| Traumatic brain injury(TBI)has a high incidence and high rate of disabling and mortality,which greatly affects patients,their families,and society.TBI is an urgent worldwide public health problem.Various complications such as Paroxysmal Sympathetic Hyperactivity(PSH)following TBI severely affect patients’ survival and recovery.The excessive sympathetic excitation after brain trauma mainly manifests as increased blood pressure,body temperature,breathing and heart rate,diaphoresis,limb rigidity,or abnormal activity.Research on the pathogenesis of excessive sympathetic excitement after TBI is essential for its prevention and treatment,but there are currently only several pathogenesis hypotheses in pathophysiological level,and mechanistic studies relating to molecular mechanisms are much needed.Studies found that neutrophils can infiltrate into the cerebrospinal fluid and brain tissue within 4 hours to several days after TBI,forming neutrophil extracellular traps(NETs)that can trap pathogens and is an important form of neutrophils’ functions.HMGB1,one of the components of NETs,is closely related to sympathetic excitation in cardiovascular diseases,but whether it plays a role in sympathetic excitation after TBI warrants further investigation.HMGB1 can mediate inflammatory responses within microglia and further promote the expression of the intracellular transcription factor AP1.AP1 is a transcription factor that can regulate DNA transcription and is closely related to the transcription of inflammation-related cytokines such as IL-1β.IL-1β within brain tissue may regulate the function of excitatory or inhibitory postsynaptic receptors,and their expression levels of counterpart transmitters,which in turn modulate sympathetic excitation.We speculate that after TBI,neutrophils infiltrate into the brain tissue,producing a large number of NETs.HMGB1,one of the components of NETs,enables the abundant production of the transcription factor AP1 within microglia.AP1 promotes the expression of the cytokine IL-1β.Microglia release large amounts of IL-1β cytokines,which alter the activity and transmitter content of neurons within the PVN,affecting the activity and function of receptors and,in turn,leading to sympathetic excitation.Therefore,the hypothesis of this study is: neutrophil NETs contribute to post TBI sympathetic excitation via the HMGB1/AP1 pathway.Both HMGB1 production and IL-1βleading to sympathetic excitation have been reported,and this study focuses on the role and mechanism of the HMGB1/AP1 pathway in sympathetic excitation after head injury.The hypotheses were explored from the perspective of animal experiments and cell experiments in parts I and II,and the conclusions were then further verified by animal experiments in part III:Part Ⅰ.Profile of NETs formation in PVN after TBI and the relationship between HMGB1/AP1 pathway and sympathetic excitationObjective: To confirm the sympathetic excitability of TBI model(Diffuse Axonal Injury,DAI)in rats;to detect the formation of NETs in PVN and the expression of HMGB1/AP1 pathway related molecules;to explore the relationship between HMGB1/AP1 pathway and sympathetic excitation after TBI.Methods: Thirty surviving rats after TBI and thirty sham-operated rats were randomly selected as the experimental and control groups,and randomly divided into five further groups(n = 6)according to five different time points(24 h,48 h,72 h,120 h,168 h).After modeling,the pattern of change in sympathetic excitation(heart rate variability,arterial blood pressure,and serum catecholamine concentrations)was recorded in the experimental and control groups.Brain histopathological sections and β-amyloid precursor protein(β-APP)immunohistochemical staining were performed to observe the brain injury;hypothalamic paraventricular nucleus(PVN)tissue specimens were subjected to CitH3 and MPO double-labelled immunofluorescence staining to determine the formation of NETs by neutrophils;HMGB1 and AP1 immunohistochemical staining to detect their expression levels within the PVN;IL-1β expression levels within the PVN were detected by WB.Results:1.Survival rate of TBI model was 82.22%.Compared with control group,the pathology of experimental group showed obvious damage to the brain tissue,β-App immunohistochemical staining of experimental group showed obvious diffuse axonal injury;2.Compared with control group,decreased heart rate variability(5.22±0.71 vs6.47±0.70,P<0.05),increased arterial blood pressure(121.54±1.33 vs 107.78±1.56,P<0.001)and serum catecholamine concentrations(508.50(493.20,607.80)vs 333.45(322.60,348.35),P<0.01)of experimental group showed similar trends of change after TBI.The peak of sympathetic excitation reached at 72 hours after modeling;3.Observation of CitH3 and MPO double-labelled immunofluorescence staining revealed that the fluorescence was obviously enhanced within the PVN after TBI,and there were colocalization of CitH3 and MPO,indicating that NETs were highly formed;4.Compared with control group,the expression of HMGB1/AP1 pathway-related molecules(HMGB1(12405.47±1817.76 vs 7760.15±851.90,P<0.05),and AP1(5750.73±460.19 vs 4461.94±332.50,P<0.05))and IL-1β(0.80±0.03 vs0.58±0.02,P<0.01)of experimental group were significantly increased in the PVN tissues of rat brains after TBI.Conclusions:1.The model is simple and reliable with a high survival rate,and the peak of sympathetic excitation reaches at 72 h after TBI modeling in rats;2.Nets highly formed in the PVN of rat brain tissue after TBI,and the expression levels of HMGB1/AP1 pathway-related molecules significantly increased;3.HMGB1,AP1,IL-1 β were increased in the PVN of rat brain tissue after TBI,which may be closely related to sympathoexcitation