Research Of Key Inflammatory Factors And The NF-κB Pathway In Encephalitis Of Tongcheng Piglets Induced By PRRSV

Porcine reproductive and respiratory syndrome(PRRS)is an immunosuppressive disease with high morbidity and infectivity.PRRS infection affects the growth and causes developmental retardation.The clinical manifestations include dyspnea,asthma,ataxia,quadriplegia,etc.The symptoms of infection in growing pigs and fattening pigs were mild,while the clinical manifestations of infection in piglets and sows were serious.At present,most studies focus on the effects of PRRSV on the respiratory and reproductive system and its pathogenic mechanism,but there are few studies on the inflammatory response caused by PRRSV entering the brain tissue and its pathogenic mechanism.Therefore,in this study,13 40-day-old Tongcheng piglets were artificially infected.Clinical symptoms and histological changes were recorded and observed,and pathological changes of brain histomorphology were observed.According to the characteristics of pathological changes,using transcriptomics techniques to screen and identify key cytokines related to inflammation and key inflammatory pathways.To investigate the pathogenesis of encephalitis caused by PRRSV.The main research results are as follows:1.Pathological changes of piglets infected with PRRSVPiglets infected with PRRSV mainly show persistent high fever,cyanosis of the skin,retarded reaction,dyspnea,anorexia,cough,asthma,convulsions,ataxia,paresis,etc.,seriously leading to death.Necropsy revealed that the lung tissue of the PRRSVinfected piglets was edema,dark red,and firm in texture;there were bleeding spots on the surface of the kidney from the size of a needle to the size of a sesame;lymph nodes were enlarged and hemorrhage;Cerebrovascular dilation,edema,congestion.Histopathological observation showed that alveolar wall capillary dilatation,alveolar epithelial cells and lymphocytes were shed in alveolar cavity.Glomerular capillaries were congested and renal tubular epithelial cells were necrotic.Lymph node follicular necrosis,increased macrophages;There are numerous lymphocytes in the brain,vascular cuff formation,glial nodule formation,neuronal necrosis,satellite appearance,and the formation of focal hemorrhage.These results indicate that PRRSV causes tissue and organ damage and causes severe inflammatory response.2.Changes of lymphocytes in brain tissue of PRRSV-infected pigletsSevere lymphocytic vascular cuffs were found by histopathological observation,but no correlation analysis of perivascular lymphocytes has been found in most of the literature.In this study,CD3(T lymphocyte marker)and CD20(B lymphocyte marker)immunohistochemical staining were performed on perivascular lymphocytes.The overall observation of tissue sections of each pig was evaluated,and the positive rates of CD3 and CD20 were statistically analyzed in 15 fields randomly selected.It was found that the lymphocytes of the infected group were significantly different from those of the control group,and the T lymphocytes were significantly higher than that of the B lymphocytes.The results of this experiment showed that PRRSV infected brain tissue with severe vascular cuff phenomenon,and the peripheral blood vessels were dominated by T lymphocyte3.Transcriptome analysis of brain tissue of PRRSV-infected pigletsHistopathological observation showed that after PRRSV infection,the brain tissues of pigs showed inflammatory pathological changes,mainly lymphocytic vascular cuff.However,the molecular mechanisms that lead to these inflammatory pathological changes are still unclear.Therefore,in this study,transcriptome sequencing was performed on the brain tissues of healthy piglets and PRRSV-infected Tongcheng pigs to analyze the association between the differentially expressed genes related to inflammation and the changes in the expression of signaling pathways and histopathology.The results showed that there were 1,955 differentially expressed genes in the PRRSV-infected group.The GO functional classification and enrichment analysis showed that the differentially expressed genes were significantly enriched in the biological process,including kinase activity and protein domain specific binding.KEGG enrichment analysis of metabolic pathways showed that the differentially expressed genes were mainly concentrated in inflammation-related signaling pathways.The screened main pathways associated with inflammation included NF-κB signaling pathway,etc.And in these inflammatory pathways,there are different genes related to inflammation,such as TNF-α,CXCL9,CXCL10,CXCL11,etc.These factors were enriched in major inflammatory pathways screened out.The results showed that PRRSV infection significantly activated inflammatory cytokines and inflammationrelated signaling pathways,suggesting that these genes and pathways are involved in the pathological process of brain tissue after PRRSV infection.4.Effect of PRRSV on the expression of cytokines in pig brain tissueFor verifying the changes of expression level of related inflammatory factor genes in brain tissues after PRRSV infection,Q-PCR technology was used to detect TNF-α,CXCL9,CXCL10,CXCL11 and CCL2.The results showed that TNF-α,CXCL9,CXCL10,CXCL11 and CCL2 in PRRSV-infected group were significantly higher than those in the control group(P < 0.05).Western blot was used to measure the protein levels of these five factors.Compared with the control group,the protein expression levels in PRRSV-infected group were significantly higher(P < 0.05).The results showed that PRRSV infection can significantly up-regulate the expression of TNF-α,CXCL9,CXCL10,CXCL11 and CCL2 in brain tissue,and participate in the formation of encephalitis.Among them,CCL2,CXCL10,CXCL9 and CXCL11 belong to lymphocyte chemokines,and the high expression of these chemokines attracts lymphocytes to gather around blood vessels and form vascular cuffs.5.Effect of PRRSV on the expression of TNFR1/NF-κB pathway related proteins in porcine brain tissueWestern blot was used to evaluate the TNFR1/NF-κB pathway.The results showed that the expression of TNFR1,IKKα,IKKβ and P65 protein in PRRSV-infected pig brain tissue was up-regulated.These results indicate that PRRSV infection can activate the NF-κB signaling pathway in brain tissue of piglets.