温阳活血利水方对被动型He...ann肾炎大鼠足细胞的影响_薛雪.pdf

中华中医药杂志(原中国医药学报)2023年2月第38卷第2期 CJTCMP,February 2023,Vol.38,No.2 603 温阳活血利水方对被动型Heymann肾炎大鼠 足细胞的影响薛雪1,2，吴健3，付彤飞3，谭瑶3，袁军1,2（1湖北中医药大学第一临床学院，武汉 430061；2湖北省中医院肾病科，武汉 430061；3湖北中医药大学中医临床学院，武汉 430061）摘要：目的：观察温阳活血利水方药对被动型Heymann肾炎（PHN）模型大鼠的干预作用，并探讨该方药的疗效机制。方法：雄性SD大鼠60只，随机分为正常对照组、PHN模型组、温阳活血利水组、他克莫司组，每组15只。以尾静脉注射Anti-Fx1A血清法构建IMN模型。检测各组大鼠24 h尿蛋白定量、血清总蛋白、血清白蛋白、谷丙转氨酶、谷草转氨酶、血肌酐、胆固醇及甘油三酯水平。采用免疫荧光法检测肾小球Klotho、瞬时受体电位通道6（TRPC6）、组织蛋白酶L（CatL）及其底物Synaptopodin表达；采用酶联免疫吸附（ELISA）法检测肾皮质Ras同源基因家族成员（Rho）A、Rho关联含卷曲螺旋蛋白激酶（ROCK）1与ROCK2的活性；采用Western blot方法检测肾皮质磷酸化LIM结构域激酶p-LIMK1、磷酸化后p-cofilin蛋白的表达。光镜下观察肾脏病理形态学变化，电子显微镜下观察肾小球足细胞足突及电子致密物变化情况。结果：与正常对照组比较，干预4周后PHN模型组24 h尿蛋白定量、血清胆固醇与甘油三酯水平显著升高（P0.01），血清白蛋白水平显著下降（P0.01）；肾小球Klotho及Synaptopodin的表达显著下调（P0.01），RhoA、ROCK1、ROCK2活性显著升高（P0.01），TRPC6、CatL、p-LIMK1/LIMK1及p-cofilin/cofilin表达显著上调（P0.01）；足细胞足突弥漫性融合，足细胞下电子致密物沉积。与PHN模型组比较，干预4周后温阳活血利水组血清白蛋白显著升高（P0.05），24 h尿蛋白定量与血清甘油三酯水平显著下降（P0.05，P0.01）；肾小球Klotho及Synaptopodin表达显著上调（P0.01），RhoA、ROCK1、ROCK2活性显著下降（P0.01），TRPC6、CatL、p-LIMK1/LIMK1与p-cofilin/cofilin表达显著下调（P0.01）；足突融合改善，足细胞下电子致密物沉积减轻。结论：Klotho/TRPC6/CatL/Synaptopodin/RhoA/ROCK/LIMK/cofilin信号通路表达异常可能参与了IMN的发病过程；温阳活血利水方药能够减少IMN大鼠蛋白尿，调整足细胞形态，其作用机制或与 调控该通路有关。关键词：温阳活血利水方；特发性膜性肾病；机制；足细胞基金资助：国家自然科学基金面上项目（No.82074364），武汉市2022年度知识创新专项基础研究项目（No.2022020801020506）Effects of Wenyang Huoxue Lishui recipe on podocytes in rats with passive Heymann nephritis XUE Xue1,2,WU Jian3,FU Tong-fei3,TAN Yao3,YUAN Jun1,2(1First Clinical College,Hubei University of Chinese Medicine,Wuhan 430061,China;2Department of Nephrology,Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan 430061,China;3Clinical College of Traditional Chinese Medicine,Hubei University of Chinese Medicine,Wuhan 430061,China)Abstract:Objective:To observe the interventional effect of Wenyang Huoxue Lishui recipe(WHLR)on passive heymann nephritis(PHN)model and to explore the therapeutic mechanism.Methods:Sixty male SD rats were randomly divided into normal control group,PHN model group,WHLR group,tacrolimus group,15 rats in each group.PHN rats were modeled 论著通信作者：袁军，湖北省武汉市武昌区花园山4号湖北省中医院肾病科，邮编：430061，电话：027-88929221E-mail：内文1.indd 6032023/2/10 16:57:23中华中医药杂志(原中国医药学报)2023年2月第38卷第2期 CJTCMP,February 2023,Vol.38,No.2 604 by injecting anti-Fx1A serum.The levels of 24 h urine total protein and serum total protein,albumin,alanine aminotransferase,aspartate aminotransferase,creatinine,cholesterol,triglyceride were detected in each group of rats.Immunofluorescence methods were used to detect