基于cAMP/AQP2/NLRP3通路探讨济生肾气丸对慢性肾脏病模型大鼠的影响  

Jisheng Shenqi Pills Treat Chronic Kidney Disease in Rats via cAMP/AQP2/NLRP3 Pathway

作　　者：崔言坤 王琳 黄艳美 陈丽梅[3] 刘春花[1] 郭怡 姜劼琳 左铮云[2] 刘超[3] 姚凤云[1,2] CUI Yankun;WANG Lin;HUANG Yanmei;CHEN Limei;LIU Chunhua;GUO Yi;JIANG Jielin;ZUO Zhengyun;LIU Chao;YAO Fengyun(College of Traditional Chinese Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Formula-Pattern Research Center,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China)

机构地区：[1]江西中医药大学中医学院,南昌330004 [2]江西中医药大学方-证研究中心,南昌330004 [3]江西中医药大学附属医院,南昌330006

出　　处：《中国实验方剂学杂志》2024年第16期52-59,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：江西省教育厅科学技术研究项目(GJJ201258);国家自然科学基金项目(82360812);江西省自然科学基金项目(20212BA216007);江西省中医药管理局科技计划项目(2022A353);江西中医药大学重点学科青年教师培养计划项目(2021jzzdxk007)。

摘　　要：目的:通过观察济生肾气丸对慢性肾脏病(CKD)模型大鼠环磷酸腺苷(cAMP)/水通道蛋白2(AQP2)/NOD样受体蛋白3(NLRP3)信号通路及其相关炎性因子的影响,探讨其改善慢性肾脏病大鼠的作用机制。方法:将60只SPF级SD大鼠随机分为空白组(15只)和造模组(45只),通过腺嘌呤灌胃28 d诱导建立CKD大鼠模型,将造模组随机分为模型组和济生肾气丸高、中、低剂量组(1.2、0.6、0.3 g·kg^(-1)·d^(-1)),空白组和模型组灌胃等体积纯净水,连续给药28 d。在给药的第14、28天收集大鼠24 h尿量,检测大鼠24 h尿蛋白含量。经末次给药后,大鼠称体质量麻醉取材,采用生化法检测大鼠血清肌酐(SCr)、尿素氮(BUN)的含量。酶联免疫吸附测定法(ELISA)检测肾组织AQP2、cAMP、精氨酸加压素(AVP)和白细胞介素-1β(IL-1β)的含量。蛋白免疫印迹法(Western blot)检测肾组织NLRP3、AQP2蛋白表达水平。苏木素-伊红(HE)染色、马松(Masson)染色及过碘酸-雪夫染色(PAS)观察大鼠肾组织病理变化。结果:与空白组比较,模型组大鼠体质量、24 h尿量显著下降,24 h尿蛋白、肾脏湿重及肾体比显著升高(P<0.01),血清SCr、BUN和肾组织IL-1β含量明显升高(P<0.05,P<0.01),肾组织AVP、cAMP、AQP2含量显著降低(P<0.01),肾组织NLRP3蛋白表达水平显著升高(P<0.01),AQP2蛋白表达水平显著降低(P<0.01),肾脏组织病理损伤严重;与模型组比较,济生肾气丸组明显改善大鼠精神状况,体质量、24 h尿量显著增加,24 h尿蛋白含量、肾脏湿重及肾体比明显下降(P<0.05,P<0.01),血清SCr、BUN水平明显降低(P<0.05,P<0.01),肾组织AVP、cAMP、AQP2含量显著升高(P<0.01),肾组织IL-1β和NLRP3蛋白表达水平明显降低(P<0.05,P<0.01),AQP2蛋白表达水平显著升高(P<0.01),肾脏组织病理损伤得到明显改善。结论:济生肾气丸可通过调控cAMP/AQP2/NLRP3信号通路减少炎性因子释放,改善肾脏功能和病理损伤,发挥防治慢性肾脏病Objective:To explore the mechanism of Jisheng Shenqi pills in ameliorating chronic kidney disease(CKD)by observing the effects of this formula on the cyclic adenosine monophosphate(cAMP)/aquaporin 2(AQP2)/NOD-like receptor protein 3(NLRP3)signaling pathway and related inflammatory mediators in the rat model of CKD.Method:Sixty SPF-grade male SD rats were randomized into a blank group(n=15)and a modeling group(n=45).The rat model of CKD was established by gavage with adenine for 28 days.The modeled rats were randomized into a model group and high-,medium-,and low-dose(1.2,0.6,0.3 g·kg^(-1)·d^(-1),respectively)Jisheng Shenqi pills groups.Rats were administrated with corresponding drugs or pure water(blank group and model group)by gavage for 28 days.On days 14 and 28,the 24 h urine output was collected from rats for measurement of the 24 h urine protein.After the last administration,rats were anesthetized and samples were collected.The biochemical methods were employed to determine the serum levels of creatinine(SCr)and blood urea nitrogen(BUN)in rats.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of AQP2,arginine vasopressin(AVP),cAMP,and interleukin-1β(IL-1β)in the renal tissue.Western blot was employed to determine the expression levels of NLRP3 and AQP2 in the renal tissue.Hematoxylin-eosin staining,Masson staining,and periodic acid-Schiff staining(PAS)were employed to observe the pathological changes in the renal tissue.Result:Compared with the blank group,the model group showcased decreased body weight and 24 h urine output,increased 24 h urine protein,kidney wet weight,and kidney-to-body weight ratio(P<0.01),elevated levels of SCr and BUN in the serum and IL-1βin the renal tissue(P<0.05,P<0.01),lowered levels of AVP,cAMP,and AQP2 in the renal tissue(P<0.01),up-regulated expression of NLRP3(P<0.01),and down-regulated expression of AQP2(P<0.01)in the renal tissue.In addition,the modeling led to severe pathological damage to the renal tissue.Compared with the model group,Jisheng Shen

关 键 词：济生肾气丸 慢性肾脏病 环磷酸腺苷(cAMP) 水通道蛋白2(AQP2) NOD样受体蛋白3(NLRP3) 

分 类 号：R2-0[医药卫生—中医学] R33R289R692