小檗碱通过铁死亡途径的对RSV感染小鼠病毒载量及肺部炎症反应的调节作用

Berberine Regulates RSV Viral Load and Pulmonary Inflammation in Mice Infected with RSV Via Iron Death Pathway

为了基于核因子E2相关因子2(Nuclear factor E2 related factor 2, Nrf2)-谷胱甘肽过氧化物酶4(Glutathione peroxidase 4,Gpx4)通路介导的铁死亡途径,探讨小檗碱对呼吸道合胞病毒(Respiratory syncytial virus,RSV)诱导的肺炎小鼠模型体内病毒载量和肺部炎症反应的影响及其作用机制,本研究利用雄性BALB/c幼龄小鼠建立RSV诱导的肺炎动物模型,并随机分为空白组、模型组、低浓度小檗碱组(30mg/kg)、中浓度小檗碱组(45mg/kg)、高浓度小檗碱组(60mg/kg)和阳性药(利巴韦林)组,每组10只。除空白组外,均采用RSV滴鼻的方法制备肺炎小鼠模型,造模后各组分别予相应的药物干预,空白组和模型组给予生理盐水。给药4d后处死小鼠,摘取肺组织并称重计算肺指数;RT-PCR检测肺组织核因子E2相关因子2(Nuclear factor E2 related factor 2,Nrf2)、谷胱甘肽过氧化物酶4(Glutathione Peroxidase 4,Gpx4)和RSV-F的mRNA表达水平;Western blot法检测肺组织Nrf2和Gpx4蛋白表达水平;ELISA法检测肺泡灌洗液中白细胞介素-6(Interleukin-6,IL-6)和肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)水平;HE染色观察小鼠肺组织病理变化。实验数据显示,与空白组相比,模型组肺指数升高,肺部Nrf2、Gpx4的mRNA表达水平降低、RSV-F的mRNA表达水平升高,肺部Nrf2和Gpx4的蛋白表达水平降低,肺泡灌洗液中IL-6、TNF-α的含量升高,数据均具有统计学意义(P<0.05),且模型组小鼠肺组织明显充血,并伴有大量炎症性细胞浸润;与模型组相比,低、中、高浓度小檗碱组肺指数均下降,肺部Nrf2、Gpx4的mRNA表达水平均降低、肺部Nrf2和Gpx4的蛋白表达水平均降低,肺泡灌洗液中IL-6、TNF-α的含量均升高,且小鼠肺组织病理损伤得到不同程度改善;与阳性药组相比,高浓度小檗碱组肺指数、肺部Nrf2和Gpx4的mRNA表达水平及蛋白表达水平、肺泡灌洗液中IL-6和TNF-α的含量都无显著差异。根据以上实验结果,推测小檗碱可以通过铁死亡途径发挥抗RSV病毒和减轻肺部炎症的作用,其主要作用机制涉及到上调Nrf2和Gpx4基因的表达水平,以及下调IL-6、TNF-α等炎症因子的表达水平,降低肺部RSV病毒载量。

In order to explore the effects of berberine on viral load and pulmonary inflammation in mice with respiratory syncytial virus(RSV)‐induced pneumonia based on the iron death pathway mediated by Nrf2‐Gpx4 pathway,male BALB/c young mice were used to establish animal model of RSV‐induced pneumonia.The mice were randomly divided into control group,RSV group,low berberine group,medium berberine group,high berberine group and ribavirin group with 10 mice in each group.Except the control group,the mouse model of pneumonia was established by RSV nasal drip.After establishment of the model,each group was given corresponding drug intervention,and the blank group and model group were given saline.Four days after administration,mice were sacrificed and lung tissues were removed and weighed to calculate the lung index.The mRNA expression of nuclear factor E2 related factor 2(Nrf2),glutathione peroxidase 4(Gpx4)and RSVF in lung tissue was detected by RT‐PCR.The expression levels of Nrf2 and Gpx4 protein in lung tissue were detected by Western blot.The levels of interleukin‐6(IL‐6)and tumor necrosis factor‐α(TNF‐α)in bronchoalveolar lavage fluid were detected by ELISA.The pathological changes of lung tissue in mice were observed under microscope by HE staining.Results showed that compared with the control group,the lung index of the RSV group were increased,the mRNA expression of Nrf2 and Gpx4 were decreased,the mRNA expression of RSV‐F increased,the protein expression of Nrf2 and Gpx4 decreased,and the contents of IL‐6 and TNF‐αin BALF increased.The data were statistically significant(P<0.05),and the lung tissue of the model group was obviously congested with a large number of inflammatory cell infiltration.Compared with the RSV group,the lung index in three‐concentration berberine groups decreased,the mRNA expression of Nrf2 and Gpx4 in the lung decreased,the mRNA expression of RSV‐F increased,the protein expression levels of Nrf2 and Gpx4 in the lung decreased,and the contents of IL‐6 and TNF‐αin BALF increased,All these changes were in dose‐dependent manner.The pathological injury of lung tissue of mice was improved in varying degrees.Compared with the positive drug group,there was no significant difference in lung index,mRNA and protein expression levels of Nrf2 and Gpx4,and contents of IL‐6 and TNF‐αin BALF in high concentration berberine group.According to above results,it is speculated that berberine can play the role of anti‐RSV virus and reducing pulmonary inflammation through iron death pathway,and its main mechanism is to up‐regulate the expression of Nrf2 and Gpx4 genes,down‐regulate the expression of inflammatory factors such as IL‐6 and TNF‐α,and reduce the load of RSV virus in the lungs.