Effects Of Different Physiological Stages And NEFA On The Formation Of Neutrophil Extracellular Traps In Dairy Cows

The basal metabolism and oxidative stress levels of dairy cows at different physiological stages are different,which theoretically leads to differences in immunity.Identifying the differences in immune levels of Nulliparous cows,Late pregnancy cows,postpartum healthy cows,postpartum ketotic cows,and mid-lactation cows at different physiological stages is of great significance for early warning and monitoring of disease occurrence at each stage.Due to the enhanced lipid metabolism in perinatal dairy cows,cows are prone to occur the negative energy balance（NEB）,causing a surge in the concentration of non-esterified fatty acids（NEFA）in the blood.Therefore,NEFA plays an important role in the immune status and inflammation of perinatal dairy cows.The function of neutrophils（Polymorphonuclear neutrophils,PMN）is closely related to immunity,and the impairment of PMN function will directly affect the immunity of dairy cows.The main functions of PMN are phagocytosis,respiratory burst and formation of neutrophil extracellular traps（NETs）.The purpose of this study was to research whether there are differences in the formation of NETs in Holstein cows at different physiological stages,and to study the mechanism of NEFA on the formation of NETs from the perspective of the release of NETs to the outside of the cell.In this study,dairy cow PMN was isolated by an improved erythrocyte lysis method,and cell viability and purity were identified by trypan blue staining and Giemsa staining;4’,6-diamidino-2-phenylindole（DAPI）)to stain the cell nuclei;2’,7’-dichlorofluorescein diacetate（DCFH-DA）was used to evaluate the generation of reactive oxygen species（ROS）;NETs morphology was observed by SYTOX Orange fluorescent staining;Quant-i T^TMPico Green ds DNA hypersensitive nucleic acid fluorescence staining method was used to quantify the generated NETs;Western Blotting was used to semi-quantitatively determine NETs composition-related proteins and related signaling pathway proteins;q PCR was used to detect m RNA expression of inflammatory factors and related signaling pathway genes.The results of the study showed that,by comparing cows at different physiological stages,the ROS generated by PMN in Late pregnancy cows was 1.3 times higher than that in Nulliparous cows（p<0.01）.PMN isolated from mid-lactation and postpartum healthy cows produced 2.1-and 2.5-fold higher NETs than Nulliparous cows,respectively（p<0.01）.Compared with cows in other stages,PMN of ketosis cows can produce higher levels of NETs and ROS,while PMN of Nulliparous cows produces lower amounts of NETs and ROS.PMN isolated from Nulliparous dairy cows were stimulated by a concentration gradient of lipopolysaccharide（LPS）,and a concentration-dependent increase in the production of ROS and NETs was found（R²=0.96 and 0.86,respectively）.Similarly,stimulation of PMN of Nulliparous cows with a concentration gradient of Phorbol myristate 13-acetate（PMA）resulted in stimulation of ROS（R²=0.99）and NETs（R²=0.78）also increased in a concentration-dependent manner.NEFA can stimulate PMN to generate NETs,and this process is accompanied by an increase in intracellular ROS levels.Western Blotting results showed that under the stimulation of NEFA,the expression of NETs constituent proteins,including histone H3,neutrophil elastase（NE）,and citrullinated histone H3,were significantly increased;and PMN produced a large number of inflammatory factors,including IL-6,IL-10,IL-18,which may be important metabolic factors of inflammation in perinatal dairy cows;Results also showed that the expressions of pore-forming GSDMD and sheared N-GSDMD on PMN membranes were increased,indicating that the formation mechanism of NETs is related to the expression of pore-forming proteins on the PMN membrane.The key proteins and genes in the signaling pathway related to GSDMD formation,including NLRP3,NF-κB,Caspase1,and Caspase4 were detected,and showed that the protein levels and m RNAs of these important signaling molecules were significantly increased.Therefore,it is concluded that due to the different metabolism of dairy cows in different physiological stages,there are differences in PMN function,and the ability to eventually form NETs is also different.The amounts of NETs and ROS generated by PMNs stimulated by LPS or PMA in a concentration gradient increases linearly,the quantitative results suggest that the production of NETs and ROS is related to the degree of inflammation caused by the stimuli.NEFA can induce the production of NETs in dairy cow PMN,which is accompanied by an increase in the production of inflammatory factors.Therefore,NEFA may also be one of the causes of metabolic inflammation in dairy cows during perinatal period.The mechanism of NEFA-induced NETs and their extracellular release is related to the expression of pore-forming protein GSDMD on PMN cell membrane.This process is mediated through the NF-κB--NLRP3-Caspase1/Caspase4--GSDMD/N-GSDMD signaling pathway.