after TBI.Part Ⅱ HMGB1 mediates microglial activation via AP1 and increases IL-1βexpression to promote sympathetic excitationObjective: Using in vitro cell experiments,excluding the interference of complicating factors in vivo,we further demonstrate that HMGB1 can activate microglia and increase IL-1β secretion through microglia by increasing AP1 expression levels,which in turn leads to sympathetic excitation.Methods: Standard microglial cell line was cultured and HMGB1 intervention(10ng/ml,50 ng/ml,100ng/ml,24 h)was applied.Then microglial activation status was determined.WB,qPCR,ELISA and other experiments were used to examine the changes in the expression levels of HMGB1/AP1 pathway molecules and the levels of IL-1β expressed and secreted by microglia.The objective was to demonstrate the ability of HMGB1 to activate microglia and to modulate the AP1 pathway and IL-1 β expression levels.Results:1.Compared with control group,after HMGB1 treatment of experimental group,the protrusions of microglia were significantly reduced and shortened,and the cell body became larger,showing an activated state.The proportion of activated microglia to the total cell number obviously rose(74.70% vs 24.52%,P<0.001);2.Compared with control group,after HMGB1 treatment of experimental group,increased transcript expression of AP1 related genes(2.45±0.34 vs 1.02±0.20,P<0.001)and increased AP1 content(0.94±0.006 vs 0.30±0.005,P<0.001)in microglial cells were detected by qPCR and WB;3.Compared with control group,after HMGB1 treatment of experimental group,increased transcriptional expression of IL-1β related genes(1.96±0.21 vs 1.01±0.15,P<0.001)in microglia was detected by qPCR,and the results of ELISA showed that the amount of IL-1β in the supernatant(162.98±13.49 vs 128.50±16.23,P<0.01)increased.Conclusions:1.HMGB1 can act on microglia to shift microglia activation,morphology,and function.2.HMGB1 can regulate the expression level of AP1 in microglia so that the expression level of AP1 in microglia has a trend of consistent elevation in transcription and translation.3.HMGB1 can regulate the level of IL-1β expression in microglia,which leads to increased expression and secretion of IL-1β in microglia.Part Ⅲ Demonstrates the contribution of the HMGB1/AP1 pathway to sympathetic excitation within the PVN after TBIObjective: To further validate the contribution of the HMGB1/AP1 pathway to sympathetic excitation within the PVN after TBI in live animals.Methods: SD rats were divided into DAI group and sham group according to whether or not they received the DAI procedure.DAI group was further divided into DAI + antiHMGB1 group(n = 6)and DAI + NS group(n = 6).24 h after DAI modeling,brain PVN nuclei were stereotaxically microinjected with anti-HMGB1 or saline,respectively.The sham group was further divided into sham + HMGB1 group(n = 6)and sham + NS group(n = 6).24 h after sham operation,brain PVN nuclei were stereotaxically microinjected with HMGB1 or saline,respectively.The sympathetic excitation indexes,such as blood pressure,heart rate variation rate,and serum catecholamine content of the rats after 72 h of modeling or sham operation were examined;AP1 and IL-1β expression levels in PVN nuclei were analyzed by WB.Results:1.Stereotaxic microinjection of HMGB1 increased the AP1 expression level in the paraventricular nucleus(PVN)of the sham group(1.19±0.04 vs 0.75±0.01,P<0.01),and HMGB1 inhibitors decreased the AP1 expression level in the paraventricular nucleus(PVN)of the DAI group(1.09±0.003 vs 1029±0.017,P<0.001);2.Stereotaxic microinjection of HMGB1 increases the expression level of IL-1β in the PVN of the sham group(1.53±0.09 vs 0.84±0.02,P<0.001),and HMGB1 inhibitors decrease the expression level of IL-1β in the PVN of the DAI group(1.38±0.05 vs 1.58±0.04,P<0.05);3.Stereotaxic microinjection of HMGB1 increased blood pressure(112.47±0.94 vs105.36±1.16,P<0.001),serum catecholamine content(321.78±14.47 vs 293.78±11.52,P<0.05),and decreased heart rate variability(5.55±0.08 vs 6.12±0.19,P<0.001)in the sham group,and HMGB1 inhibitors decreased blood pressure(107.98±1.15 vs 114.40±0.91,P<0.001),serum catecholamine content(293.24±11.76 vs 320.95±7.96,P<0.05),and increased heart rate variability(5.79±0.05 vs 5.21±0.07,P<0.001)in the DAI group.Conclusions:1.HMGB1 within the PVN of brain tissue can regulate the expression level of AP1 after TBI and further lead to changes in the amount of IL-1β expression.2.HMGB1 within the PVN of brain tissue can regulate heart rate variability,blood pressure,and serum catecholamine content after TBI and alter the body’s sympathetic excitation.This innovative proposal highlights the role and mechanism of the HMGB1/AP1 pathway within the PVN in sympathetic hyperexcitability after brain injury.At present,there are very few studies on the underlying molecular mechanisms of sympathetic hyperexcitability after brain injury,and no clear reports on the regulatory pathways.Studies have shown that the PVN plays an important role in the regulation of sympathetic activity,but the relevant mechanisms of action are not well understood.Recent studies have shown that PVN can regulate sympathetic activity through HMGB1,AP1,IL-1 β and other diseases such as myocardial infarction.But this study is the first to link this related molecule with altered sympathetic activity after head injury and to innovatively investigate the key intermolecular reciprocal regulatory relationship,composing a related molecular pathway to elaborate the underlying molecular mechanism.This study is important for elucidating and enriching the pathogenesis of sympathetic hyperexcitability after traumatic brain injury. |
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