SU5416在小鼠急性肺损伤中的作用及其机制研究

Effect and mechanism of vascular endothelial growth factor inhibitor SU5416 on acute lung injury in mice

目的探讨血管内皮生长因子抑制剂Z-3-[(2,4-二甲基吡咯-5-烃基)亚甲基]-2-吲哚满酮(SU5416)在小鼠急性肺损伤(ALI)中的作用及其机制。方法将30只SPF级雄性C57BL/6小鼠随机分为模型组(0.9%氯化钠溶液灌胃)、SU5416组(SU5416灌胃)、地塞米松组(地塞米松灌胃)、SU5416+地塞米松组(SU5416+地塞米松灌胃)、空白对照组(0.9%氯化钠溶液灌胃),各6只。模型组、SU5416组、地塞米松组、SU5416+地塞米松组采用脂多糖(LPS)滴鼻制备小鼠ALI模型。观察并比较5组小鼠支气管肺泡灌洗液(BALF)中性粒细胞、淋巴细胞计数;使用试剂盒检测肺组织超氧化物歧化酶(SOD)、活性氧簇(ROS)活性;ELISA法检测肺组织炎症因子IL-1β、IL-6、TNF-α、MCP-1水平;Western blot法检测肺组织bcl-2、TLR4、VEGF、NF-κB蛋白水平;观察肺组织病理形态。结果 5组小鼠BALF中性粒细胞、淋巴细胞计数比较差异均有统计学意义(均P<0.05)。5组小鼠肺组织SOD、ROS活性比较差异均有统计学意义(均P<0.05),模型组小鼠肺组织SOD活性明显下降,ROS活性明显上升。5组小鼠肺组织IL-1β、IL-6、TNF-α、MCP-1水平比较差异均有统计学意义(均P<0.05),模型组小鼠肺组织IL-1β、IL-6、TNF-α、MCP-1水平均明显上升。5组小鼠肺组织bcl-2、TLR4、VEGF、NF-κB蛋白水平比较差异均有统计学差异(均P<0.05),模型组小鼠肺组织在LPS刺激下抗凋亡蛋白bcl-2的表达下调,TLR4、VEGR、VEGFR2和pNF-κB的表达上调。SU5416组与地塞米松组小鼠肺组织的肺泡结构破坏、水肿,肺泡毛细血管充血、出血,肺泡间隔增厚,炎症细胞浸润等病理状态较模型组改善明显。SU5416与地塞米松均能明显逆转以上LPS刺激下小鼠ALI的改变,两者无明显差异,且两者联用的逆转改变明显强于其中一种单独使用。结论 SU5416对小鼠ALI具有保护作用,其作用机制可能是通过减轻炎症反应、氧化应激以及调控TLR4/VEGF/NF-κB信号通路实现。

Objective To investigate the effect of vascular endothelial growth factor inhibitor SU5416 on acute lung injury in mice and the underlying mechanism. Methods Thirty C57BL/6 mice were randomly divided into 5 groups. Acute lung injury was induced by tranasal administration of LPS in mice. The mice in model group, SU5416 group, dex group, SU5416+dex group and blank control group were given normal saline (NS), SU5416, dexamethasone, SU5416+dexamethasone and NS by gavage respectively. Bronchoalveolar lavage fluid (BALF) was obtained by lung perfusion, and the number of neutrophils and lymphocytes was measured by automatic hematology analyzer. HE staining and electron microscopy were used to examine the pathological changes in lung tissues, and the concentrations of inflammatory factors IL-1β, IL-6, TNF-ɑ, MCP-1, and the activities of SOD/ROSin lung tissues were detectedby ELISA. The expression of related proteins (bcl-2/TLR4/VEGF/NF-κB) was detected by Western blot. Results SU5416 and dexamethasone significantly decreased the number of neutrophils, lymphocytes in BALF, and the concentrations of IL-1β, IL-6, TNF-ɑand MCP-1;inhibited ROS and increased SODactivity in lung tissues. SU5416 and dexamethasone also attenuated alveolar structural damage, edema, alveolar capillary congestion, bleeding, alveolar septum thickening, inflammatory cell infiltration and other pathological conditions in lung tissues. SU5416 had regulatory effect on the expression of TLR4, VEGF, VEGFR, Bcl-2 and the phosphorylation of NF-κB. Both SU5416 and dexamethasone significantly reversed the changes of ALI induced by LPS in mice. There was no significant difference between SU5416 and dexamethasone,and the combined effects of two were significantly stronger than those used alone. Conclusion SU5416 has a protective effect on acute lung injury in mice, which may be achieved by controlling inflammation, oxidative stress and regulating TLR4/VEGF/NF-κB pathway.