| Ketosis cause perinatal cows hyperketonemia.The increase of blood acetoacetic(ACAC)and β-hydroxybutyric acid(BHBA)concentrations is one of the main clinical manifestations of hyperketonemia.Perinatal cows suffering from ketosis will have negative energy balance and low immunity,and they will also cause complications such as mastitis and endometritis.Neutrophils immune defense as a first line of defense when exogenous bacteria invade cells,neutrophils infection reaches at a first time,for the immune defense through a variety of ways.It is traditionally believed that neutrophils clear bacteria by phagocytosing pathogenic microorganisms and thereby forming phagocytic vesicles.Recently,it was discovered that in addition to the traditional methods of phagocytosis and degranulation,neutrophil extracellular traps(NETs)were discovered in 2004 by Brinkmann et al.NETs are cell non-apoptotic deaths.After being stimulated by pathogens,the NETs release a net-like structure from neutrophils,destroying the pathogens,and the main component of the network structure is DNA.In recent years,it has been found that the overproduction of NETs will not only have a defensive effect,but may also cause the body to produce some innate immune diseases.Autophagy is a kind of self-digestion pathway commonly found in living organisms.Through autophagy pathway,it can maintain the metabolic balance of living body and keep the intracellular environment in a stable state.Autophagy plays an important role in NETs.Inhibiting autophagy will block the growth of intracellular chromatin.Therefore,the purpose of this study was to investigate whether highconcentration ACAC could induce autophagy accumulation and induce the overproduction of NETs,and whether autophagy could affect the production of NETs induced by ACAC.By simulating the pathological concentration of ketosis,we detected whether neutrophil accumulation in neutrophils could induce neutrophil autophagy accumulation at different concentrations of ACAC(0 m M,0.25 m M,0.5 m M,1 m M,2 m M)and high concentration ACAC(2 m M)at different time(0 h,1 h,2 h,4 h,8 h,12 h).The immunofluorescence assay was used to qualitatively analyze the fluorescence intensity of autophagic protein and found that the fluorescence intensity of LC3 protein after ACAC induction was significantly higher than that of blank control group.At the same time we use Western blotting method LC3 Ⅱ protein quantitative analysis,and immunofluorescence results are consistent results,a high concentration of initiator may ACAC autophagy accumulation.Second,we studied the formation of NETs under high concentration of ACAC.The removal of ACAC-induced release of neutrophils into the extracellular nets of DNA was observed by laser confocal microscopy.At the same time,we used LPS,PMA as a positive control of the experiment.And we also used Western blotting,ELISA and other experimental methods to detect other components of NETs.The results showed that high concentrations of ACAC can induce excessive NETs.Finally,we investigated whether autophagy affects the formation of NETs induced by high concentrations of ACAC.First,autophagy inhibitors,3-MA and LY294002,were added to detect neutophilicity after ACAC stimulation by Western blotting,immunofluorescence,and ELISA assays.The phagocyte induced autophagy and NETs production were qualitatively and quantitatively analyzed.The results showed that autophagy can affect the production of high concentrations of ACAC-induced NETs.Therefore,it is clear that high concentration of ACAC induces autophagy and induces the over-production of NETs,providing a theoretical basis for revealing the mechanism of innate immune suppression in cows with ketosis and improving immune function in transitional cows. |
