Apoptosis Induced By Lipid-associated Membrane Proteins From Mycoplasma Hyopneumoniae Is Mediated By Caspase3in St. Jude Porcine Lung Epithelial Cell Line

Mycoplasma hyopneumoniae is the causative agent of Mycoplasma Pneumoniae ofSwine (MPS), which causes a chronic respiratory disease of pigs. The main symptoms ofthis disease are cough and asthma, with meat or shrimp-like consolidation in the leadingedge of lobus cardiacus of lung as pathological features. The disease has high morbidityand low mortality. Lipoproteins (lipid associated membrane proteins, LAMPs) aresignificant structural of mycoplasma, which play an important role in the interactions withhost. In recent years there have been many reports proved that lipoproteins may beimportantly contributing in the pathogenesis of mycoplasma. In order to explore the roleLAMPs play in the pathogenic of M. hyopneumoniae, following experiments weredesigned.Firstly, the interaction between LAMPs and St. Jude Porcine Lung Epithelial CellLine (SJPL) was confirmed. To investigate the inhibition of LAMPs from M.hyopneumoniae of different virulence on SJPL cells LAMPs were extracted from differentculture of M. hyopneumoniae by Trion-X114, the suppression ratio was detected throughMTT assay. The results demonstrate that LAMPs from five different strains have theinhibited effect on SJPL cells. Cell morphology was changed observed by an opticalmicroscope. The number of dead cells increased when the concentration of LAMPs stimuliincreased. At different concentration intervals cell inhibitions (%) were measured by MTTassay. Moreover, the concentrations of IC50were compared. All of five kinds LAMPs cando toxic action on SJPL cells, and the containment procedure was agreed with thevirulence of M. hyopneumoniae. The inhibited effects caused by strain NJ,168and XLW-1were stronger than strain attenuate168and J, which the minimum IC50was0.13mg/mlbelonged to strain NJ, IC50was0.2mg/ml belonged to strain XLW-1and the maximumIC50was0.56mg/ml belonged to strain J. After considering of the inhibited effect on SJPLcells, the concentration of IC50and the characters and growth state of strains, LAMPsextracted from M. hyopneumoniae strain XLW-1were choose to act with SJPL cells, andIC500.2mg/ml to be the concentration of the induction of apoptosis in the laterexperiments.Therefore, some experiments related to the typical characteristics of apoptosis weretaken to detect whether LAMPs could induce SJPL cells apoptosis, including DAPI,TUNEL, AO-EB and Annexin-V-PI. The chromosome crimpled, nuclei cleavage andapoptotic bodies could be observed after cells been treated with0.2mg/ml LAMPs. The genome between nucleosome was cut off from each other, it’s exposed-3’OH stump couldcombine FITC labeled dUTPs, which showing green fluorescence specifically. AO-EBstaining was used to distinguish between apoptotic cells induced by LAMPs and live cells.Furthermore, the rate of apoptosis was detected by flow cytometry. LAMPs could induce36.5%±11.66%apoptotic cells of porcine alveolar epithelial cell while stimulated for24h.To further demonstrate the regulatory mechanism of apoptosis in SJPL cells andexplore the possible pathway that leads to apoptosis induced by LAMPs, the activity ofcaspase was examined, some signaling proteins through western blot assay and the releaseof Cyt c. The results indicated that caspase3has involvement in apoptotic pathway.Moreover, after treated with LAMPs, some proteins related in the pathway of apoptosischanged. PARP was cut into two fragments, p38MAPK was phosphorylated, theexpression of pro-apoptosis protein Bax raised while the expression of anti-apoptosisprotein Bcl-2was inhibited. Cyt c was released from mitochondria to the cytoplasm so thatcan detect it with specific antibody labeled with FITC showing fluorescent in thecytoplasm specially. LAMPs can induce NO and superoxide released. The production has apositive correlation with the concentration of LAMPs. The maximum concentration of NOwas20.13μM when the concentration of LAMPs was0.5mg/ml and the concentration ofsuperoxide was tripled after treated with0.2mg/ml LAMPs for1h.This study demonstrated that LAMPs from five different strains of M. hyopneumoniaecan do toxic action on SJPL cells, and the containment procedure was agreed with thevirulence of M. hyopneumoniae. LAMPs from M. hyopneumoniae XLW-1strain couldinduce apoptosis in SJPL cells and parts of its apoptosis signal pathway dependent oncaspase3and LAMPs could stimulate cells to generate ROS and NO productions. Theoutcome of this study could contribute to quest the pathogenesis of mycoplasma, while theunderstanding of apoptosis pathway may help people find out right targets for treatingmycoplasma infection.