白细胞介素22对肝星状细胞活化的影响及其机制  

Effect of interleukin-22 on hepatic stellate cell activation and its mechanism

作　　者：高君 陈欢 刘燕 张峰 诸葛宇征[1] GAO Jun;CHEN Huan;LIU Yan;ZHANG Feng;ZHUGE Yuzheng(Department of Gastroenterology,Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University,Nanjing 210008,China)

机构地区：[1]南京医科大学鼓楼临床医学院消化内科,南京210008

出　　处：《临床肝胆病杂志》2024年第11期2229-2237,共9页Journal of Clinical Hepatology

基　　金：国家自然科学基金(81900552);南京市卫生科技发展专项资金项目-杰出青年基金项目(JQX20005)。

摘　　要：目的探讨肝星状细胞活化过程中白细胞介素(IL)22发挥的作用及影响机制。方法选取人肝星状细胞系LX-2细胞为研究对象,以转化生长因子(TGF)β1诱导LX-2细胞构建肝星状细胞活化模型,以梯度浓度的IL-22处理LX-2细胞,通过Western Blot、q RT-PCR检测活化标志物Ⅰ型胶原蛋白(COL1A1)、α-平滑肌肌动蛋白(α-SMA)表达水平以确定适宜的药物工作浓度、时间;通过Western Blot、q RT-PCR及免疫荧光方法检测经IL-22处理的活化肝星状细胞中成纤维细胞因子诱导早期反应蛋白14(Fn14)、内质网应激(ERS)及其活化标志物水平;以衣霉素(TM)诱导LX-2细胞ERS,通过Western Blot、q RT-PCR检测经IL-22处理后LX-2细胞ERS及其活化标志物水平;使用肿瘤坏死因子样细胞凋亡弱诱导剂(TWEAK)、小干扰RNA分别上/下调Fn14,再检测磷酸化肌醇需求蛋白1α(p-IRE1α)、肌醇需求蛋白1α(IRE1α)、转录因子剪接型X-盒结合蛋白1(XBP1s)、COL1A1和α-SMA基因及蛋白水平;在IL-22处理TGF-β1诱导的LX-2细胞的基础上加用TWEAK上调Fn14,通过Western Blot、免疫荧光方法检测Fn14、ERS及其活化标志物水平。计量资料两组间比较采用成组t检验;多组间比较采用单因素方差分析,进一步两两比较采用Sidak’s多重比较检验。结果与TGF-β1组相比,TGFβ1+IL-22组COL1A1、α-SMA的蛋白和m RNA表达水平均下降,且在IL-22浓度为10 ng/m L以上作用24小时时效果更加显著(P值均<0.01);与TGF-β1组相比,TGF-β1+IL-22组Fn14、p-IRE1α、XBP1s表达水平均下降(P值均<0.05);与TM组相比,TM+IL-22组p-IRE1α、XBP1s、COL1A1和α-SMA表达水平均下降(P值均<0.05);与沉默对照(NC)组相比,Fn14 si RNA组p-IRE1α、XBP1s、COL1A1和α-SMA表达水平均下降(P值均<0.05);与正常对照组相比,TWEAK组Fn14、p-IRE1α、XBP1s、COL1A1和α-SMA表达水平均上升(P值均<0.01);与TGF-β1+IL-22组相比,TGF-β1+IL-22+TWEAK组Fn14、p-IRE1α、XBP1s、COL1A1和α-SMA表达水平均上升(Objective To investigate the effect of interleukin-22(IL-22)on the activation of hepatic stellate cells(HSCs)and its mechanism.Methods The human HSC LX-2 cells were selected for the study,and the LX-2 cells induced by TGF-β1 were used to establish a model of HSC activation.LX-2 cells were treated with IL-22 at gradient concentrations,and Western blot and qRTPCR were used to measure the expression levels of the activation markers COL1A1 andα-SMA and determine the appropriate working concentration and time of the drug.Western blot,qRT-PCR,and immunofluorescence assay were used to determine the levels of Fn14 and the markers for endoplasmic reticulum stress(ERS)and activation in activated HSCs treated by IL-22.ERS in LX-2 cells was induced by tunicamycin(TM),and Western blot and qRT-PCR were used to measure the levels of markers for ERS and activation in LX-2 cells treated by IL-22.TNF-like weak inducer of apoptosis(TWEAK)and small interfering RNA were used to upregulate and downregulate Fn14,and then the mRNA and protein expression levels of p-IRE1α,IRE1α,XBP1s,COL1A1,andα-SMA were measured.After LX-2 cells induced by TGF-β1 were treated by IL-22,TWEAK was used to upregulate Fn14,and Western blot and immunofluorescence assay were used to measure the levels of Fn14 and the markers for ERS and activation.The independent-samples t-test was used for comparison of continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the Sidak’s multiple comparison test was used for further comparison between two groups.Results Compared with the TGF-β1 group,the TGF-β1+IL-22 group had significant reductions in the protein and mRNA expression levels of COL1A1 andα-SMA,with a more significant effect after treatment with 10 ng/mL IL-22 for 24 hours(all P<0.01).Compared with the TGF-β1 group,the TGF-β1+IL-22 group had significant reductions in the expression levels of Fn14,p-IRE1α,and XBP1s(all P<0.05).Compared with the TM group,the TM+IL-22 group had significant

关 键 词：肝纤维化 白细胞介素22 肝星状细胞 内质网应激 

分 类 号：R575.2[医药卫生—消化系统]