摘要
目的探讨分拣蛋白(Sortilin)参与调控氧化型低密度脂蛋白(ox-LDL)诱导的人肝窦内皮细胞(HLSECs)内吞功能障碍的作用机制。方法体外培养HLSECs,构建Sortilin慢病毒过表达载体(LV-Sortilin)及慢病毒空载体(LV-Con)并转染HLSECs。将细胞分为正常对照(NC)组、ox-LDL组、LV-Sortilin组和LV-Con组。LV-Sortilin及LV-Con感染HLSECs后,加入100μg/ml ox-LDL培养HLSECs 24 h,记为ox-LDL+LV-Sortilin组和ox-LDL+LV-Con组。分别用10μmol/L的EX527和1μmol/L的AS1842856处理LV-Sortilin和LV-Con感染的HLSECs 24 h后,记为ox-LDL+LV-Sortilin+EX527组、ox-LDL+LV-Con+EX527组、ox-LDL+LV-Sortilin+AS1842856组、ox-LDL+LV-Con+AS1842856组。荧光显微镜、RT-PCR和Western blot判断转染效率,CCK-8检测HLSECs活力,RT-PCR和Western blot检测各组Sortilin、沉默信息调节因子1(SIRT1)、叉头盒蛋白O1(FoxO1)、小窝蛋白1(CAV-1)mRNA和蛋白表达。结果LV-Sortilin重组体感染细胞72 h后,LV-Sortilin、LV-Con组细胞出现大量绿色荧光,NC组细胞无绿色荧光。LV-Sortilin组Sortilin mRNA和蛋白表达高于NC组(P<0.05)。与NC组比较,ox-LDL组Sortilin、SIRT1、FoxO1 mRNA和蛋白表达降低(P<0.05),CAV-1 mRNA和蛋白表达升高(P<0.05),Ac-FoxO1蛋白表达升高(P<0.05)。与ox-LDL组、ox-LDL+LV-Con组比较,ox-LDL+LV-Sortilin组Sortilin、SIRT1、FoxO1 mRNA和蛋白表达升高(P<0.05),CAV-1 mRNA和蛋白表达降低(P<0.05),Ac-FoxO1蛋白表达降低(P<0.05)。与ox-LDL+LV-Sortilin组比较,ox-LDL+LV-Sortilin+EX527、ox-LDL+LV-Sortilin+AS1842856组SIRT1、FoxO1 mRNA和蛋白表达降低(P<0.05),CAV-1 mRNA和蛋白表达升高(P<0.05),Ac-FoxO1表达升高(P<0.05)。结论Sortilin通过SIRT1/FoxO1途径调控CAV-1表达进而调控ox-LDL诱导的HLSECs内吞功能障碍,过表达Sortilin可抑制该病理反应。
Objective To investigate the molecular mechanism of Sortilin involved in regulating of oxidized low-density lipoprotein(ox-LDL)induced endocytosis dysfunction in human liver sinusoidal endothelial cells(HLSECs)M.Iethods In vitro culture of HLSECs,construction of Sortilin lentivirus overexpression vector(LV-Sortilin)and lentivirus empty vector(LV-Con),and transfection of HLSECs were performed.Then the cells were divided into normal control(NC)group,ox-LDL group,Sortilin overexpression group(LV-Sortilin)and Lentiviral empty vector group(LV-Con).After LV-Sortilin and LV-Con infection in HLSECs,HLSECs were incubated for 24 h by adding 100μg/ml ox-LDL,which were divided into ox-LDL+LV-Sortilin group and ox-LDL+LV-Con group.The LV-Sortilin and LV-Con infected HLSECs were treated with 10μmol/L of EX527 and 1μmol/L of AS1842856 for 24 h,respectively,and were divided into ox-LDL+LV-Sortilin+EX527 group,ox-LDL+LV-Con+EX527 group,ox-LDL+LV-Sortilin+AS1842856 group,and ox-LDL+LV-Con+AS1842856 group.Transfection efficiency was determined through fluorescence microscopy,RT-PCR and Western blot.CCK-8 was used to detect the activity of HLSECs.RT-PCR and Western blot were used to detect the mRNA and protein expression levels of Sortilin,Silent information regulator of transcription 1(SIRT1),Forkhead box protein O1(FoxO1),Caveolin-1(CAV-1)in HLSECs of each group.Results After LV-Sortilin recombinant infecting cells for 72 h,HLSECs in LV-Sortilin and LV-Con groups showed a large amount of green fluorescence,while NC group showed no green fluorescence.The mRNA and protein expression of Sortilin were increased in LV-Sortilin group compared with NC group(P<0.05).Compared with NC group,the mRNA and protein expression of Sortilin,SIRT1 and FoxO1 were decreased(P<0.05),the mRNA and protein expression of CAV-1 were increased(P<0.05),and the protein expression of Ac-FoxO1 was increased(P<0.05)in ox-LDL group.Compared with the ox-LDL group and ox-LDL+LV-Con group,the mRNA and protein expression of Sortilin,SIRT1 and FoxO1were increased(P<O.05),the mRNA and protein expression of CAV-1 were decreased(P<0.05),and the protein expression of Ac-FoxO1 was decreased(P<0.05)in ox-LDL+LV-Sortilin group.Compared with ox-LDL+LV-Sortilin group,the mRNA and protein expression of SIRT1 and FoxO1were decreased(P<0.05),the mRNA and protein expression of CAV-1 were increased(P<0.05),and the protein expression of Ac-FoxO1was increased(P<0.05)in ox-LDL+LV-Sortilin+EX527 group and ox-LDL+LV-Sortilin+AS1842856 group.Conclusion Sortilin regulates the expression of CAV-1 through the SIRT1/FoxO1 pathway and then regulates ox-LDL induced endocytosis dysfunction in HLSECs.Overexpression of Sortilin inhibits this pathological reaction.
作者
张延燕
刘静
田利民
余静
赵文淑
张雅娟
刘亚倩
司惠玲
仇菊梅
张琦
ZHANG Yanyan;LIU Jing;TIAN Limin(The First Clinical Medical College of Gansu University of Chinese Medicine,Lanzhou 730000,China)
出处
《中国糖尿病杂志》
CAS
CSCD
北大核心
2023年第5期369-375,共7页
Chinese Journal of Diabetes
基金
国家自然科学基金(82160166、81960173)
甘肃省重点研发计划(22YF7FA096)
甘肃省人民医院院内科研基金(22GSSYA-1)
兰州市人才创新创业项目(2021-RC-136)
兰州市卫生健康科技发展项目(2021005)
甜蜜医生培育项目(2021SD01)。
关键词
分拣蛋白
沉默信息调节因子1/叉头盒蛋白O1
氧化型低密度脂蛋白
肝窦内皮细胞
内吞功能
Sortilin
Silent information regulator of transcription 1/Forkhead box protein Ol
Oxidized low-density lipoprotein
Liver sinusoidal endothelial cells
Endocytosis function
