鼻滴脂多糖对新生小鼠肺发育的影响及机制探讨

Effect of lipopolysaccharide via intranasal instillation on lung development in neonatal mice and its mechanism

目的:观察鼻滴脂多糖(lipopolysaccharide,LPS)对新生小鼠肺发育的影响及NF-κB、MIP-1α和VEGFR-2的变化,探讨出生后肺部炎症在支气管肺发育不良发病中的作用及可能的机制。方法:新生1 d内的C57BL/6小鼠20只随机分为生理盐水组和LPS组:LPS组于出生后1 d(P_1)到出生后13 d(P_(13))每天鼻滴LPS 25 mg/kg,生理盐水组P_1到P_(13)每天鼻滴等量生理盐水。第14天收集2组小鼠肺组织标本进行检测,运用HE染色观察各组小鼠肺组织形态学改变,免疫组化观察CD31的表达并进行肺微血管密度计数,qRT-PCR检测VEGFR-2、MIP-1αmRNA水平变化,Western blot检测P65、IκBα、磷酸化P65(Ser536)、磷酸化IκBα(Ser32)及VEGFR-2蛋白水平的变化。结果:与生理盐水组相比,LPS组HE染色表现为肺泡增大,血管周围可见炎性细胞浸润。LPS组放射性肺泡计数(4.533±0.166)低于生理盐水组(8.377±0.290)(P=0.000),肺泡平均截距(54.890±2.074)高于生理盐水组(32.750±0.787)(P=0.000)。CD31免疫组化示LPS组肺微血管密度(3.387±0.007)低于生理盐水组(5.631±0.014)(P=0.000)。q RT-PCR示LPS组VEGFR-2 m RNA表达下降(P=0.000),MIP-1αm RNA表达升高(P=0.000)。Western blot示LPS组P65蛋白、磷酸化P65(Ser536)及磷酸化IκBα(Ser32)蛋白水平升高,IκBα蛋白及VEGFR-2蛋白水平降低(P=0.000)。结论:鼻滴脂多糖抑制新生小鼠肺发育,出生后肺部炎症可能参与支气管肺发育不良的发生,其机制可能和VEGFR-2表达下降、NF-κB激活上调MIP-1α有关。

Objective :To investigate the effect of lipopolysaccharide(LPS)via intranasal instillation on the lung development in neonatal mice,and to explore the role of postnatal pulmonary inflammation in the development of bronchopulmonary dysplasia and its mechanism.Methods :A total of 20C57BL/6mice on postnatal day 1(P1)were randomly divided into LPS group arid saline group.The LPS group was given LPS(25mg/kg/day)via intranasal instillation from P1 to postnatal day 13(P13),while the saline group was given an equal volume of saline via intranasal instillation from P1 to P13.The mice were randomly sacrificed on postnatal day 14(P14)for col- lection of the lung tissue.The morphological changes in the lung tissue were observed by HE staining.The expression of cluster of differentiation 31(CD31)was measured by immunohistochemistry to determine pulmonary microvasctilar density.The mRNA expression of vascular endothelial growth factor receptor 2(VEGFR-2)and macrophage inflammatory protein-1 alpha(MIP-1α)was determined by quantitative real-time PCR (qRT-PCR).The protein expression of nuclear factor kappa-B p65subunit (P65),nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha(IκBα),phosphorylated P65(Ser536),phosphorylated Iκ- Bα(Ser32),and VEGFR-2wag measured by Western blot. Results:HE staining showed that compared with the saline group,the LPS group showed alveolar enlargement and inflammatory cell infiltration around blood vessels;the LPS group had a significantly lower radial alveolar count(4.533±0.166vs.8.377±0.290,P=0.000)and a significantly higher mean linear intercept (54.890±2.074 vs. 32.750±0.787,P=0.000).The immunohistochemistry results of CD31showed that the LPS group had a significandy lower microvascular density than the salitie group(3.387±0.007vs.5.631±0.014,P=0.000).qRT-PCR showed that compared with the saline group,the LPS group had significantly lower mRNA expression of VEGFR-2and significantly higher mRNA expression of MIP-1α(P=0.000;P=0.000).Western blot showed that the LPS group had signifieandy increased protein expression of P65, Ser536,and Ser32 and signifieandy reduced protein expression,of IκBα and VEGFR-2compared with the saline group (all P=0.000). Conclusion:LPS via intranasal instillation can inhibit the lung development in neonatal mice and postnatal pulmonary inflammation may be involved in the development of bronchopulmonary dysplasia.The mechanism may be related to downregulation of VEGFR-2, activation of NF-κB,and upregulation of MIP-1α.