苦参碱抗PCV2诱导小鼠肺炎的作用及其机制研究

Effect and mechanisms of matrine inhibits PCV2-induced pneumonia in mice

为探讨苦参碱在小鼠体内的抗炎作用及其机制,本研究利用猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染诱导小鼠肺炎模型,将32只昆明小鼠均分为空白对照组、PCV2组、苦参碱处理组(40mg/kg)和利巴韦林阳性对照组(40mg/kg)。除空白对照组外,其他各组小鼠每只腹腔注射0.5mL PCV2,攻毒后的第5天开始腹腔注射对应药物,每天给药一次,连续给药5d。在攻毒后的第11天,观察小鼠肺脏的病理组织学变化,qPCR检测PCV2 Cap基因,IL-1β、IL-6、IL-8和TNF-α基因的mRNA表达,Western blot检测相应炎症因子、TLR4/MyD88/NF-κB信号通路及NLRP3炎症小体关键蛋白的表达。结果表明:1)苦参碱可显著抑制PCV2感染诱发的肺间质增宽现象。2)苦参碱可显著降低肺脏PCV2病毒载量(P<0.05)及炎症因子IL-1β(P<0.05)、IL-6(P<0.0001)、IL-8(P<0.0001)和TNF-α(P<0.0001)的mRNA和蛋白(P<0.0001)的相对表达量。3)苦参碱可显著降低TLR4(P<0.0001)、MyD88(P<0.0001)、p-IκBα(P<0.0001)、NF-κB p65(P<0.01)、NLRP3(P<0.0001)、ASC(P<0.0001)和Caspase-1(P<0.0001)的蛋白表达量,升高IκBα(P<0.001)的蛋白表达量。综上,苦参碱可通过抑制TLR4/MyD88/NF-κB信号通路的活化以及NLRP3炎症小体的激活,来发挥抗PCV2诱导的小鼠肺炎作用,为进一步揭示苦参碱的抗病毒抗炎作用提供数据支撑,为苦参碱作为新兽药开发奠定基础。

In order to investigate the anti-inflammatory effect and mechanism of matrine in mice,32Kunming mice were divided into blank control group,PCV2group,matrine treatment group(40 mg/kg)and positive control group(ribavirin 40mg/kg),respectively.Except for the normal control group,mice in the other groups were intraperitoneally injected with 0.5mL PCV2.Starting on the 5th day after PCV2inoculation,the corresponding drugs were injected intraperitoneally once a day for five days.On the 11th day after challenge,the histopathological changes of mouse lungs were observed,and PCV2 Cap gene,the pro-inflammatory cytokines(IL-1β,IL-6,IL-8,TNF-α)in the lung tissues of each group were detected by qPCR.Western blot was used to detect inflammatory cytokines,key proteins related to TLR4/MyD88/NF-κB pathway and NLRP3inflammasome.The results showed that:1)Matrine significantly inhibited pulmonary widening induced by PCV2infection.2)Matrine significantly reduced the expression of PCV2 Cap gene(P<0.05)and the inflammatory factors including IL-1β(P<0.05),IL-6(P<0.0001),IL-8(P<0.0001)and TNF-α(P<0.0001)mRNA and protein(P<0.0001).3)Matrine inhibited the protein expression levels of TLR4(P<0.0001),MyD88(P<0.0001),p-IκBα(P<0.0001),NF-κB p65(P<0.01),NLRP3(P<0.0001),ASC(P<0.0001)and Caspase-1(P<0.0001),while increased IκBαexpression(P<0.001).In conclusion,matrine can inhibit the activation of TLR4/MyD88/NF-κB signaling pathway and NLRP3inflammasome to relieve pneumonia induced by PCV2in mice,which provide data support for further revealing the antiviral and anti-inflammatory mechanisms of matrine,and lay a foundation for matrine as a new veterinary medicine development.