干预肝细胞氧化应激感受器Keap1对肝星状细胞基质金属蛋白酶-2表达及其活性调节的影响-东南大学学报医学版

干预肝细胞氧化应激感受器Keap1对肝星状细胞基质金属蛋白酶-2表达及其活性调节的影响

单位：东南大学医学院病理学与病理生理学系, 江苏南京 210009

分类号：R362

出版年·卷·期（页码）：2019·38·第六期（947-953）

摘要：

目的：建立稳定敲低肝细胞内氧化应激感受器Kelch样ECH相关蛋白1（Kelch-like ECH-associated protein1，Keap1）的模型，探讨稳定下调Keap1的缺氧肝细胞对小鼠肝星状细胞（HSCs）基质金属蛋白酶-2（MMP-2）活性及其调控分子基质金属蛋白酶抑制因子-2（TIMP-2）、MMP-14、小窝蛋白-1（Cav-1）的影响。方法：根据本组建立的方法，构建Keap1下调的稳转肝细胞株，缺氧条件下培养Keap1下调的肝细胞，通过蛋白质印迹法和QPCR验证肝细胞中Keap1mRNA是否下调以及其下游分子和肿瘤坏死因子α（TNF-α）的表达水平。利用上述肝细胞培养上清液干预小鼠肝星状细胞株（HSC-T6），分别收集小鼠HSC-T6细胞培养上清液和细胞，通过蛋白质印迹法检测小鼠HSC-T6细胞的Keap1、抗氧化应激分子核因子E2相关因子2（Nrf2）、MMP-2等蛋白表达，明胶酶谱法检测小鼠HSC-T6细胞培养液上清中MMP-2活性。结果：稳定敲低肝细胞内氧化应激感受器Keap 1后，Nrf2表达增高，肿瘤坏死因-α（TNF-α）表达减少。与对照组相比，敲低Keap1的肝细胞培养液上清使小鼠HSC-T6细胞的MMP-2、Cav-1、MT1-MMP、TIMP-2蛋白表达减少而MMP-2的活性增高。结论：缺氧条件培养稳定敲低Keap1的肝细胞，肝细胞损伤程度减轻，合成及释放TNF-α减少。肝细胞培养上清液对HSC-T6细胞内吞相关分子Cav-1表达的刺激作用降低，减轻了Cav-1对膜表面的MMP-2活化的干扰，使MMP-2活性得以升高。

Objective: To establish an interventional model of knockdown Kelch-like ECH-associated protein 1 (Keap1) in hepatocytes and investigate the influence of hypoxic hepatocytes with Keap1 knock-down on the matrix metalloproteinase-2 (MMP-2), and the corresponding regulatory molecules such as matrix metalloproteinase inhibitor (TIMP-2), membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP14), caveolin-1 (Cav-1) of the hepatic satellite cells(HSCs). Methods: According to the method established by our group, the hepatocytes with Keap1 knock-down was constructed, and were cultured under hypoxic conditions. The expression levels of Keap1 and nuclear factor erythroid 2 related factor 2 (Nrf2), tumor necrosis factor-α (TNF-α) its downstream molecules of the hepatocytes were measured by Western blotting and QPCR. Hepatic stellate cells(HSC-T6) were cultured in the above-mentioned hepatocyte culture supernatant, then HSC-T6 cells and culture supernatant were collected. Expression of Keap1, Nrf2 and MMP-2 of the HSC-T6 cells were detected by Western blotting, the activity of MMP-2 in supernatant of HSC-T6 cells was measured by gelatin zymography. Results: After stable knock-down of Keap1,the expression of Nrf2 was increased and the expression of TNF-α was decreased in the hepatocytes. Compared with the control group,the culture supernatant of hepatocytes with Keap1 knock-down reduced the expression of MMP-2, Cav-1, MT1-MMP, TIMP-2 protein and increased the activity of MMP-2 in HSC-T6 cells. Conclusion: When hepatocytes with Keap1 knock-down are under hypoxic conditions, the degree of hepatocyte injury is reduced, and the synthesis and release of TNF-α are decreased,which may reduce the stimulation of Cav-1 in endocytic molecules of HSC-T6 cells. Cav-1 interferes with MMP-2 activation on the membrane surface, in this condition the MMP-2 activity is increased.

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