Exploration Of The Molecular Mechanism Of Cellular Apoptosis Induced By Pasteurella Multocida PmCQ2 In Mice

Psteurella multocida(P.multocida,Pm)is a Gram-negative opportunistic that can infect a variety of animals and humans.It can be divided into five capsular serotypes: A,B,D,E,and F according to the different capsular antigens.P.multocida type A is one of the pathogens causing bovine respiratory disease syndrome,which mainly causes bovine lung infection,namely P.multocida pneumonia,which has caused serious losses to our country’s breeding industry.During the pathological process of bovine lung injury caused by P.multocida,a variety of immunological reactions such as apoptosis occurred in host cells.Apoptosis plays an important role in both physiological and pathological conditions,and is one of the important mechanisms of pathogenic microorganisms.However,less research has been done on the mechanism by which Pasteurella multocida type A(PmA)induces host cell apoptosis.Therefore,this study mainly explored the mechanism of PmA-induced apoptosis of host cells.Firstly,mice were infected with PmCQ2 intranasally to establish a model of Pasteurella multocida pneumonia.After 40 hours of infection,the lung tissues of the mice in the control group and the infected group were collected under sterile conditions for real-time quantitative PCR(qRT-PCR),Western Blotting(WB),immunohistochemistry(IHC),and TUNEL signal detection.It was preliminarily determined that PmCQ2 induced apoptosis of mouse lung tissue cells.Then,a P.multocida infection model was established in vitro using peritoneal extraction of macrophages(PEMs),which was further verified by qRT-PCR,WB and flow cytometry,and the results showed that PmCQ2 caused the apoptosis of PEMs.Finally,transcriptomic sequencing was performed on cells in the infected and control groups,and KEGG Pathway analysis of differentially expressed genes(DEGs)was performed to screen and validate cell death-related pathways(such as apoptosis)in order to provide evidence for Pasteurella multocida infection.The exploration of pathogenesis and the selection of therapeutic targets provide a certain theoretical basis.1 PmCQ2 induced apoptosis in lung tissues of miceIn order to explore whether host cells undergo apoptosis after PmCQ2 infection,this study used PmCQ2 intranasal-infected mice as an animal infection model,and collected mouse lung tissue for related research.Firstly,qRT-PCR was used to detect that the expression level of apoptotic effector molecule Caspase3 was significantly up-regulated in the lung tissues of the infection group,and the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl2(Bax/Bcl2)was also significantly up-regulated.IHC detection showed that Cleaved-Caspase3 and Bax were strongly positive in the lung tissues of the infected group.At the same time,a strong positive signal of TUNEL was detected in the lungs of the same batch of mice.Then,the expression level of Bcl2 was significantly decreased and Bax/Bcl2 was significantly up-regulated in the infected group by WB.These data suggest apoptosis of lung tissue cells after PmCQ2 infection in mice.2 PmCQ2 induced apoptosis in PEMs of miceIn order to further explore the apoptosis induced by PmA infection,this study used PmCQ2-infected mouse PEMs as a model for in vitro experiments.First,in this study,the expression of Caspase3 and the ratio of Bax/Bcl2 were up-regulated in PEMs infected with PmCQ2 at different time points(8 h,12 h,16 h,24 h)by qRT-PCR,and there were significant changes at 12 h and 16 h.Annexin V/PI assay and mitochondrial membrane potential detection showed that apoptosis rate of macrophages significantly increased and mitochondrial membrane potential significantly decreased after P.multocida infection.In addition,we detected the expression of Cleaved-Caspase3 in the cells by WB,and found that compared with the control group,the expression of Cleaved-Caspase 3 was significantly increased after PmCQ2 infected macrophages for 16 h,At the same time,it was detected that the expression of Bcl2 was significantly down-regulated,and the value of Bax/Bcl2 was significantly up-regulated.The above results were consistent with the results of mouse lung tissue studies,indicating that PmCQ2 induced the apoptosis of mouse PEMs after infection,and the phenomenon was more obvious when the infection time was 16 h.3 Transcriptome sequencing and analysis of PEMs in miceIn order to explore the molecular mechanism of apoptosis induced by P.multocida of bovine origin,this study performed transcriptome sequencing analysis of mouse PEMs before and after PmCQ2 infection.By analyzing the gene expression levels,3990 DEGs were counted,of which 2026 genes were up-regulated and 1964 were down-regulated.On this basis,KEGG Pathway enrichment analysis was performed on DEGs.The results showed that DEGs were mainly enriched in TNF Signaling Pathway,MAPK Signaling Pathway,NF-kappa B Signaling Pathway,Necroptosis,Apoptosis,p53 Signaling Pathway,suggesting that these molecular pathways may be involved in the apoptosis process induced by PmCQ2.4 PmCQ2 induces apoptosis in PEMs of mice through SMAC/DIABLO-IAP/XIAF-Caspase3/7 pathwayBased on transcriptome sequencing results,DEGs significantly enriched in apoptosis pathways were analyzed,and related DEGs were verified at the cellular level by qRT-PCR and WB.The results showed that the expressions of p-JNK,DIABLO,Cleaved-Caspase3 and Caspase7 on the mitochondrial pathway were significantly increased after PmCQ2 infection in PEMs of mice,while the expression of Bcl2 was significantly decreased,suggesting that PmCQ2-infected PEMs mainly activate SMAC/DIABLO-IAP/XIAF-Caspase3/7 pathway to mediate apoptosis.