Study Of Mycoplasma Gallisepticum-induced Inflammation,Oxidative Stress And Apoptosis In HD11 Cells And Chicken Bursa

Mycoplasma gallisepticum(MG)also known as Mycoplasma septicum,can cause chronic respiratory disease.MG infection is widespread all over the world,and caused huge economic losses in terms of appetite,weight loss,and lower egg production in poultry.In severe cases,it can also cause the death of chicks.Studies have reported that the chicken bursa of fabricius(BOF)plays an important role in the defense of MG infection.At the same time,our laboratory has also proved that MG infection can cause inflammation after HD11 infection.However,the relevant molecular mechanisms and targets have not been studied in depth.Th is experiment will explain the changes brought about by MG infection from three aspects:oxidative stress,inflammatory damage and cell apoptosis.The research will provide new ideas and targets for subsequent drug treatment and prevention.(1)Study on the Inflammation,Oxidative Stress and Apoptosis of HD11 Cells Induced by MG InfectionIn the in vitro experiment part,MG-induced HD11 cell injury model test,the final MG concentration was 400MOI,and the HD11 cells were collected and sampled for 3,6,an d 12 h.Scanning electron microscopy,ELISA,Real-Time PCR,mitochondrial membrane potential,ROS detection,Tunel green fluorescence and Western Blot were selected to explore the effects of MG infection on HD11 cells caused by inflammation,apoptosis and oxidative stress.Under an optical microscope,normal HD11 cells have a round elliptical shape.As time grew,the infected group gradually showed cell deformation,shrinkage,vacuolization in the cytoplasm,cell disintegration,and floating death.Under the electron microscope,it was observed that the mitochondria in the cells were swollen and vacuolated,the nuclear membrane was broken,and the chromatin condensed.Inflammatory results revealed that compared with the control group,the m RNA levels of NF-k B,MYD88,i NOS2,IL-1β,and IL-6 gene increased significantly at 3,6,and12 hours(p<0.05),and the increase in m RNA level of IFN-γwas significant at 6 and 12 h(p<0.05),and the protein expression level showed that the expression of TLR-2,P-P65/P65,and COX-1increased significantly(p<0.05).In the oxidative stress indicators,compared with th e control group,GSH-Px,CAT,MDA,SOD,and LDH all showed significant changes at 6 h in the infection group(p<0.05).The fluorescence intensity of ROS detected by flow cytometry showed that at three different time points,the reactive oxygen species in t he cells of the infection group showed a significant upward trend(p<0.05).Among apoptosis-related indicators,compared with the control group,the m RNA expression levels of Caspase-9,Cytcle-C,Bcl-2,P53,Caspase-9 and Bax in the infected group showed significant changes at 6 h(p<0.05).The protein content of Cytcle-C,Caspase-3 and Bax increased significantly(p<0.05).The results of TUNEL assay showed that cell apoptosis occurred significantly at 6 and 12 h(p<0.05).In the mitochondrial membrane potential,the membrane potential decreased at all the three time points,which indicated that the cells had undergone apoptosis.(2)Study on the Inflammatory Damage,Oxidative Stress and Apoptosis of the Bursal of Fabricius Caused by MG InfectionIn the in vivo experiment part,the method of constructing MG infection model was used to infect the chicks in the early stage of the experiment.The MG concentration was 1×10~9 CCU/m L,and each 0.2 m L was inoculated.The chickens were dissected on the first,third,and seventh days of infection,and the bursa was sampled.Scanning electron microscopy,histopathological section,ELISA,Real-Time PCR,immunofluorescence labeling,TUNEL assay and western blotting were used to explore the inflammatory damage,apoptosis and oxidative stress caused by MG infection in chickens BOF.The results of pathological tissue section and electron microscope showed that 7 days after MG infected chickens,the histocellular structure showed obvious lesions,lymphocytes infiltrated in the medulla area of the bursa of fabric,necrotic lymphocytes increased,and the disappearance of lymphocytes caused vacuolation in the medulla of BOF.Mitochondrial swelling or vacuolization,deepening of chromatin,nuclear membrane rupture,and the appea rance of apoptotic bodies appear in the relevant lymphocytes in the BOF under electron microscopy.The immunofluorescence results showed that the CD8~+content in the infection group decreased significantly(p<0.05),indicating that the immune function of the bursa of fabric was impaired.In inflammation-related indicators,compared with the control group,the m RNA expression levels of TNF-α,IL-1β,IL-6,and i NOS in the infection group increased significantly on the 3rd and 7th days,and the NO content also increased in the three days.Significant increase(p<0.05).In terms of protein level,the protein content of i NOS increased significantly on the 7th day(p<0.05).Among the indicators of oxidative stress,on the 3rd and 7th days of infection,CAT,SOD,G SH-The content of px and GSH decreased significantly(p<0.05),and the content of G-GT increased significantly(p<0.05).The anti-apoptotic-gene Bcl-2 m RNA expression of the infected group was significantly decreased during these three days(p<0.05)compared to the control group.On the 3rd and 7th days of infection,the m RNA expression of Bax,Caspase-9,Caspase-3,Cytcle-C increased significantly(p<0.05).The protein expression level showed that on the 7th day,the pro-apoptotic factors such as Bax,Caspase-9,Caspase-3,and Cytcle-C all increased significantly(p<0.05),and the anti-apoptotic factor Bcl-2 decreased significantly(p<0.05).TUNEL assay results showed that the number of cell apoptosis increased significantly on the 3rd and 7th day(p<0.05),which indicated that MG infection induced apoptosis in the BOF.In conclusion,this study proved that MG infection induces inflammation in HD11 and the bursa of Fabricius through the TLR2-NFκB signaling pathway,and at the same time induces oxidative stress and apoptosis in cells and the body through the mitochondrial apoptosis pathway.MG infection damages HD11 cells And the integrity of the bursa tissue structure,this study will lay the relevant foundation for subsequent drug screening and the study of the mechanism of action.