潜阳育阴颗粒调控NR3C2/ROS/ERK信号通路抑制醛固酮诱导足细胞损伤的机制  

Mechanism of Qianyang Yuyin Granules Regulating NR3C2/ROS/ERK Pathway to Alleviate Aldosterone-induced Podocyte Injury

作　　者：李茵 袁方 许骏尧 宁澄[1] 王艺璇 钱丽超 李海涛[2] 李婕 LI Yin;YUAN Fang;XU Junyao;NING Cheng;WANG Yixuan;QIAN Lichao;LI Haitao;LI Jie(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine,Nanjing 210022,China)

机构地区：[1]南京中医药大学附属医院,南京210029 [2]南京中医药大学药学院,南京210023 [3]南京中医药大学附属南京中医院,南京210022

出　　处：《中国实验方剂学杂志》2024年第13期95-105,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年基金项目(81904113);江苏省自然科学基金青年基金项目(BK20181095);江苏省中医院第三批高峰学术人才培养对象项目(y2021rc33);江苏省第六期333高层次人才培养工程项目;2022年度江苏省科协青年科技人才托举工程项目(JSTJ2022-026);南京中医药大学自然科学基金项目(XZR2021050);国家中医临床研究基地开放课题项目(JD2023SZ16);2021年度南京中医药大学自然科学基金项目(XZR2020002);2022年度省中医药科技发展计划项目(MS202227);江苏省中医药科技发展计划项目-青年人才项目(QN202310)。

摘　　要：目的:探讨潜阳育阴颗粒对醛固酮干预的小鼠足细胞的保护机制。方法:将30只C57BL/6J小鼠随机分为5组:正常组、模型组、潜阳育阴颗粒低、高剂量组、螺内酯组,每组6只;除正常组外其余小鼠均植入微型渗透泵持续注入醛固酮(300μg·kg^(-1)·d^(-1)),诱导小鼠肾损伤,造模后给药组分别给予潜阳育阴颗粒低剂量、高剂量(2.6、5.2 g·kg^(-1)·d^(-1)),螺内酯(18 mg·kg^(-1)·d^(-1))连续干预28 d。苏木素-伊红(HE)染色及马松(Masson)染色观察小鼠肾脏病理改变;蛋白免疫印迹法(Western blot)检测小鼠肾脏组织足细胞裂孔膜结构蛋白Nephrin、足细胞丝结蛋白Desmin、B细胞淋巴瘤-2(Bcl-2)、B细胞淋巴瘤-2相关X蛋白(Bax)、剪切的胱天蛋白酶-3(cleaved Caspase-3)、核受体亚家族3C组成员2(NR3C2)、细胞外调节蛋白激酶(ERK)、磷酸化细胞外调节蛋白激酶(p-ERK)的表达水平;原位末端标记法(TUNEL)染色观察小鼠肾脏组织的凋亡水平;检测小鼠肾脏组织超氧化物歧化酶(SOD)水平;体外研究分为5组:正常组、模型组(醛固酮浓度200 nmol·L^(-1))、潜阳育阴颗粒低、中、高质量浓度组(25、50、100 mg·L^(-1))。细胞增殖与活性检测(CCK-8)法检测不同浓度潜阳育阴颗粒对醛固酮诱导的足细胞相对活力的影响;Western blot检测足细胞中Nephrin、Desmin、Bax、Bcl-2、cleaved Caspase-3、NR3C2、p-ERK/ERK表达水平;流式细胞术结合异硫氰酸荧光素(Annexin V-FITC)/碘化丙啶(PI)检测足细胞的凋亡水平;2,7-二氯二氢荧光素二乙酸酯荧光探针法(DCFH-DA)观察足细胞的活性氧(ROS)水平。结果:与正常组比较,模型组小鼠肾脏结构病变、出现纤维化情况,凋亡程度升高(P<0.01),SOD水平显著下降(P<0.01);醛固酮浓度在200 nmol·L^(-1)时足细胞活性明显下降(P<0.05),模型组足细胞形态病变、细胞间微丝结构排列错乱,凋亡水平升高(P<0.01),细胞内ROS水平显著升高(P<0.01),小鼠肾脏组织�Objective:To investigate the protective mechanism of Qianyang Yuyin granules(QYYY)on aldosterone-induced podocyte injury.Method:A total of 30 C57BL/6J mice were randomly divided into five groups:control group,model group,QYYY low dose(QYYY-L)group,QYYY high dose(QYYY-H)group,and spironolactone(SPL)group,with six mice in each group.Except for the control group,mice were implanted with osmotic minipumps and injected continuously with aldosterone(300μg·kg^(-1)·d^(-1))to induce renal injury.The drug administration group was given low and high doses(2.6,5.2 g·kg^(-1)·d^(-1))of QYYY and SPL(18 mg·kg^(-1)·d^(-1))for 28 days.The renal pathological changes of mice were observed by hematoxylin-eosin(HE)staining and Masson staining.The expression levels of Nephrin,Desmin,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X(Bax),cleaved Caspase-3,nuclear receptor subfamily 3 group C member 2(NR3C2),extracellular regulated protein kinases(ERK),and phospho-ERK(p-ERK)in kidney tissue were detected by Western blot.The apoptosis levels of kidney tissue were detected by TdT-mediated dUTP nick and labeling(TUNEL)staining,and the superoxide dismutase(SOD)levels were detected.In vitro,the mice were divided into five groups:Control group,model group(aldosterone concentration of 200 nmol·L^(-1)),QYYY-L group,QYYY medium dose(QYYY-M)group,and QYYY-H group(25,50,and 100 mg·L^(-1)).The effect of different concentrations of QYYY on the relative viability of aldosterone-induced podocytes was detected by cell proliferation and viability assay(CCK-8).The expressions of Nephrin,Desmin,Bax,Bcl-2,cleaved Caspase-3,NR3C2,and p-ERK/ERK were detected by Western blot.AnnexinV-FITC/PI flow cytometry was used to detect the apoptosis levels of podocytes.Reactive oxygen species(ROS)in podocytes were observed by DCFHDA.Result:Compared with the control group,the model group showed structural pathological changes and fibrotic conditions in the kidney,increased apoptosis levels(P<0.01),and decreased SOD levels(P<0.01).Aldosterone concentration at 200 nmol·

关 键 词：潜阳育阴颗粒 足细胞 醛固酮 肾损害 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33