The Role And Mechanism Of Neutrophil Extracellular Traps In The Sympathetic Hyperactivity After Traumatic Diffuse Axonal Injury

Traumatic brain injury(TBI)is one of the most lethal injuries in peace or during the wartime.Severe craniocerebral injury such as diffuse axonal injury(DAI)would cause patient's sympathetic nerve excited abnormally,which often characterized as paroxysmal sympathetic hyperactivity(PSH)in clinical.Extremely high heart rate and body temperature,hypertension,shortness of breath,sweating a lot,increased muscle tone with plasma catecholamine levels such as norepinephrine are cardinal symptoms.The clinical syndrome often leads to the aggravation of patients' intracranial conditions,perplexing extracranial complications,complicating treatment,prolonging hospitalization,worsening prognosis and even result in death.The pathological mechanism of sympathetic hyperactivity after TBI is still unclear,several studies show that the hypothalamus Paraventricular nucleus(PVN)is the important neuroendocrine regulating center.In a variety of pathological conditions,the activation and inflammatory factors' secretion of the microglial change the molecular environment in the PVN of Central nervous system(CNS),which is the key to the regulation of sympathetic hyperactivity.Recent years,the pathological and physiological role of the neutrophil extracellular traps(NETs)in the CNS have drawn more and more attention by the researchers.Our previous study shows that,the ROS levels in the experimental animals' PVN increased significantly within three days after TBI,which coincide with the delay onset of sympathetic activation clinically.As the elevated ROS level is a classic way inducing the formation of NETs,we propose the following hypothesis for this study:After TBI,neutrophils entered PVN chemotactically and activated,which lead to the continuous rise of intracellular ROS level.Then inducing neutrophils release NETs or NETs ingredients change like LL37.NETs formation promotes LL37/P2X7/MST1 pathway causing microglia activated and IL-1beta released,and thus regulating level of neurotransmitters and their functions,enhance the activity of the sympathetic neurons,cause the sympathetic hyperactivity eventually.On the basis of previous research group's result,this study intends to verify hypothesis as following:1.Verify the pattern of sympathetic nerve activity in rats and detect the formation and level of NETs in the PVN region after DAI damage.Ascertain their correlation and determine the optimal observation time.2.Verify the formation of NETs in peripheral blood and the PVN region during the optimal observation time in DAI rats.3.Verify the NETs promote the activation of microglia through the Hippo/MST1 pathway and then regulate sympathetic hyperactivity through Cell experiments.Part I: levels of neutrophil extracellular traps(NETs)in the PVN region and post-sympathetic excitatory patterns of DAI ratsObjective: 1.To verify the rule of sympathetic hyperactivity after DAI in rats,and to determine the optimal observation time for follow-up studies;2.Detect the formation of NETs in the PVN region after DAI in rats.Methods: 1.70 male SD rats were randomly divided into DAI group(n=40)and control group(n=30).After brain damage conducted by diffuse axonal injury model,DAI group and control group were divided into 5 subgroups according to 9h,24 h,48h,72 h and 96 h,respectively.2.Detect the rats' plasma levels of metanephrine(MN)and normetanephrine(NMN)in each group to determine the dynamic changes of their SNS activity through ELISA.3.Western-blot was used to detect the expression of NETs formation marker Cit H3 in the PVN region of DAI rats.Results:1.The plasma level of MN in DAI group increased slightly at 9h,significantly at24h(P<0.05),climbing to the peak at 72 h,and then steady slowly at 96 h,but still higher than that of the control group(P<0.01).While the plasma level of NMN in DAI group started to increase at 9h(P< 0.05),then decreased slightly at 24 h,continued to decline at48 h,but peaked at 72 h,slowly down at 96 h at last,still higher than that of the control group(P<0.01).2.The NETs marker Cit H3 began to appear in the PVN region around 24 h after DAI injury in rats,but to a lesser extent,and it further increased around 72 h,then showed a further upward trend.Conclusions:1.DAI cause rats a sympathetic hyperactivity state.Rats' plasma catecholaminebaseline levels increased and peaked at 72 h,then gradually leveled off.2.The formation of NETs in the PVN region occurred about 24 h after DAI,and further increased at 72 h.3.72 h after DAI in rats was the optimal observation