STAT3通过血小板反应蛋白4诱导足细胞凋亡及炎症反应  

STAT3-activated thrombospondin-4 induces apoptosis and inflammatory response in podocytes

作　　者：庄一波 杨勇[1] 宁晶晶[1] ZHUANG Yibo;YANG Yong;NING Jingjing(Department of Pediatrics,the First People’s Hospital of Changzhou,Changzhou 213004,China)

机构地区：[1]常州市第一人民医院儿科,江苏常州213004

出　　处：《南京医科大学学报（自然科学版）》2022年第8期1087-1092,1106,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：常州市卫生健康青苗人才培养工程项目(CZQM2020056)。

摘　　要：目的:探讨血小板反应蛋白4(thrombospondin-4,TSP-4)对高糖(high glucose,HG)刺激足细胞增殖、凋亡、氧化应激及炎症反应的潜在影响,揭示TSP-4在糖尿病肾脏病(diabetic kidney disease,DKD)发生发展中的作用。方法:(1)通过实时荧光定量聚合酶链反应和蛋白质印迹法检测HG诱导的足细胞中TSP-4和信号转导因子和转录激活因子3(signal transducer and activator of transcription3,STAT3)的表达水平;(2)在HG诱导的足细胞中干扰TSP-4后,通过细胞计数试剂盒(cell counting kit-8,CCK-8)和5-乙炔基-2′脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine,EdU)测定增殖能力;通过流式细胞术测定足细胞的凋亡和细胞周期分布;使用试剂盒检测活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的相对含量。此外,通过酶联免疫吸附法检测炎症因子白细胞介素(interleukin,IL)-1β、肿瘤坏死因子(tumor necrosis factor,TNF)-α和IL-6的表达水平;(3)应用染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)验证转录因子STAT3与TSP-4的启动子区域的关系,并通过双荧光素酶报告基因检测进一步验证。结果:(1)TSP-4和p-STAT3在HG诱导的足细胞中呈时间依赖性上调(P<0.05);(2)干扰TSP-4可以逆转HG诱导的足细胞凋亡、氧化应激、炎症反应和受抑制的增殖(P<0.05);(3)STAT3可以结合TSP-4的启动子区域,从而诱导转录,对TSP-4启动子活性具有正向调控作用。结论:干扰TSP-4可减轻HG诱导的足细胞凋亡、氧化应激和炎症反应,对足细胞具有保护作用;转录因子STAT3对TSP-4基因启动子活性具有正向调控作用。Objective:To investigate the potential influence of thrombospondin-4(TSP-4)on the proliferation,apoptosis,oxidative stress and inflammatory response in podocytes stimulated by high glucose(HG),thus revealing the function of TSP-4 in the development of diabetic kidney disease(DKD).Methods:(1)Relative levels of TSP-4 and signal transducer and activator of transcription3(STAT3)in HG-induced podocytes were examined by real-time quantitative PCR(qRT-PCR)and Western blot.(2)After intervention of TSP-4 in HG-induced podocytes,proliferative ability was examined by cell counting kit-8(CCK-8)and 5-Ethynyl-2’-deoxyuridine(EdU)assay.The apoptosis and cell cycle distribution of podocytes were determined by flow cytometry.Relative contents of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT)were detected by commercial kits.In addition,the expression levels of inflammatory factors interleukin(IL)-1β,tumor necrosis factor(TNF)-αand IL-6were measured by enzyme-linked immunosorbent assay.(3)The interaction between the transcription factor STAT3 and the promoter region of TSP-4 was validated by chromatin immunoprecipitation(ChIP)and further confirmed by dual-luciferase reporter assay.Results:(1)TSP-4 and p-STAT3 were time-dependently upregulated in HG-induced podocytes(P<0.05).(2)Knockdown of TSP-4could reverse the inhibited proliferation,apoptosis,oxidative stress and inflammatory response in HG-induced podocytes(P<0.05).(3)STAT3 could bind the promoter region of TSP-4,thus inducing the transcription,and potitively regulating the TSP-4 promoter activity.Conclusion:Knockdown of TSP-4 alleviates HG-induced apoptosis,oxidative stress and inflammatory response in podocytes,thus showing a protective effect on podocytes.Transcription factor STAT3 has positive function to the promoter activity of TSP-4.

关 键 词：足细胞 糖尿病肾脏病 血小板反应蛋白-4 细胞凋亡 炎症反应 

分 类 号：R587.2[医药卫生—内分泌]