| Background:Acute myocardial infarction(ami)is a common cardiovascular disease due to the acute continuous ischemia and hypoxia of coronary arteries,which further causes the necrosis of myocardial cells.Its morbidity and mortality are increasing year by year.Arrhythmia is a common clinical manifestations of AMI,and ventricular arrhythmia after infarction(ventricular arrhythmias,VAs),especially ventricular tachycardia,ventricular tachycardia,VT),ventricular fibrillation,ventricular fibrillation,VF),is the important cause of death and disability AMI patients.Therefore,the early treatment and prevention of post-infarction VAs has become an important measure to reduce the mortality of AMI patients,and how to find the drug targets for the prevention and treatment of AMI arrhythmia and the development of related drugs for the treatment of AMI arrhythmia have become the focus of research at home and abroad.At present,the widely recognized mechanisms of arrhythmia after AMI include:autonomic nerve remodeling after AMI,myocardial electrophysiological remodeling,ventricular remodeling,and the imbalance of distribution,density and activity of sym-pathetic and parasympathetic nerves after myocardial infarction.Among them,the enhancement of peripheral sympathetic activity is the most important pathophysiologi-cal response and clinical feature after the occurrence of AMI.Other studies have shown that the central nervous system also participates in the regulation of sympathe-tic activity.However,current studies on central pathogenesis mainly focus on central cardiovascular regulatory regions,such as the hypothalamic paraventricular nucleus(PVN)and rostral ventrolateral medulla(RVLM),which mainly regulate AMI injury through relevant signaling pathways such as inflammation and oxidative stress.PVN is an important center for regulating humoral balance and sympathetic excitation.It can regulate cardiovascular function by feeling the changes of cerebrospinal fluid in the periventricular ventricles,pressure in the brain stem region,blood volume and chemical signal changes,playing an important role in the regulation of sympathetic activity,also known as "sympathetic outflow tract".Relevant studies have pointed out that injection of inflammatory factors into the lateral ventricle can stimulate the excitability of the sympathetic nerves to a certain extent,and the increase of inflammatory factors expression has a great relationship with the enhancement of sympathetic nerve excitability.Therefore,if a target can be found in the central nervous system to regulate peripheral sympathetic activity,it will certainly provide a new approach for the clinical treatment of AMI arrhythmia.High Mobility Group Protein(HMG)is a non-group dna-binding Protein,widely distributed in the nucleus of higher eukaryotes,and can be divided into HMGA,HMGB and HMGBN families.There are three members of HMGB family,namely HMGB1,HMGB2 and HMGB3,among which HMGB1 is the most abundant.Due to its highly conserved sequence in the evolutionary process,it exists in almost all eukar-yotic cells with up to 99%homology,and is the only protein that can be released to play its extracellular biological activities.Human HMGB1 gene loci exist in the long arm of chromosome 13 1 zone 2,relative molecular weight of 25 kd,distribution is very extensive,a variety of organs,such as lymphoid tissue,brain,liver,lung,heart,spleen,kidney,etc.)are expressed in the cells,in addition to mainly exist in the cytop-lasm in the liver,brain,in most organizations exist in nucleus.Under normal physiol-ogical conditions,the binding of HMGB1 to DNA in the nucleus is loose,but when the cell suffers mechanical damage or necrosis,HMGB1 is easy to be released from the damaged nucleus to the outside of the cell,thus inducing inflammatory response.Because HMGB1 plays an important role in the pathogenesis of inflammatory disease-ss it is also considered to be an endogenous danger signal from dead cells of the body.Mitogen-activated protein kinase(MAPK)signaling pathways play an important role in maintaining the normal physiological activities of the brain,such as gene reproduction and differentiation,and death.At present,the most studied MAPK pathways in mammals are erk 1/2,JNKs and p38 pathways.Studies have proved that ERK 1/2 signaling pathway plays an important role in regulating sympathetic nerve activity in heart failure after myocardial infarction in PVN.Therefore,we speculated that HMGB1 might be expressed in PVN after AMI,and micropump injection of HMGB1 inhibitor(anti-hmgbl-polyclonal antibody,anti-hmgbl polyclonal antibody,hereinafter referred to as anti-HMGB1-Ab),to observe the effect of HMGB1 on peripheral sympathetic activity,better explain the relationship between HMGB1 and arrhythmia after myocardial infarction,and