time for sympathetic hyperactivity after brain injury.Part ii: observation of the formation of NETs in peripheral blood and the PVN region after DAIObjective: To verify the formation of NETs in peripheral blood and the PVN region at the optimal observation time(72h)for sympathetic hyperactivity after DAI in rats.Methods: 16 male SD rats were randomly divided into DAI group(n=10)and control group(n=6).Samples were collected 72 hours after DAI strike.Then detect the formation of NETs in peripheral blood of rats by flow cytometry.Immunofluorescence was used to verify the formation of NETs marker in the PVN region at the optimal observation time.Results:1.Cit H3+ and MPO+ double positive were used as the marker of NETs formation cells.The ratio of NETs in peripheral blood of rats was analyzed by flow cytometry.The results indicated that the average generation rate of NETs in peripheral blood of DAI group was 40%,while the average generation rate of the control group was 16.7%.The difference was statistically significant(P < 0.05).2.By immunofluorescence staining of Cit H3,a specific expression protein of NETs in the PVN region of rats,the fluorescence results showed that,compared with the control group,DAI group had almost no Cit H3 fluorescence in the PVN region,and in DAI group,Cit H3 fluorescence in the PVN region Significantly increased expression.Conclusion:1.The formation of NETs in peripheral blood in DAI group was significantly higher than that of the control group.2.72 h After DAI injury,NETs were formed in the PVN region of rats,while there is almost no NETs formed in the PVN region of control group.Part iii: NETs activated microglia through the Hippo/MST1 pathway to induce central inflammatory response in DAI ratsObjective: To clarify that NETs formation activates the Hippo/MST1 pathway by producing LL37 to activate the microglial cell membrane receptor P2X7,which activates microglia cells and promotes the secretion of inflammatory factors.Methods: Extracted DAI rats' peripheral blood neutrophils and co-cultured with rat microglia.The interaction between LL37 and P2X7 in the target cell system was detected by immunoprecipitation.After PAD4 inhibitor YW3-56 was used to inhibit NETs formation and Hippo/MST1 pathway inhibitor XMU-MP-1 was used to inhibit MST1 activation,the m RNA and protein levels of MST1 and YAP in the cell system were detected by q PCR and Western blot.ELISA was used to detect the change of IL-1beta in the cell system at last.Results:1.There was an interaction between P2X7 and LL37 in the co-culture system of DAI rats' peripheral blood neutrophils and glial cells.2.In the co-culture of rat neutrophils with microglia,the neutrophils of DAI rats can promote the transcription and expression of MST1 in the system,and the transcription and expression of MST1 in the system decreased after the addition of PAD4 inhibitor YW3-56 to inhibit the formation of NETs.3.In the co-culture of rat neutrophils with microglia,the neutrophils of DAI rats could promote the transcription and expression of downstream protein YAP of Hippo/MST1 pathway in the system,and the transcription expression of YAP in the system decreased after the addition of PAD4 inhibitor yw3-56.After the addition of Hippo/MST1 pathway inhibitor XMU-MP-1,the transcription expression of YAP in this cell system decreased also.4.IL-1beta content in the co-culture system of neutrophils and microglia cells in DAI rats was higher than that in the control group.After addition of PAD4 inhibitor YW3-56 to inhibit the formation of NETs,the content of IL-1beta decreased.After inhibition of MST1 activation by the addition of Hippo/MST1 pathway inhibitor xmu-mp-1,IL-1beta decreased in this cell system.Conclusion:1.In the co-culture system of rat neutrophils and neuroglial cells,LL37 in NETs interacts with the microglial cell membrane receptor P2X7.2.Inhibit the NETs formation by PAD4 inhibitor YW3-56 can inhibit the expression of MST1 and its downstream effector protein YAP in the Hippo/MST1 pathway,and further inhibit the activation of microglia to release IL-1beta.3.The inhibition of MST1 activation Hippo/MST1 pathway inhibitor XMU-MP-1can inhibit the expression of Hippo/MST1 pathway effector protein YAP in the co-culture system of rat neutrophils and microglia,and further inhibit the activation of microglia cells to release IL-1beta.4.In the co-culture system of rat neutrophils and neuroglial cells,the formation of NETs by neutrophils would release LL37 to promote the activation of membrane receptorP2X7 in microglial and through Hippo/MST1 pathway to further activate it to release inflammatory factors.