Research Of Underlying Mechanism Of Chronic Pain Related Myocardial Ischemic Susceptibility

BackgroundSince chronic pain is not in isolated pathophysiological condition,WHO came up with a conclusion,chronic pain is a kind of disease.In 2018,WHO clarified chronic pain as a systemic disease.Long-last pain leads body into a malignant stress state,which exerts pervasive influences on human bodies.Chronic pain often delineates the decline of physical fitness or the threat against organs health,which might incur derived diseases,abuse and side effect of analgesics and many other problems.Nowadays,the occurrence of chronic pain is almost about 30% worldwide,which means that there are 3 people in 10 are suffered from chronic pain in average.How to charge the pain effectively,take precautional methods against pain-derived diseases and prevent the abuse and side effect of drugs,etc.those problem are of wild practical significance and crucial clinical importance.However,for now,chronic pain influence towards other remote organs,such as the function of heart,remains obscure.Epidemiological studies have already shown clearly that,the morbidity of heart diseases increases in patients under chronic pain condition followed with a worse prognosis,nevertheless underlying pathophysiological mechanisms are not well known.Common analgesics,such as non-steroid anti-inflammatory drugs,could increase the concurrence of heart diseases which constrict its application.Therefore,it is urgent to find out the outset anddevelop process of chronic pain-derived heart diseases and seek potential prevention and cure methods.Myocardial ischemia/reperfusion injury is an imperative clinical problem,improving the vulnerability of heart against ischemia is an important direction towards clinical treatment.Clinical data delineate that,chronic pain elevates the morbidity of myocardial ischemia patients which sheds light on the possibility of chronic pain contributing to the incidence of ischemia vulnerability.Hence,this study aimed to discuss biological mechanisms of increased vulnerability against myocardial ischemia under chronic pain condition.Our previous related study has demonstrated that,using classic chronic compression of the dorsal root ganglion(CCD)model to mimic chronic pain,lipid peroxidation results in reactive carbonyl species accumulation organs and cells,like 4HNE,which destroys proteins,triggers carbonyl stress.Chronic neuropathic pain enhanced reactive carbonyl species accumulation could cause exacerbated cardiac damage and cardiomyocytes death during MI/R injury.Researches have showed that necrosis is of great proportion in cardiomyocytes death during MI/R injury.It necessary to further discuss the way cardiomyocyte death during MI/R injury under chronic pain condition.Necroptosis,referred as a controllable way of cell death,has been getting more and more attention in MI/R injury.It has already been confirmed that Necroptosis including RIP1,RIP3 complex,necrosome,mediating downstream MLKL activation and its cascading signaling.Thus,to explore the prevention measures of myocardial Necroptosis under chronic pain condition is a new strategy in cardiopretection.AimsThis study was based on previous studies,emphasis on discuss:1.To verify the impact of chronic neuropathic pain on MI/R susceptibility.2.To analyze the role of myocardial Necroptosis in chronic pain related myocardial ischemic vulnerability.3.To clarify the effect of increased TNF-? in both plasma and myocardial tissue under chronic neuropathic pain condition and to analyze the underlying signaling of RIP1-RIP3-MLK and RIP3-CaMKII.4.To clarify whether exogenous melatonin could suppress TNF-? in plasma and myocardial tissue,subsequently,inhibit RIP1-RIP3-MLKL and RIP3-CaMKII pathway,resulting in decreasing in oxidative stress,myocardial Necroptosis and relieving the pain and realizing cardioprotection.Methods1.This study used male C57 mice and applied classic mice spared nerve injury(SNI)to build chronic pain model(SNI group),sham operation group served as control group(Con group).Some experiments was used C57 background RIP3 knockout mice(RIP3 KO)to build SNI model,served wild type C57 mice(WT)as control.2.The nociception of SNI mice assessed by Von Frey filaments,which pressed and kept bent in the middle area of the plantar surface of the hindpaw.3.Four weeks after SNI surgery,mice were subjected to myocardial ischemia/reperfusion injury(ischemia for 30 min following with reperfusion for 4 h)in vivo.4.Cardiac function was assessed by echocardiography.Quantitative determination of LDH level in blood circulation.TTC staining was used to present infarct size and EBD dyeing was used to detect myocardial Necroptosis extent.5.Quantitative determination of plasma TNF-? level;immunohistochemistry was used to assay myocardial TNF-?.6.Western blotting was used to detect RIP1,RIP3,MLKL,phospho-MLKL,CaMKII and phospho-CaMKII expression.Co-immunoprecipitation assays was used to indicate the interaction of RIP1-RIP3.Results1.Chronic pain led to exacerbated MI/R injuryWT mice were used to establish mice SNI model.Ipsilateral hind paw withdrawal mechanical threshold(PWMT)was analyzed by Von Frey filament test.After SNI surgery,the PWMT of mice was obviously decreased and sustained for at least 4 weeks,which showed neuropathic pain models are manufactured.4 weeks after SNI surgery,SNI-WT and mice were subjected to were subjected to 30 minischemia followed by 4 h reperfusion in vivo.Compared with Con group,the myocardial injury was obvious severe in SNI