急性中耳炎中IL-27通过信号通路PI3K对肺炎链球菌清除的影响

Effect of IL-27 on the clearance of streptococcus pneumoniae through the PI3K pathway in acute otitis media

目的探讨白细胞介素-27(IL-27)对急性中耳炎(AOM)中肺炎链球菌的清除作用以及其对磷脂酰肌醇-3-激酶(PI3K)信号的调节作用。方法采用肺炎链球菌感染建立急性中耳炎小鼠模型。将实验小鼠随机分为正常组、模型组、实验组、对照组。实验组在感染肺炎链球菌前12 h耳后注射200μL的浓度为5μg/mL的IL-27;对照组在感染肺炎链球菌前12 h耳后注射200μL的浓度为5μg/mL的PI3K特异性抑制剂2-(4-吗啉基)-8-苯基-4H-1-苯并吡喃-4-酮[2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-on,LY294002],正常组、模型组注射等剂量的生理盐水。模型组、实验组、对照组小鼠每只耳鼓膜前下方注射5μL肺炎链球菌,正常组注射等剂量的生理盐水。在感染注射5 d后,进行听性脑干反应(ABR)测试,检测中耳灌洗液(MELF)中炎性细胞和细菌数量,ELISA测定MELF中肿瘤坏死因子α(TNF-α)、IL-6的表达,苏木精-伊红染色(HE)检测各组小鼠中耳组织的病理变化,Western blot检测各组小鼠中耳组织中磷酸化磷脂酰肌醇-3激酶(p-PI3K)、磷酸化丝/苏氨酸蛋白激酶(p-AKT)的表达。结果与正常组相比,模型组小鼠ABR阈值、炎性细胞以及细菌数、TNF-α、IL-6以及p-PI3K、p-AKT的表达明显升高;中耳组织AOM损伤病理明显;与模型组相比,实验组和对照组小鼠ABR阈值、炎性细胞以及细菌数、TNF-α、IL-6以及p-PI3K、p-AKT的表达明显降低,中耳组织AOM损伤病理明显减轻;差异均具有统计学意义(P<0.05)。结论在肺炎链球菌感染的急性中耳炎中,IL-27能通过抑制PI3K信号的磷酸化来调节中性粒细胞对病菌的清除。

Objective To explore the effect of IL-27 on pneumococcal clearance by regulating PI3K signaling pathway in acute otitis media(AOM).Methods A mouse model of AOM was established with Streptococcus pneumoniae infection.The mice were randomly divided into normal group,model group,experimental group and control group.Twelve hours before the infection with Streptococcus pneumoniae,the mice in the experimental group received retroauricular injection with 200μL of IL-27(5μg/mL),those in the control group with the same amount of PI3K specific inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-on(LY294002)(5μg/mL),and those in the normal group and the model group with the same amount of normal saline.The mice in the model group,the experimental group and the control group were each injected with 5μL of Streptococcus pneumoniae in the front and bottom of the eardrum,and the normal group was given the same dose of normal saline.Five days after the infection injection,an auditory brainstem response(ABR)test was performed.The number of inflammatory cells and bacteria in middle ear lavage fluids(MELF)was detected.ELISA was used to determine the expression of TNF-αand IL-6 in MELF.Hematoxylin-eosin(HE)staining was used to detect the pathological changes in the middle ear tissue.Immunohistochemistry and Western blot were used to detect the expressions of phosphorylation of phosphatidylinositol 3-kinase(p-PI3K)and phosphorylated serine/threonine kinase(p-AKT)in the middle ear tissue.Results Compared with the normal group,the ABR threshold,the numbers of inflammatory cells and bacteria,the expressions of TNF-α,IL-6,p-PI3K,and p-AKT in the model group mice were significantly increased,and the AOM lesion pathology in the middle ear tissue was significantly increased.Compared with the model group,the ABR threshold,the numbers of inflammatory cells and bacteria,the expressions of TNF-α,IL-6,p-PI3K,and p-AKT in the experimental and control mice were significantly reduced,and the pathology of AOM damage in the middle ear tissue was significantly reduced.The differences were all statistically significant(all P<0.05).Conclusion In AOM with Streptococcus pneumoniae infection,IL-27 can regulate the neutrophil clearance of pathogens by inhibiting the phosphorylation of PI3K signal.