Actinobacillus pleuropneumoniae (APP)為造成豬隻纖維素性、出血性、壞死性胸膜肺炎主要病原，Actinobacillus pleuropneumoniae exotoxin (Apx)為APP重要毒力因子之一，分為I~IV型，其中ApxI具有強烈的溶血性及細胞毒性。ApxI除可誘發細胞傷害外，亦刺激豬肺泡巨噬細胞(PAM) MAPK p38、JNK活化，進而調控前炎症細胞素IL-1β、IL-8及TNF-α之分泌。因NF-κB參與許多炎症相關基因的調控，而NF-κB於ApxI誘發前炎症細胞素表現中所扮演之角色尚不清楚，本研究主要目的為探討ApxI刺激PAM是否活化NF-κB，釐清NF-κB是否調控IL-1β、IL-8及TNF-α之生成及NF-κB與MAPK p38、JNK間是否相互調控。首先以APP血清型第10型所分泌ApxI作用於PAM，在不同時間點觀察NF-κB活化情形。以西方轉漬法分析IκB降解及免疫螢光染色偵測NF-κB位移，發現ApxI感作PAM 15分鐘，IκB即開始被降解，至30分鐘降解最顯著；免疫螢光染色則發現，ApxI感作60分鐘時可見明顯NF-κB轉移至細胞核。後續實驗則探討NF-κB是否參與IL-1β、IL-8及TNF-α表現之調控，先將PAM與NF-κB抑制劑isohelenin作用1小時，再以ApxI刺激2小時後，以即時定量PCR (RT-qPCR)及ELISA分析前炎症細胞素RNA及蛋白質表現量，發現isohelenin可有效抑制IL-1β、IL-8及TNF-α mRNA之表現及蛋白質生成。以上結果證實，ApxI刺激PAM可誘發IκB降解及NF-κB活化，且NF-κB參與前炎症細胞素IL-1β、IL-8及TNF-α表現之調控，而p38、JNK與NF-κB間的相互關係則有待進一步研究。
Actinobacillus pleuropneumoniae (APP) causes fibrinous, hemorrhagic necrotizing pleuropeneumonia in pigs. Actinobacillus pleuropneumoniae exotoxin (Apx) is one of the major virulence factors of APP. Among the four types of Apx identified, ApxI elicits its most significant effects on hemolysis and cytolysis. ApxI induces MAPK p38 and JNK activation and subsequent expression of proinflammatory cytokines IL-1β, IL-8, and TNF-α in porcine alveolar macrophages (PAM). NF-κB regulates the expression of many genes which participate in inflammatory and immune responses. However, up to now, the role of NF-κB in ApxI-induced proinflammatory cytokine expression remains unidentified. The goals of this study were to determine whether ApxI induces NF-κB activation, to assess the role of NF-κB in the expression of proinflammatory cytokines, and to investigate the correlation between NF-κB and MAPK. ApxI-derived from APP was used to stimulate PAM for different time periods. Western blot analysis and immunofluorescence assay were performed to detect IκB degradation and NF-κB translocation, respectively. IκB degradation was noted after 15 min of stimulation, and most apparent at 30 min. In immunofluorescence assay, NF-κB translocation from cytosol to nucleus was observed after 60 min of ApxI treatment. In addition, inhibitor isohelenin was used to investigate the role of NF-κB in regulating IL-1β, IL-8, and TNF-α expression. PAM pre-treated with isohelenin followed by ApxI stimulation were subjected to real-time quantitative PCR (RT-qPCR) and ELISA analysis to determine mRNA and protein levels of proinflammatory cytokines, respectively. Isohelenin attenuated the mRNA expression and protein synthesis of IL-1β, IL-8, and TNF-α. In conclusion, NF-κB is activated in PAM upon ApxI stimulation and plays a role in ApxI-induced IL-1β, IL-8, and TNF-α expression. The relationship between NF-κB and MAPK (p38 and JNK) requires further investigation.