博落回提取物对副猪嗜血杆菌诱导的猪肺泡巨噬细胞炎症反应及信号通路影响的研究

The effect of Macleaya cordata extract on the inflammation and signaling pathways induced by Glaesserella parasuis in porcine alveolar macrophages

博落回(M.cordata)是一种常用的中药,具有抗炎、抗病毒、抗氧化、抗菌、杀虫和抗肿瘤等多种生物学活性。为了探究博落回提取物对副猪嗜血杆菌(G.parasuis)诱导的猪肺泡巨噬细胞(PAM)炎症反应及NF-κB和MAPK信号通路的影响,本研究采用12.5 ng/mL、25 ng/mL、50 ng/mL、100 ng/mL和200 ng/mL的博落回提取物分别预处理PAM 4 h,再利用1×107 cfu/mL副猪嗜血杆菌刺激PAM 12 h。以博落回提取物溶剂(TSB培养液)预处理PAM再感染副猪嗜血杆菌作为感染对照组。分别提取各组细胞的RNA和蛋白质,将RNA反转录成cDNA作为模板,采用qRT-PCR检测各组细胞中IL-1α、IL-1β、IL-6、IL-8和TNF-αmRNA的转录水平。通过western blot检测各组细胞中NF-κB信号通路蛋白p65、p-p65、IκBα和p-IκBα及MAPK信号通路蛋白JNK、p-JNK、ERK、p-ERK、p38和p-p38的表达量。结果显示,与空白对照组相比,感染对照组细胞炎性因子IL-1α、IL-1β、IL-6、IL-8和TNF-αmRNA的转录水平均显著升高(P<0.05),而不同浓度博落回提取物预处理均能够显著抑制副猪嗜血杆菌诱导炎性细胞因子mRNA的转录水平(P<0.05),且与博落回提取物的浓度呈正相关。Western blot结果显示,与空白对照组相比,感染对照组NF-κB信号通路蛋白p-p65和p-IκBα及MAPK信号通路蛋白p-JNK、p-ERK和p-p38的表达水平均显著升高(P<0.05),而不同浓度的博落回提取物预处理后均能够显著抑制副猪嗜血杆菌诱导的p-p65、p-IκBα、p-JNK、p-ERK和p-p38蛋白的表达水平(P<0.05),且基本与博落回提取物的浓度呈正相关;各组细胞中NF-κB信号通路蛋白p65和IκBα及MAPK信号通路蛋白JNK、ERK和p38的表达水平均无显著差异。本研究首次证实博落回提取物通过抑制PAM中NF-κB和MAPK信号通路的活化从而抑制副猪嗜血杆菌诱导PAM的炎性反应,为副猪嗜血杆菌病的防治提供了一种新的思路。

Macleaya cordata,a commonly used traditional Chinese medication,has been shown to possess several biological activities,such as anti-inflammatory,antiviral,antioxidative,antimicrobial,insecticidal,and antitumor activities.The aim of this study was to verify the effect of M.cordata extract on inflammation and NF-κB and MAPK signaling pathways induced by Glaesserella parasuis in porcine alveolar macrophages(PAMs).In this study,PAMs were pretreated with 12.5ng/mL,25ng/mL,50ng/mL,100ng/mL,and 200ng/mL of M.cordata extract for 4 hours.PAMs was then stimulated with 1×107cfu/mL G.parasuis for 12 hours.The PAMs pretreated with M.cordata extract solvent(TSB culture medium)were reinfected with G.parasuis and used as the infection control group.The RNA and protein lysate of cells were extracted respectively.The extracted RNA was reversed into cDNA.The mRNA transcription levels of IL-1α,IL-1β,IL-6,IL-8,and TNF-αwere then detected by qRT-PCR.The NF-κB signaling pathway proteins p65,p-p65,IκBαand p-IκBαand the MAPK signaling pathway proteins JNK,p-JNK,ERK,p-ERK,p38 and p-p38 in each group were detected by western blot.The results showed that the mRNA transcription levels of inflammatory factors IL-1α,IL-1β,IL-6,IL-8,and TNF-αwere significantly increased(P<0.05)in the infection control group compared with those in the blank control group,while the mRNA transcription level for these factors above induced by G.parasuis was significantly inhibited in the groups pretreated with the different concentrations of M.cordata extracts(P<0.05),and this inhibitory effect was positively correlated with the concentration of the M.cordata extract.Western blot results showed that the protein expression levels of NF-κB signaling pathway proteins p-p65 and p-IκBαand MAPK signaling pathway proteins p-JNK,p-ERK and p-p38 were significantly increased(P<0.05)in the infection control group compared with those in the blank control group,but the expression levels of p-p65,p-IκBα,p-JNK,p-ERK and p-p38 proteins were significantly inhibited after pretreatment with different concentrations of M.cordata extracts(P<0.05),which were basically positively correlated with the concentration of M.cordata extract.There were no significant differences in the expression levels of NF-κB signaling pathway proteins p65 and IκBαand MAPK signaling pathway proteins JNK,ERK and p38 in all groups.This study confirmed firstly that M.cordata extract could regulate the inflammatory response of PAMs induced by G.parasuis by inhibiting the activation of NF-κB and MAPK signaling pathways and the expression of inflammatory factors,which provide a new idea for the prevention and treatment of G.parasuis.