FoxO1在同型半胱氨酸诱导的肾脏足细胞损伤及凋亡中的作用研究

摘要：目的　探讨叉头状转录因子O1（FoxO1）在同型半胱氨酸（Hcy）诱导的肾脏足细胞损伤及凋亡中的作用。方法　采用高蛋氨酸饮食喂养胱硫醚β-合成酶（Cbs）基因正常Cbs+/+小鼠和单基因敲除Cbs+/-小鼠各10只。8周后处死，收集肾组织，PAS染色观察小鼠肾小球形态学变化。体外培养足细胞，分为Control组和Hcy组（用含80 μmol/L Hcy的细胞培养液干预48 h）。将携带绿色荧光蛋白（GFP）的过表达FoxO1腺病毒（Ad-FoxO1）和干扰FoxO1腺病毒（Sh-FoxO1）转染足细胞，分为对照组、Ad-GFP组、Ad-FoxO1组、Sh-NC组、Sh-FoxO1组、Ad-GFP+Hcy组、Ad-FoxO1+Hcy组、Sh-NC+Hcy组、Sh-FoxO1+Hcy组。Western blot检测小鼠肾组织和足细胞裂隙膜蛋白（Podocin和Nephrin）、FoxO1及凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱氨酸蛋白酶12（Caspase12）蛋白表达；qPCR检测小鼠肾组织和足细胞中FoxO1 mRNA的表达。结果　PAS染色结果显示，Cbs+/+组小鼠肾小球结构正常，基底膜清晰且分布均匀，而Cbs+/-组小鼠肾小球基底膜则呈现节段性增厚，系膜基质增多；与Cbs+/+组小鼠比较，Cbs+/-组小鼠肾组织中Podocin、Nephrin和FoxO1蛋白、FoxO1 mRNA的表达明显降低（P＜0.01）。与Control组比较，Hcy组FoxO1 mRNA和蛋白表达显著降低（P＜0.01）；过表达FoxO1后，与Ad-GFP+Hcy组相比，Ad-FoxO1+Hcy组Podocin和Nephrin蛋白表达均明显升高，Bax/Bcl-2比值、Caspase12蛋白表达均明显降低（P＜0.05）；而干扰FoxO1后，与Sh-NC+Hcy组相比，Sh-FoxO1+Hcy组Podocin和Nephrin蛋白表达均明显降低，Bax/Bcl-2比值、Caspase12蛋白表达均明显增高（P＜0.05）。结论　FoxO1可减轻Hcy诱导的小鼠肾脏足细胞损伤及凋亡。

The role of FoxO1 in renal podocyte injury and apoptosis induced by homocysteine

Abstract: Objective　To discuss the role of forkhead box O1 (FoxO1) in the injury and apoptosis of podocytes induced by homocysteine (Hcy). Methods　Ten Cbs+/+ mice with normal cysteine β -synthase (Cbs) gene and 10 Cbs+/- mice with single gene knockout were fed a high methionine diet. After 8 weeks, the mice were sacrificed to collect kidney tissues. Morphological changes of glomerulus were observed by PAS staining. Podocytes were cultured in vitro and divided into the control group and the Hcy group (treated with cell culture medium containing 80 μmol/L Hcy for 48 h). Podocytes were transfected with Ad-FoxO1 overexpressed adenovirus and Sh-FoxO1 interfering adenovirus carrying green fluorescent protein (GFP), and they were divided into the control group, the Ad-GFP group, the Ad-FoxO1 group, the Sh-NC group, the Sh-FoxO1 group, the Ad-GFP+Hcy group, the Ad-FoxO1+Hcy group, the Sh-NC+Hcy group and the Sh-FoxO1+Hcy group. Western blot assay was used to detect the expression of Podocin and Nephrin, FoxO1 and apoptosis-related B lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cystine proteinase 12 (Caspase12) proteins in renal tissues and podocytes of mice. The expression of FoxO1 mRNA in mouse kidney tissue and podocytes was detected by qPCR. Results　PAS staining results showed that the glomerular structure was normal, the basement membrane was clear and distributed evenly in Cbs+/+ mice, while the glomerular basement membrane presented intermittent thickening and the mesangial matrix increased in Cbs+/- mice. Compared with Cbs+/+ mice, the expression levels of Podocin, Nephrin and FoxO1 protein and FoxO1 mRNA were significantly decreased in Cbs+/- mice (P＜0.01). Compared with the control group, the expression of FoxO1 protein and mRNA in podocytes were significantly decreased in the Hcy group after treated with Hcy (P＜0.01). After overexpression of FoxO1 in podocytes, compared with the Ad-GFP+Hcy group, the protein expression of Podocin and Nephrin were significantly increased, both the ratio of Bax/Bcl-2 and the protein expression of Caspase12 were significantly decreased in the Ad-FoxO1+Hcy group (P＜0.05). After FoxO1 interference, compared with the Sh-NC+Hcy group, Podocin and Nephrin protein expressions were significantly decreased, Bax/Bcl-2 ratio and Caspase12 protein expression were significantly increased in the Sh-FoxO1+Hcy group (P＜0.05). Conclusion　FoxO1 can reduce Hcy-induced renal podocyte injury and apoptosis in mice.