白细胞介素-22通过抗氧化轴Nrf2-keap1-ARE抑制酒精性肝纤维化
Antioxidant axis Nrf2-keap1-ARE in inhibition of alcoholic liver fibrosis by IL-22.
作者信息
Ni Ya-Hui, Huo Li-Juan, Li Ting-Ting
机构信息
Li-Juan Huo, Ya-Hui Ni, Ting-Ting Li, Department of Gastroenterology, First Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China.
出版信息
World J Gastroenterol. 2017 Mar 21;23(11):2002-2011. doi: 10.3748/wjg.v23.i11.2002.
AIM
To explore the effect of interleukin (IL)-22 on model of alcoholic liver fibrosis hepatic stellate cells (HSCs), and whether this is related to regulation of Nrf2-keap1-ARE.
METHODS
HSC-T6 cells were incubated with 25, 50, 100, 200 and 400 μmol/L acetaldehyde. After 24 and 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect proliferation of HSCs to choose the best concentration and action time. We used the optimal concentration of acetaldehyde (200 μmol/L) to stimulate HSCs for 24 h, and treated the cells with a final concentration of 10, 20 or 50 ng/mL IL-22. The cell proliferation rate was detected by MTT assay. The cell cycle was analyzed by flow cytometry. The expression of nuclear factor-related factor (Nrf)2 and α-smooth muscle antigen was detected by western blotting and immunocytochemistry. The levels of malondialdehyde (MDA) and glutathione (GSH) were measured by spectrophotometry.
RESULTS
In the MTT assay, when HSCs were incubated with acetaldehyde, activity and proliferation were higher than in the control group, and were most obvious after 48 h treatment with 200 μmol/L acetaldehyde. The number of cells in G0/G1 phases was decreased and the number in S phase was increased in comparison with the control group. When treated with different concentrations of IL-22, HSC-T6 cell activity and proliferation rate were markedly decreased in a dose-dependent manner, and cell cycle progression was arrested from G1 to S phase. Western blotting and immunocytochemistry demonstrated that expression of Nrf2 total protein was not significantly affected. Expression of Nrf2 nuclear protein was low in the control group, increased slightly in the model group (or acetaldehyde-stimulated group), and increased more obviously in the IL-22 intervention groups. The levels of MDA and GSH in the model group were significantly enhanced in comparison with those in the control group. In cells treated with IL-22, the MDA level was attenuated but the GSH level was further increased. These changes were dose-dependent.
CONCLUSION
IL-22 inhibits acetaldehyde-induced HSC activation and proliferation, which may be related to nuclear translocation of Nrf2 and increased activity of the antioxidant axis Nrf2-keap1-ARE.
目的
探讨白细胞介素（IL）-22对酒精性肝纤维化肝星状细胞（HSCs）模型的影响，以及这是否与核因子相关因子2（Nrf2）- Kelch样环氧氯丙烷相关蛋白1（Keap1）-抗氧化反应元件（ARE）的调节有关。
方法
将HSC-T6细胞分别用25、50、100、200和400μmol/L乙醛孵育。24和48小时后，采用噻唑蓝（MTT）比色法检测HSCs的增殖情况，以选择最佳浓度和作用时间。采用最佳浓度的乙醛（200μmol/L）刺激HSCs 24小时，然后分别用终浓度为10、20或50 ng/mL的IL-22处理细胞。用MTT比色法检测细胞增殖率。采用流式细胞术分析细胞周期。采用蛋白质免疫印迹法和免疫细胞化学法检测核因子相关因子（Nrf）2和α-平滑肌肌动蛋白的表达。采用分光光度法测定丙二醛（MDA）和谷胱甘肽（GSH）水平。
结果
MTT比色法检测结果显示，与对照组相比，乙醛孵育的HSCs活性及增殖能力增强，且在200μmol/L乙醛处理48小时后最为明显。与对照组相比，G0/G1期细胞数量减少，S期细胞数量增加。用不同浓度的IL-22处理后，HSC-T6细胞活性和增殖率呈剂量依赖性显著降低，细胞周期进程从G1期阻滞于S期。蛋白质免疫印迹法和免疫细胞化学法检测结果显示，Nrf2总蛋白表达无明显变化。对照组Nrf2核蛋白表达较低，模型组（或乙醛刺激组）略有增加，IL-22干预组增加更明显。与对照组相比，模型组MDA和GSH水平显著升高。用IL-22处理的细胞，MDA水平降低，但GSH水平进一步升高。这些变化呈剂量依赖性。
结论
IL-22抑制乙醛诱导的HSCs激活和增殖，这可能与Nrf2核转位及抗氧化轴Nrf2-Keap1-ARE活性增强有关。
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