Study On The Role Of Pyroptosis In Liver Injury Caused By Fowl Cholera In Chickens

Pasteurella multocida(P.multocida)is mainly divided into five serotypes A,B,D,E and F.Among them,Pasteurella multocida type A often causes fowl cholera(FC)in chickens.Fowl cholera is characterized by hemorrhagic sepsis and acute liver necrosis and has a high mortality,which has a huge impact on the economy of the poultry industry.Pyroptosis is a kind of programmed death with inflammatory reaction,which has important research significance.At present,the research on the mechanism of liver injury caused by P.multocida infection in poultry mainly focuses on inflammation,apoptosis and necrotic apoptosis,but it is unclear whether pyroptosis is involved.Therefore,this study discusses its occurrence and role of pyroptosis in the liver injury model of broilers induced by P.multocida,in order to provide theoretical reference for clinical liver injury repair of fowl cholera.In this study,the experimental chickens were randomly divided into infection group and control group.The total amount of bacteria injected with P.multocida bacterial solution per feather flipper in the infection group was 8.75×10~5CFU.Chickens in the control group were injected with an equal volume of sterile normal saline.The tissue samples were collected by dynamic autopsy after infection.The pathological changes of liver tissue were observed,and the expression of pyroptosis related proteins was detected by immunohistochemical method in liver tissue.Autopsy was performed after infection with P.multocida.And the results showed that liver volume began to increase after 12h of infection.The liver surface was densely covered with a large number of gray white necrosis points from the tip of needles to the size of millet after 24h of infection.Under microscope,there were coagulation necrosis foci with uneven size in the hepatic lobules.There were disintegrating hepatocytes with nuclear fragmentation and karyolysis in the necrotic area,accompanied by massive heterophilic granulocyte infiltration.Immunohistochemical results showed that the expressions of pyroptosis related proteins Caspase-1,GSDMD and NLRP3 increased extremely significantly in the necrotic area of liver tissue(P<0.01).In order to further verify whether hepatocytes undergo pyroptosis after P.multocida infection,chicken primary hepatocytes were used in this study.In order to obtain the best primary hepatocytes,the primary hepatocytes from 1-day-old,15-day-old,30-day-old and adult chickens were isolated and cultured by shearing and semi in situ perfusion methods.The results showed that the cells isolated from 30 day old and adult chickens by semi in situ perfusion had high purity,large number and good condition,which were suitable for in vitro experimental research.And the primary cells were identified by oil red O staining,PAS staining,immunohistochemical staining and indirect immunofluorescence staining.The results showed that the isolated cells have the function of secreting glycogen and storing lipids,confirmed that the isolated cells are hepatocytes.The classic pathway of pyroptosis,namely NLRP3/GSDMD/Caspase-1 signal pathway,was selected to detect the pyroptosis related factors in liver tissue and hepatocytes of broilers infected with P.multocida.The primary hepatocytes of 30-day-old broilers were selected for in vitro experiment.The primary hepatocytes growing to70%-80%were infected with P.multocida at the concentration of 7.00×10~7CFU/m L,1m L per well.The supernatants and cells were collected at 6h,12h and 24h after infection,respectively.After detecting the LDH content of cell supernatant,it was found that the LDH content in the infected group increased significantly,significantly at 6h and 12h after infection,and very significantly at 24h after infection.The results showed that the primary hepatocytes were damaged.The transcriptional and expression levels of pyroptosis related factors in broiler liver tissues and primary hepatocytes were detected by q PCR and ELISA methods.The results of q PCR detection showed that compared with the control group,the transcriptional levels of NEK7,Caspase-1,IL-18 and IL-1βin the infected group were all up-regulated,significantly up-regulated at 12h,36h and 48h after infection.And the transcriptional levels of NLRP3 were general decreased.However,the transcriptional levels of NEK7,NLRP3,Caspase-1,IL-18 and IL-1βin primary hepatocytes of infection group were all up-regulated.The transcriptional levels of NEK7was up-regulated extremely significantly up-regulated at 12h of infection(P<0.01),the transcriptional levels of NLRP3 was up-regulated extremely significantly at 6h and 24h of infection(P<0.01),the transcriptional levels of Caspase-1 was up-regulated extremely significantly at 6h and 12h of infection(P<0.01),the transcriptional levels of IL-18 and IL-1βwas all up-regulated extremely significantly(P<0.01).The results of ELISA detection showed that compared with the control group,the expression of IL-1βand IL-18 in liver tissue and primary hepatocytes in the infection group was up-regulated.The results of Western Blot detection showed that compared with the control group,the expression of NLRP3 protein in liver tissue of infection group decreased extremely significantly at 12h and 24h(P<0.01),and increased extremely significantly at 36h and48h(P<0.01).While the expression of GSDMD protein increased extremely significantly at 12h,24h and 36h after infection(P<0.01).The above results suggest that pyroptosis is involved in liver injury caused by P.multocida infection in broilers.The primary hepatocytes of 30-day-old broilers and adult broilers isolated and cultured by semi in situ perfusion method were the best.There are typical pathological changes appeared in the liver of broilers infected with P.multocida.Through in vivo and in vitro experiments,it was found that pyroptosis was involved in liver injury caused by P.multocida infection in broilers.