白细胞介素22对乙醛诱导下肝星状细胞增殖活化的影响及其机制

Effect of interleukin-22 on proliferation and activation of hepatic stellate cells induced by acetaldehyde and related mechanism

目的研究白细胞介素22（IL-22）对乙醛诱导的肝星状细胞（HSC）活化增殖的影响，并探讨抗氧化轴Nrf2-keapl—ARE在该作用中的地位。方法体外培养HSC—T6，采用不同浓度（25、50、100、200、400μmol／L）的乙醛刺激24、48h后，四甲基偶氮唑盐法测定细胞增殖率以筛选出最佳造模条件。用最适浓度的乙醛（200μmol／L）处理HSC—T624h后，再分别用不同浓度的IL-22（10、20、50ng／m1）干预24h，四甲基偶氮唑盐法测定细胞增殖情况，同时采用Westernblot和免疫细胞化学检测Nrf2、α-平滑肌肌动蛋白的表达情况，分光光度法检测培养上清液中丙二醛（MDA）、谷胱甘肽（GSH）含量的变化。采用SPSS17．0软件进行统计学分析，结果以均数±标准差（X±s）表示，P〈0．05为差异有统计学意义。多组样本均数的两两比较采用One-wayANOVA分析。结果乙醛刺激后HSC增殖活化明显增强，以200μmol／L作用48h最为明显。加用梯度浓度的IL-22干预后，表现为剂量依赖性地抑制HSC活化增殖，IL-2210、20、50ng／ml组增殖移植率分别为14％、25％、35％垆值均〈0．05）。Westernblot和免疫组织化学检测结果均显示各组HSC内Nrf2总蛋白表达无明显差异，而Nrf2核蛋白在空白对照组表达很少；当乙醛刺激后表达增加（与空白组相比，P〈0．05），加用梯度浓度IL-22干预后，Nrf2核蛋白表达进一步增加垆值均〈0．05）。分光光度法显示，与空白组相比，乙醛刺激后培养上清液中MDA、GSH水平均升高，加用梯度浓度的IL-22干预后，MDA水平显著下降，而GSH水平进一步明显升高（P值均〈0．05），二者均存在剂量依赖性。结论乙醛诱导HSC活化增殖，成功建立了酒精性肝纤维化的体外模型；IL-22对乙醛诱导的HSC活化增殖有明显的抑制作用，其机制可能与促进HSC内Nrf2核转位及下游靶基因GSH表达、增强机体抗氧化轴Nrf2-keapl-ARE的活性有关。

Objective To investigate the effect of interleukin-22 （IL-22） on the activation and proliferation of hepatic stellate cells （HSCs） induced by acetaldehyde, as well as the role of the antioxidant axis Nrf2-keapl-ARE. Methods Hepatic stellate cell-T6 （HSC-T6） cells were cultured in vitro, and after 24 and 48 hours of acetaldehyde stimulation at various concentrations （25, 50, 100, 200, and 400 μmol/ L）, MTT assay was used to measure cell proliferation rate to screen out the optimal conditions for model establishment. HSC-T6 cells were treated first with the optimal concentration of acetaldehyde （200 μmol/L） for 24 hours and then with different concentrations of IL-22 （10, 20, and 50 ng/ml） for 24 hours. MTT assay was used to measure cell proliferation, Western blot and cell immunohistochemistry were used to measure the expression of nuclear factor erythroid 2-related factor 2 （Nrf2） and α-smooth muscle actin （ct-SMA）, and spectrophotometry was used to measure the changes in the content of malondialdehyde （MDA） and reduced glutathione （GSH） in culture supematant. SPSS 17.0 was used for statistical analysis and data were expressed as mean＋SD. P 〈 0.05 was considered statistically significant. A one-way analysis of variance was used for comparison of means between any two groups. Results HSCs had significantly enhanced proliferation and activation after being treated with acetaldehyde, especially at 200μmol/L for 48 hours. After the intervention with gradient concentrations of IL-22, the proliferation and activation of HSCs were inhibited in a dose- dependent manner, and the proliferation and migration rates in the 10, 20, and 50 ng/ml IL-22 groups were 14%, 25%, and 35%, respectively （all P 〈 0.05）. The results of Western blot and immunohistochemistry showed that there was no significant difference in the expression of Nrf2 total protein in HSCs between groups, while there was extremely low expression of Nrf2 nucleoprotein in the blank control group. There was increased expression of Nrf2 nucleoprotein after acetaldehyde stimulation （compared with the blank control group, P 〈 0.05）, and after the intervention with gradient concentrations of IL-22, the expression of Nrf2 nucleoprotein was further increased （all P 〈 0.05）. The results of spectrophotometry showed that compared with the blank control group, the model group had increased levels of MDA and GSH in culture supernatant after acetaldehyde stimulation; after the intervention with gradient concentrations of IL-22, there was a significant reduction in the MDA level and a significant increase in the GSH level in a dose-dependent manner （all P 〈 0.05）. Conclusion The activation and proliferation of HSCs induced by acetaldehyde helps with the successful establishment of an in vitro model of alcoholic liver fibrosis. IL-22 effectively inhibits the activation and proliferation of HSCs induced by acetaldehyde, and its mechanism may be related to promoting Nrf2 nuclear translocation in HSCs and expression of the downstream target gene GSH and increasing the activity of the antioxidant axis Nrf2-keapl-ARE.