柴黄益肾颗粒对单侧输尿管梗阻小鼠肾纤维化的作用及其机制  

Effects of Chaihuang Yishen Granule on renal fibrosis in unilateral ureteral obstruction mice and its mechanisms

作　　者：谢柯欢 张浩军[2] 谭睿陟 粟宏伟 刘鹏 李平[2] 王丽 Xie Ke-Huan;Zhang Hao-Jun;Tan Rui-Zhi;Su Hong-Wei;Liu Peng;Li Ping;Wang Li(Center of Integrated Traditional Chinese and Western Medicine Research,Affiliated Chinese Medicine Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China;Clinical Medical Research Institute,China-Japan Friendship Hospital,Beijing 100029,China;Department of Urology Surgery,Affiliated Chinese Medicine Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China;Department of Nephrology,Shunyi Hospital,Beijing Traditional Chinese Medicine Hospital,Beijing 101320,China)

机构地区：[1]西南医科大学附属中医医院中西结合研究中心,四川泸州646000 [2]北京中日友好医院临床医学研究所,北京100029 [3]西南医科大学附属中医医院泌尿外科,四川泸州646000 [4]北京顺义区中医医院肾病科,北京101320

出　　处：《解放军医学杂志》2024年第7期804-813,共10页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(82274489,81904174);四川省科技计划资助项目(2022YFS0621)。

摘　　要：目的探讨柴黄益肾颗粒对单侧输尿管梗阻(UUO)小鼠肾纤维化的作用及其机制。方法将24只8周龄C57BL/6雄性小鼠随机分为对照组、模型组及柴黄益肾颗粒低、高剂量组(n=6)。对照组仅暴露且游离右侧肾输尿管;模型组行UUO建立UUO动物模型;柴黄益肾颗粒低、高剂量组按模型组方法建立UUO小鼠模型并分别予以3.8、7.6 g/kg柴黄益肾颗粒灌胃,7 d后麻醉处死小鼠收集肾标本。采用HE、Masson染色观察肾脏病理变化及纤维化程度,天狼星红染色观察胶原沉积情况。采用Western blotting检测α-平滑肌肌动蛋白(α-SMA)、纤连蛋白(FN)、Ⅰ型胶原蛋白(Col-Ⅰ)、糖原合成酶激酶-3β(GSK-3β)、β-连环蛋白(β-catenin)相关蛋白的表达水平。采用RT-PCR检测A33及GSK-3β、β-catenin mRNA水平的变化。同时,以正常小鼠肾小管上皮细胞(TCMK1)作为正常组,并利用转化生长因子-β(TGF-β)刺激TCMK1建立体外纤维化模型,柴黄益肾颗粒含药血清处理建立体外用药模型;并通过转染A33过表达质粒在TCMK1细胞内过表达A33,观察纤维化相关指标及A33及GSK-3β、β-catenin mRNA的表达变化。结果RT-PCR检测结果显示,与对照组比较,模型组A33水平明显升高,而柴黄益肾颗粒高、低剂量组明显降低(P<0.05)。Western blotting检测结果显示,模型组α-SMA、FN、Col-Ⅰ等纤维化相关因子的表达水平明显高于对照组(P<0.05);与模型组比较,柴黄益肾颗粒低、高剂量组α-SMA、FN、Col-Ⅰ的表达水平明显降低(P<0.05)。HE、Masson、免疫组化染色结果显示,与对照组比较,模型组肾脏结构损伤严重,胶原沉积明显增加,且GSK-3β、β-catenin蛋白表达水平明显升高(P<0.01);与模型组比较,柴黄益肾颗粒低、高剂量组肾脏结构良好,肾脏损伤及纤维化均明显好转,GSK-3β、β-catenin蛋白表达水平明显降低(P<0.05)。体外实验结果证实,与正常组比较,A33过表达可促进TCMK1细胞纤维化相关�Objective To investigate the effects of Chaihuang Yishen Granules on renal fibrosis in unilateral ureteral obstruction(UUO)mice and its underlying mechanisms.Methods Twenty-four 8-week-old male C57BL/6 mice were randomly divided into control group,model group,and low and high dose groups of Chaihuang Yishen Granules(6 in each group).In control group,only right kidney ureter was exposed and dissected.In model group,the UUO animal model was established by UUO.In low and high dose groups,mice were administered intragastrically at doses of 3.8 and 7.6 g/kg of Chaihuang Yishen Granules respectively,following the model group's method to establish the UUO model.After 7 days,the mice were euthanized and renal samples were collected.HE and Masson staining were used to observe pathological changes and fibrosis degree of the kidneys in each group,Sirius red staining was used to observe collagen deposition.The expression levels ofα-smooth muscle actin(α-SMA),fibronectin(FN),typeⅠcollagen(Col-Ⅰ),glycogen synthase kinase 3β(GSK-3β),andβ-catenin related proteins were detected using Western blotting.Changes in A33 and GSK-3β,β-catenin mRNA levels were measured by RT-PCR.Additionally,a normal transformed C3H mouse kidney-1(TCMK1)was used as control(normal group);an in vitro fibrosis model was established using TCMK1 stimulated with Transforming Growth Factor‐β(TGF‐β);and an in vitro drug model was established using TCMK1 treated with serum containing Chaihuang Yishen Granules.A33 was overexpressed in TCMK1 cells using a transfection with an A33 overexpression plasmid,and changes in fibrosis-related indicators and the expression of A33 and GSK-3β,β-catenin mRNA were observed.Results RT-PCR results showed that,compared with control group,A33 level was significantly increased in model group,while it was significantly reduced in both low and high dose groups of Chaihuang Yishen Granules(P<0.05).Western blotting showed that the expression levels of fibrosis-related factors such asα-SMA,FN,Col-Ⅰin model group were

关 键 词：柴黄益肾颗粒 肾纤维化 WNT/Β-CATENIN信号通路 

分 类 号：R285.5[医药卫生—中药学]