增强的中性粒细胞呼吸爆发对黏液型铜绿假单胞菌生物膜及其藻酸盐的影响

Effect of enhanced neutrophil respiratory burst on mucoid Pseudomonas aeruginosa biofilm and its alginate

目的:在体内探讨中性粒细胞呼吸爆发增强后对黏液型铜绿假单胞菌(Pseudomonas aeruginosa,P.a)生物膜(biofilm,BF和其重要的胞外基质藻酸盐的影响。方法:建立大鼠气道黏液型P.a BF感染模型,分别用呼吸爆发增强剂(PMA)(BF-PMA组)和生理盐水(BF-对照组)干预,平板计数法检测BF内活菌数目,扫描电镜观察BF的结构;硫酸-咔唑法测定BF内藻酸盐含量;HE染色观察2组肺组织病理损伤情况、ELISA分析炎症反应水平。结果:扫描电镜显示BF-PMA组的BF结构较对照组更复杂、更厚,且BF-PMA组的BF活菌数目明显高于对照组(P=0.000);BF-PMA组BF内藻酸盐合成明显高于对照组(P=0.004);HE染色发现BF-PMA组肺组织病理改变更严重,且炎症反应更重,γ干扰素(interferon-γ,IFN-γ)水平较对照组高(P=0.002),IFN-γ与白介素-10(interleukin-10,IL-10)的比值也高于对照组(P=0.023)。结论:在体内中性粒细胞呼吸爆发增强后可能通过促进黏液型P.a BF的重要胞外基质藻酸盐的合成,导致黏液型P.a BF清除更难,同时伴随更严重的病理损害和炎症反应。

Objective:To investigate the effect of enhanced neutrophil respiratory burst on mucoid Pseudomonas aeruginosa biofilm and alginate,the main in extracellular matrix,in vivo. Methods:A rat model of mucoid Pseudomonas aeruginosa biofilm infection in the airway was established,and the rats were treated with the respiratory burst enhancer PMA(BF-PMA group) or normal saline(control group). The plate count method was used to measure the number of live bacteria in biofilm;a scanning electron microscope was used to observe the structure of biofilm;the sulfuric acid-carbazole method was used to measure the content of alginate in biofilm;HE staining was used to observe pathological injury of lung tissue;ELISA was used to measure the levels of inflammatory factors.Results:The scanning electron microscope showed that compared with the control group,the BF-PMA group had a significantly more complex structure and significantly greater thickness of the biofilm,as well as a significantly higher number of live bacteria in the biofilm(P=0.000). The BF-PMA group had a significantly higher level of alginate synthesis in biofilm than the control group(P=0.004). HE staining showed that compared with the control group,the BF-PMA group had significantly greater pathological changes of lung tissue,a significantly higher degree of inflammatory response in lung tissue,and a significantly higher level of interferon gamma(IFN-γ)(P =0.002),as well as a significantly higher ratio of IFN-γ to interleukin-10(P =0.023). Conclusion:Enhanced neutrophil respiratory burst in vivo may promote the synthesis of alginate,an important substance in the extracellular matrix of mucoid Pseudomonas aeruginosa biofilm,and make it more difficult to eliminate mucoid Pseudomonas aeruginosa biofilm. It is often associated with severe pathological injury and inflammatory response.