HIF-2α通过抑制炎症反应减轻大鼠脑出血损伤

HIF-2α attenuates intracerebral hemorrhage injury in rats by inhibiting inflammatory response

摘要:

目的：探讨缺氧诱导因子2α（hypoxia-inducible factor 2α，HIF-2α）在脑出血损伤后（intracerebral hemorrhage，ICH）的作用，并明确其是否参与调控脑出血后的炎症反应。方法：健康雄性SD大鼠95只，其中35只随机分配用于以下时间点分析：Sham、12 h、24 h、48 h、3 d、5 d和7 d，每组5只；另外60只随机分为Sham组（假手术组，注射等量生理盐水）、ICH组（脑出血模型组组，胶原酶诱导的脑出血模型）、Vehicle组（空载体组组，建模前注射空载慢病毒载体）和Oe-HIF-2α组（HIF-2α过表达组，建模前注射HIF-2α过表达慢病毒载体）用于脑含水量检测、神经功能评分、免疫印记分析和免疫荧光检测。通过脑含水量和Garcia神经功能评分评估脑损伤的严重程度；免疫印迹检测HIF-2α及肿瘤坏死因子-α（tumor necrosis factor α，TNF-α）、白细胞介素-18（interleukin 18，IL-18）和白细胞介素-1β（interleukin 1β，IL-1β）的表达水平，免疫荧光检测病灶周边髓过氧化物酶（myeloperoxidase，MPO）的表达。结果：HIF-2α表达在ICH后24 h（0.555 4±0.070 2，P=0.000）开始上调并于3 d（2.368 4±0.346 6，P=0.000）达到峰值，随后降低；与ICH组相比较，Oe-HIF-2α组脑3 d时含水量明显降低（0.793 5±0.002 5，P=0.000），神经功能明显改善（14.700 0±0.674 9，P=0.000）；与ICH组相比，Oe-HIF-2α组TNF-α（1.350 4±0.191 5，P=0.000）、IL-1β（1.158 4±0.070 8，P=0.000）、IL-18（0.784 2±0.073 9，P=0.000）等炎性介质在3 d时表达明显降低；Oe-HIF-2α组（3.500 0±0.534 5）病灶周边区域MPO表达明显低于ICH组（5.125 0±0.991 0，P=0.002）。结论：脑出血后，HIF-2α表达上调并且通过抑制TNF-α、IL-1β、IL-18等炎症因子的表达，抑制炎症反应减轻大鼠脑出血损伤。

Abstract:

Objective：To investigate the role of hypoxia-inducible factor 2α（HIF-2α） in intracerebral hemorrhage（ICH） injury，and to determine whether HIF-2α is involved in the regulation of inflammatory response after ICH. Methods：There were 95 healthy male Sprague-Dawley rats，and 35 of them were randomly assigned to seven groups for different data collection time points：Sham，12 h，24 h，48 h，3 d，5 d，and 7 d，with 5 rats in each group. The other 60 rats were randomly divided into Sham group（sham operation group with injection of the same amount of normal saline），ICH group（ICH model group with collagenase-induced ICH），Vehicle group（empty vector group with injection of empty lentiviral vector before modeling），and Oe-HIF-2α group（HIF-2α over－expression group with injection of HIF-2α overexpression lentiviral vector before modeling） for brain water content mea－surement，neurological function scoring，Western blot analysis，and immunofluorescence detection. The severity of brain injury was assessed by brain water content and Garcia neurological deficit scores. The expression levels of HIF-2α，tumor necrosis factor α（TNF-α），interleukin 18（IL-18），and interleukin 1 beta（IL-1β） were determined by Western blot，and the expression of myeloper－oxidase（MPO） was determined by immunofluorescence assay. Results：The expression of HIF-2α began to increase at 24 hours after ICH（0.555 4±0.070 2，P=0.000），peaked on day 3（2.368 4±0.346 6，P=0.000），and then decreased. Compared with the ICH group on day 3，the Oe-HIF-2α group had significantly decreased brain water content（0.793 5±0.002 5，P=0.000），significantly improved neurological function（14.700 0±0.674 9，P=0.000），and significantly reduced expression of inflammatory mediators including TNF-α（1.350 4±0.191 5，P=0.000），IL-1β（1.158 4±0.070 8，P=0.000），and IL-18（0.784 2±0.073 9，P=0.000）；the expression of MPO in the surrounding area of lesion was significantly lower in the Oe-HIF-2α group than in the ICH group（3.500 0±0.534 5 vs. 5.125 0±0.991 0，P=0.002）. Conclusion：After ICH，HIF-2α is upregulated，and it can suppress the expression of inflammatory factors includ－ing TNF-α，IL-1β，and IL-18 to reduce inflammatory response and ICH injury in rats.