Jagged/Notch信号通路在铜负荷大鼠肝纤维化发生过程中的作用

Jagged/Notch signaling pathway contributes to the progress of hepatic fibrosis induced by copper overload in rats

目的探讨Jagged/Notch信号通路在铜负荷大鼠肝纤维化发生发展中的作用。方法雄性SD大鼠36只随机分为对照组、模型组,模型组按造模后不同时间分为8、12、16、20、24周5个亚组,每组6只。模型组喂饲含1.5g/kg硫酸铜的粉状饲料和0.185%硫酸铜去离子水,观察大鼠一般情况,全自动生化分析仪测定各组大鼠肝组织血清丙氨酸氨基转移酶(ALT)、血清天冬氨酸氨基转移酶(AST)、透明质酸(HA)、层粘蛋白(LN)、Ⅲ型前胶原(PCⅢ)水平;比色法测定大鼠肝脏羟脯氨酸(Hyp)水平;HE、Masson染色法观察大鼠肝脏病理学改变;实时荧光定量PCR法检测TGF-β1、Notch1、Notch3、Hes1、Hes5mRNA表达情况;Western blot检测Jagged1、Notch3、Hes1蛋白表达情况,免疫组化方法检测Hes1、a-SMA表达情况。结果与正常组相比,模型组大鼠肝组织ALT、AST、HA水平自造模第8周开始升高,PCⅢ、LN、Hyp水平自第12周开始升高,差异有统计学意义(P<0.05),肝组织炎症及纤维化程度进行性加重,TGF-β1、Notch3、Hes1mRNA及Jagged1、Notch3、Hes1蛋白表达显著升高,差异有统计学意义(P<0.05);Hes1蛋白在模型组纤维化区域大量表达,与肌成纤维细胞标志蛋白a-SMA表达区域高度重合,两者表达量呈同步增加关系(r=0.952,P<0.05)。结论 Jagged1/Notch3/Hes1信号通路可能参与铜负荷大鼠肝纤维化的发生与发展,并与肝纤维化的严重程度密切相关。

Objective To investigate the role of Jagged/Notch signaling pathway contributing to the progress of hepatic fibrosis induced by copper overload in rats.Methods Thirty-six male Sprague-Dawley rats were randomly divided into normal group（n=6） and model group（n=36）.The model group was further divided into five subgroups of 8 weeks,12 weeks,16 weeks,20 weeks,and 24 weeks with six rats in each subgroup,according to different time after modeling.The model group was fed with powdered diet containing 1.5g/kg of copper sulfate and deionized water containing 0.185% copper sulfate.General condition of the rats was observed.Serum levels of alanine aminotransferase（ALT）,serum aspartate aminotransferase （AST）,hyaluronic acid （HA）,laminin （LN）,type of Ⅲ procollagen（PC）,and hydroxyproline content in liver tissues were tested dynamically according to different time points mentioned above.Liver pathological changes were observed with HE and Masson staining.Expressions of TGF-changes were observed ere tes and Jagged1/Notch3/Hes1 proteins were detected dynamically with RT-PCR and Western Blot.Immunohistochemistry was used to detect expressions of Hes1 and a-SMA.Results The serum levels of ALT,AST,HA and PCⅢ,LN,Hyp after 8 and 12 weeks of modeling were significantly higher than those in control group（P〈0.05）.The degree of inflammation and fibrosis increased progressively.The expressions of TGF-β1,Notch3,Hes1 mRNA and Jagged1,Notch3,Hes1 protein increased（P〈0.05）.The Hes1 protein was expressed in a large amount in the fibrotic region,which coincided greatly with the a-SMA （myofibroblast marker protein） expression,and the two proteins increased synchronously（r=0.952,P〈0.05）.Conclusion Jagged1/Notch3/Hes1 signaling pathway is possibly involved in the occurrence and development of liver fibrosis in rats with copper overload,and is closely related to the severity of liver fibrosis.