滋肾丸对糖尿病肾病小鼠肾小管上皮细胞焦亡及上皮-间充质转化的影响  

Effect of Zishenwan on Pyroptosis and Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells in Diabetic Nephropathy Mice

作　　者：郭晓媛[1] 张程斐 吴悠 吴丽丽[3] 秦灵灵[4] 刘铜华[3] GUO Xiao-yuan;ZHANG Cheng-fei;WU You;WU Li-li;QIN Ling-ling;LIU Tong-hua(Dongfang Hospital Affiliated to Beijing University of Chinese Medicine,Beijing 100078,China;Second Clinical Medical College Beijing University of Chinese Medicine,Beijing 100078,China;Key Laboratory of Health Cultivation of the Ministry of Education,Beijing University of Chinese Medicine,Beijing 100029,China;Technology Department,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学东方医院,北京100078 [2]北京中医药大学第二临床医学院,北京100078 [3]北京中医药大学教育部中医养生学重点实验室,北京100029 [4]北京中医药大学,北京100029

出　　处：《中国实验方剂学杂志》2021年第21期27-36,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医药管理局中医药防治慢性病国际合作项目(GZYYGJ2019034)。

摘　　要：目的:研究滋肾丸(ZSW)对糖尿病肾病(DN)小鼠肾小管上皮细胞焦亡及上皮-间充质转化(EMT)的作用及机制,为ZSW治疗DN提供科学依据。方法:自发性糖尿病db/db小鼠随机分为模型组、达格列净组(1.0 mg·kg^(-1))及ZSW高、中、低剂量(6.0,3.0,1.5 g·kg^(-1))组,非糖尿病db/m小鼠为正常组。模型组和正常组给予等体积去离子水灌胃,其余各组使用相应药物干预12周。每2周检测尾静脉空腹血糖(FBG);每4周检测尿白蛋白/肌酐(ACR),N-乙酰β-D-氨基葡萄糖苷酶(NAG)和胱抑素C(CysC);给药12周后摘眼球取血,血清检测尿素氮(BUN)和肌酐(SCr);光镜及投射电镜观察肾组织病理学改变;免疫组化法观察分析肾小管上皮EMT标志物表达强度;原位末端标记法(TUNEL)染色观察分析肾小管上皮细胞的核损伤水平;蛋白免疫印迹法(Western blot)检测EMT标志物、核苷酸寡聚化结构域样受体蛋白3(NLRP3)炎症小体相关蛋白及焦亡相关炎症因子的蛋白表达水平。实时荧光定量聚合酶链式反应(Real-time PCR)检测上述蛋白的mRNA表达水平。结果:与正常组比较,模型组FBG,BUN,血SCr及尿ACR,NAG酶,CysC显著升高,肾组织出现病理损伤,肾小管上皮细胞EMT标志物表达强度、蛋白及mRNA表达显著改变,肾小管上皮细胞TUNEL染色阳性率、肾组织焦亡相关炎症因子和NLRP3炎症小体相关蛋白及mRNA表达显著升高(P<0.01);与模型组比较,ZSW及达格列净组FBG,BUN,血SCr及尿ACR,NAG酶,CysC显著降低(P<0.01),肾组织病理损伤不同程度减轻,肾小管上皮细胞EMT标志物表达强度显著改变(P<0.01),蛋白及mRNA表达不同程度改变(P<0.05,P<0.01),肾小管上皮细胞TUNEL染色阳性率及肾组织焦亡相关炎症因子的蛋白及mRNA表达显著降低(P<0.01),NLRP3炎症小体相关蛋白及mRNA表达不同程度降低(P<0.05,P<0.01)。结论:ZSW抑制肾小管上皮细胞焦亡及EMT,可能是其治疗DN的作用机制。Objective:To study the efficacy and mechanism of Zishenwan(ZSW)against pyroptosis and epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells in diabetic nephropathy(DN)mice,so as to provide evidence for the treatment of DN with ZSW.Method:The db/db mice with spontaneous diabetes were randomly divided into the model group,dapagliflozin(1.0 mg·kg^(-1))group,and high-,medium-,and low-dose(6.0,3.0,1.5 g·kg^(-1))ZSW groups.The non-diabetic db/m mice were classified into the normal group.The ones in the model and normal groups were given an equal volume of deionized water by gavage,while those in the other groups were intervened with the corresponding drugs for 12 weeks.The fasting blood glucose(FBG)level was tested at tail vein once every two weeks.The levels of urine albumin-creatinine ratio(ACR),β-N-acetyl-D-glucosaminidase(NAG),and cystatin C(CysC)were detected once every four weeks.After 12 weeks of administration,the blood sampled from eyeballs was used for measuring the blood urea nitrogen(BUN)and serum creatinine(SCr).The pathological changes in renal tissues were observed by light microscopy and transmission electron microscopy.The expression of EMT markers in the renal tubular epithelium was analyzed by immunohistochemistry(IHC).The in situ terminal end-labeling(TUNEL)staining was conducted to analyze the nuclear damage of renal tubular epithelial cells.The protein and mRNA expression levels of EMT markers,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and pyroptosis-related inflammatory cytokines in renal tissues were separately assayed by Western blot and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Result:Compared with the normal group,the model group displayed significantly increased FBG,BUN,serum SCr,ACR,NAG,and CysC(P<0.01),impaired renal tissues,altered EMT marker expression intensities and levels(P<0.01),and elevated TUNEL-positive rate and protein and mRNA expression levels of pyroptosis-related inflammatory cyt

关 键 词：滋肾丸 糖尿病肾病 焦亡 上皮-间充质转分化 核苷酸寡聚化结构域样受体蛋白3(NLRP3)炎症小体 

分 类 号：R2-0[医药卫生—中医学] R22