新生期肺炎链球菌肺炎对VA的影响及其与肺部免疫及气道反应性的关系研究

The effect of neonatal pneumococcal pneumonia on vitamin A and its relationship with pulmonary immunity and airway responsiveness

目的研究新生期肺炎链球菌肺炎(Streptococcus pneumoniaepneumonia,S.pp)对维生素A(VA)水平的影响及其与肺部免疫及气道反应性的关系。方法 Balb/c小鼠出生后第7天鼻腔滴注2×10~7CFU肺炎链球菌5μl建立新生期S.pp模型,对照组滴注等量PBS,新生期肺炎链球菌肺炎补充VA组(S.pp+VA)感染后口饲补充VA,每天1次,连续4天,对照组给予等量VA溶解溶质菜籽油。收集感染后第7、14、21、28天肺组织、血清、肝脏组织标本,高效液相色谱仪(HPLC)监测组织中VA水平。小鼠成年后肺组织病理切片HE染色观察肺组织病理改变,体积描记法检测小鼠肺功能,ELISA检测支气管肺泡灌洗液(BALF)上清IL-4、IL-5、IL-13、IL-17A、INF-γ水平,流式细胞术检测肺组织中的CD4+T细胞亚群Th1、Th2及Foxp3^+Treg。结果 S.pp组小鼠肺组织VA持续降低至感染后4周,血清VA降低直至感染后2周恢复正常,而肝脏中VA水平无统计学差异。S.pp+VA组肺组织炎性细胞浸润较S.pp组显著减轻,S.pp+VA组BALF中IL-4、IL-5、IL-13及IL-17A水平显著低于S.pp组(P<0.05),IFN-γ则显著增高(P<0.01),肺组织Th1、Foxp3^+Treg显著高于S.pp组(P<0.01),Th2水平明显降低(P<0.01),且Th1/Th2比值显著增高(5.7 vs 1.7,P<0.001))。S.pp+VA组小鼠气道反应性显著低于S.pp组(P<0.001)。结论新生期肺炎链球菌肺炎致肺部VA水平持续降低至成年期,肺炎后补充VA能减轻新生期肺炎链球菌肺炎后肺组织炎性细胞浸润,促进Foxp3^+Treg表达、纠正Th1/Th2失衡,抑制成年期气道高反应性形成。

To investigate the effect of neonatal Streptococcus pneumoniae pneumonia（S.pp） on vitamin A status and its relationship with pulmonary immunity and airway responsiveness,neonatal（1-week-old） Balb/c mice were infected intranasally in conscious with 2 × 10^7 CFU of S.pneumoniae（D39） in 5 μl of PBS（S.pp group）,while,mock-infected mice were intranasally with 5 μl of PBS（control group）.Mice in S.pp＋VA group were infected with S.pneumoniae and then administrated orally with a dose of 20 IU/g of VA for 4 consecutive days,while mice in control group was administrated the same dose of VA dissolved solute（rapeseed oil）.To evaluate the effect of neonatal S.pp on vitamin A status in mice,we monitored vitamin A levels in lung,serum,and liver on 7,14,21,28 days post infection by HPLC; five weeks after infection,lung tissue slices were made and stained with HE to observe the lung pathology.Airway hyperresponsiveness（AHR） was measured in conscious and unrestrained mice by means of whole-body plethysmography.The levels of IL-4,IL-5,IL-13,IL-17 A and IFN-γ in BALF were examined by ELISA; and the levels of Th1,Th2 and Foxp3~＋Treg were determined by flow cytometry.Here we found that after neonatal S.pp,the VA status in lung decreased until the fourth week after infection,and the serum VA decreased until the 14 th day after infection,while the VA level in the liver showed no significant difference.Data showed that the lung inflammatory cells infiltration in S.pp ＋ VA group was alleviated,as compared with neonatal S.pp group;concentration of IFN-γ in BALF of S.pp＋VA group was significantly higher than that in the neonatal S.pp group（P〈0.01）,while concentrations of IL-4,IL-5,IL-13 and IL-17 A were significantly lower（P〈0 05）.Th1 and Foxp3~＋Treg cells were significant higher in S.pp＋VA group as compared with the neonatal S.pp group（P〈0.01）,while Th2 cells in S.pp＋VA group were significantly lower than that in the neonatal S.pp group（P〈0.01）.Furthermore,the ratio of Th1/Th2 was significantly increased in S.pp ＋ VA group as compared with the neonatal S.pp group（P〈0.001）.Besides,we found that airway hyperresponsiveness in S.pp ＋ VA group was significantly lower than that in the neonatal S.pp group（P〈0.001）.Taken together,neonatal S.pp can decrease lung VA significantly; supplementation of VA after neonatal S.pp can reduce lung inflammatory cells infiltration,improve the expression of Foxp3~＋Treg cells in lung,correct the imbalance of Th1/Th2 and inhibit the formation of airway hyperresponsiveness.