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白细胞介素22对肝星状细胞活化的影响及其机制

高君 陈欢 刘燕 张峰 诸葛宇征

引用本文:
Citation:

白细胞介素22对肝星状细胞活化的影响及其机制

DOI: 10.12449/JCH241116
基金项目: 

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)

利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:高君负责实验设计与操作,起草论文;陈欢、刘燕负责实验操作;张峰、诸葛宇征提供课题思路,指导实验内容,指导撰写文章并最后定稿。
详细信息
    通信作者:

    诸葛宇征, yuzheng9111963@aliyun.com (ORCID: 0000-0002-3829-5831)

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

Research funding: 

National Natural Science Foundation of China (81900552);

Nanjing Health Science and Technology Development Special Fund Project-Outstanding Youth Fund (JQX20005)

More Information
  • 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、qRT-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、qRT-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、qRT-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mRNA表达水平均下降,且在IL-22浓度为10 ng/mL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 siRNA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(P值均<0.05)。  结论  IL-22通过抑制Fn14负调控肝星状细胞ERS进而抑制其活化增殖。

     

  • 图  1  IL-22对肝星状细胞活化的影响

    注: a,Western Blot分析LX-2细胞中的活化标志物α-SMA和COL1A1在梯度浓度IL-22处理条件下的表达情况;b,使用ImageJ软件对图a条带进行灰度值分析;c,qRT-PCR分析LX-2细胞中的活化标志物α-SMA和COL1A1在梯度浓度IL-22处理条件下的表达情况;d,qRT-PCR分析LX-2细胞中的活化标志物α-SMA和COL1A1在不同处理时间下的表达情况。

    Figure  1.  Effect of IL-22 on LX-2 cells activation

    图  2  IL-22抑制肝星状细胞中Fn14的表达和ERS

    注: a,Western Blot分析LX-2细胞经不同分组(NC、TGF-β1、TGF-β1+IL-22)处理24 h后,相关目的蛋白表达情况;b,qRT-PCR分析LX-2细胞经同a图方法处理后XBP1s、Fn14基因表达量;c、d为免疫荧光分析LX-2细胞同a图方法处理后Fn14、XBP1s表达量(×400);e,Western Blot分析LX-2细胞经不同分组(NC、TM、TM+IL-22)处理24 h后相关目的蛋白表达情况;f,qRT-PCR分析LX-2细胞经同e图方法处理后XBP1s、COL1A1、α-SMA基因表达量。

    Figure  2.  IL-22 inhibits Fn14 expression and ERS in HSCs

    图  3  Fn14调控IRE1α-XBP1s信号通路

    注: a、b,Western Blot、qRT-PCR分析不同Fn14 siRNA的沉默效果,筛选沉默效率最佳的S3;c,Western Blot分析沉默Fn14表达对LX-2细胞相关蛋白表达影响;d,qRT-PCR分析沉默Fn14表达对LX-2细胞XBP1s、COL1A1、α-SMA基因表达影响;e,Western Blot分析TWEAK蛋白处理对LX-2细胞相关蛋白表达影响;f,qRT-PCR分析TWEAK蛋白处理对LX-2细胞Fn14、XBP1s、α-SMA基因表达影响;g,免疫荧光分析TWEAK蛋白处理对LX-2细胞XBP1s、Fn14表达量影响(×400)。

    Figure  3.  Fn14 regulates the IRE1α-XBP1s signaling pathway

    图  4  IL-22通过抑制Fn14/ERS信号通路抑制肝星状细胞活化

    注: a,Western Blot分析不同分组处理对LX-2细胞相关蛋白表达影响;b、c为免疫荧光分析同a处理后Fn14、XBP1s表达情况(×400);d,使用ImageJ软件对a图条带进行了灰度值分析。

    Figure  4.  IL-22 inhibits HSCs activation by inhibiting Fn14 and negatively regulating the IRE1α-XBP1S signaling pathway

    表  1  目标基因的引物序列

    Table  1.   Primer sequences of target genes

    基因 正向引物(5'-3') 反向引物(5'-3')
    β-actin GGCACCACACCTTCTACAATGAG GGATAGCACAGCCTGGATAGCA
    α-SMA CCTGTGTTGTGGTTTACACTGG GGGGGAATTATCTTTCCTGGTCC
    COL1A1 GAGGGCCAAGACGAAGACATC CAGATCACGTCATCGCACAAC
    XBP1s TGGATTCTGGCGGTATTGACTC GAACTGGGTCCTTCTGGGTAGA
    Fn14 GCTCTGAGCCTGACCTTCGT TCTCTCCTGCGGCATCGT
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  • 收稿日期:  2024-03-03
  • 录用日期:  2024-05-17
  • 出版日期:  2024-11-25
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