控制血糖通过下调KLF6而抑制STZ所致的1型糖尿病大鼠肾小管上皮细胞损伤  

Blood glucose control inhibits STZ-induced renal tubular epithelial cell damage in type 1 diabetic rats by down-regulating KLF6

作　　者：张会芳 向珈谊 梁露群 王丹 周星丞 张小欢 毛彦稳 王圆圆 郭兵 ZHANG Hui-fang;XIANG Jia-yi;LIANG Lu-qun;WANG Dan;ZHOU Xing-chen;ZHANG Xiao-huan;MAO Yan-wen;WANG Yuan-yuan;GUO Bing(Department of Pathophysiology,School of Basic Medicine,Guizhou Medical University,Guizhou Provincial Key Laborato-ry of Pathogenetis&Drug Research on Common Chronic Disease,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学基础医学院病理生理教研室,贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州贵阳550025

出　　处：《中国病理生理杂志》2021年第11期1921-1932,共12页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82060141,No.81960141);贵州省科技创新平台计划(黔科中引地[2019]4008号)。

摘　　要：目的:用胰岛素(INS)控制1型糖尿病(DM)大鼠血糖,观察糖尿病肾病(DKD)发病过程中大鼠肾小管上皮细胞核转录因子Krüppel样因子6(KLF6)的表达和作用。方法:(1)将SD大鼠随机分为正常对照(NC)组、DM组和INS治疗组(INS组)。采用链脲佐菌素(STZ)复制1型DM大鼠模型,INS组于造模成功4周后给予INS(22 U·kg^(−1)·d^(−1))治疗6周。于造模后第10周处死所有大鼠,检测相应生化指标;HE和天狼星红染色观察肾组织病理学变化;免疫组织化学染色法检测肾组织KLF6的表达;Western blot检测大鼠肾组织中KLF6、过氧化物酶体增殖物活化受体γ(PPARγ)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、α-平滑肌肌动蛋白(α-SMA)、Ⅲ型胶原蛋白(ColⅢ)和Bax蛋白的表达水平;RT-qPCR检测PPARγ的mRNA表达变化。(2)将大鼠近端肾小管上皮NRK-52E细胞予以正常糖(NG)或高糖(HG)培养以及在HG环境中培养24 h后置于NG中培养24 h(HGtoNG),或转染KLF6 siRNA(si-KLF6)后HG培养及转染KLF6过表达质粒(OE-KLF6)后NG培养,分为NG组、HG组、HGtoNG组、NG+vector组、NG+OE-KLF6组和HG+si-KLF6组,共6组。Western blot检测KLF6、PPARγ、钙黏蛋白(E-cadherin)、纤维连接蛋白(FN)、IL-6和Bax表达的变化,流式细胞术检测细胞凋亡情况。结果:(1)与NC组相比,DM组大鼠肾重/体重、血糖、糖化血红蛋白、甘油三酯、血尿素氮和24 h尿蛋白明显升高,肾小管灶性萎缩,系膜及间质轻度节段增生,并伴有胶原的明显沉积,肾组织KLF6、IL-6、TNF-α、α-SMA、ColⅢ和Bax蛋白表达增多,KLF6阳性染色主要定位在肾小管胞质及胞核,而PPARγ的mRNA和蛋白表达水平均降低。与DM组相比,INS治疗后,上述生化指标均降低,肾组织病理形态学显示肾纤维化病变减轻,KLF6、IL-6、TNF-α、α-SMA、ColⅢ和Bax蛋白表达均有所降低,而PPARγ的mRNA和蛋白表达水平均升高。(2)过表达KLF6可使NRK-52E细胞中PPARγ和E-cadherin的表达降低,FN和IAIM:To observe the expression and effect of nuclear transcription factor Krüppel-like factor 6(KLF6)in rat renal tubular epithelial cells during the onset of diabetic nephropathy when insulin(INS)was given to control blood glucose in rats with type 1 diabetets mellitus(DM).METHODS:(1)The SD rats were randomly divided into nor-mal control(NC)group,DM group and INS treatment group(INS group).Streptozotocin(STZ)was used to replicate type 1 diabetic rat model.The rats in INS group were treated with INS at 22 U·kg^(−1)·d^(−1) for 6 weeks after modeling,and all the rats were sacrificed at the 10th week.The serum and urine were collected 24 h before the death to detect the biochemical in-dexes,and the pathological changes of renal tissues were observed by HE and Sirius red staining.The expression of KLF6 in kidney tissues was detected by immunohistochemistry,and the protein levels of KLF6,peroxisome proliferator-activated receptorγ(PPARγ),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),α-smooth muscle actin(α-SMA),collagen type Ⅲ(Col Ⅲ)and Bax in kidney tissues were detected by Western blot.RT-qPCR was used to detect the mRNA expres-sion of PPARγ.(2)Rat proximal renal tubular epithelial NRK-52E cells were cultured in vitro and divided into normal glu-cose(NG)group(cultured with NG),high glucose(HG)group(cultured with HG),HG to NG group(cultured in HG en-vironment for 24 h and then in NG environment for 24 h),NG+vector group(transfected with vector in NG environment),NG+KLF6-overexpressing plasmid(OE-KLF6)group(transfected with OE-KLF6 in NG environment)and HG+KLF6 siR-NA(si-KLF6)group(transfected with si-KLF6 in HG environment).The protein levels of KLF6,PPARγ,E-cadherin,fi-bronectin(FN),IL-6 and Bax were determined by Western blot.The apoptotic rate was determined by flow cytometry.RE⁃SULTS:(1)Compared with NC group,the kidney/body weight ratio,blood glucose,glycosylated hemoglobin,triglycer-ide,blood urea nitrogen and 24-h urine protein in DM group were significantly increased.Focal atrophy of

关 键 词：糖尿病肾病 胰岛素 炎症 Krüppel样因子6 过氧化物酶体增殖物活化受体Γ 

分 类 号：R587.2[医药卫生—内分泌] R363.2[医药卫生—内科学]