生长停滞特异性蛋白6对脓毒症小鼠急性肺损伤的保护研究

Protective effects of GAS6 on acute lung injury in septic mice

目的研究生长停滞特异性蛋白6（Gas6）对脓毒症急性肺损伤小鼠的保护作用，并探讨其作用机制。方法取90只BALB／c雄性小鼠，按随机数字表法分为假手术组、脓毒症组、脓毒症＋生理盐水组、脓毒症＋Gas6干预（1μg）组、脓毒症＋Gas6干预（5μg）组、脓毒症＋Gas6干预（10μg）组，每组15只。脓毒症和脓毒症＋Gas6干预组行盲肠结扎穿孔术（cecal ligation and puncture，CLP），Gas6干预组术后立即尾静脉注射Gas6。假手术组仅行开腹，不予以结扎穿孔。术后24h收集血液，酶联免疫吸附法（ELISA）检测血清中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）水平；处死小鼠，留取肺组织，测定肺组织湿／干质量比值（W／D）；观察肺组织病理学改变，计算肺组织病理评分；蛋白免疫印迹法（Westernblotting）检测肺组织胞核NF-κB p65及胞浆IκB-α蛋白表达。统计学方法采用SPSS23．0统计软件，两组样本比较采用独立样本t检验，多组间比较采用单因素方差分析检验。结果与假手术组相比较，脓毒症组小鼠血清TNF-α（P=0．000）、IL-1β（P=0．000）、肺组织湿／干重比值（W／D）（P=0．000）、肺组织病理评分（P=0．000）、肺组织胞核NF-κBp65蛋白含量（P=0．006）的表达均增加，而肺组织胞浆IκB-α（P=0．001）表达明显下降。与脓毒症＋生理盐水组相比，脓毒症＋Gas6（1、5、10μg）组小鼠24h后血清中TNF-α（P=0．000）、IL-1β（P=0．000）、肺W／D（P=0．000）、肺组织病理评分（P=0．000）、肺组织胞核NF-κBp65蛋白含量（P=0．000）的表达水平降低，而胞浆IκB-α（P=0．009）表达水平明显上升并呈一定的剂量依赖关系。结论Gas6能够有效减轻脓毒症所致的肺损伤，其分子作用机制可能与抑制炎性反应以及下调NF-κBp65胞核内转位有关。

Objective To explore the protective effect of GAS6 （growth arrest-specific gene 6 protein） on lung injury in septic mice so as to elucidate the mechanism. Methods Ninty male BALB/c mice were divided into 6 groups （ n = 15 ） ： sham operation group, CLP group, CLP ＋ vehicle group, CLP ＋ GAS6 group （ 1 μg）, CLP ＋ GAS6 group （5μg）, CLP ＋ GAS6 group （ 10 μg）. Mice in CLP groups and GAS6 groups were made to be sepsis models by CLP. GAS6 was administrated after CLP in GAS6 groups by intravenous injection. And mice in sham group were only treated with laparotomy without CLP. And 24 h later, all survived mice were sacrificed to obtain blood and lung tissue. Serum levels of TNF-α, IL-1 were measured by using ELISA. The lung wet/dry ratio was calculated. Histopathological changes of lung tissues were observed under light microscope. NF-κB and IκB-α were assayed by Western blotting. Data were analyzed by SPSS 23.0. Statistical analyses were performed using independent sample t-test to comparebetween two groups or one-way analysis of variance test to compare among mu｜tipie groups. Results The serum levels of TNF-α （ P =0. 000 ）, IL-1β （ P = 0. 000 ）, the lung W/D （ P = 0. 000 ）, pulmonary pathological change （P = 0. 000） , the levels of NF-κB （P = 0. 006） in CLP group were higher than those in sham group, while IκB-α （ P = 0. 001 ） in CLP groups was lower than those in sham group. The GAS6 significantly reduced the serum levels of TNF-α （ P = 0. 000）, IL-1β （ P = 0. 000）, the lung W/D （ P = 0. 000 ） , pathogenesis of lung tissue （ P = 0. 000） and the levels of NF-κB （ P = 0. 000） in septic mice. The IκB-α （P = 0. 009） was increased after treatment with GAS6 in a dose-dependent manner. Conclusions GAS6 has protective effect on septic lung injury in mice, and its molecular mechanism may be associated with inhibiting inflammatory response and suppressing NF-κB nuclear translocation.