explore its possible signal transduction pathway,so as to provide theoretical support and new targets for drug development for the treatment and prevention of arrhythmia after myoccardial infarction.Therefore,we hypothesized that after AMI,PVN may exist in the expression of HMGB1,through central bipolar casing in PVN of rat after implantation of trace injection pump HMGB1 inhibitors,to observe the effects of HMGB1 foreign weeks sympathetic nervous activity,to better explain the relationship between HMGB1 and arrhythmia after myocardial infarction and discuss the possible signal transduction pathways,for the treatment and prevention of the cardiac arrhythmia after myocardial infarction to provide theoretical support and drug developed newtargets.Objective:In this study,the expression of HMGB1 in PVN and its effect on peripheral symp-athetic activity after AMI were studied by means of micro-pump administration after implantation of bipolar casing in PVN,and the possible mechanism of action was discussed.The expression of HMGB1 and ERK proteins in central PVN of rats in each group was observed from the perspective of morphology and molecular biology;The changes of peripheral sympathetic activity were observed from the functional point of view.It was confirmed that after the occurrence of AMI in rats,the central PVN could participate in the regulation of arrhythmia after AMI through the upregula-tion of HMGB1 expression,and this process was probably completed through the ERK signal transduction pathway.Methods:I.PVN bipolar casing implantationTwo SD rats were placed in a cage,and after 7 days of adaptation,central PVN internal bipolar cannula implantation was performed.By implanting the catheter at one time,the sealing problem of the subsequent multiple dosing can be solved.The sealing of the catheter is good,which can effectively prevent the leakage of the liquid from causing the error of the experimental data.2.Establishment and grouping of rat AMI modelThe rats were divided into the following four groups:I-Control group;II-Sham group:After thoracotomy,the anterior descending branch of the left coronary artery in rats was only threaded without ligation;III-MI group:The AMI model was construct-ed by permanent ligation of the anterior descending branch of the left coronary artery;MI group was further divided into MI+IgG group and MI+Anti-HMGB1-Ab group(The lOug anti-HMGB1-Ab was diluted into a 10u1 TBS(pH=7.4)buffer,and lOul was injected each time with a micro pump at the rate of 0.5 L/min.The antibody was injected every other day for 7 consecutive days.The measurement and frequency of IgG injection in MI+IgG group were the same as before.)3.On the 7th day after AMI,the induction rate and susceptibility score of arrhy-thmia were determined by electrical stimulation with an electrical stimulator support-ed by ventilator.4.On the 7th day after AMI,the left lumbar abdomen was cut vertically to expose the kidney and isolate the sympathetic nerve of the left kidney.5.At the end of the body experiment,methylene blue was injected bilaterally thr-ough central PVN,and then the rats were decapitated and their brains were taken to make frozen sections to determine whether central PVN catheterization was successful.6.After the end of the body experiment,the heart was immediately removed,and the heart tissue was collected for Masson staining to detect the success of the rat model of acute myocardial infarction.Meanwhile,the myocardial infarction area of the rats was measured,and 30-50%of the rats were included in the experimental group.7.After the end of the body experiment,the left ventricular blood of each group was extracted and the plasma NE concentration was detected by ELISA.8.The expression of HMGB1 in PVN was detected by Immunohistochemistry.9.Detection of HMGB1 and ERK proteins expression by Western blot was used to detect the expression in PVN of rats in each group.Results:A total of 115 rats took part in the experiment,78 rats were included in the study(Thirty-one died during PVN catheterization and preparation of the mi model,and six were not included in the experiment because mi area was less than 30%)and were divided into 4 groups:group I:Control group(n=15).II groups:Sham group(n=15).? groups:MI+IgG group(n?26);IV groups:MI+Anti-HMGB1-Ab group(n=22).1.Referring to the stereotaxic map of paxinos-watson rat brain,and according to the PVN position parameters,the PVN internal bipolar casing implantation was performed after accurate positioning with the PVN stereotaxic device.2.In AMI building process,observe the ligature the following myocardial dark,local reduced volatility as the standard,ecg monitoring tip ?,?,lead aVL and aVF,lead at least two of the ST segment elevation 0.2 mv,prompt myocardial infarction building success.3.After conducting