抑制SGLT对软脂酸诱导的内皮细胞损伤影响研究  

Effect of suppressed SGLT on palmitic acid-induced endothelial dysfunction

作　　者：李春盈 苏悦 陈霞[2] 何航辉 郑超[3] LI Chunying;SU Yue;CHEN Xia;HE Hanghui;ZHENG Chao(Department of Nephrology,Ningbo Yinzhou NO.2 Hospital,Ningbo 315040,China)

机构地区：[1]宁波市鄞州区第二医院肾内科,315040 [2]温州医科大学 [3]浙江大学医学院附属第二医院内分泌科

出　　处：《浙江医学》2020年第20期2146-2151,共6页Zhejiang Medical Journal

基　　金：国家自然科学基金资助项目(81670777)。

摘　　要：目的探讨采用软脂酸(PA)诱导人脐静脉内皮细胞胰岛素抵抗下,钠葡萄糖共转运体(SGLT)的表达变化,及抑制SGLT表达后对内皮细胞损伤的影响和作用机制。方法将人脐静脉内皮细胞分为空白对照组(DMSO组)、PA组、PA+Insulin组、PA+根皮苷(PH)组、PA+PH+LY组。DMSO组细胞加入DMSO处理,作对照用;PA组用0.3 mmol/L PA干预18 h建立胰岛素抵抗模型;PA+Insulin组、PA+PH组在PA组处理的基础上再分别给予100 nmol/L胰岛素、PH干预30 min;PA+PH+LY组预先加入100 nmol/L磷脂酰肌醇-3激酶(PI3K)抑制剂LY294002孵育30 min,后再加入PH干预30 min。采用Western blot法检测各组细胞SGLT1、SGLT2、磷酸化胰岛素受体底物1(p-IRS-1)、磷酸化AKT(p-AKT)、磷酸化eNOS(p-eNOS)蛋白表达水平,采用RT-PCR法检测SGLT1、SGLT2 mRNA表达水平,采用硝酸盐还原酶法检测NO浓度和葡萄糖消耗量。采用siRNA转染内皮细胞以沉默SGLT1和SGLT2,分为DMSO组、PA组和PA+si-Ctrl组、PA+si-SGLT1组、PA+si-SGLT2组,其中PA+si-Ctrl组细胞用无意义片段转染,采用Western blot法检测p-IRS-1、p-AKT、p-eNOS蛋白表达水平。结果PA组细胞SGLT1、SGLT2蛋白和mRNA表达水平较DMSO组增加(均P<0.05),PA+PH组细胞SGLT1、SGLT2蛋白和mRNA表达水平较PA组降低(均P<0.05)。PA组细胞葡萄糖消耗量与NO浓度均低于DMSO组(均P<0.05),PA+Insulin组、PA+PH组细胞葡萄糖消耗量与NO浓度均高于PA组(均P<0.05)。PA组细胞p-IRS-1、p-AKT、p-eNOS蛋白表达水平及NO浓度均低于DMSO组(均P<0.05)。PA+PH组细胞p-AKT、p-eNOS蛋白表达水平及NO浓度均较PA组升高(均P<0.05),但p-IRS-1蛋白表达水平比较差异无统计学意义(P>0.05)。PA+PH+LY组细胞p-AKT、p-eNOS蛋白表达水平均较PA+PH组降低(均P<0.05),但p-IRS-1蛋白表达水平及NO浓度比较差异均无统计学意义(均P>0.05)。PA+si-SGLT1组、PA+si-SGLT2组细胞p-AKT、p-eNOS蛋白表达水平均明显高于PA组和PA+si-Ctrl组(均P<0.05)。与DMSO组相比,p-IRS-1�Objective To investigate the effect of sodium-glucose linked transporter(SGLT)on palmitic acid(PA)-induced endothelial dysfunction and related mechanism.Methods The cultured human umbilical vein endothelial cells(HUVECs)were divided into DMSO group,PA group,PA+Insulin group,PA+PH group and PA+PH+LY group.The DMSO group was treated with DMSO as control.The insulin resistance(IR)model was established by incubation with 0.3 mmon/L PA for 18 h in HUVECs;PA+insulin group and PA+PH group were treated with 100 nmol/L insulin and phloridzin(PH)for 30 min on the basis of PA group treatment,PA+PH+LY group was pre incubated with 100 nmon/L phosphatidylinositol-3 kinase(PI3K)specific inhibitor(LY294002)for 30 min,then intervened with PH for 30 mins.The expression of SGLT and the phosphorylation of AKT,eNOS,IRS-1was detected by Western blot.The levels of SGLT mRNA were detected by RT-PCR.Nitric oxide(NO)concentration was determined by nitrate reductase method,and glucose consumption was determined by glucose reductase method.SGLT1 and SGLT2 on HUVECs were silenced by siRNA transfection,and then the cells were divided into DMSO group,PA group and PA+si-Ctrl group,PA+si-SGLT1 group and PA+si-SGLT2 group.The expression levels of p-IRS-1,p-AKT and p-eNOS were detected by Western blot.Results The expression levels of SGLT1 and SGLT2 protein and mRNA in PA group were higher than those in DMSO group(all P<0.05).The levels of p-IRS-1,p-AKT,p-eNOS protein and NO concentration in PA group were lower than those in DMSO group(all P<0.05).The levels of p-AKT,p-eNOS and NO concentration in PA+pH group were higher than those in PA group(all P<0.05),however,there was no significant difference in the expression of p-IRS-1(P>0.05).The protein levels of p-AKT and p-eNOS in PA+PH+LY group were lower than those in PA+pH group(all P<0.05);however,there was no significant difference in the expression of p-IRS-1 protein and NO concentration(all P>0.05).The levels of p-AKT and p-eNOS in PA+si-SGLT1 and PA+si-SGLT2 groups were significantly higher tha

关 键 词：胰岛素抵抗 钠葡萄糖共转运体 根皮苷 一氧化氮 内皮细胞功能 

分 类 号：R587.1[医药卫生—内分泌]