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

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),小鼠肾脏组织及足细胞中Nephrin、Bcl-2、p-ERK/ERK蛋白表达明显下降(P<0.05,P<0.01),Desmin、Bax、cleaved Caspase-3、NR3C2蛋白表达明显上升(P<0.05,P<0.01);与模型组比较,潜阳育阴颗粒各剂量改善小鼠肾脏结构损伤和纤维化情况,减轻小鼠肾脏凋亡水平(P<0.05,P<0.01),提高肾脏SOD含量(P<0.05,P<0.001),改善足细胞活性(P<0.05,P<0.01),恢复足细胞足突结构,改善足细胞凋亡(P<0.01),下调足细胞ROS水平(P<0.01),上调小鼠肾脏组织和足细胞中Nephrin、Bcl-2、p-ERK/ERK蛋白表达(P<0.05,P<0.01),下调Desmin、Bax、cleaved Caspase-3、NR3C2蛋白表达(P<0.05,P<0.01)。结论:潜阳育阴颗粒通过调节NR3C2/ROS/ERK信号通路改善醛固酮诱导的小鼠足细胞损伤。

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·L^(-1)showed a significant decrease in podocyte activity(P<0.05).Podocytes in the model group showed structural pathological changes,disordered arrangement of intercellular microfilaments,increased apoptosis levels(P<0.01),and increased intracellular ROS levels(P<0.01).The protein expressions of Nephrin,Bcl-2,and p-ERK/ERK in kidney tissue and podocytes were decreased(P<0.05,P<0.01).The protein expressions of Desmin,Bax,cleaved Caspase-3,and NR3C2 were increased(P<0.05,P<0.01).Compared with the model group,QYYY alleviated the structural damage and fibrosis of the kidney,decreased the apoptosis levels(P<0.05,P<0.01),and enhanced the SOD content of the kidney(P<0.05,P<0.01).QYYY improved the activity of podocytes(P<0.05,P<0.01),restored the foot process structure of podocytes,and decreased apoptosis levels(P<0.01)and ROS levels of podocytes(P<0.01).The protein expressions of Nephrin,Bcl-2,and p-ERK/ERK in kidney tissue and podocytes were increased(P<0.05,P<0.01),and the protein expressions of Desmin,Bax,cleaved Caspase-3,and NR3C2 were downregulated(P<0.05,P<0.01).Conclusion:QYYY improves aldosterone-induced podocyte injury by regulating the NR3C2/ROS/ERK pathway.