The Existence And Mechanism Of Necroptosis In The Liver Injury Of Ducks Infected By P.multocida

The bacterial liver disease has always been the key point of the liver diseases research in China.Pasteurellosis is caused by Pasteurella multocida(P.multocida).When infected with P.multocida,human would present with cirrhosis and chronic liver disease,and ducks would suffer from fowl cholera,which appear multi-focal necrosis diffusely spreading over the liver surface.However,there's a little information about the mechanism of liver damage in ducks.It will appear a lot of cell death patterns including necrosis,apoptosis,necroptosis and autophagy after infected with bacteria like Brucella abortus,Salmonella Typhimurium.Necroptosis is a cell death controlled tightly during various diseases process,such as ischemia–reperfusion,alcoholic liver disease and avian typhoid.Inhibition of signaling pathways of necroptosis,tissue damage by the mentioned above diseases will be correspondingly reduced.Necroptosis attracts much attention for its potentiality as a new treatment target.RIPK3 and MLKL are the key molecules of necroptosis.The high expressions of MLKL will eventually cause necroptosis,following the releasing of MMP9,HMGB1 and inflammatory factors(TNF-?,IL-6).In this study,we will investigate the cell death patterns of the liver,and the role of necroptosis signaling pathway in liver damage after infected with P.multocida.After 3 weeks old ducks infected with P.multocida,liver lesions typically consist of multifocal necrosis diffusely spreading over the surface and incision.Histopathology results showed lots of inflammatory cells infiltration,as well as a mass of multi-focal necrosis associated with colonies of bacteria.The colonies of bacteria appeared in the hepatic sinus,necrotic area and the cytoplasm of hepatocytes,heterophilic granulocytes and sinusoidal endothelial cells.The distribution of bacteria in the liver related with the histopathological changes of the liver.The serum levels of T-pro,Glob and Alb were obviously higher than the control group.The body was actively producing antibodies against infection.HE staining showed a large number of necrotic lesions in liver at 24 h.p.i.There were strong positive signal using IHC staining to detect the expressions of Caspase 3,Cleaved Caspase 3,MLKL in liver.As the key molecule of apoptosis,the high expressions of Caspase 3 suggested the occurrence of apoptosis.The high expressions of MLKL suggested the occurrence of necroptosis.There were simultaneous apoptosis and necroptosis in the liver damage.Peritoneal macrophages infected with P.multocida showed obviously vacuolization,swelling,cell membrane rupture and cell death.In the peritoneal macrophage and liver of ducks infected with P.multocida,the gene transcription levels of RIPK3,MLKL were up-regulated.The protein expression of MLKL obviously increased in the liver at 24 h.p.i and 48 h.p.i.RIPK3/MLKL signaling pathway of necroptosis was activated.The upstream molecules of necroptosis signaling pathway,including TLR4,TRIF,RIPK1,TNF-? and IFN-?,were detected in the liver infected with P.multocida.The mRNA expression of TLR4 was up-regulated,but there was no significant difference at all time points.The mRNA expression of TRIF was down-regulated at 12 h.p.i,and obviously up-regulated at 48 h.p.i.The mRNA expression of RIPK1 was up-regulated in the liver at 6h.p.i,as well as in the infected macrophages.The protein expression of RIPK1 significantly increased in the liver at 24 h.p.i.The liver produced and released lots of inflammatory factors during liver injury.The mRNA expression of TNF-? and IFN-? were obviously up-regulated at 48 h.p.i.But the mRNA expression of IFN-? was down-regulated at 12 h.p.i.The protein expression of IL-6,HMGB1,MMP9,and TIMP1 obviously increased in liver at 24 h.p.i and 48 h.p.i,and the changes of expression level were along with the degree of liver injury.Conclusions: In the liver injury of ducklings infected with P.multocida,there were necrosis,apoptosis and necroptosis involved.RIPK3/MLKL signaling pathway of necroptosis signaling pathway was activated,and released TNF-?,IL-6,HMGB1 and MMP9 were involved in the liver injury.