基于NF-κB通路探索柴银颗粒治疗甲型H1N1流感的作用机制

Mechanism of Chaiyin(柴银)Granules in the Treatment of H1N1 Influenza A Based on NF-κB Pathway

目的:基于核转录因子-κB(NF-κB)信号通路研究柴银颗粒治疗甲型流感的分子机制。方法:将30只Balb/c小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、柴银颗粒1.26、2.52 g/kg组,每组6只,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,感染后2 h各组灌胃给予相应药物或蒸馏水,连续给药5 d。小鼠于感染后第6 d摘眼取血,测定肺指数,用苏木精-伊红染色(HE)观察肺部病变情况;ELISA法测定血清中干扰素-γ(IFN-γ)、白细胞介素-4(IL-4)、IL-6含量;Real-time PCR法检测动物肺组织病毒载量及NF-κB信号通路相关基因表达水平;Western blot法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加(P<0.01),肺病毒载量的相对表达显著升高(P<0.01),血清IFN-γ、IL-4、IL-6含量显著升高,肺组织Nfkb、Ikba mRNA表达上调,磷酸化P65、IκBα蛋白表达上调(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,柴银颗粒各组均可明显降低血清IFN-γ、IL-6含量(P<0.05或P<0.01),升高IL-4含量(P<0.01),可下调NF-κB信号通路相关基因或蛋白表达。结论:柴银颗粒对感染甲型流感小鼠具有保护作用,可能与调控NF-κB信号通路发挥抗炎及免疫调节作用有关。

Objective:To study the mechanism of Chaiyin(柴银)Granules in the treatment of influenza A based on the nuclear factor-κB(NF-κB)signaling pathway.Methods:Thirty Balb/c mice were randomLy divided into a blank group,a model group,an oseltamivir phosphate group(0.026 g/kg),and Chaiyin Granule groups(2.52 g/kg and 1.26 g/kg),with 6 mice in each group.After 3 days of adaptive feeding,the model group and each medication group were given nasal drops of influenza virus,while mice in the blank group were given nasal drops of the same amount of normal saline.Two hours after modeling,each group was given intragastric administration based on the corresponding drug or distilled water for 5 consecutive days.On the 6 th day after the infection,the eyeball blood was taken,the lung index was measured,and the pulmonary lesions were observed by hematoxylin-eosin staining(HE).The content of interferon-gamma(IFN-γ),interleukin-4(IL-4),and interleukin-6(IL-6)in the serum was determined by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(real-time PCR)was used to detect the viral load in the lung tissue and the expression levels of NF-κB signal pathway-related genes.Western blotting was used to detect the protein expression levels in the lung tissue of each group.Results:After modeling,the lung index was increased significantly in the model group as compared with the blank group(P<0.01),and the relative expression of pμLmonary viral load was increased significantly(P<0.01),and the serum expression levels of IFN-γ,IL-4,and IL-6 were significantly increased.In lung tissue,the mRNA expression of Nfκb and Iκbαwere up-regulated,and the protein expression of p-P65 and p-IκBαwere up-regulated(P<0.01).As compared with the model group,HE staining showed that the pathological condition of the lung tissue in the medication groups was improved after administration.In the Chaiyin Granule groups,the serum expression levels of IFN-γ and IL-6 were significantly decreased(P<0.05 or P<0.01),and the expression level of IL-4 was increased(P<0.01).The Results of real-time PCR and Western blot showed that Chaiyin Granules exerted an anti-influenza effect by down-regulating the expression of genes or proteins related to the NF-κB signaling pathway.Conclusion:Chaiyin Granules have a protective effect on mice infected with H1 N1 influenza A,and the mechanism may be related to its anti-inflammatory and immunomodulatory effects by regulating the NF-κB signal pathway.