| Summary: | 碩士 === 國立中興大學 === 獸醫病理生物學研究所 === 100 === Actinobacillus pleuropneumoniae (AP) is one of the important respiratory pathogens that causes hemorrhagic, necrotizing, and fibrinous pleuropneumonia in porcine. Actinobacillus pleuropneumoniae exotoxin (Apx) belonging to repeats-in-toxin (RTX) family, is a virulence factor and plays an important role in pathogenesis of pleuropneumonia. Another RTX member, Mannheimia haemolytica leukotoxin has been reported to cause intracellular calcium elevation in bovine alveolar macrophage and cytolysis. It is not clear whether Apx could induce intracellular calcium increase in porcine alveolar macrophage (PAM) and whether calcium signaling participates in Apx-mediated effects. The objectives of this study are to explore (i) whether ApxI induces intracellular calcium increase in PAM, (ii) the role of calcium in ApxI induced cell membrane damage, mitochondrial activity decrease, and in apoptosis, and (iii) the relationship between mitogen-activated protein kinase (MAPK) p38 and JNK and calcium elevation. PAM were collected from 4-5 weeks old SPF piglets. AP serotype 10 was cultured and supernatant collected as crude ApxI preparation. PAM were stimulated by different concentrations of ApxI and intracellular calcium detected with FluoForte assay kit. The results showed that ApxI induced an elevated intracellular calcium concentration in a short time period. Cells were pre-incubated with four different calcium inhibitors, amiloride, BAPTA-AM, EDTA, and nifedipine, followed by ApxI stimulation. The results showed that EDTA inhibited intracellular calcium elevation induced by ApxI, indicating a calcium influx from extracellular space. LDH release, XTT assay, and Hoechst dye staining were used to detect the effects of calcium inhibitors on ApxI-induced membrane damage, mitochondrial activity decrease, and apoptosis, respectively. The results showed that all calcium inhibitors tested did not diminish ApxI-induced cytolysis and apoptosis, but EDTA could significantly prevent ApxI-induced mitochondrial damage. Finally, p38 and JNK activation induced by ApxI was inhibited by pretreatment of BAPTA-AM and EDTA, suggesting a possible role of calcium in p38 and JNK activation. In conclusion, Inhibition of calcium influx from extracellular space could reduce mitochondrial toxicity and p38 and JNK activation, but not apoptosis or cell membrane damage, induced by ApxI.
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