摘要
目的 探讨质膜膜泡关联蛋白(PLVAP)通过Kelch样环氧氯丙烷相关蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/Maf蛋白,调控高糖(HG)诱导人肝窦内皮细胞(HLSECs)内吞功能障碍的分子机制。方法 体外培养HLSECs,分为正常对照(NC)组、HG组、PLVAP过表达重组载体(LV-PLVAP)组、慢病毒空载体(LV-CON)组、HG+LV-PLVAP+Nrf2抑制剂(ML385)组、HG+LV-PLVAP+Maf抑制剂(Mafenide)组、HG+LV-CON+ML385组、HG+LV-CON+Mafenide组。CCK-8法检测细胞活性,荧光显微镜观察LV-PLVAP转染效率,免疫荧光检测PLVAP、Nrf2荧光表达,RT-PCR和Western blot检测各组PLVAP、Keap1、Nrf2、Maf、小窝蛋白1(CAV-1)m RNA和蛋白表达。结果 LV-PLVAP、LV-CON组出现大量绿色荧光,NC组无绿色荧光。免疫荧光结果显示,PLVAP在细胞膜中表达;Nrf2在细胞质表达,HG组Nrf2在细胞核表达。与NC组比较,HG组PLVAP、Keap1 mRNA和蛋白表达降低(P<0.05或P<0.01),Nrf2、Maf、CAV-1 mRNA和蛋白表达升高(P<0.01)。与HG组比较,HG+LV-PLVAP组PLVAP、Keap1、Nrf2 mRNA和蛋白表达升高(P<0.01),Maf、CAV-1 mRNA和蛋白表达降低(P<0.01)。与HG+LV-PLVAP组比较,HG+LV-PLVAP+ML385、HG+LV-PLVAP+Mafenide组CAV-1 mRNA和蛋白表达升高(P<0.05或P<0.01)。结论 PLVAP通过Keap1/Nrf2/Maf通路调控CAV-1表达,进而调控HG诱导的HLSECs内吞功能障碍,过表达PLVAP可减缓该病理进程。
Objective To explore the molecular mechanism of plasmalemmal vesicle-associated protein(PLVAP)regulating high glucose(HG)induced endocytosis dysfunction in human liver sinusoidal endothelial cells(HLSECs)through Kelch-like epichlorohydrin ECH-associated protein 1(Keap1)/nuclear factor erythroid 2-related factor 2(Nrf2)/Maf protein.Methods HLSECs were cultured in vitro and divided into normal control(NC)group,high glucose(HG)group,PLVAP overexpression recombinant vector(LV-PLVAP)group,lentivirus empty vector(LV-CON)group,HG+LV-PLVAP+Nrf2 inhibitor(ML385)group,HG+LV-PLVAP+Maf inhibitor(Mafenide)group,HG+LV-CON+ML385 group and HG+LV-CON+Mafenide group.Cell activity was detected by CCK-8 assay.The transfection efficiency of LV-PLVAP was observed by fluorescence microscopy.The fluorescence expression of PLVAP and Nrf2 was detected by immunofluorescence.RT-PCR and western blot were used to detect the mRNA and protein expression of PLVAP,Keap1,Nrf2,and Maf,Caveolin-1(CAV-1).Results A lot of green fluorescence appeared in LV-PLVAP and LV-CON groups,however,no green fluorescence was shown in NC group.Immunofluorescence results showed that PLVAP was expressed in cell membrane;Nrf2 was expressed in cytoplasm,and in HG group,Nrf2 was expressed in nucleus.Compared with NC group,the expression of PLVAP,Keap1 mRNA and protein decreased in HG group(P<0.05 or P<0.01),while the expression of Nrf2,Maf and CAV-1 mRNA and protein increased in HG group(P<0.01).Compared with HG group,the expression of PLVAP,Keap1,Nrf2 mRNA and protein increased in HG+LV-PLVAP group(P<0.01),while the expression of Maf,CAV-1 mRNA and protein decreased in HG+LV-PLVAP group(P<0.01).Compared with HG+LV-PLVAP group,the expression of CAV-1 mRNA and protein increased in HG+LV-PLVAP+ML385 and HG+LV-PLVAP+Mafenide groups(P<0.05 or P<0.01).Conclusions PLVAP regulates the expression of CAV-1 through the Keap1/Nrf2/Maf pathway and then regulates HG induced endocytosis dys-function in HLSECs.Over expression of PLVAP alleviates this pathological reaction.
作者
郭文惠
潘友卓
雷皓月
鲁珣
张琦
GUO Wenhui;PAN Youzhuo;LEI Haoyue(The First Clinical Medical College of Gansu University of Chinese Medicine,Lanzhou 730000,China)
出处
《中国糖尿病杂志》
北大核心
2025年第11期856-862,共7页
Chinese Journal of Diabetes
基金
国家自然科学基金(82160166)
甘肃省科技计划项目任务书(22YF7FA096)
甘肃省人民医院院内科研基金项目(22GSSYA-1)。
关键词
质膜膜泡关联蛋白
内吞功能障碍
小窝蛋白1
人肝窦内皮细胞
Plasmalemmal vesicle-associated protein
Endocytosis dysfunction
Caveolin-1
Human liver sinusoidal endothelial cells
