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内质网应激蛋白激酶RNA样ER激酶（PERK）通路对肝星状细胞激活及Ⅰ型胶原蛋白表达的影响
DOI: 10.12449/JCH240516
Effect of the protein kinase RNA-like endoplasmic reticulum kinase pathway in endoplasmic reticulum stress on hepatic stellate cell activation and collagen type I expression
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摘要:
目的 探讨内质网应激蛋白激酶RNA样ER激酶（PERK）/真核生物翻译起始因子2α（eIF2α）信号通路对肝星状细胞（HSC）活化的影响。 方法 收集11例肝穿刺病理提示S1～S4肝纤维化患者和9例肝血管瘤、肝腺瘤患者术后周围正常肝组织病理切片，进一步行组织免疫组化检测PERK、eIF2α、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（CHOP）表达情况；使用不同浓度的内质网应激诱导剂毒胡萝卜素（0、125、250、500、1 000 nmol/L）作用于人HSC-LX2，使用qRT-PCR检测PERK mRNA及Western Blot检测PERK、肌醇需要酶1（IRE1）、激活转录因子6（ATF6）、CHOP及α平滑肌肌动蛋白（α-SMA）表达水平。使用慢病毒转染构建PERK稳定过表达LX-2组及对照组，并通过qRT-PCR检测PERK、eIF2α、α-SMA mRNA，Western Blot检测PERK、p-eIF2α、α-SMA蛋白表达，免疫荧光检测胶Ⅰ型原蛋白（COL1A1）表达。符合正态分布的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LSD-t检验；不符合正态分布的计数资料，两组间比较采用Mann-Whitney U秩和检验。 结果 与正常肝组织相比，肝纤维化患者肝组织中PERK、eIF2α及CHOP表达升高，差异均有统计学意义（Z=-3.56、t=-5.75、Z=-3.52，P值均<0.001）。不同浓度毒胡萝卜素作用后，与溶媒组相比，内质网相关蛋白PERK、CHOP、IRE1、ATF6及α-SMA蛋白表达显著升高（P值均<0.05）。与对照空载慢病毒组相比，PERK稳定过表达组PERK mRNA、eIF2α mRNA、α-SMA mRNA表达及PERK、p-eIF2α、α-SMA蛋白表达明显升高（P值均<0.05）。细胞免疫荧光结果提示，PERK过表达组COL1A1表达升高（P<0.05）。 结论 PERK过表达可诱导LX-2细胞α-SMA、胶原蛋白COL1A1表达，提示PERK信号通路可能是HSC活化的重要机制之一。 Abstract:Objective To investigate the effect of the protein kinase RNA-like endoplasmic reticulum kinase （PERK）/eukaryotic initiation factor 2α （eIF2α） pathway in endoplasmic reticulum stress on the activation of hepatic stellate cells （HSC）. Methods Pathological sections of normal liver tissue after surgery were collected from 11 patients with hepatic fibrosis （S1-S4） and 9 patients with hepatic hemangioma and hepatic adenoma confirmed by liver biopsy， and immunohistochemistry was used to measure the protein expression levels of PERK， eIF2α， and C/EBP homologous protein （CHOP）. Human HSC-LX2 cells were treated with different concentrations of the endoplasmic reticulum stress inducer thapsigargin （0， 125， 250， 500， and 1 000 nmol/L）， and qRT-PCR was used to measure the mRNA expression level of PERK， while Western blot was used to measure the protein expression levels of PERK， inositol requiring protein 1 （IRE1）， activating transcription factor 6 （ATF6）， CHOP， and α-smooth muscle actin （α-SMA）. The method of lentivirus transfection was used to construct a PERK stable overexpression LX-2 group and a control group； qRT-PCR was used to measure the mRNA expression levels of PERK， eIF2α， and α-SMA， Western blot was used to measure the protein expression levels of PERK， phosphorylated eIF2α （p-eIF2α）， and α-SMA， and immunofluorescence assay was used to measure the expression of collagen type I alpha 1 （COL1A1）. The independent samples t-test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. Results Compared with normal liver tissue， the liver tissue of patients with hepatic fibrosis had significantly higher expression levels of PERK， eIF2α， and CHOP （Z=-3.56， t=-5.75， Z=-3.52， all P<0.001）. Compared with the solvent group， the groups treated with different concentrations of thapsigargin had significant increases in the expression levels of the endoplasmic reticulum-associated proteins PERK， CHOP， IRE1， ATF6， and α-SMA （all P<0.05）. Compared with the control group， the PERK stable overexpression group had significant increases in the mRNA expression levels of PERK， eIF2α， and α-SMA and the protein expression levels of PERK， p-eIF2α， and α-SMA （all P<0.05）， and immunofluorescence assay showed a significant increase in the expression level of COL1A1 in the PERK stable overexpression group （P<0.05）. Conclusion PERK overexpression can induce the expression of α-SMA and COL1A1 in LX-2 cells， suggesting that the PERK signaling pathway might be one of the important mechanisms of HSC activation. -
图 1 肝纤维化患者与非肝纤维化患者中PERK信号通路表达情况（免疫组化，×400）
Figure 1. Expression of PERK pathway in patients with and without hepatic fibrosis（Immunohistochemical，×400）
图 2 不同浓度Tg诱导后内质网应激相关蛋白表达情况
注： a，不同浓度Tg干预不同时间细胞光学显微镜下结果（×40）；b，qRT-PCR结果；c、d，Western Blot结果。
Figure 2. Expression of endoplasmic reticulum stress-related proteins after induction with different concentrations of Tg
图 3 不同浓度Tg诱导下α-SMA表达情况
注： a，qRT-PCR结果；b，Western Blot结果。
Figure 3. The expression of α-SMA induced by different concentrations of Tg
图 4 转染PERK过表达慢病毒后PERK信号通路基因及蛋白表达情况
注： a，光学显微镜下结果（×40）；b，qRT-PCR结果；c，Western Blot结果。
Figure 4. Expression of PERK pathway genes and proteins after transfection with PERK-overexpressing lentivirus
图 5 PERK过表达促进HSC活化
注： a，免疫荧光结果（×100）；b，qRT-PCR结果；c，Western Blot结果。
Figure 5. Overexpression of PERK promotes the activation of hepatic stellate cells
表 1 引物序列
Table 1. Primer sequences
引物 序列 PERK R： 5′-CATTGGGGTCAGAACCGT-3′ F： 5′-ATCGCAGAGGCAGTGGAG-3′ eIF2α R： 5′-TCACACGTAGTAGCAAAAGAACC-3′ F： 5′-GCCGCTAAACTTGCATATCTTCA-3′ α-SMA R： 5′-GCAGGGTGGGATGCTCTT-3′ F： 5′-GGGTGATGGTGGGAATGG-3′ β-actin R： 5′-CCACATAGGAATCCTTCTGACC-3′ F： 5′-CTTCGCGGGCGACGAT-3′ 
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表 2 肝纤维化患者与非肝纤维化患者病理切片细胞着色比例及染色强度得分
Table 2. The final score of cell staining proportion and staining intensity in hepatic fibrosis and non-hepatic fibrosis pathological sections
指标 非肝纤维化 患者（n=9） 肝纤维化 患者（n=11） 统计值 P值 PERK 0.50（0.10～1.13） 6.10（4.30～7.73） Z=-3.56 <0.001 eIF2α 1.46±0.89 4.83±1.52 t=-5.75 <0.001 CHOP 0.50（0.00～0.75） 3.00（2.00～4.75） Z=-3.52 <0.001
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