Role Of MAPK/mitochondria Apoptosis Pathway In Mycoplasma Ovipneumoniae-induced Oxidative Damage Of Sheep Bronchial Epithelial Cells

Background:Mycoplasma ovipneumoniae(M.ovipneumoniae,MO)is a mycoplasma species that mainly infects sheep and goats,which is the pathogen that causes sheep contagious pleuropneumonia.M.ovipneumoniae primarily infects sheep bronchial epithelial cells,in which the H2O2 produced by M.ovipneumoniae is abled to induce sedimentation of producation reactive oxygen species(ROS),and sequentially a damage of infected sheep bronchial epithelial cells.ROS can cause mitochondrial damage,mitochondria is an important organelle of the respiratory chain,which is more sensitive to ROS,and mitochondrial damage can lead to apoptosis.And ROS is an activator of mitogen-activated protein kinases(MAPK)signaling pathway.The primary responsibility of the MAPK signaling pathway is the transmission of signals from the outside the sheep bronchial epithelial cells to various organelles.However,whether the M.ovipneumoniae induces sheep bronchial epithelial cells to induce oxidative stress and apoptosis through MAPK/mitochondrial apoptotic pathways still unknown.Methods and contents:Using an air-liquid interface(ALI)epithelial culture model generated from primary bronchial epithelial cells of Ningxia Tan sheep(ovis aries),the main content contains several points as below by DCFH-DA and JC-1 probe loading,WST-8,Western Blot,AnnexinV-FITC/PI staining flow cytometry and TUNEL stain.1.the mechanisms of M.ovipneumoniae-induced sheep bronchial epithelial cells oxidative stress were first investigated by accessing productions of ROS,malondialdehyde(MDA)and antioxidant enzymes,the GSH/GSSG levels and the mitochondrial membrane potential in M.ovipneumoniae-infected ALI epithelial cells.2.The main effect modeling of the M.ovipneumoniae-induced oxidative stress in host cells by accessing productions of ROS in ALI epithelial cells.3.The mechanisms of M.ovipneumoniae-induced ROS Sheep bronchial epithelial cells apoptosis were investigated by accessing productions of ROS,the mitochondrial memebrane potential,cell apoptosis and activities of mitogen-activated protein kinases(MAPK)signaling,mitochondria damage and the activity of caspase signaling cascade,using sheep bronchial epithelial cells cultured on an air-liquid interface model with or without ROS scavenger NAC and mitochondria-targeted antioxidant MiTo-TEMPO were apically infected with M.ovipneumoniae.4.The mechanisms of M.ovipneumoniae-induced sheep bronchial epithelial cells apoptosis by accessing productions of ROS,the mitochondrial memebrane potential,cell apoptosis and activities of mitogen-activated protein kinases(MAPK)signaling,mitochondria damage and the activity of caspase signaling cascade,using sheep bronchial epithelial cells cultured on an air-liquid interface model with or without MEK/ERK signaling inhibitor(PD032590),JNK signaling inhibitor(SP600125)and P38 MAPK signaling inhibitor(SB203580)were apically infected with M.ovipneumoniae.Results:1 An infection of M.ovipneumoniae showed an ability to induce ROS production and cell apoptosis in ALI sheep bronchial epithelial cells,and this process was accompanied by mitochondria damage,an increased intracellular concentration of MDA and the level of GSSG,a reduced expression of antioxidant CAT,GSS,T-SOD and Mn-SOD,and a decreased level of GSH and GSH/GSSG ratio.2 The sheep bronchial epithelial cells cultured on an air-liquid interface model infected with M.ovipneumoniae for a period of time.Then,adding USG medium with antibiotic after removed ex-medium of M.ovipneumoniae for a period of time.The production of ROS returned to the level of removal of pathogens.It hints the M.ovipneumoniae-induced ROS is the main trigger of oxidative stress in bronchial epithelial cells.3 The results from studies of molecular mechanism showed that M.ovipneumoniae-induced apoptosis and the damage of mitochondrial integrity was in part owing to ROS induced by pathogen infection,which in turn stimulated an oxidative stress in host cells,and sequentially triggered MAPK signaling cascade by activating ERK,JNK and P38 MAPK signaling.The M.ovipneumoniae-activated MARK signaling pathway further led mitochondrial damage and release of Cytochrome C(Cyt-C)into the cytoplasm,sequentially inhibited the expression of anti-apoptotic BCL-2 protein,and enhanced the activation of caspase signaling cascade,which ultimately led an apoptotic cell death of host cells infection with M.ovipneumoniae bacteria.4 An evoked expression of ERK,JNK and P38 MAPK signaling,including phosphorylation of MEK,ERK,p90RSK,JNK1/2,AP-1 TAK1,MKK3/6,P38 MAPK and so on,in sheep bronchial epithelial cells infected with M.ovipneumoniae could be remarkable inhibited by MiTo-TEMPO(P<0.05,P<0.01).It means that the M.ovipneumoniae induced host cells-produced ROS activated MAPK signaling pathway through activating ERK,JNK and P38 MAPK signaling.Conclusion:By using an ALI sheep bronchial epithelial cell culture model,the potential mechanism of M.ovipneumoniae-induced cell apoptosis was investigated in this study.The results demonstrated that an infection of M.ovipneumoniae led an increased production of ROS and lipid peroxides,along with a reduced level of antioxidants;the oxidative stress of host cells in turn activated MAPK signaling pathway which induced mitochondrial damage and Cyt-C release,sequentially inhibited the expression of anti-apoptotic protein,ultimately activated caspase signaling cascade and apoptosis of M.ovipneumoniae-infected sheep bronchial epithelial cells.Results from this study thus suggest that M.ovipneumoniae-induced ROS and MAPK signaling-mediated mitochondrial apoptotic pathway may play an important regulatory role in the cellular pathogenesis of M.ovipneumoniae-infected sheep pulmonary epithelial cells.