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苦参碱抗PCV2诱导小鼠肺炎的作用及其机制研究
孙娜1,张司寰1,岑龙座1,曹志刚1,张华1,孙盼盼2,孙耀贵1,范阔海2,尹伟1,李宏全1*
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(1.山西农业大学 动物医学学院/中兽医药现代化山西省重点实验室, 山西 太谷 030801;2.山西农业大学 实验动物管理中心, 山西 太谷 030801)
摘要:
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(40 mg/kg)和利巴韦林阳性对照组(40 mg/kg)。除空白对照组外,其他各组小鼠每只腹腔注射0.5 mL 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5 d。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-8TNF-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<0.000 1)、IL-8(P<0.000 1)和TNF-α(P<0.000 1)的mRNA和蛋白(P<0.000 1)的相对表达量。3)苦参碱可显著降低TLR4(P<0.000 1)、MyD88(P<0.000 1)、p-IκBα(P<0.000 1)、NF-κB p65(P<0.01)、NLRP3(P<0.000 1)、ASC(P<0.000 1)和Caspase-1(P<0.000 1)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。
关键词:  苦参碱  猪圆环病毒2型  TLR4/MyD88/NF-κB  NLRP3炎症小体
DOI:10.11841/j.issn.1007-4333.2022.10.13
投稿时间:2022-03-10
基金项目:国家自然科学基金面上项目(32172904);山西省高等学校科技创新项目(2021L108);山西省中央引导地方科技发展资金项目(YDZJSX2021A035)
Effect and mechanisms of matrine inhibits PCV2-induced pneumonia in mice
SUN Na1,ZHANG Sihuan1,CEN Longzuo1,CAO Zhigang1,ZHANG Hua1,SUN Panpan2,SUN Yaogui1,FAN Kuohai2,YIN Wei1,LI Hongquan1*
(1.Shanxi Key Laboratory for Modernization of TCVM/College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China;2.Laboratory Animal Center, Shanxi Agricultural University, Taigu 030801, China)
Abstract:
In order to investigate the anti-inflammatory effect and mechanism of matrine in mice, 32 Kunming mice were divided into blank control group, PCV2 group, matrine treatment group(40 mg/kg)and positive control group(ribavirin 40 mg/kg), respectively. Except for the normal control group, mice in the other groups were intraperitoneally injected with 0. 5 mL PCV2. Starting on the 5th day after PCV2 inoculation, 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 NLRP3 inflammasome. The results showed that: 1)Matrine significantly inhibited pulmonary widening induced by PCV2 infection. 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. 000 1), IL-8(P<0. 000 1)and TNF-α(P<0. 000 1)mRNA and protein(P<0. 000 1). 3)Matrine inhibited the protein expression levels of TLR4(P<0. 000 1), MyD88(P<0. 000 1), p-IκBα(P<0. 000 1), NF-κB p65(P<0. 01), NLRP3(P<0. 000 1), ASC(P<0. 000 1)and Caspase-1(P<0. 000 1), while increased IκBα expression(P<0. 001). In conclusion, matrine can inhibit the activation of TLR4/MyD88/NF-κB signaling pathway and NLRP3 inflammasome to relieve pneumonia induced by PCV2 in 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.
Key words:  matrine  PCV2  TLR4/MyD88/NF-κB  NLRP3 inflammasome