GDC-0449在CCl_(4)联合二乙酰氨基芴诱导肝纤维化大鼠模型中的作用  

Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene

作　　者：胡永红 肖准 付亚东 梁悦 张霖璋 刘伟[1,2] 慕永平[1,2] 刘成海[1,2] 刘平[1,2,3] 陈佳美[1,2] HU Yonghong;XIAO Zhun;FU Yadong;LIANG Yue;ZHANG Linzhang;LIU Wei;MU Yongping;LIU Chenghai;LIU Ping;CHEN Jiamei(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Institute of Liver Diseases,Shanghai Institute of Traditional Chinese Medicine,Shanghai 201203,China;Key Laboratory of Liver and Kidney Diseases,Ministry of Education,Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shanghai 201203,China;Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院上海市中医药研究院肝病研究所,上海201203 [2]肝肾疾病病证教育部重点实验室上海市中医临床重点实验室,上海201203 [3]上海中医药大学交叉科学研究院,上海201203

出　　处：《临床肝胆病杂志》2022年第2期334-341,共8页Journal of Clinical Hepatology

基　　金：国家自然科学基金(81973613,81603465);上海市科技启明星项目(19QA1408900);王宝恩肝纤维化基金(CFHPC2020010);上海中医药大学附属曙光医院四明青年基金(SGKJ-202004)。

摘　　要：目的研究Hedgehog信号通路抑制剂维莫德吉(GDC-0449)对CCl_(4)联合二乙酰氨基芴(2-AAF)诱导的大鼠肝纤维化的干预作用。方法将18只Fisher344雌性大鼠随机分为正常组、CCl_(4)联合2-AAF模型组(CCl_(4)/2-AAF组)和GDC-0449组,每组6只。CCl_(4)/2-AAF组和GDC-0449组大鼠按2 mL/kg体质量皮下注射30%CCl_(4)-橄榄油溶液,每周2次,持续造模6周;自第7周始,在注射CCl_(4)-橄榄油溶液同时予2-AAF(100 mg·kg^(-1)·d^(-1))灌胃,同时GDC-0449组大鼠按25 mg·kg^(-1)·d^(-1)灌胃干预,正常组大鼠予等体积橄榄油溶液注射及生理盐水灌胃,9周末处死取材。HE染色和天狼猩红(SR)染色观察各组大鼠肝组织病理及胶原沉积变化,SR阳性染色面积半定量分析和Ishak评分评估纤维化程度;碱水解法测定肝组织羟脯氨酸(Hyp)水平;免疫组化、Western Blot、实时荧光定量聚合酶链式反应(qRT-PCR)检测肝组织α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col-Ⅰ)、Ⅳ型胶原(Col-Ⅳ)、细胞角蛋白(CK)19、CK7、上皮细胞黏附分子(Epcam)及Hedgehog信号通路表达变化;免疫荧光共染观察CK19和卵圆细胞标志物6(OV6)共定位情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与正常组相比,CCl_(4)/2-AAF组大鼠肝组织炎性细胞聚集、胶原纤维沉积明显,存在明显的假小叶结构;肝组织Hyp水平和胶原阳性面积比显著增加(P值均<0.05),Ishak评分显著上升(P<0.05);α-SMA、Col-Ⅰ、Col-Ⅳ、Epcam、CK19、CK7、跨膜转运蛋白Smoothened(Smo)、Hedgehog配体Desert Hedgehog(Dhh)、Indian Hedgehog结合膜受体Patched(Ptch2)、胶质瘤相关致癌基因(Gli1、Gli2、Gli3)的表达显著增多(P值均<0.05);免疫荧光共染显示CCl_(4)/2-AAF组大鼠肝组织内CK19阳性细胞均表达OV6,且较正常组显著增多。与CCl_(4)/2-AAF组相比,GDC-0449组肝组织炎性细胞聚集、胶原沉积明显减少;肝组织Hyp水平、胶原阳性面积比显著�Objective To investigate the intervention effect of GDC-0449,a hedgehog signaling pathway inhibitor,on rats with liver fibrosis induced by carbon tetrachloride(CCl_(4))combined with 2-acetylaminofluorene(2-AAF).Methods A total of 18 female Fisher344 rats were randomly divided into normal group,CCl_(4)/2-AAF group,and GDC-0449 group,with 6 rats in each group.The rats in the CCl_(4)/2-AAF group and the GDC-0449 group were given subcutaneously injected 30%CCl_(4)-olive oil solution at a dose of 2 mL/kg twice a week for 6 weeks to induce liver fibrosis;since week 7,in addition to the injection of CCl_(4)-olive oil solution,the rats in these two groups were given 2-AAF(100 mg/kg/d)by gavage,and the rats in the GDC-0449 group were given GDC-0449(25 mg/kg/d)by gavage,while those in the normal group were given an equal volume of olive oil solution by injection and normal saline by gavage.All rats were sacrificed at the end of week 9,and related samples were collected.HE staining and sirius red(SR)staining were used to observe the changes in liver histopathology and collagen deposition,and the semi-quantitative analysis of SR-positive area and Ishak score were used to evaluate fibrosis degree;the alkaline hydrolysis method was used to measure the level of hydroxyproline(Hyp)in liver tissue;immunohistochemistry,Western blot,and qRT-PCR were used to measure the expression ofα-smooth muscle actin(α-SMA),type I collagen(Col-I),type IV collagen(Col-IV),cytokeratin 19(CK19),cytokeratin 7(CK7),the epithelial cell adhesion molecule Epcam,and the hedgehog signaling pathway in liver tissue;double immunofluorescence staining was used to observe the colocalization of CK19 and the oval cell marker OV6.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the normal group,the CCl_(4)/2-AAF group had marked inflammatory cell aggregation and collagen deposition in liver tissue,

关 键 词：肝硬化 HEDGEHOG信号通路 大鼠 近交F344 

分 类 号：R575.2[医药卫生—消化系统]