Effects Of Targeting Blockade Of Nuclear Factor-?B In The Hypothalamus Paraventricular Nucleus On Sympathetic Hyperinnervation After Myocardial Infarction

BackgroundMyocardial infarction(MI)is one of the most serious diseases that threaten human health.MI is most common in Europe and America.In the United States,about 0.05%of the population suffers MI every year,what's more,the incidence of MI in China has shown a significant upward trend in recent years.MI is a serious life-threatening complication that can lead to shock,heart failure,ventricular aneurysm and arrhythmia.Especially the malignant ventricular arrhythmia(VAs)in arrhythmia is the most serious,which is one of the main causes of death during the recovery period of MI patients.The occurrence of VAs is associated with excessive regeneration and remodeling of cardiac sympathetic nerves after MI.The sympathetic and vagus nerves control the heart activity together,and any instability of cardiac electrophysiological activity(such as sympathetic activity or inhibition of vagus inhibition)can lead to the occurrence of VAs in the clinic,which threatens the lives of patients.Recently,related studies have shown that cardiac autonomic remodeling characterized by excessive sympathetic regeneration is an important factor in the development of early VAs in MI.Therefore,exploring the molecular mechanism of sympathetic regeneration after MI can provide a new theory for the treatment of VAs after MI.Nuclear factor-?B(NF-?B),known as an important nuclear transcription factor,is involved in the process of neural cell development,growth and apoptosis.Our previous study found that the activation of NF-?B in myocardial tissue after MI was a key link in cardiac sympathetic regeneration,inhibiting NF-?B activity in myocardial tissue,significantly inhibiting sympathetic nerve regeneration after MI,and improving MI cardiac sympathetic remodeling.However,our previous studies on nerve regeneration and arrhythmia after MI are only limited to heart itself,and we know little about the upstream of cardiovascular regeneration regulation mechanism after MI-the central nervous system(CNS).There are many cardiovascular regulatory centers in the brain(the area of the CNS where neurons are concentrated and are involved in controlling cardiovascular activity),hypothalamus plays an important role in regulating cardiovascular activities.The hypothalamic paraventricular nucleus(PVN)is the regulative central of cardiovascular activity and autonomic nervous system.The frequency of spontaneous action potentials was increased in PVN pre-sympathetic neurons after MI,and further caused cardiac sympathetic activation,which suggested that PVN can regulate the excitation sympathetic nerves,and regulate the cardiovascular activity of MI rats.In recent years,there have been many studies about the effect of PVN on sympathetic activity in rats with hypertension and heart failure,but few study about the effect of PVN on sympathetic remodeling in rats after MI.Therefore,we hypothesizing that targeting blockade of NF-?B signaling pathway in PVN can attenuate cardiac sympathetic hyperinnervation and decrease the incidence of malignant arrhythmias after MI.ObjectiveThe aim of this study was to investigate whether targeting blockade NF-?B signaling pathway in PVN,can regulate the sympathetic nerve regeneration,malignant arrhythmia after MI.MethodsExperiment 1To determine the dynamic changes of NF-?B expression in PVN after MI,we performed 30 male Sprague-Dawley(SD)rats(260-280 g)with left anterior descending coronary artery ligation to establish MI model,which was divided into 5 groups:MI-Od,MI-1d,MI-3d,MI-5d,MI-7d,rats in MI-Od group were sacrificed immediately after MI,and others were sacrificed 1 day,3 days,5 days and 7 days after MI.The dynamic expression of NF-?B and I?B? was detected by Western Blot.Experiment 2To explore the relationship between NF-?B and interleukin-1?(IL-1?)in PVN,we designed the following experiment:18 SD rats were randomly divided into 3 groups,A:naive group,the animals in this group were anesthetized only;B:LPS group,after anesthesia,two small holes were drilled into the top of the skull through the animal brain stereotactic device,and then LPS was administered to the PVN on both sides via a micro-syringe(12.5?g LPS were dissolved in 50nl normal saline),50nl per side;C:LPS+gevokizumab group(LPS+gevo group),the rats in this group were injected with LPS in both sides of PVN,the method and dosage were the same as group B,30 minutes later,injected the IL-1? inhibitor gevokizumab(50 ?l of physiological saline containing 10 ?g of gevokizumab).After 2 hours,the brains were removed for subsequent Western Blot experiments.Experiment 3In order to explore the role and mechanism of NF-?B signaling pathway in PVN in sympathetic remodeling after MI,two holes were drilled in rats skull through the brain stereotaxic instrument,then place and fix the stainless steel PVN double,restore for 7 days,then left anterior descending coronary artery was firstly established,and then PDTC was delivered to inhibit NF-?B activity through the PVN in the cannula for 7 days.On the 7th day after MI,the ventricular pressure volume test was used to detect the hemodynamic state of the rats and the electrical stimulation test was performed to detect the susceptibility to arrhythmia.After the experiment,the brain tissues and myocardial tissues were removed,and some brain tissues were separated to detect the expression of NF-?B signaling pathway-related protein in PVN,and the other part was used to detect the expression level of Fra-LI through immunohistochemistry.Left ventricular was isolated from myocardial tissue,and immunofluorescence assay was used to detect tyrosine hydroxylase(TH)and growth associated protein-43(GAP-43)positive nerve fibers in the periinfarct area to analyze sympathetic regeneration.Results1.The level of NF-?B in PVN increased significantly at 3 days after MI,and decreased slightly on the 7th day,but remained at high level,suggesting that the NF-?B pathway in PVN was activated after MI.2.NF-?B,IL-1? and nerve growth factor(NGF)protein expression levels were increased in rats PVN in LPS group.After administration of IL-1? inhibitor,NF-?B remained at high level,but the expression levels of IL-1? and NGF protein were significantly decreased,suggesting that IL-1? involved in NF-?B pathway in rat PVN.3.The NF-KB/IL-1? signaling pathway in PVN is activated after MI,and this phenomenon can be blocked by targeting infusion NF-?B inhibitor PDTC in PVN.MI+PDTC group compared with MI+PBS group,Western Blot results showed that the expression levels of NF-?B,IL-1? and NGF protein in PVN were decreased,and immunohistochemistry experiment showed that the sympathetic activity in PVN was also weakened.Immunofluorescence experiment showed decreased TH and GAP-43 positive nerve fiber density in infarcted area;programmed electrical stimulation test and ventricular pressure volume measurement suggest a decrease in susceptibility to arrhythmia and improvement of cardiac function.ConclusionTaken together,our data showed that targeted inhibition NF-?B signaling pathway in PVN can attenuate sympathetic regeneration after MI by down-regulating IL-1? and NGF protein expression levels.This result suggested that targeted inhibition NF-?B in PVN may be a new target for attenuating cardiac sympathetic remodeling and reducing the incidence of arrhythmia after MI.