基于PRRSV致炎作用探讨苦参碱调控IL-1β的机制

It is reported that Matrine has anti-inflammatory effect and inhibits IL-1β expression. However the regulation mechanisms remains unclear. Our previous study has demonstrated that Matrine has a significant therapeutic effect on interstitial pneumonia in mice induced by PRRSV/PCV2 co-infection. So far, LPS was normally used as an inflammatory inducer for studying the anti-inflammatory effect and mechanism. The virus infection was rarely used as an inflammatory inducer for this purpose. In this proposal, the 5ˊUTR transcripts of PRRSV will be used as an inducer to induce the inflammatory response in porcine pulmonary alveolar macrophage (PAM) as an in vitro model and interstitial pneumonia in weaning piglets induced by PRRSV infection will be used as an in vivo model to investigate the anti-inflammatory mechanisms of Matrine. qPCR and ELISA will be used to assess the effect of Matrine on IL-1β expression both in vitro and in vivo models. The piglets lung pathological changes will be observed by histopathologic examination and transmission electron microscope. The effect of Matrine on the counts of various white blood cells in bronchoalveolar lavage (BAL) will be determined by flow cytometry. Western blot will be used to detect the effect of Matrine on the key proteins during the expression of proIL-1β and mature IL-1β. In summary, our study will be focus on elucidating the regulation mechanism of Matrine on IL-1β expression both in cell model and target animal model, basing on the inflammatory response induced by PRRSV.

研究表明，苦参碱具有抗炎作用并可抑制IL-1β的产生，但其调控IL-1β产生的机制目前仍不清楚。申请人研究发现苦参碱对PRRSV/PCV2感染诱导的小鼠间质性肺炎有显著治疗作用。目前，药物抗炎作用及机制的研究多以LPS作为致炎因子，鲜有以病毒感染诱导炎症反应为研究模型。因此，本项目以PRRSV 5ˊUTR转录物诱导猪肺泡巨噬细胞（PAM）炎症反应为体外模型、以PRRSV感染断奶仔猪造成间质性肺炎为体内模型探讨苦参碱的抗炎机制；采用qPCR和ELISA检测苦参碱在体内外对IL-1β产生的影响；病理组织切片和透射电镜观察苦参碱对仔猪肺脏病理变化的改变；流式细胞术检测苦参碱对支气管肺泡灌洗液（BAL）中各种白细胞含量的影响；Western blot检测苦参碱对proIL-1β和成熟IL-1β产生过程中关键蛋白的作用。基于PRRSV致炎作用，从细胞模型和靶动物模型阐释苦参碱调控IL-1β表达机制。

猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）感染主要引起猪的繁殖障碍和间质性肺炎，给养猪生产造成了极大的经济损失。前期研究证明苦参碱具有抗PRRSV的作用，对PRRSV/PCV2共感染昆明小鼠诱导的间质性肺炎有显著的治疗作用，还可以抑制LPS诱导的小鼠急性肺损伤。炎症是临床常见的病理过程，而IL-1β是最主要的多效性炎症因子之一。因此，为阐明苦参碱的抗炎作用机理，本项目分别建立原代猪肺泡巨噬细胞（Porcine alveolar macrophages，PAM）炎症模型和PRRSV感染断奶仔猪间质性肺炎模型，评价苦参碱的抗炎作用。针对IL-1β前体和成熟IL-1β的产生过程，阐释苦参碱的抗炎机制。.结果表明：4 μg PRRSV 5'UTR RNA和1 μg/mL LPS共刺激PAM 12 h可显著诱导IL-1β的表达，可用于评价苦参碱抗炎作用及机制研究。qPCR和western blot结果表明苦参碱显著抑制IL-1β在PAM细胞上的表达。通过颈部肌肉注射和滴鼻PRRSV病毒，成功建立PRRSV诱导断奶仔猪间质性肺炎模型。经肺组织HE染色，苦参碱处理组可显著缓解PRRSV诱导的间质性肺炎症状。qPCR检测肺脏灌洗液PRRSV N基因的表达量，提示苦参碱可显著抑制PRRSV在肺脏中的复制。Western blot检测肺脏组织IL-1β的表达量，结果表明，苦参碱可显著抑制IL-1β在肺脏中的表达。以上结果表明苦参碱在细胞模型和在体模型均具有抑制IL-1β表达的作用。针对苦参碱的抗炎机制，qPCR和western blot 检测IL-1β产生过程中的相关基因和蛋白表达量，结果表明：苦参碱可显著抑制NOD2、MyD88、NLRP3、Caspase-1、p-IκBα以及细胞核中p65蛋白的表达，升高胞浆蛋白中p65蛋白的表达，但是对TLR4、DHX36和RIPK2的蛋白表达没有显著影响，免疫荧光显示苦参碱可抑制ASC斑点的形成。这些结果提示苦参碱通过抑制MyD88/NF-κB信号通路以及NLRP3炎症小体的激活干扰IL-1β的分泌。.本项目证实苦参碱在PRRSV 5'UTR RNA和LPS共刺激PAM的炎症模型及PRRSV诱导断奶仔猪间质性肺炎模型中均具有显著的抗炎作用。其抗炎