡。

Objective：To explore the effect of end stage renal disease （ESRD） serum on apoptosis in human umbilical vein endothelial cells and the intervention action by total flavonoids of astragalus in this process.Method：The serums of 22 healthy volunteers and 25 patients with ESRD undergoing regular hemodialysis were collected.Simulating ESRD environment in vitro,human umbilical vein endothelial cells （HUVECs） were selected as the subject.Then they were divided into blank control group （including 10％ fetal calf serum）,normal control group （including 10％ healthy serum）,ESRD group （including 10％ ESRD serum）,low dose group,medial dose group and high dose group with total flavonoids of astragalus （0.5,1.0,2.0 g ·L-1 total flavonoids of astragalus,respectively）.After being culturing for 24 hours,the morphological change of endothelial cells were observed by microscopy,survival rate was detected by MTT test,the expression of C/EBP homologous protein （CHOP） was examined by immunohistochemical staining,the content of 4-hydroxynonenal （HNE） was detected by ELISA,and the morphological change of apoptosis was observed by TUNEL method.Result：Comparing with the normal control group,HUVECs survival rate was lower in ESRD group （P ＜0.01）,CHOP,4-HNE （P ＜ 0.05） and apoptosis index （P ＜ 0.01） were increased.The total flavonoids of astragalus could significantly increase the survival rate of HUVECs （P ＜ 0.05）,and decrease the expression of CHOP （P ＜ 0.05）.Besides,better results were found in medial dose group than low dose group （P ＜ 0.01）,but no statistically significant difference were found between high dose group and middle does group.Morever,the total flavonoids of astragalus could decrease 4-HNE and the apoptosis index （P ＜ 0.05）,and the effect is dose-dependent （P ＜ 0.05）.Positive correlation were found between CHOP and 4-HNE （P ＜ 0.01）.Conclusion：ESRD serum could induce HUVECs apoptosis,and the mechanism may be related to endoplasmic reticulum stress.Total flavonoids of astragalus could inhibit endothelial cell apoptosis induced by ESRD serum through alleviating endoplasmic reticulum stress.