chronic pain mice group,referring to enlarged myocardial infarct size,increased LDH,further depressed left ventricular ejection fraction(EF%)and fractional shortening(FS%),further more,the mortality induced by MI/R injury of SNI was distinctly increased compared with control group.the results above suggests that chronic pain sensitizes heart to MI/R injury and lead to chronic pain related myocardial ischemic susceptibility.2.Chronic pain induced enhanced myocardial NecroptosisAfter verified chronic pain related myocardial ischemic susceptibility,we found that myocardial Necroptosis was enhanced in SNI-WT mice compared with Con-WT by applying EBD staining,which indicated that enhanced myocardial necrosis is an important component of chronic pain induced susceptibility to MI/R injury.While testing the level of TNF-? in plasma,an obvious increase was showed in SNI-WT mice compared with Con-WT group.After reperfusion for 4 h,TNF-? in myocardial tissue,detected by immunohistochemistry also elevated in SNI-WT mice compared with Con-WT mice.Western blotting results showed that RIP1 and RIP3 expression fortified in SNI-WT mice hearts(compared with Con-WT,P<0.05,n=5).Co-IP assays revealed that MI/R-induced cardiac RIP1-RIP3 interaction(necrosome formation)were further strengthen in SNI mice compared with control group.These signaling mechanism results indicated that myocardial Necroptosis play an important role in myocardial ischemia vulnerability.Chronic pain incurred TNF-? elevated is a possible trigger of cardiomyocyte death.3.RIP3 deficiency attenuated MI/R injury in chronic pain miceSo in order to clarify the effect of Necroptosis,RIP3 KO mice were used to establish SNI model(SNI-RIP3 KO group),compared with chronic pain WT mice(SNI-WT group),and then subjected them to MI/R injury.Analyzing the myocardial Necroptosis signaling related protein expression,we found that after MI/R surgery.Compared with SNI-WT mice,the level of myocardial phospho-MLKL and phospho-CaMKII markedly suppressed in SNI-RIP3 KO mice.RIP3 deficiency clearly inhibits MI/R-induced increase in ROS production in SNI mice by DHE staining,and myocardial Necroptosis was inhibited shown by EBD dyeing(vs.SNI-WT,P<0.05,n=5).These data further proved that RIP1/3-MLKL/CaMKII mediating myocardial Necroptosis participated in myocardial ischemia vulnerability under chronic pain condition;specifically inhibit these signaling pathways could effectively mitigate mice myocardial injury.4.Melatonin suppressed enhanced myocardial Necroptosis under chronic pain conditionMelatomin mainly produced by the pineal gland,and it exerts analgesia and cardioprotection effect.We next discussed the influence of exogenous melatonin on chronic pain and chronic pain related myocardial ischemic susceptibility.Different dose of melatonin,20,40,60 mg/kg,i.p.,was applied to SNI mice.The PWMT was elevated while low dosage(20,40mg/kg,i.p.)failed to meliorate allodynia,while high dosage of melatonin treatment(60 mg/kg,i.p.)could elevate,which suggested that melatonin has allodynia effect to some extent.However,we found that low dosage of melatonin treatment(20 mg/kg,i.p.)administrated to mice 10 min before reperfusion)could effectively reduce cardiac TNF-? IHC staining and plasma TNF-? level,lower the expression of RIP1 and RIP3,weaken the interaction of RIP1-RIP3,disturb the formation of necrosome,and inhibit the phosphorylaiton of MLKL and CaMKII.Further,we found that CaMKII mediated myocardial ROS production was suppressed and EBD dying suggested that melatonin treatment effectively reduced myocardial Necroptosis in response to MI/R injury.Results from the experiments above shows that chronic pain induces myocardial ischemic susceptibility,oxidative stress and myocardial Necroptosis enhances and cardiac RIP1-RIP3 mediating MLKL/CaMKII pathway is of great importance.Melatomin exerts inhibition of RIP1-RIP3 mediating MLKL/CaMKII pathway,oxidative stress and myocardial Necroptosis.5.Melatonin alleviated chronic pain related myocardial ischemic susceptibilityIn vivo animal experiments confirmed that,melatonin treatment could effectively relieve M/IR injury in SNI mice,minimized cardiac infarct size,decreased LDH release,and restore cardiac function,finally,markedly improved survival rate of SNI mice after MI/R injury.Conclusions1.Chronic pain sensitizes to myocardial ischemic susceptibility.2.Myocardial Necroptosis is an important method of cardiomyocyte death in chronic neuropathic pain incurred myocardial ischemia injury,increased level of TNF-? plays a crucial role therein.3.RIP1-RIP3 interaction and its downstream MLKL/CaMKII signaling plays an important role inmediating myocardial Necroptosis under chronic neuropathic pain condition.4.Applying exogenous melatonin suppresses TNF-?,RIP1-RIP3 mediating MLKL/CaMKII signaling,and myocardial Necroptosis.5.Melatonin meliorates chronic pain induced myocardial ischemic vulnerability,and has analgesia effect.The current study verifies that chronic neuropathic pain referred as a malignant basic factor contributes to myocardial Necroptosis during ischmiea/reperfusion injury,which is characterized by ischemia vulnerability.The strategy of our study is worth to be noticed as a new way to protect heart under chronic pain condition.We apply melatonin to inhibiting myocardial neroptosis,so in order to discuss the way in cardioprotection and analgesia effect as well,which provides to clinical patients with novel experiment basis of organ protection and precaution of chronic pain related cardiovascular complications.