UⅡ/UT系统对LPS刺激枯否细胞TRAF6和ROS表达以及自噬与凋亡的影响

Effect of UⅡ/UT System on LPS-stimulated TRAF6 and ROS Expres- sion;Autophagy and Apoptosis in Kupffer Cells

目的在分析urotensin（UⅡ）／urotensin Ⅱ receptor（uT）系统对LPS（lipopolysaccharide）刺激枯否细胞（Kupffcr cell，KC）肿瘤坏死因子受体相关因子6（TNF receptor．associated factor6，TRAF6）表达和活性氧簇（reactive oxygen species，ROS）释放效应的基础上，探讨该系统对细胞自噬和凋亡的影响。方法采用胶原酶灌注、不连续密度梯度离心和选择性贴壁等方法分离培养SD大鼠原代KC。将其随机分为A组：LPS（-）urantide（-）；B组：LPS（-）urantide（＋）；C组：LPS（＋）urantide（-）；D组：LPS（＋）urantide（＋）。细胞mRNA和蛋白质表达分别采用real—timePCR和Western印迹检测，ROS水平采用2’，7’-二氯荧光素二乙酸酯（DCFH—DA）试剂盒检测。结果细胞TRAF6mRNA、TRAF6蛋白、ROS含量、LC3mRNA、LC3-Ⅱ蛋白和p62mRNA相对表达水平均为C组较A组和B组高，D组较C组低（均为P〈0．01）；P62蛋白、CLEAVEDCASPASE-3蛋白相对表达水平均为C组较A组和B组高，D组较c组高（均为P〈0．01）。此外，BCL-2蛋白相对表达水平为c组较A组和B组低，D组较c组低（均为P〈0．01）。以上差异均有显著统计学意义。结论UⅡ／UT系统介导LPS刺激KC炎症和氧化应激反应可能与细胞自噬增强和凋亡抑制有关。

Objective To investigate the effect of urotensin （UⅡ） /urotensin Ⅱreceptor （UT） system on the expression of lipopolysaceharide （LPS） -stimulated TNF receptor-associated factor 6 （TRAF6） in Kupffer cells, and the effects of this system on autophagy and apoptosis of Kupffer cells based on the release effect of reactive oxygen species （ROS） . Methods The prima- ry Kupffer cells of SD rats were isolated and cultured by collagenase digestion, discontinuous densi- ty gradient centrifugation and selective adherence. The ceils were treated with urantide before LPS stimulation. They were randomly divided into group A： LPS （ - ） urantide （ - ） ; group B： LPS （-） urantide （＋）; group C： LPS （＋） urantide （-）; group D： LPS （＋） urantide （ ＋ ） . The mRNA and protein expressions of the cells were detected by real-time PCR and Western blotting respectively. The ROS level was detected by the 2＇, 7＇-dichlorofluorescein diacetate （DCFH-DA） kit. Results The relative expression levels of TRAF6 mRNA, TRAF6 protein, ROS, LC3 mRNA, LC3-Ⅱ protein and p62 mRNA were much higher in group C than in groups A and B, and lower in group D than in group C （allP 〈0.01 ） . The relative expression levels of P62protein and cleaved caspase-3 protein were higher in group C than in groups A and B, and higher in group D than in group C （allP 〈 0.01 ） . In addition, the relative expression level of BCL-2 protein was lower in group C than in groups A and B, and lower in group D than in group C （allP 〈 0. 01 ）. Conclusion The inflammatory and oxidative stress responses mediated by U Ⅱ/UT system may be associated with autophagy increase and apoptosis decrease in LPS-stimulated Kupffer cells.