programmed electrophysiological stimulation on rats,we found that no spontaneous VAs were observed during the electrode placement and no death was observed in the electrophysiological study.The incidence of VAs in the Control group and Sham group was 10.0%,significantly lower than that in the MI+IgG group(13/26,50%)and the MI+anti-hmgbl-ab group(8/22,36.4%).The susceptibility score of arrhythmia in the Control group and Sham group was close to 0,while that of the MI+IgG group was 3.4±0.85,and that of the MI+anti-HMGB1-Ab group was 1.5±0.56.The above results showed that the induction rate and susceptibility index of arrhythmia in rats after MI were significantly increased,while the susceptibility to arrhythmia after MI was significantly improved after anti-hmgbl-ab was given in PVN.4.We used the biological function experiment system to record the activity of peripheral renal sympathetic nerve,and found that there were no significant differences in RSNA level between two groups,while the expression of RSNA in the MI+IgG group and the MI+anti-HMGBl-Ab group had certain changes compared with the basic value.Compared with the MI+IgG group,the RSNA of the MI+anti-HMGB1-Ab group was significantly decreased(0.06±0.01 uv-s vs 0.11±0.01 uv-s,p<0.05).5.We confirmed the success of PVN bipolar catheterization by injecting methylene blue into the PVN bipolar catheterization with a micro pump and then making frozen sections of brain tissue.6.Masson staining was used to further confirm the success of myocardial infarction modeling and to evaluate the infarct area.The results showed that the myocardial cells were red and the fibrous tissue was blue.7.We also used ELISA to determine the plasma norepinephrine level of the rats in each group,and found that although the plasma norepinephrine level of the rats in the MI+anti-HMGB1-Ab group was higher than that in the Sham group(17.101±0.490 pg/mL vs.14.949 ± 0.562 pg/mL,p<0.001),it was significantly lower than that in the rats in the MI+IgG group(17.101±0.490 pg/mL vs.21.047±1.358 pg/mL,p<0.001).The above results showed that inhibition of HMGB1 expression in PVN could reduce peripheral sympathetic activity which was abnormally increased after MI.8.Immunohistochemical test results showed that HMGB 1 in PVN was rarely seen in Control group and Sham group,and the density of HMGB 1 positive nucleus in PVN was increased in MI+IgG group compared with Control group and Sham group.The density of HMGB 1 positive nucleus in PVN in the MI+anti-HMGB1-Ab group was obviously decreased than that in the MI+IgG group.9.Western Blot was used to detect whether HMGB1 in PVN was involved in the activation of peripheral sympathies after the regulation of MI through the ERK signaling pathway.Image J software was used for WB strip recovery analysis,and the gray value of target protein/internal reference-actin was normalized.The results showed that the level of HMGB1 protein in PVN in the MI+IgG group was significantly higher than that in the Control group and Sham group(2.66±0.43 vs 0.77±0.12,p<0.001).2.66±0.43 vs 1.08±0.23,p<0.001),while the HMGB1 protein level in the MI+anti-HMGB1-Ab group was obviously decreased than that in the MI+IgG group(1.43±0.24vs 2.66±0.43,p<0.001).We also found that the protein level of p-ERK in PVN of the MI+IgG group was obviously increased than that of the Control group and Sham group(1.81±0.45 vs 0.77± 0.22,p<0.001).1.81±0.45 vs 0.93 ± 0.23,p<0.001).However,the expression of p-ERK protein in the anti-HMGB1-Ab group was obviously decreased than that in the MI+IgG group(1.30 ± 0.23 vs 1.81±0.45,p<0.001).The above results showed that AMI can lead to up-regulation of HMGB 1 protein expression in PVN and activation of ERK signaling pathway.However,after the administration of HMGB 1 inhibitor,the expression level of ERK signaling pathway significantly decreased,indicating that the regulation of peripheral sympathetic activity of HMGB1 in PVN is completed through the ERK signaling pathway.Conclusions:l.The AMI model was prepared by ligating the anterior descending branch of the left coronary artery of the rats,which could up-regulate the expression of HMGB1 in the central PVN of the rats after AMI,accompanied by enhanced peripheral sympathetic activity and increased susceptibility to ventricular arrhythmia.2.After the occurrence of AMI,micro-pump injection of anti-hmgbl-ab through PVN can reduce the expression of HMGB1 in PVN,reduce the activity of peripheral sympathetic nerve and susceptibility to ventricular arrhythmia,and provide a new therapeutic target for the prevention and treatment of ventricular arrhythmia after AML3.The effect of HMGB1 on peripheral sympathetic activity in PVN after AMI in rats may be accomplished through the ERK signaling pathway. |
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