脂多糖滴鼻对新生小鼠肺微血管发育的影响

Influence of lipopolysaccharide via intranasal instillation on pulmonary microvascular development in neonatal mice

目的:探讨脂多糖(lipopolysaccharide,LPS)滴鼻对新生小鼠肺微血管发育的影响及机制。方法:将32只新生24 h内的C57BL/6小鼠按随机数字表法分为生理盐水组和LPS组,每组16只。LPS组采用鼻内滴入LPS 25 mg/kg,持续20 d,生理盐水组用等体积生理盐水代替。每组分别于实验的14 d、21 d收集肺组织标本进行检测,采用苏木素-伊红(HE)染色观察肺组织病理改变,免疫组化法检测肺组织中CD31、血管内皮生长因子(vascular endothelial growth factor,VEGF)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin-1β,IL-1β)表达,ELISA法检测肺组织匀浆中巨噬细胞炎性蛋白-1α(macrophage inflamma-tory protein-1α,MIP-1α)和单核细胞趋化蛋白-1(monocyte chemoattractant pro-tein-1,MCP-1)含量,RTPCR检测肺组织中VEGF及以上炎症因子基因的表达情况。结果:LPS组小鼠肺组织的病理改变表现为肺部炎症细胞浸润,肺泡简单化。LPS组14 d和21 d肺部微血管密度计数(microvessel density,MVD)均明显低于生理盐水组(F=7.646,P=0.000;F=9.596,P=0.000);肺组织VEGF蛋白表达也明显低于生理盐水组(F=10.375,P=0.000;F=7.091,P=0.000);LPS组TNF-α、IL-1β、MIP-1α和MCP-1蛋白表达明显升高(IL-1β:F=5.949,P=0.000;F=8.386,P=0.000;TNF-α:F=9.132,P=0.000;F=7.375,P=0.000;MIP-1α:F=4.248,P=0.003;F=4.827,P=0.000;MCP-1:F=5.990,P=0.000;F=4.107,P=0.001);LPS组VEGF、TNF-α、IL-1β、MIP-1α和MCP-1 mRNA表达水平分别与其蛋白表达水平趋势一致。结论:LPS诱导的出生后肺部炎症可导致新生小鼠肺微血管发育紊乱,其机制可能与炎症因子和趋化因子表达升高及VEGF表达下降有关。

Objective:To investigate the influence of lipopolysaccharide(LPS)via intranasal instillation on pulmonary microvascular development in neonatal mice and the possible mechanism.Methods:A total of 32 neonatal C57 BL/6 mice within 24 hours after birth were divided into normal saline group and LPS group using a random number table,with 16 mice in each group.The mice in the LPS group were given intranasal instillation of LPS 25 mg/kg for 20 consecutive days,and those in the normal saline group were given an equal volume of normal saline.Lung tissue samples were collected on days 14 and 21 of experiment,and HE staining was performed to observe lung pathological changes.Immunohistochemistry was used to measure the protein expression of cluster of differentiation 31(CD31),vascular endothelial growth factor(VEGF),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)in lung tissue;ELISA was used to measure the content of macrophage inflammatory protein-1α(MIP-1α)and monocyte chemoattractant protein-1(MCP-1)in lung homogenate;real-time PCR was used to measure the mRNA expression of VEGF and the above inflammatory cytokines in 1 ung tissue.Results:Lung pathological changes in the LPS group were pulmonary inflammatory cell infiltration and alveolar simplification.Compared with the normal saline group on days 14 and 21,the LPS group had significantly lower pulmonary microvessel density(F=7.646 and 9.596,both P=0.000)and protein expression of VEGF in lung tissue(F=10.375 and 7.091,both P=0.000).On days 14 and 21,the LPS group had significant increases in the protein expression of TNF-α(F=9.132 and 7.375,both P=0.000),IL-1β(F=5.949 and 8.386,both P=0.000),MIP-1α(F=4.248 and 4.827,P=0.003 and 0.000),and MCP-1(F=5.990 and 4.107,P=0.000 and 0.001),and the mRNA expression of VEGF,MIP-1α,MCP-1,TNF-α,and IL-1βin the LPS group showed a similar changing trend to their protein expression.Conclusion:LPS-induced postnatal pulmonary inflammation may lead to the disturbance of pulmonary microvascular development in neonatal mice,possibly by upregulating inflammatory factors and chemokines and downregulating VEGF expression.