the expression of glomerular Klotho,transient receptor potential channel 6(TRPC6),cathepsin L(CatL)and its substrate synaptopodin in each group of podocytes.Enzyme-linked immunosorbent assay(ELISA)was adopted to measure the activities of Ras homologgene family member(Rho)A and Rho-activated kinase(ROCK)1 and ROCK2 of the renal cortex.And Western blot method was used to detect the expression of phosphorylated-LIMK1(p-LIMK1),p-cofilin protein of the renal cortex.The pathological changes of rat kidney tissue were observed under ordinary light microscope,and the changes of glomerular podocyte foot processes and electron dense deposits were observed under electron microscope.Results:Corresponding drugs were given by gavage for 4 weeks,compared with normal control group,the level of 24 h urine total protein,serum cholesterol and triglycerides significantly increased(P0.01)as well as the level of serum albumin decreased(P0.01);expression of glomerular Klotho and Synaptopodin were down-regulated(P0.01)as well as expression of TRPC6,CatL,RhoA,ROCK1,ROCK2,p-LIMK1/LIMK1 and p-cofilin/cofilin were up-regulated in the PHN model group(P0.01).The effacement of the podocyte foot processes was diffuse,and electron dense deposits were deposited under the podocytes.Corresponding drugs were given by gavage for 4 weeks,compared with the PHN model group,the level of serum albumin significantly increased(P0.05)as well as the level of 24 h urine total protein,serum triglycerides decreased(P0.05,P0.01)in WHLR group;expression of glomerular Klotho and Synaptopodin were up-regulated(P0.01)as well as expression of TRPC6,CatL,RhoA,ROCK1,ROCK2,p-LIMK1/LIMK1 and p-cofilin/cofilin significantly were down-regulated in WHLR group(P10 mg为造模成功9。实验结束后随机选取造模大鼠和正常对照组大鼠各2只，予以包埋、固定，光镜下能够观察到免疫复合物在造模大鼠肾小球毛细血管袢上皮下沉积，电镜下可见电子致密物沉积于毛细血管袢上皮下，毛细血管不均匀增厚呈钉突样改变，基底膜增厚，足突融合，表示造模成功。4.干预与取材 正常对照组与PHN模型组给予蒸馏水6 mLkg-1d-1灌胃，温阳活血利水组给予 12 g/d中药制剂灌胃，他克莫司组给予1 mgkg-1d-1他克莫司混悬液灌胃。连续干预4周，每周检测24 h尿蛋白定量。干预结束后，1.5%戊巴比妥钠30 mg/kg麻醉大鼠，腹主动脉取血，分离出血清；剖腹取肾脏分离肾皮质，保存于液氮罐中。分离左肾切块后，分别送冰冻切片、石蜡切片与电镜检查。5.观察指标及检测方法5.1 一般情况 每周观测大鼠的体质量、尿量、饮水、摄食、毛发光泽及反应状态等。5.2 血生化指标检测 全自动生化分析仪检测各组大鼠血肌酐、总胆固醇、甘油三酯、血清总蛋白及白蛋白水平、血清谷草转氨酶及谷丙转氨酶水平；贝克曼Au5821全自动生化分析仪检测第0、1、2、3、4周各组大鼠24 h尿蛋白定量。5.3 Western blot检测肾皮质中磷酸化LIM结构域激酶1（phosphorylated-LIM domain kinase 1，p-LIMK1）、LIMK1、p-cofilin及cofilin蛋白 表达 蛋白含量按BCA蛋白浓度测定试剂盒测定。取总蛋白10 g经SDS-PAGE凝胶电泳后，转移至0.45 m PVDF膜上。含5%脱脂牛奶的TBST 4 封闭2 h，洗膜后加入一抗（p-LIMK1：1 750；LIMK1：11 000；p-cofilin：11 000；cofilin：1900；-actin：150 000）孵育过夜。再用1 3 000的辣根过氧化物酶标记的