糖肾康调控NLRP3-Caspase-1-IL-18/IL-1β轴改善糖尿病肾病足细胞焦亡的机制研究  

Study on themechanism of Tangshenkang regulating NLRP3-Caspase-1-IL-18/IL-1β axis to improve podocyte pyroptosis in diabetic kidney disease

作　　者：周乐[1] 韩聪 高冉冉 鲁容辰 高逸爽 鲍孝云 王一川[1] 李伟[1] ZHOU Le;HAN Cong;GAO Ranran;LU Rongchen;GAO Yishuang;BAO Xiaoyun;WANG Yichuan;LI Wei(Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,China)

机构地区：[1]山东中医药大学附属医院肾内二科,济南250014 [2]山东中医药大学中医学院 [3]山东中医药大学第一临床医学院

出　　处：《环球中医药》2024年第12期2409-2417,共9页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(82174179);山东省自然科学基金(ZR2021LZY041);山东省中医药科技项目(2020Q012);济南市科技计划项目(201907107);中国医师协会循证专业委员会肾科学组“PRO·润”基金;山东省医药卫生科技发展计划项目(202103050260)。

摘　　要：目的基于肾虚湿瘀理论探讨以益肾活血清泄为原则组方制备的糖肾康颗粒通过调控NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)-胱天蛋白酶-1(Caspase-1)-白介素(interleukin,IL)-18/IL-1β轴改善糖尿病肾病足细胞损伤的机制。方法动物实验:将18只db/db小鼠随机分为模型组、缬沙坦组、糖肾康组,每组6只。6只db/m小鼠作为对照组。糖肾康组给予1.17 g/(kg·d)灌胃,缬沙坦组予缬沙坦悬液10.4 mg/(kg·d)灌胃,对照组及模型组予等量生理盐水灌胃。疗程8周,观察db/db小鼠一般情况及糖代谢情况,通过肾脏组织苏木素—伊红、马松与过碘酸雪夫氏染色及透射电镜观察肾脏病理变化,通过生化分析或酶联免疫吸附测定法检测肾损伤指标包括尿白蛋白/肌酐、血肌酐、尿素氮、胱抑素C、β2微球蛋白、N-乙酰-β-D-氨基葡萄糖苷酶,Western blot及免疫荧光检测db/db小鼠肾脏肾病蛋白Nephrin表达情况,以及细胞焦亡炎性因子NLRP3、Caspase-1、IL-18及IL-1β的表达变化。细胞实验:采用30 mmol/L葡萄糖培养基诱导小鼠足细胞48小时建立足细胞损伤模型。制备糖肾康大鼠含药血清,采用噻唑蓝法筛选出糖肾康含药血清的最佳作用浓度。采用流式细胞仪检测糖肾康对高糖诱导的足细胞凋亡率的影响,收集各组细胞培养上清液,检测乳酸脱氢酶水平,Western blot法观察NLRP3、Caspase-1,酶联免疫吸附测定法检测IL-18及IL-1β的表达变化。结果糖肾康可改善db/db小鼠糖代谢,与模型组比较,糖肾康组及缬沙坦组的尿白蛋白/肌酐均明显降低(P<0.01),亦改善了N-乙酰-β-D-氨基葡萄糖苷酶(P<0.01)、β2微球蛋白(P<0.05,P<0.01)、血肌酐(P<0.01)、尿素氮(P<0.01)及胱抑素C(P<0.01)等肾损伤指标;糖肾康组Nephrin的表达明显增加(P<0.01),且优于缬沙坦组(P<0.05)。糖肾康可明显降低小鼠肾组织细胞焦亡炎性因子NLRP3(P<0.05)、Caspase-1(P<0.01)、IL-18(P<0.01)及IL-1β(P<0Objective To explore the mechanism of Tangshenkang regulating NLRP3-Caspase-1-IL-18/IL-1β axis to improve podocyte pyroptosis in diabetic kidney disease based on the theory of kidney deficiency,dampness and stasis.Methods 18 db/db mice were randomly divided into model group,valsartan group,and Tangshenkang group,with 6 mice in each group.Six db/m mice were used as the control group.The Tangshenkang group was given 1.17 g/(kg·d)by gavage,the valsartan group was given 10.4 mg/(kg·d)of valsartan suspension by gavage,and the control group and the model group were given equal amounts of physiological saline.After 8 weeks of treatment,the general condition and glucose metabolism of db/db mice were monitored,the pathological changes of the kidney tissues were observed through the methods of hematoxylin eosin(HE),masson,periodate Schiff reaction(PAS)and transmission electron microscopy.Renal injury indicators such as urinary albumin/creatinine(UACR),blood creatinine(Scr),urea nitrogen(BUN),cystatin C(CysC),β2 microglobulin(β2-MG),N-Acetyl-β-D-glucosaminidase(NAG)were detected through biochemical analysis.The methods of ELISA,Western blot and immunofluorescence were used to detect the expression of Nephrin,as well as the pyroptotic inflammatory factors nod like receptor protein 3(NLRP3),aspartate proteolytic enzyme-1(Caspase-1),interleukin-18(IL-18)and IL-1βchanges in the kidneys of db/db mice.Cell experiment:Mouse podocyte(MPC5)cells were induced by 30 mmol/L glucose medium for 48 hours to establish the podocyte injury model.The optimal concentration of Tangshenkang drug-containing serum was selected by MTT method.The effect of Tangshenkang on the apoptosis rate of podocytes induced by high sugar was detected by flow cytometry.The cell culture supernatant in each group was collected to detect the levels of lactate dehydrogenase(LDH).NLRP3 and Caspase-1 were detected by Western blot,and the expressions of IL-18 and IL-1β were detected by ELISA.Results Tangshenkang can improve the general condition and glucose meta

关 键 词：糖尿病肾病 糖肾康颗粒 足细胞焦亡 肾虚湿瘀 益肾活血清泄法 

分 类 号：R285.5[医药卫生—中药学]