内毒素在小鼠过敏性哮喘模型所起作用的实验研究

Experimental study on role of endotoxin in induction of murine model of allergic asthma

目的建立小鼠过敏性哮喘模型,在过敏原的致敏和激发阶段给予不同剂量内毒素(即脂多糖LPS),探讨内毒素在哮喘模型中的作用机制。方法建立BALB/c小鼠哮喘模型,设立实验组和对照组开展后续实验。实验组包括:鸡卵清白蛋白(OVA)致敏,OVA激发组;OVA致敏,LPS激发组;OVA致敏,OVA+LPS联合激发组;LPS暴露于OVA致敏前,OVA激发组;对照组包括试剂对照组和健康对照组。各实验组的LPS剂量均分为高、中、低三个剂量组。通过测定小鼠气道阻力和肺顺应性来反映其肺功能改变;流式细胞术(FCM)测定FITC、PE阳性细胞数,表征CD4+CD2+5调节性T淋巴细胞(以下简称Treg细胞)的相对比例;荧光实时聚合酶链反应定量测定肺脏和脾脏Foxp3mRNA表达的水平;ELISA法测定血浆总IgE和OVA特异性IgE水平,抗体夹心ELISA法测定支气管肺泡灌洗液(BALF)中Th2细胞因子(IL-4和IL-5)的水平;计数BALF中白细胞总数和嗜酸性粒细胞等各类白细胞所占百分比;常规病理切片观察肺组织改变情况。结果结果表明,与哮喘模型组小鼠相比,在LPS各处理组中,总体上能诱导Treg细胞的产生,改善肺功能,降低IgE滴度,降低Th2介导的免疫反应,同时诱导肺部炎症。尤其是在致敏阶段腹腔注射内毒素可以缓解由过敏原诱导的气道炎症和改善肺功能,诱导肺组织和脾组织Foxp3mRNA表达的增加。结论LPS抑制了Th2介导的过敏反应,可能与Foxp3表达量的增加相关,后者诱导Treg细胞增殖,对哮喘症状起到缓解作用。

Objective To study the effect of lipopolysaccharide （LPS） on allergen sensitization and challenge in allergic asthma model of mice based on a murlne model of allergic asthma. Methods BALB/c mice were separated into four experimental groups and two control groups. Those were allergen （OVA） sensitization and challenge one, OVA sensitization and exposure to LPS one, OVA sensitization and exposure to LPS during OVA challenge one, and exposure to LPS during OVA sensitization and OVA challenge one. There were also reagent group and healthy one as control. Each experimental group was treated with three doses of LPS. Lung functional disorder was demonstrated by assessing resistance of airway and pulmonary compliance, Expression of CD4^＋CD25^＋ regulatory T cells （Treg cells） was measured by flow cytometry （FCM）. Expressions of transcription factor, Foxp3 mRNA, which was extracted from lung and spleen issues, were assayed by real-time quantitative polymerase chain reaction （real-time PCR）. Total and OVA-specific IgE levels in blood plasma were quantified by ELISA assay. Inflammation was assessed by determining total and differential leukocyte counts and T-helper type 2 cytokine （IL-4 and IL-5） levels in bronehoaleolar lavage fluid （BALF）. Lung tissues were sliced and stained to observe the changes of histology by light microscope. Results The results showed that LPS might shift cytokine response toward a Thl response and away from a Th2 response, might reduce the level of plasma IgE and induce proliferation of Treg cells, thereby confer benefits on lung function of experimental animals, More significant immunoprotection roles in the groups treated with LPS were found through peritoneal injection before allergen sensitization. Less severe inflammatory response, less lung function injury and higher expression of Foxp3 were noticed in the group. Conclusion It was suggested that endotoxin might restrain Th2 response and might be related to the expression increase of Foxp3, hence subsequently might alleviate asthmatic symptoms.