高同型半胱氨酸经TRPC6/NF-κB诱导肾小球足细胞铁死亡的机制

Mechanism of iron death induced by high homocysteine via TRPC6/NF⁃κb in glomerular podiatocytes

目的探讨TRPC6/NF-κB在高同型半胱氨酸(Hcy)介导下诱导肾小球足细胞铁死亡的机制。方法对小鼠肾脏足细胞进行体外培养,将其分成对照组(0μmol/LHcy)和Hcy组(80μmol/LHcy),干预细胞48 h后,采用Western blot检测GPX4、SLC7A11铁死亡相关蛋白以及TRPC6、NFκb蛋白表达情况;实时荧光定量PCR(qRT-PCR)与免疫荧光检测TRPC6的表达水平;通过流式细胞术检测足细胞凋亡率;采用丙二醛(MDA)试剂盒测定细胞内MDA活性水平。转染TRPC6干扰片段及TRPC6阴性对照(NC)后qRT-PCR分组为Control、si-NC及si-TRPC6(si-TRPC6-1、si-TRPC6-2、si-TRPC6-3),Western blot分组为Control、Hcy、si-NC+Hcy、si-TRPC6+Hcy,采用qRT-PCR检测TRPC6 mRNA的表达;采用Western blot检测GPX4、SLC7A11、NF-κb及TRPC6的表达水平;采用流式细胞术检测干扰后足细胞凋亡水平。结果(1)与Control组相比,Hcy组中铁死亡相关蛋白GPX4、SLC7A11表达水平减低,凋亡水平增加(P<0.05);(2)与Control组相比,Hcy组TRPC6蛋白、mRNA水平及免疫荧光表达均增加,MDA水平增高,NF-κb信号通路蛋白表达增加,两组间比较均差异有统计学意义(P<0.05);(3)与si-NC组相对比,si-TRPC6(si-TRPC6-1、si-TRPC6-2、si-TRPC6-3)组中TRPC6的mRNA表达水平降低,其中si-TRPC6-3干扰效果最佳(P<0.05);转染TRPC6NC与TRPC6干扰片段并给予Hcy干预后,与Hcy组相比,si-NC+Hcy组GPX4、SLC7A11、NFκb、TRPC6表达无差异;与si-NC+Hcy组相比,si-TRPC6+Hcy组中铁死亡相关蛋白:GPX4、SLC7A11表达增高,NFκb、TRPC6表达降低,凋亡率降低(P<0.05)。结论本研究证实了在Hcy作用下TRPC6/NFκb可以调控肾足细胞铁死亡,是导致肾损伤的机制之一。

Objective To investigate the mechanism of iron death induced by TRPC6/NF⁃κB in glomerular podiocytes mediated by high homocysteine(Hcy).Methods Mouse glomerulopocytes were cultured in vitro and divided into Control group(0μmol/L Hcy)and Hcy group(80μmol/L Hcy).After 48h of intervention,Western blot was used to detect the expression levels of iron death related proteins GPX4 and SLC7A11 and TRPC6 and NF⁃κB.Real-time quantitative fluorescence PCR(qRT-PCR)and immunofluorescence were used to detect the expression of TRPC6.The level of podocyte apoptosis was detected by flow cytometry.Malondialdehyde(MDA)assay kit was used to determine intracellular MDA levels.After transfection of TRPC6 interference fragment and TRPC6 negative control(NC),qRT-PCR was divided into Control,si-NC and si-TRPC6(Si-TRPC6-1,Si-TRPC6-2,Si-TRPC6-3).Western Blot was divided into Control,Hcy,si-NC+Hcy,si-TRPC6+Hcy.The expression of TRPC6 mRNA was detected by qRT-PCR.The expression levels of GPX4,SLC7A11,NF⁃κB and TRPC6 were detected by Western Blot.The level of podocyte apoptosis after interference was detected by flow cytometry.Results(1)Compared with Control group,the expression levels of iron death related proteins GPX4 and SLC7A11 in Hcy group were decreased,and the apoptosis rate was increased(P<0.05).(2)Compared with Control group,TRPC6 protein,mRNA levels and immunofluorescence expression were increased in Hcy group.The level of MDA and the expression of NF⁃κB signaling pathway protein increased in Hcy group,and the comparison between the two groups had statistical significance(P<0.05).(3)Compared with the si-NC group,the mRNA expression level of TRPC6 in si-TRPC6(Si-TRPC6-1,Si-TRPC6-2,Si-TRPC6-3)group was decreased,and the interference effect of Si-TRPC6-3 was the best(P<0.05).After transfecting TRPC6 NC and TRPC6 interference fragment and administering Hcy,there was no difference in GPX4,SLC7A11,NF⁃κB and TRPC6 expression in si-NC+Hcy group compared with Hcy group.Compared with the si-NC+Hcy group,the si-TRPC6+Hcy group had higher expression of iron death related proteins,GPX4 and SLC7A11,lower expression of NF⁃κB and TRPC6,and decreased apoptosis rate(P<0.05).Conclusion This study confirmed that TRPC6/NF⁃κB can regulate iron death of renal podocytes under the induc⁃tion of Hcy,which is one of the mechanisms leading to kidney injury.