不同浓度脂多糖对支气管哮喘大鼠模型建立的影响及调节机制

Effect of E. coli lipopolysaccharide on tbe allergic airway inflammation of asthma rat

目的观察不同浓度大肠埃希菌脂多糖(E、coli LPS)对年幼期哮喘大鼠模型建立的影响及其调控机制。方法清洁级SD大鼠45只,随机分为对照组(A组)、哮喘组(B组)、LPS组(分为C1组、C2组、C3组),每组9只。用卵白蛋白(OVA)致敏与激发建立大鼠哮喘模型。光镜、电镜观察肺组织病理及超微结构的变化;支气管肺泡灌洗液(BALF)中嗜酸粒细胞(EOS)及其它炎症细胞计数;酶联免疫吸附试验(ELISA)法测定血清OVA-sIgE与BALF中TGF-β_1含量;TUNEL法检测肺组织EOS凋亡。结果①光镜电镜观察:光镜下B组见支气管及血管周围、肺间质及肺泡腔内炎性细胞浸润,支气管黏膜下水肿,黏液腺增生,黏膜皱折增多,部分黏膜上皮细胞脱落,可见气道黏液栓;C1组、C3组上述改变无明显减轻;C2组上述现象明显减轻。电镜下B组见EOS数量增多,周围有大量浆细胞聚集;C1组无明显改变;C2组凋亡的EOS增多;C3组中性粒细胞和凋亡的EOS均增多。②BALF中炎症细胞计数;B组BALF中细胞总数、EOS绝对计数和EOS占细胞总数的百分比(EOS%)均高于A组(均P<0.01);C2组均低于B组(均P<0.01)。③血清OVA-sIgE和BALF中TGF-β_1含量;血清OVA-sIgE检测,B组高于A组(P<0.01);C2组、C3组明显低于B组(均P<0.01);但C1组与B组比较无统计学意义(P>0.05)。BALF中TGF-β_1含量测定,B组与A组比较,两组有显著性差异(P<0.05);C2组、C3组明显高于B组(均P<0.01)。④肺组织EOS凋亡率检测结果,B组与A组比较,两组有显著性差异(P<0.05);C2组、C3组与B组比较EOS凋亡率均明显增高(均P<0.01);但C1组与B组比较无统计学意义(P>0.05)。相关分析结果,BALF中TGF-β_1水平与EOS数有非常显著性负相关(r=-0.48 3,P<0.01);EOS绝对计数与血清OVA-sIgE水平显著性正相关(r=0.634,P<0.01)。结论 E.coli LPS(>10μg/ml)通过降低OVA-sIgE水平,增加TGF-β_1分泌,诱导EOS凋亡,从而可能减轻变态反应症状,但过高浓度E.coli LPS(100μg/ml)可能会促使中性粒细胞增高。

Objective To observe the immune regulation of E. coli lipopolysaccharide （LPS） on the airway allergic inflammation of asthma rat model. Methods Forty-Eve Sprague Dawley （SD） rats were randomly divided into control group（A）,asthma group（B） and LPS group（C）. Group C were divided into three subgroups of 0.1 μg/ml（C1 ）, 10 μg/ml（C2） and 100μg/ml （C3）. Asthma model rats were sensitized with ovalbumin（OVA） and AI（OH）3, and repeatedly exposed to aerosolized OVA. The histopathology and ultrastructure change of pulmonary tissues observed by light microscope （LM） and transmission electron microscope（ TEM ）. EOS and other inflamation cells count in bronehoalveolar lavage fluid （BAI.F）were performed, Levels of OVA-sIgE in serum, TGF-β1 in BALF were measured by sandwich EHSA;Apoptosis of EOS in pulmonary tissue were detected by TUNEL. Results （1）Observation of LM and TEM： LM showed many inflammatory cells infiltration around the bronchus, vascular, bronchus mucus increased, airway epithelium damage anddes quamated,airway mucous plug in group B; group C1,C3 showed less lightened; group C2 showed obviously lightened. TEM showed EOS recruitment in group B;group C1 were similar with group A;group C2 showed increased EOS apoptosis,group C3 showed both neutrophil and apoptotie EOS increased. （2） Inflammationary cells count in BALF： compared with groupA, the total cellular score, EOS absolute count and EOS% in group B were all significantly increased（ P 〈0.01 ）;compared with group B, the total cellular score.EOS absolute count and EOS% were all significantly decreased in group C2. （2）Levels of OVA-sIgE and TGF-β1 ：Levels of OVA-sIgE in serum of group B were significantly higher than group A （ P 〈0.01） ;group C2,group C3 were all significantly lower than group B （ P 〈0.01）. Levels of TGF-β1 in BALF of group C2,group C3 were all significantly higher than group A （ P〈0.01） ,and those in group A and B had significant diference（ P 〈0.05）. （4）Apoptotic EOS detection： the percentages of apoptotic EOS in group B were significantly lower than those in group A （ P 〈0.01） ;compared with group B,group C2 and group C3 were increased（ P 〈0.01）. Correlative analysis, the level of TGF-β1 in BALF was significantly negative correlation with EOS absolute count （ r = -0. 483, P 〈0.01） ;EOS absolute count in BALF was significantly positive correlation with the levels of OVA-sIgE in serum（ r =0. 634, P 〈0.01 ）. Conclusions E. eoli LPS can decrease OVA-sIgE, increase TGF-β1 production, induce EOS apoptosis; but excess LPS exposure（100 μg/ml） may induce neutrophil increase.