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  

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

作　　者：张宏红 谢琳[1,2,3] 盛思琪 卢冠军 姜怡邓 杨安宁[1,2,3] 李桂忠[1,2,3] ZHANG Honghong;XIE Lin;SHENG Siqi;LU Guanjun;JIANG Yideng;YANG Anning;LI Guizhong(Basic Medical College of Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Metabolic Cardiovascular Diseases Research of National Health Commission;Ningxia Key Laboratory of Vascular Injury and Repair Research;Department of Urology,General Hospital of Ningxia Medical University)

机构地区：[1]宁夏医科大学基础医学院,750004 [2]国家卫生健康委代谢性心血管疾病研究重点实验室 [3]宁夏血管损伤与修复研究重点实验室 [4]宁夏医科大学总医院泌尿外科

出　　处：《天津医药》2022年第1期53-58,共6页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(82060139);宁夏回族自治区重点研发计划重点项目(2018BEG02004);宁夏回族自治区重点研发计划重点项目(2020BFH02003)。

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80μmol/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、AdFoxO1+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 m RNA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。Objective To discuss the role of forkhead box O1(FoxO1)in the injury and apoptosis of podocytes induced by homocysteine(Hey).Methods Ten Cbs^(+/+)mice with normal cysteineβ-synthase(Cbs)gene and 10 Cbs^(+/-)mice with single gene knockout were fed a high methionine dieth.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 Hey group(treated with cell culture medium containing 80μmol/L Hey 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-FoxOl+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),Bel-2 associated X protein(Bax)and cystine proteinase 12(Caspase 12)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 Hey group after treated with Hey(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 Caspase

关 键 词：足细胞 叉头框蛋白O1 细胞凋亡 胱硫醚Β合酶 同型半胱氨酸 肾损伤 

分 类 号：R692[医药卫生—泌尿科学]