| Background:Myocardial ischemia/reperfusion(I/R)injury refers to the phenomenon in which acute myocardial ischemia occurs,and when the blood flow to the ischemic tissue is restored,the myocardial injury will be further aggravated after the blood returns to the heart,including severe heart failure,increased myocardial infarction area and fatal reperfusion injury.It is the one of the main reasons for the poor clinical treatment of ischemic heart disease.Therefore,an in-depth study on the pathogenesis of MI/R injury and exploration of new targets and drugs for protecting the myocardium have become hot research topics.Melatonin is an indole hormone secreted by the pineal gland.It is widely distributed in the brain.Melatonin has a wide range of physiological effects such as neuro-endocrine-immune regulation,hypnosis,sedation,analgesia,anticonvulsant,anti-tumor,anti-aging and other broad biological effects.In addition,the protective role in cardiovascular diseases has been confirmed.In humans and other mammals,there are two types of melatonin membrane receptors: melatonin receptor 1(MT1)and melatonin receptor 2(MT2)subtypes.Melatonin receptors are widely distributed in the brain.MT1 is mainly distributed in the suprachiasmatic nucleus and hippocampal dentate gyrus,while MT2 is mainly distributed in the hypothalamic paraventricular nucleus.At present,the protective effect of melatonin on myocardial I/R injury has become a hot research topic.However,the mechanism of melatonin against myocardial I/R injury has not been fully elucidated.Inflammation is an important link in the process of myocardial I/R injury,and there is overexpression of inflammatory cytokines when myocardial I/R injury.Nuclear transcription factor-κB(NF-κB)plays an important role in the occurrence and development of inflammatory immunity and cardiovascular disease.It is the central mediator of stress stimulation that regulates inflammation in various tissues.Myocardial I/R injury is accompanied by changes in the autonomic nerves,and the paraventricular nucleus(PVN)of the hypothalamus is an important autonomic control center.It plays an important role in the supervision of the cardiovascular system by regulating the energy balance and body fluid balance of the neuroendocrine and autonomic nerves.After myocardial I/R injury,NF-κB in PVN is activated,and sympathetic nerve activity is increased.Melatonin can cross the blood-brain barrier and enter the center,inhibiting NF-κB activity.It can ultimately regulate sympathetic nerve excitability by inhibiting inflammatory response.In this study,we used the method of silent expression of MT2 in PVN area to reveal whether MT2 mediates the myocardial protection of melatonin.We have found that MT2 in the PVN area could mediate melatonin to regulate the NF-κB pathway,which in turn affects the inflammatory response in the PVN area,and could ultimately regulate peripheral sympathetic nerve activity to produce myocardial protection.We will take the melatonin receptor as the starting point in the PVN area to explore the mechanism of melatonin in reducing myocardial I/R injury.It will provide a scientific theoretical basis for a comprehensive explanation of the myocardial protective effect and clinical application of melatonin,and will offer new perspectives.Objecitives:We focus on the role of MT2 in the PVN area in the protection of myocardial I/R injury by melatonin,the central mechanism that MT2 mediated melatonin against myocardial I/R injury and produced myocardial protection.Our goal is to provide a new target for the treatment of myocardial I/R injury and lay the foundation and ideas for clinical application of melatonin.Methods:1.In vivo experimental methodsIn this experiment,C57BL/6J mice were used to ligate the left coronary artery(30min/24 h)to construct a myocardial I/R injury model,and melatonin was administered by intraperitoneal injection and PVN stereotactic micropump infusion.(1)q PCR method was used to detect the expression of melatonin receptor in PVN area after myocardial I/R injury group;(2)Western blot method was used to detect the expression of MT1,MT2,IL-1β,IL-10,NF-κB p65 protein in PVN area;(3)Transcriptome sequencing(RNA-Seq)technology was used to screen PVN region sh-RNA to silence and express MT2 differentially expressed genes;(4)M-mode ultrasound imaging system was used to detect left ventricular ejection fraction(EF)and Left ventricular short axis shortening fraction(FS);(5)Evan’s blue-TTC double staining method was used to determine the percentage of myocardial infarction area;(6)ELISA method was used to observe plasma norepinephrine(NE)and melatonin The levels of IL-1β,IL-10,IL-6 and TNF-α in PVN area;(7)Immunofluorescence method was used to detect tyrosine hydroxylase(TH)in myocardial tissue and MT2 expression level in PVN area of brain tissue.2.In vitro experimental methodsAstrocytes were primary cultured and then transfected with sh-RNA.We established an OGD/R injury model of astrocytes and detected the changes of MT2 and NF-κB p65.Results:1.MT2 expression in PVN region was up-regulated after myocardial I/R injury was established in C57BL/6J mice;astrocytes were cultured in vitro,and MT2 protein levels increased after OGD/R injury.2.In the myocardial I/R injury model,we found that pretreatment with intraperitoneal Objectives:injection and infusion of melatonin in the PVN area stereotactic micropump could improve myocardial function and reduce the area of myocardial infarction.After silenced MT2 in PVN area with sh-RNA,the effect of melatonin is also partially offset.MT2 in the PVN region might mediate the protection of melatonin from MI/R injury effect.3.In myocardial I/R injury model mice,the sympathetic nervous system activity increased excessively,plasma NE levels increased significantly,and the heart’s sympathetic nerve excitability increased.Melatonin could reduce TH expression in myocardial tissues after MI/R,lower plasma NE levels,and reduce sympathetic nerve excitability.When MT2 was knocked out in the PVN region,the inhibitory effect of melatonin on sympathetic nerve activity after MI/R injury was partially offset.Melatonin reduced sympathetic nerve excitability after MI/R injury through PV2 MT2.4.The sh-RNA model of C57BL/6J mice was constructed,and after blocking MT2 in the PVN area,the difference genes in the brain PVN area of sh-MT2 mice and sh-GFP mice were screened by RNA-seq experiment.The results showed that inflammation,immune response,neutrophil chemotaxis and other inflammatory processes are involved in the regulation,and most of the genes involved in these processes are related to the inflammatory response,suggesting that MT2 may have an effect by affecting the inflammatory response in the PVN area.5.After the MI/R model was established,the levels of pro-inflammatory factors IL-1β,IL-6,and TNF-α in the PVN region increased significantly,while the level of anti-inflammatory factors IL-10 has decreased,and melatonin could reduce IL-1β,IL-6and TNF-α levels,but increased IL-10 level.When MT2 is silently expressed in the PVN region,this regulatory effect of melatonin on inflammatory factors was blocked.Melatonin could reduce inflammatory factor levels in PVN region after MI/R through MT2.6.After astrocytes were damaged by OGD/R,NF-κB p65 levels were significantly increased,and melatonin could be used to reduce the levels.When sh-RNA was used to silence astrocytes MT2,and melatonin was applied after OGD/R injury,we observed that NF-κB p65 protein levels have increased.Melatonin could inhibit NF-κB levels in astrocytes via MT2.Conclusion:The above results indicate that melatonin reduces myocardial I/R injury,which is related to MT2 in the PVN area: after myocardial I/R injury,the expression of MT2 in the PVN area is up-regulated,and NF-κB is activated to release a large number of inflammatory factors,which makes the sympathetic nerve over-activation.Melatonin can inhibit the level of pro-inflammatory cytokines,promote the level of anti-inflammatory cytokines,reduce the activity of sympathetic nerves and myocardial I/R damage,and finally achieve myocardial protection. |
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