益肾通络方调控TLR4/MyD88/NF-κB信号通路改善糖尿病肾病小鼠细胞焦亡的机制研究  

Mechanism of Yishen Tongluo Formula regulating the TLR4/MyD88/NF-κB signaling pathway to ameliorate pyroptosis in diabetic nephropathy mice

作　　者：张轶斐 曹梓静 张泽钰 白雪慧 唐靖怡 席俊羽 王嘉奕 谢亦冉 吴宇琪 郭玺 刘忠杰[1] 刘伟敬[1] ZHANG Yifei;CAO Zijing;ZHANG Zeyu;BAI Xuehui;TANG Jingyi;XI Junyu;WANG Jiayi;XIE Yiran;WU Yuqi;GUO Xi;LIU Zhongjie;LIU Weijing(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;Beijing Puren Hospital,Beijing 100062,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]北京普仁医院

出　　处：《北京中医药大学学报》2025年第1期21-33,共13页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.82374382,No.82074361,No.82274293);中央高校基本科研业务费专项资金资助项目揭榜挂帅项目(No.2023-JYB-JBZD-037);国家中医药管理局高水平医院临床研究和成果转化能力提升试点项目揭榜挂帅项目(No.DZMG-XZYY-23002);创新中药关键技术国家重点实验室开放课题(No.ZSL-003-02)。

摘　　要：目的研究益肾通络方通过调控Toll样受体4(TLR4)/髓系分化初级反应蛋白质88(MyD88)/核转录因子-κB(NF-κB)信号通路,改善糖尿病肾病小鼠肾脏细胞焦亡的机制。方法采用随机数字表法将60只C57BL/6雄性小鼠分为对照组(10只)和干预组(50只),干预组采用链脲佐菌素(STZ)腹腔注射(50 mg/kg)构建糖尿病肾病小鼠模型。造模成功后,采用随机数字表法将干预组进一步分为模型组、司美格鲁肽组(40μg/kg)和益肾通络方高(15.6 g/kg)、中(7.8 g/kg)、低(3.9 g/kg)剂量组。益肾通络方高、中、低剂量组灌胃相应剂量药物,司美格鲁肽组皮下注射司美格鲁肽注射液,对照组和模型组灌胃蒸馏水,连续12周。每4周监测各组小鼠随机血糖、生化法测定24 h尿蛋白含量;治疗后,采用生化法测定血清肌酐、尿素氮含量;称量小鼠肾脏质量,计算肾脏系数;苏木素-伊红、过碘酸-希夫、过碘酸六胺银和马松染色观察肾脏组织病理变化;酶联免疫吸附测定法检测尿液中β2微球蛋白(β2-MG)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤分子-1(KIM-1)水平;蛋白质印迹法和实时荧光PCR法检测肾脏组织中核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(NLRP3)、胱天蛋白酶-1(Caspase-1)、焦孔素D(GSDMD)、白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)蛋白和mRNA表达;免疫组织化学法检测肾脏组织中TLR4、MyD88、NF-κB阳性染色面积占比。结果与对照组比较,模型组小鼠随机血糖,24 h尿蛋白,血清肌酐、尿素氮含量及肾脏系数均升高,尿液β2-MG、NGAL、KIM-1水平升高,肾组织NLRP3、Caspase-1、GSDMD、IL-1β和IL-18蛋白及mRNA表达升高,TLR4、MyD88、NF-κB蛋白阳性染色面积占比增多(P<0.05),肾组织可见肾小球肥大等病理改变。与模型组比较,益肾通络方高剂量组治疗12周后随机血糖降低(P<0.05);益肾通络方高、中剂量组小鼠24 h尿蛋白,血Objective To investigate the mechanism of Yishen Tongluo Formula in ameliorating renal pyroptosis in diabetic nephropathy mice by regulating the toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB)signaling pathway.Methods Sixty C57BL/6 male mice were randomly divided into control(10 mice)and intervention groups(50 mice)using random number table method.The diabetes nephropathy model was established by intraperitoneally injecting streptozotocin(50 mg/kg).After modeling,the intervention group was further divided into model,semaglutide(40μg/kg),and high-,medium-,and low-dose Yishen Tongluo Formula groups(15.6,7.8,and 3.9 g/kg,respectively)using random number table method.The high-,medium-,and low-dose Yishen Tongluo Formula groups were administered corresponding doses of medication by gavage,the semaglutide group received a subcutaneous injection of semaglutide injection,and the control group and model groups were administered distilled water by gavage for 12 consecutive weeks.Random blood glucose levels of mice in each group were monitored,and the 24-h urinary protein content was measured using biochemical method every 4 weeks;after treatment,the serum creatinine and urea nitrogen levels were measured using biochemical method.The weight of the kidneys was measured,and the renal index was calculated.Hematoxylin and eosin,periodic acid-Schiff,periodic Schiff-methenamine,and Masson staining were used to observe the pathological changes in renal tissue.An enzyme-linked immunosorbent assay was used to detect urinary β2-microglobulin(β2-MG),neutrophil gelatinase-associated lipocalin(NGAL),and kidney injury molecule-1(KIM-1)levels.Western blotting and real-time fluorescence PCR were used to detect the relative protein and mRNA expression levels of nucleotide-binding domain leucinerich repeat and pyrin domain-containing receptor 3(NLRP3),Caspase-1,gasdermin D(GSDMD),interleukin-1β(IL-1β),and interleukin-18(IL-18)in renal tissue.Immunohistochemistry was used to detect the propo

关 键 词：糖尿病肾病 益肾通络方 细胞焦亡 Toll样受体4/髓样分化因子88/核转录因子-κB信号通路 小鼠 

分 类 号：R285.5[医药卫生—中药学]