Transcriptomic Analysis Of Mouse Lung After Acute Infection With Toxoplasma Gondii And Functional Study Of Differentially Expressed Genes

ObjectiveIn order to provide targets for the diagnosis and treatment of Toxoplasma pneumonia,the mouse model of acute Toxoplasma infection was established,the pathological changes of lung tissues were observed,the transcriptomic sequencing and bioinformatics analys is of lung tissues were carried out,and the functions of key differentially expressed genes(DEGs)were further studied.Methods1.The mouse model of acute Toxoplasma infection was established by intraperitoneal injection of Toxoplasma gondii RH strain tachyzoite.Mouse ascites smears were stained with Wright-Giemsa staining and then observed with light microscope.Pathological sections of mouse lung,liver and spleen were made,HE staining and observation were carried out.DNA was extracted from mouse lung,liver and spleen tissues respectively.Toxoplasma gondii B1 gene fragments were amplified from the mouse lung,liver and spleen tissue DNA by PCR respectively.The PCR products were sequenced,and then were matched with the target gene sequence using the Snap Gene software.2.RNA was extracted from mouse lung tissue to construc t a c DNA library,then the c DNA library was used for transcriptome sequencing.3.The DEGs were screened from the transcriptome sequencing data.GO enrichment analysis,KEGG enrichment analys is and KDA analys is were performed based on the whole DEGs.4.The RAW264.7 cell infection model was constructed with Toxoplasma gondii at a MOI=5:1(Toxoplasma gondii: RAW264.7=5:1).Then the cytokines were add into the cell model respectively and combinedly.Giemsa staining was used to observe the effects of IFN-γ,CXCL10 on the invas ion and proliferation of Toxoplasma gondii.The content of NO in cell lysate was determined by colorimetry.Results1.Typical symptoms of Toxoplasma infection was firstly observed post infection 4 days,and the symptons were aggravated on the 5th day.A large number of Toxoplasma tachyzoites and neutrophils,lymphocytes,macrophages were observed on the mouse ascites smears.Different pathological changes were observed in the lung,liver and spleen tissues of mice in the treatment group,and typical acute inflammatory pathological changes were observed in the lung tissues.The 194 bp Toxoplasma gondii B1 fragment was amplified from the DNA of lung,liver,spleen tissues of mice in the treatment group and sequencing results were 100% matched to the target sequence.2.Ten key DEGs which are TNF,CXCL10,IL10,CCL2,IFN-γ,CXCL9,CXCR2,CCR5,CCL4 and CCL7 were screened from mouse lung transcriptome sequencing data by GO enrichment analys is,KEGG enrichment analysis and KDA analysis.3.Gimsa staining showed that the percentage of Toxoplasma-infected RAW264.7 cells and the number of Toxoplasma in the infected cells were significantly decreased in the IFN-γ group compared with the control group(p<0.05),while no significant differences were found in the percentage of Toxoplasma-infected cells and the number of Toxoplasma in the infected cells between the CXCL10 group and the control group(p>0.05),and no significant differences were found in the percentage of Toxoplasmainfected cells and the number of Toxoplasma in the infected cells between the IFN-γ+ CXCL10 group and the IFN-γ group(p>0.05).4.The NO content in the lysate of RAW264.7 cell infection model was increased in the IFN-γ group compared with the control group(p<0.01),while no significant difference was found in the NO content between the CXCL10 and the control group(p>0.05),and no significant difference was found between the IFN-γ+CXCL10 group and IFN-γ group in the NO content(p>0.05).Conclusion1.The mouse model of acute infection with Toxoplasma gondii was successfully established by intraperitoneal injection of Toxoplasma gondii RH strain tachyzoites.Toxoplasma gondii can infect mouse lung,liver and spleen,and cause acute inflammatory pathological changes in lung tissues.2.Ten key DEGs which are TNF,CXCL10,IL10,CCL2,IFN-γ,CXCL9,CXCR2,CCR5,CCL4 and CCL7 were screened out by transcriptome sequencing and bioinformatics analys is,and they may play an important role in the mouse lung after acute infection with Toxoplasma gondii.3.IFN-γ treatment promoted the resistance of RAW264.7 cells to the invas ion and proliferation of Toxoplasma gondii,while CXCL10 treatment in the RAW264.7 cells had no significant effect on the invasion and proliferation of Toxoplasma gondii.