基于NOX4/ROS/p38信号通路探讨三焦祛湿方对膜性肾病小鼠的抗氧化应激作用  

Mechanism of Sanjiao Qushi Prescription on anti-oxidative sress of mice with membranous nephropathy based on NOX4/ROS/p38 signaling pathway

作　　者：赵亚云 方敬[1] 刘海平[1,3] 陈静洁 杨帆 陈志强[2] ZHAO Ya-yun;FANG Jing;LIU Hai-ping;CHEN Jing-jie;YANG Fan;CHEN Zhi-qiang(Hebei University of Chinese Medicine,Shijiazhuang 050091,China;Hebei Hospital of Traditional Chinese Medicine,Shijiazhuang 050011,China;Hebei Key Laboratory of Integrative Medicine of Liver-Kidney Patterns,Hebei University of Chinese Medicine,Shijiazhuang050091,China)

机构地区：[1]河北中医学院,河北石家庄050091 [2]河北省中医院,河北石家庄050011 [3]河北中医学院河北省中西医结合肝肾病证研究重点实验室,河北石家庄050091

出　　处：《中草药》2022年第4期1076-1083,共8页Chinese Traditional and Herbal Drugs

基　　金：河北省政府资助省级临床医学优秀人才项目(2016034829);河北省中医药管理局科研计划项目(2021039);河北省研究生创新资助项目(XCXZZBS2021021)。

摘　　要：目的研究三焦祛湿方对阳离子化牛血清白蛋白(cationic bovine serum albumin,C-BSA)诱导的膜性肾病(membranous nephropathy,MN)小鼠的作用及其对烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphate oxidase 4,NOX4)/活性氧(reactive oxygen species,ROS)/p38信号通路的影响,以阐明其抗氧化应激的作用机制。方法将60只小鼠随机分为对照组10只和造模组50只,造模组尾iv 6.5 mg/kg C-BSA建立MN小鼠模型,随机分为模型组及三焦祛湿方低、高剂量(3.71、7.42 g/kg)组和盐酸贝那普利(1.3 mg/kg)组,各给药组ig相应药物干预4周,检测各组小鼠24 h尿蛋白;腹主动脉取血,检测血清丙二醛(malondialdehyde,MDA)水平和超氧化物歧化酶(superoxide dismutase,SOD)活性;取肾组织进行苏木素-伊红(HE)、Masson、过碘酸-六胺银(PASM)染色及透射电镜(TEM),观察肾组织病理形态变化,采用免疫荧光法检测肾组织免疫球蛋白G(immunoglobulin G,IgG)沉积和ROS表达情况;采用Western blotting法检测肾组织NOX4、p38和磷酸化p38(p-p38)蛋白表达情况。结果与对照组相比,模型组小鼠24 h尿蛋白显著升高(P<0.01);血清SOD活性显著降低,MDA水平显著升高(P<0.01);肾脏病理改变明显,可见肾小球体积增大,基底膜增厚,嗜复红蛋白沉积,“钉突”形成,上皮下可见电子致密物沉积,足突融合,IgG沿毛细血管襻颗粒状沉积;肾组织ROS表达显著增多(P<0.01),NOX4、p-p38蛋白表达水平显著升高(P<0.01)。与模型组相比,各给药组小鼠24 h尿蛋白显著降低(P<0.01);血清SOD活性明显增强,MDA水平显著降低(P<0.05、0.01);肾组织病理损害明显改善;肾组织ROS表达显著减少(P<0.01),NOX4和p-p38蛋白表达水平显著降低(P<0.05、0.01)。结论三焦祛湿方能够减少尿蛋白,减轻肾脏病理损伤,缓解肾组织氧化应激,其作用机制可能与调控NOX4/ROS/p38信号通路有关。Objective To study the effects of Sanjiao Qushi Prescription(三焦祛湿方)on cationic bovine serum albumin(C-BSA)induced membranous nephropathy(MN)mice model and nicotinamide adenine dinucleotide phosphatase oxidase4(NOX4)/reactive oxygen species(ROS)/p38 signaling pathway,and elucidate the mechanism of anti-oxidative stress.Methods Sixty mice were randomly divided into control group(n=10)and model group(n=50),mice were iv C-BSA(6.5 mg/kg)by tail to establish MN model,which were randomly divided into model group,low-,high-dose Sanjiao Qushi Prescription(3.71,7.42 g/kg)groups and benazepril hydrochloride(1.3 mg/kg)group.Mice were ig corresponding drugs for four weeks,24 h urine protein were detected.Serum was collected,superoxide dismutase(SOD)activity and malondialdehyde(MDA)level were measured.Renal tissue was collected,pathological morphology changes were observed by light microscope with hematoxylineosin(HE),Masson and periodic acid-silver metheramine(PASM)staining and transmission electron microscope(TEM).Deposition of immunoglobulin G(IgG)and ROS expression in renal tissue was observed by fluorescence immunoassay.Western blotting was used to detect NOX4,p38 and p-p38 protein expressions in renal tissue.Results Compared with control group,24 h urine protein of mice in model group was significantly increased(P<0.01),SOD activity was decreased and MDA level was increased(P<0.01)in model group;Kidney exhibited enlarged volume of glomerular,significantly thickened glomerular basement membrane,polymyoglobin deposition and spike formation from the light microscope,epithelium deposition of electron dense,foot process fusion were observed from transmission electron microscope;Granular deposition of IgG along the capillary wall was observed by immunofluorescence staining;ROS expression in kidney was significantly increased(P<0.01);NOX4 and p-p38 protein expressions were significantly increased(P<0.01).Compared with model group,24 h urine protein in each administration group was significantly decreased(P<0.01),SOD a

关 键 词：三焦祛湿方 膜性肾病 氧化应激 烟酰胺腺嘌呤二核苷酸磷酸氧化酶4 活性氧 p38 

分 类 号：R285.5[医药卫生—中药学]