基于p38 MAPK/NF-κB通路探索荆防合剂治疗甲型H1N1流感的作用机制

Mechanism of Jingfang(荆防)Mixture in Treatment of H1N1 Influenza A Based on p38 MAPK/NF-κB Pathway

目的:基于p38丝裂原活化蛋白激酶(p38 MAPK)/核转录因子-κB(NF-κB)信号通路研究荆防合剂治疗甲型流感的分子机制。方法:将36只Balb/c雌鼠随机分为正常对照组、模型对照组、磷酸奥司他韦0.026 g/kg组、荆防合剂3.9、7.8、15.6 g/kg组,每组6只,适应性饲养3 d后进行造模处理,模型对照组和各给药组滴鼻流感病毒,正常对照组滴鼻等体积的生理盐水。小鼠于感染后第7 d摘取眼球血,测定肺指数,用苏木精-伊红染色(HE)法观察肺部病变情况;用酶联免疫吸附法(ELISA)测定血清中干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)、IL-4、IL-10含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测肺组织病毒载量及p38 MAPK/NF-κB信号通路相关基因表达,蛋白免疫印迹技术(Western blot)检测肺组织中蛋白表达。结果:造模后,与正常对照组相比,模型对照组肺指数显著增大,肺病毒载量的相对表达量显著升高(P<0.01),血清IFN-γ、IL-2、IL-10含量显著升高,IL-4含量显著降低(P<0.01),肺组织p38 Mapk、Nfkb p65、Ikba mRNA及蛋白表达显著上调(P<0.01);与模型对照组相比,给药后,HE染色显示肺组织病理状况得以改善,荆防合剂可明显降低血清IFN-γ、IL-2、IL-6含量(P<0.05或P<0.01),升高IL-4含量(P<0.01),Real-time PCR和Western blot结果显示荆防合剂可通过下调肺组织p38 MAPK/NF-κB信号通路相关基因或蛋白表达从而发挥抗甲型流感作用。结论:荆防合剂对感染甲型流感小鼠具有保护作用,可能与调控p38 MAPK/NF-κB信号通路,发挥抗炎及免疫调节等作用有关。

Objective:To study the molecular mechanism of Jingfang(荆防)Mixture in the treatment of influenza A based on p38 mitogen-activated protein kinase(p38 MAPK)/nuclear factor-κB(NF-κB)signaling pathway.Methods:A total of 36 Balb/c female mice were randomly divided into blank group,model group,oseltamivir phosphate group(0.026 g/kg),and Jingfang Mixture groups(3.9,7.8 and 15.6 g/kg),6 in each group.After 3 days of adaptive feeding,mice in model group and each medication group were given intranasal delivery of influenza virus,while mice in blank group received intranasal delivery of the same amount of normal saline.On the 7th day after infection,blood was taken from mouse eyeball.Lung indexes were measured,and pulmonary lesions were observed by hematoxylin-eosin(HE)staining.The serum contents of interferon-gamma(IFN-γ),interleukin-2(IL-2),interleukin-4(IL-4)and interleukin-10(IL-10)were determined by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(real-time PCR)was performed to detect the viral load in lung tissue and the expression of p38 MAPK/NF-κB signaling pathway-related genes.Western blot was used to determine the protein expression in the lung tissue.Results:After modeling,compared with blank group,the model group presented increased lung indexes(P<0.01)and up-regulated relative expression of viral load in lung tissue(P<0.01).The levels of IFN-γ,IL-2 and IL-6 were increased,while the levels of IL-4 was decreased(P<0.01)in serum.The mRNA and proteins expressions of p38 MAPK,NF-κb p65,and IκB-αwere up-regulated.Compared with the conditions in model group,HE staining showed that the pulmonary lesions after administration were alleviated.Jingfang Mixture decreased the serum levels of IFN-γ,IL-2 and IL-6(P<0.05 or P<0.01)and increased the level of IL-4(P<0.01).The real-time PCR and Western blot revealed that Jingfang Mixture played an anti-influenza effect by down-regulating the expression of genes or proteins related to p38 MAPK/NF-κB signaling pathway.Conclusion:Jingfang Mixture has a protective effect on mice infected with H1N1 influenza A,and the mechanism may be associated with its anti-inflammatory and immunomodulatory effect through regulating p38 MAPK/NF-κB signaling pathway.