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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
DOI: 10.12449/JCH241116
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。
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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。 Abstract: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 qRT-PCR were used to measure the expression levels of the activation markers COL1A1 and α-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， and α-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 and α-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 reductions in the expression levels of p-IRE1α， XBP1s， COL1A1， and α-SMA （all P<0.05）. Compared with the silenced control group， the Fn14 siRNA group had significant reductions in the expression levels of p-IRE1α， XBP1s， COL1A1， and α-SMA （all P<0.05）. Compared with the normal control group， the TWEAK group had significant increases in the expression levels of Fn14， p-IRE1α， XBP1s， COL1A1， and α-SMA （all P<0.01）. Compared with the TGFβ1+IL-22 group， the TGF-β1+IL-22+TWEAK group had significant increases in the expression levels of Fn14， p-IRE1α， XBP1s， COL1A1， and α-SMA （all P<0.05）. Conclusion IL-22 negatively regulates ERS in HSCs by inhibiting Fn14， thereby inhibiting the activation of HSCs. -
Key words:
- Hepatic Fibrosis /
- Interleukin-22 /
- Hepatic Stellate Cells /
- Endoplasmic Reticulum Stress
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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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