黄芪总皂苷抑制胆管反应抗胆汁性肝纤维化的作用机制研究  

Study on the Mechanism of Astragalosides Against Biliary Fibrosis by Inhibiting Ductular Reaction

作　　者：方静 胡永红 梁悦 慕永平[1,2] 刘伟 刘平[1,2,3] 吕莹 陈佳美[1,2] Fang Jing;Hu Yonghong;Liang Yue;Mu Yongping;Liu Wei;Liu Ping;Lyu Ying;Chen Jiamei(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Institute of Liver Disease,Shanghai Institute of Traditional Chinese Medicine,Key Laboratory of Liver and Kidney Diseases of the Ministry of Education,Key Clinical Laboratory of Shanghai Traditional Chinese Medicine,Shanghai 201203,China;Institute of Interdisciplinary Medicine,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201203 [2]上海市中医药研究院肝病研究所,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：《世界科学技术-中医药现代化》2023年第7期2327-2335,共9页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会面上项目(81973613):绞股蓝总苷调控TNFα/Gli1通路抗肝纤维化的效应机制,负责人:陈佳美;上海中医药大学附属曙光医院“四明青年基金”项目(SGKJ-201906):绞股蓝总皂苷影响赖氨酰氧化酶样蛋白-2表达干预肝纤维化的效应机制,负责人:吕莹。

摘　　要：目的研究黄芪总皂苷(ASTs)抑制胆管反应改善胆汁性肝纤维化的部分作用机制。方法将24只SD大鼠随机分为假手术组,胆管结扎组和ASTs干预组,每组8只。胆管结扎造模后第2周首日,ASTs组给予ASTs 160 mg·kg^(-1)·d^(-1)体重灌胃,每天1次,连续给药3周。假手术组和模型组大鼠予以同体积的双蒸水灌胃。第4周末处死取材。HE染色和天狼猩红染色观察各组大鼠肝组织病理及胶原沉积情况,天狼猩红染色阳性面积半定量分析和羟脯氨酸含量评估肝组织纤维化程度;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)、结蛋白(Desmin)、细胞角蛋白(CK)19、CK7、上皮细胞黏附分子(Epcam)、OV6及赖氨酰氧化酶(LOX)家族蛋白表达变化。体外采用丁酸钠诱导肝祖细胞株WB-F344细胞向胆管上皮细胞表型分化,给予ASTs干预,4天后收集细胞。qRT-PCR法检测细胞CK19、LOXL1和LOXL2表达变化。结果与BDL模型组比较,ASTs组血清ALT和AST活性显著降低(P<0.01);肝组织病理损伤和胆管增生明显减轻,Hyp含量和天狼猩红阳性面积比均显著降低(P<0.01);免疫组化染色显示,ASTs组肝组织α-SMA、Desmin、CK19、CK7、Epcam和OV6阳性表达显著减少;且α-SMA、CK7、LOX和LOXL1的mRNA表达显著降低;Epcam和LOXL1的蛋白表达显著减少。体外结果显示,丁酸钠诱导后细胞CK19、LOXL1和LOXL2的mRNA表达显著升高(P<0.01);而与丁酸钠组比较,ASTs组细胞CK19、LOXL1和LOXL2的mRNA表达显著降低(P<0.05)。结论ASTs通过抑制胆管反应改善胆汁性肝纤维化,其作用机制可能与下调LOXL1的表达有关。Objective To study the partial mechanism of astragalosides(ASTs)against biliary fibrosis through inhibiting ductular reaction.Methods Rats were randomly divided into sham operation group,bile duct ligation(BDL)group and ASTs group(n=8 in each group).On the first day of the second week after BDL,the rats in ASTs group were given intragastric administration of ASTs for 3 weeks(160 mg·kg^(-1)·d^(-1),once a day).Rats in sham operation group and BDL group were given the same volume of water.At the end of the fourth week,all rats were euthanasia.HE staining and sirius red staining were used to observe the pathological changes and collagen deposition.The degree of liver fibrosis was evaluated by semi-quantitative analysis of positive area of sirius red staining and the content of hydroxyproline.The expression changes of α smooth muscle actin(α-SMA),Desmin,cytokeratin(CK)19,CK7,epithelial cell adhesion molecule(Epcam),OV6 and lysyl oxidase(LOX)family proteins in liver tissue were detected by immunohistochemistry,Western blot,and real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).In vitro,hepatic progenitor cell line WB-F344 cells were induced by sodium butyrate to differentiate into biliary epithelial cells,intervented of ASTs,and collected after 4 days.The expression changes of CK19,LOXL1 and LOXL2 of cells were detected by qRT-PCR.Results Compared with BDL group,serum ALT and AST activities in ASTs group were significantly decreased(P<0.01).Histopathological injury of liver tissue was significantly reduced,Hyp content and percentage of positive area of sirius red were significantly decreased(P<0.01).Immunohistochemical staining showed that the positive expressions ofα-SMA,Desmin,CK19,CK7,Epcam and OV6 were significantly decreased in the ASTs group.The mRNA expressions ofα-SMA,CK7,LOX and LOXL1 were significantly decreased.The protein expressions of Epcam and LOXL1 were significantly reduced.In vitro results showed that the gene expressions of CK19,LOXL1 and LOXL2 were significantly increased a

关 键 词：黄芪总皂苷 胆汁性肝纤维化 胆管反应 赖氨酰氧化酶样蛋白 

分 类 号：R285.5[医药卫生—中药学]