藏药十二味翼首散通过抑制NF-κB介导的NLRP3炎症小体/细胞焦亡通路改善甲流病毒感染小鼠急性肺损伤

Tibetan medicine Shierwei Yishou San alleviates acute lung injury in influenza A virus-infected mice by inhibiting the NF-κB-mediated NLRP3 inflammasome/pyroptosis pathway

关键词:  十二味翼首散  甲型H1N1流感病毒  急性肺损伤  NLRP3炎症小体  细胞焦亡

Key words:  Shierwei Yishou San (SYS)  influenza A (H1N1) virus  acute lung injury  NLRP3 inflammasome  pyroptosis

摘要:

目的 探讨十二味翼首散对甲型H1N1流感病毒诱导急性肺损伤的保护作用，并阐明其潜在的分子机制。方法 将108只BALB/c小鼠随机分为6组：溶媒对照组、模型对照组、低剂量组、中剂量组、高剂量组和阳性对照组，每组18只。除溶媒对照组外，其余各组均采用H1N1-PR8毒株滴鼻感染建立流感病毒感染小鼠急性肺损伤模型；给药组分别灌胃给予十二味翼首散（低、中、高剂量）以及阳性药物磷酸奥司他韦。通过生存率、体重、肺组织病毒载量/滴度、炎症反应和病理损伤程度来评价对甲流病毒感染急性肺损伤的药效作用。利用网络药理学预测十二味翼首散改善H1N1诱导肺损伤的潜在机制，并利用上述体内药效实验的肺组织标本验证相关通路机制关键基因与蛋白表达。结果 药效实验显示，十二味翼首散对H1N1感染小鼠的生存率和体重有一定保护作用，可明显抑制肺组织病毒复制，改善小鼠体内的炎症反应和病理损伤。网络药理学预测提示，十二味翼首散四味核心药材和H1N1、肺损伤有关的潜在机制可能与NOD样受体通路中NLRP3炎症小体/细胞焦亡通路相关。机制验证表明，十二味翼首散抑制NF-κB活化，下调NLRP3、Caspase-1、GSDMD的mRNA及蛋白表达，并抑制Caspase-1活化及其介导的GSDMD切割。结论 十二味翼首散可通过抑制NF-κB介导的NLRP3炎症小体/细胞焦亡通路，改善H1N1流感病毒诱导的急性肺损伤。

Abstract:

OBJECTIVE To investigate the protective effect of Shierwei Yishou San (SYS) on acute lung injury induced by the influenza A (H1N1) virus, and to elucidate its potential molecular mechanism. METHODS 108 BALB/c mice were randomly divided into 6 groups: vehicle control group, model control group, low-dose group, mid-dose group, high-dose group, and positive control group, with 18 mice in each group. Except for the vehicle control group, all other groups were infected intranasally with the H1N1-PR8 strain to establish an acute lung injury model in influenza virus-infected mice. The drug-treated groups were administered SYS (low, mid, and high doses) or the positive drug oseltamivir phosphate by gavage. The pharmacodynamic effects on acute lung injury induced by influenza A virus infection were evaluated by survival rate, body weight, viral load/titer in lung tissues, inflammatory response, and the degree of pathological damage. Network pharmacology was used to predict the potential mechanisms by which SYS alleviates H1N1-induced lung injury, and lung tissue specimens from the aforementioned in vivo pharmacodynamic experiments were used to verify the expression of key genes and proteins in the relevant pathway mechanisms. RESULTS Pharmacodynamic experiments showed that SYS had a certain protective effect on the survival rate and body weight of H1N1-infected mice, significantly inhibited viral replication in lung tissues, and ameliorated inflammatory responses and pathological damage in mice. Network pharmacology prediction suggested that the potential mechanisms associated with the four core herbs of SYS, H1N1, and lung injury might be related to the NLRP3 inflammasome/pyroptosis pathway within the NOD-like receptor pathway.Mechanistic studies demonstrated that Shierwei Yishou San attenuated NF-κB activation, reduced the mRNA and protein expression of NLRP3, Caspase-1, and GSDMD, and inhibited Caspase-1 activation as well as the subsequent cleavage of GSDMD.CONCLUSION SYS can ameliorate H1N1 influenza virus-induced acute lung injury by inhibiting the NF-κB-mediated NLRP3 inflammasome/pyroptosis pathway.