消渴通痹颗粒对高糖诱导的HUVEC屏障功能的影响及保护作用  

Protective Effect of Xiaoke Tongbi Granules on High Glucose-induced Endothelial Barrier Dysfunction in Human Umbilical Vein Endothelial Cells(HUVEC)

作　　者：张欢 丁纪茹 陈耀 袁有才[2] 段玉红[2] 张效科[2] Zhang Huan;Ding Jiru;Chen Yao;Yuan Youcai;Duan Yuhong;Zhang Xiaoke(Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610075;Shaanxi University of Traditional Chinese Medicine,Xianyang 712046)

机构地区：[1]成都中医药大学附属医院,成都610075 [2]陕西中医药大学,咸阳712046

出　　处：《中药药理与临床》2020年第3期168-175,共8页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(编号:81774304)。

摘　　要：目的:观察益气活血中药消渴通痹颗粒对高糖诱导的人脐静脉内皮细胞(HUVEC)屏障功能及紧密连接蛋白(ZO-1)、闭合蛋白(Occludin)表达的影响,探讨消渴通痹颗粒保护糖尿病周围神经病变内皮细胞屏障功能的作用及机理。方法:将不同浓度的消渴通痹颗粒含药血清和5.5 mmol/L葡萄糖作用于HUVEC 24 h,MTT法检测细胞的存活率,确定消渴通痹颗粒含药血清的给药浓度。根据葡萄糖的浓度和消渴通痹颗粒含药血清的浓度将实验分为正常对照组,高糖模型组和消渴通痹颗粒22.5 g/kg含药血清组。各组细胞传代8代~15代时,应用HRP检测HUVEC单层通透性;流式细胞术检测HUVEC凋亡;免疫荧光法、流式细胞术检测紧密连接蛋白ZO-1、Occludin的表达。结果:与正常对照组相比,5%、10%消渴通痹颗粒22.5 g/kg含药血清组的存活率明显升高(P<0.05),15%、20%消渴通痹颗粒22.5 g/kg含药血清组的存活率明显下降(P<0.05或P<0.01),故而确定消渴通痹颗粒含药血清的浓度为5%、10%、15%。与正常对照组相比,高糖模型组单层通透性增加,凋亡率显著上升,ZO-1、Occludin蛋白的表达减少、荧光信号减弱、边缘粗糙、细胞与细胞之间的连接有断裂(P<0.05或P<0.01)。与高糖模型组相比,5%、10%消渴通痹颗粒22.5 g/kg含药血清组单层通透性降低,凋亡率下降,ZO-1、Occludin的蛋白表达上调、荧光信号加强、边缘趋于光滑、细胞与细胞之间连接紧密(P<0.05或P<0.01);15%消渴通痹颗粒22.5 g/kg含药血清组单层通透性降低,凋亡率下降,Occludin蛋白表达下调、荧光信号减弱(P<0.05或P<0.01),ZO-1蛋白表达变化不明显(P>0.05)。结论:益气活血中药消渴通痹颗粒改善了高糖诱导的HUVEC细胞屏障功能,其机制可能与增加紧密连接蛋白ZO-1、Occludin的表达有关。Objective:To observe the protective effect of Yiqihuoxue traditional medicine Xiaoke Tongbi Formula on high glucose-induced HUVEC barrier dysfunction and expressions of tight junction protein ZO-1 and Occludin,and to explore its mechanism on endothelial barrier dysfunction in diabetic peripheral neuropathy.Methods:HUVEC were treated with various concentrations of serum containing Xiaoke Tongbi Formula and 5.5 mmol/L glucose for 24 h.MTT assay was used to determine the cell viability,to determine the concentration of drug.According to the various concentrations of serum containing Xiaoke Tongbi Formula and glucose,HUVEC were divided into the control group,the high-glucose model group and serum containing Xiaoke Tongbi Formula groups.Primary HUVEC were sub-cultured to passage 8 to 15.Cell monolayer integrity was measured by HRP.Apoptosis of HUVEC was evaluated by flow cytometry.Expressions of tight junction proteins ZO-1 and Occludin were detected by immunofluorescence assay and flow cytometry.Results:Compared with the control group,the cell viability in 22.5 g/kg serum containing Xiaoke Tongbi Formula group(5%and 10%)was increased(P<0.05),the cell viability in 22.5 g/kg serum containing Xiaoke Tongbi Formula group(15%and 20%)was decreased(P<0.05).Therefore,the doses of serum containing Xiaoke Tongbi Formula were set 5%,10%and 15%,respectively.Compared with the control group,the cell monolayer integrity was increased in high glucose model group,the rate of apoptosis was increased,the expressions of ZO-1 and Occludin proteins were down regulated,the fluorescence signal was weakened,cell edge was observed rough and fractures were observed in cell junctions(P<0.05 orP<0.01).Compared with high glucose model group,the cell monolayer integrity was decreased in 22.5 g/kg serum containing Xiaoke Tongbi Formula groups(5%and 10%),the rate of apoptosis was decreased,the expressions of ZO-1 and Occludin proteins were up regulated,the fluorescence signal was enhanced,the cell edge was observed smooth and cells were tightly conn

关 键 词：消渴通痹颗粒 糖尿病周围神经病变 内皮细胞屏障 紧密连接蛋白 闭合蛋白 

分 类 号：R285.5[医药卫生—中药学]