TNF-α拮抗剂对大鼠缺血再灌注肺组织中致炎性细胞因子及HIF-1α表达的影响

Effects of Tumor Necrosis Factor-α Antagonist on Proinflammatory Cytokines and Hypoxia Inducible Factor-1α in Lung Ischemia-Reperfusion Injury in Rats

目的观察肿瘤坏死因子-α(TNF-α)拮抗剂TNFRI-Fc融合蛋白对大鼠肺缺血-再灌注损伤过程中肺内致炎性细胞因子释放与缺氧诱导因子-1α(HIF-1α)表达的影响。方法选择体质量250～350g的雄性SD大鼠54只,随机分为3组,假手术组(C组)6只仅行开胸,不行缺血及再灌注处理;;Ⅰ组与Ⅱ组各24只,均行左肺缺血和再灌注处理,Ⅱ组在肺门夹闭前及肺门开放前5min经颈静脉留置针推注溶于生理盐水中的可溶性TNFRI-Fc融合蛋白(3μg/㎏)。Ⅰ组与Ⅱ组于再灌注后1、3、6、12h各处死6只动物,C组于Ⅰ组、Ⅱ组再灌注后1h同时处死动物,均取左肺上2/3制成匀浆ELISA法检测TNF-α、白细胞介素(IL)-8、IL-6含量,下1/3通过免疫组化分析HIF-1α蛋白表达水平。结果Ⅰ组TNF-α、IL-8、IL-6含量在1h即较C组明显升高;;Ⅱ组各时间点肺组织匀浆内TNF-α、IL-8、IL-6含量较Ⅰ组明显降低,且TNF-α达峰时间明显错后;;HIF-1α蛋白主要表达于肺泡上皮细胞的胞核或胞浆,再灌注后6h达高峰,表达水平Ⅱ组较Ⅰ组明显降低(P<0.01),C组HIF-1α极少量表达。Ⅰ组和Ⅱ组的HIF-1α蛋白表达与TNF-α水平成正比。结论TNF-α拮抗剂通过有效中和TNF-α并阻断其生物学活性,抑制致炎性细胞因子释放、减弱HIF-1α蛋白表达,起到减轻缺血再灌注损伤的作用。

Objective To investigate the effects of tumor necrosis factor-α （TNF-α） antagonist （TNFRI-Fc） on the release of proinflammatory cytokines and the expression of hypoxia-inducible factor-1α （HIF-1α） in lung ischemia-reperfu- sion injury （I/R） in rats. Methods Fifty-four male SD rats （250 - 350 g） were randomly divided into sham operation group （group C, n=6）, ischemia-reperfusion （I/R） group （group Ⅰ , n=24） and TNFRI-Fc group （group Ⅱ , n=24）. Rats were killed 1, 3, 6 and 12 h after reperfusion （6 rats in each time point） respectively in group I and group Ⅱ. Values of TNF-α, IL-8 and IL-6 were detected by ELISA method using pulmonary homogenate of the left upper 2/3 lung tissue of rats. The HIF-1α ex- pression was detected by immunohistochemistry using left lower 1/3 pulmonary homogenate. Results The levels of TNF-1α, IL-6 and IL-8 were significantly higher in group Ⅰ than those of group C and group Ⅱ （P 〈 0.05）. But levels of TNF-1α, IL-6 and IL-8 were significantly lower in group Ⅱ than those of group I, and the peak level of TNF-1α was significantly pushed back. The expressions of HIF-1α were mainly in the nucleus or cytoplasm of alveolar epithelial cells, and reached the peak 6 h after reperfusion. The expression levels of HIF-1α were significantly lower in group Ⅱ than those of group Ⅰ （P 〈 0.01）. The very lower expression of HIF-1α was found in groupC. The expressions of HIF-1α were proportional to the level of TNF-1α in group I and group Ⅱ. Conclusion The TNF-1α antagonist can effective neutralize TNF-1α to block the biological activity, inhibit the release of proinflammatory cytokines and attenuate the expression of HIF-1α to protect the lung function against ischemia-reperfusion injury.