| Background:Ischemia heart disease (IHD) is a critical killer that jeopardizes human health. Restoringthe supply of blood is of particular significance for the treatment of IHD. However,myocardium often suffers severer insults after the restoration of blood, which is calledischemia reperfusion injury (IRI). IRI is a crucial cause of the bad prognosis of IHDpatients and those who undergo cardiac surgery. Currently, there are a multitude of drugsthat protect against myocardial IRI. However, an ideal pharmaceutical cardioprotectivestrategy has not been achieved because of the side effects, drug safety and ethicalproblems. In recent years, a series of natural substances have been discovered to be safeand non-toxic with potent anti-inflammatory, anti-oxidative and anti-apoptotic features,and thus have become hot spots in the protection against myocardial IRI. Curcumin (Cur) is extracted from the tuber of Zingiberaceae and Araceae plants. Previous studies havefound that Cur has been developed to be an extremely valuable traditional Chinesemedicine due to its multiple cardiovascular benefits. However, the specific mechanism ofCur’s fighting against myocardial IRI has not been well elucidated, which limits thefurther development and widespread clinical application of Cur.Janus activated kinase signal transducer2and activator of transcription3(JAK2/STAT3)is a vital signaling pathway of JAK/STAT family. Previous study has found that ischemiapreconditioning (IPC), ischemia post-treatment and the anti-IRI effect of some drugs arerelevant to the activation of JAK2/STAT3pathway. Notably, we speculate that Curprobably fight against myocardial IRI via JAK2/STAT3pathway since tumor-relatedstudies have revealed the pharmaceutical effect of Cur is closely linked to the modulationof JAK2/STAT3pathway.Silent information regulator of transcription1(SIRT1) is a nicotinamide adeninedinucleotide (NAD+)-dependent histonedeacetylase (HDAC). Recent research has foundthat SIRT1regulates the proliferation, apoptosis, differentiation, aging and metabolism ofcells in a tissue-specific manner, and plays a crucial role in the modulation ofcardiovascular diseases. Forkhead box transcription factor O1(FOXO1) is an importanttarget of SIRT1. SIRT1can regulate the myocardial apoptosis pathway by modulating thedeacetylation level of FOXO1. Both IPC and resveratrol fight against myocardial IRI viathe activation of SIRT1. Cur and resveratrol are polyphenols that can regulate the activityof SIRT1. Besides, activating SIRT1can alleviate OSI and maintain the mitochondrialfunction. Studies in tumor, liver and blood have manifested that there exists cross-talkingbetween JAK2/STAT3pathway and SIRT1pathway. However, their relationship in thecardiac pathophysiological process remains unclear.Above all, we propose that Cur may fight against myocardial IRI by activatingJAK2/STAT3pathway and/or SIRT1pathway, and SIRT1pathway may cross-talk withJAK2/STAT3pathway and eventually exert cardioprotective effect through attenuatingmitochondrial oxidative stress and activating the anti-apoptotic pathway. This project is toemploy a series of research models and experimental methods to elucidate the specific mechanism of JAK2/STAT3and SIRT1-mediated anti-IRI of Cur. This aim of thisresearch is to obtain a new target of drugs that fight against myocardial IRI and providetheoretical basis for studying Cur-based pharmaceutical cardioprotective strategy.Objective:1. To explore whether Cur can fight against myocardial IRI，and to explore whether Curexerts cardioprotection via the regulation of JAK2/STAT3pathway.2. To explore whether Cur exerts cardioprotection via the regulation of SIRT1pathway.3. To explore the cross-talking or upstream and downstream relationship of JAK2/STAT3pathway and SIRT1pathway in normal cardiomyocytes or in the process of Cur’sfighting against myocardial IRI.Methods:Part I: Exploring the effect of Cur against myocardial IRI and whether JAK2/STAT3pathway is involved in the protective effect of CurTo observe the effect of Cur on the isolated SD rat hearts and neonatal ratcardiomyocytes which were subjected to IRI or SIRI, and to define the anti-myocardialIRI effect of Cur. Meanwhile, the effect of Cur on the related molecules of JAK2/STAT3pathway was explored. Further, JAK2/STAT3pathway was blocked by the specificinhibitor AG490or JAK2siRNA in isolated rat hearts or cardiomyocytes, and then toobserve the effect on Cur’s fighting against myocardial IRI. Finally, to explore whetherCur exerts its cardioprotection through the regulation of JAK2/STAT3pathway.1. Firstly, the effect of different concentrations of Cur on the isolated SD rat heartssubjected to IRI and on JAK2/STAT3pathway was explored. The rats were randomlydivided into4groups:①IR;②Cur0.25μM+IR;③Cur0.5μM+IR;④Cur1μM+IR. Thehearts were subjected to ischemia for45min, followed by60min of reperfusion. Recordthe cardiac hemodynamic parameters, including heart rate (HR), left ventricular developedpressure (LVDP), the highest rate of change of pressure development (+dP/dtmax) andcoronary flow (CF). Meanwhile, detect the lactate dehydrogenase (LDH) release in thecoronary effluent and the myocardial infarct size. The apoptosis rate of cardiomyocyteswas analyzed by TUNEL staining. Then, the samples were analyzed for the expression of JAK2, p-JAK2, STAT3and p-STAT3by Western Blot.2. Then, the effect of different concentrations of Cur on rat cardiomyocytes subjected tosimulated ischemia reperfusion injury (SIRI) and on JAK2/STAT3pathway was explored.The cells were randomly divided into4groups:①SIR;②Cur1μM+SIR;③Cur5μM+SIR;④Cur10μM+SIR. The cells were treated with Cur for2h, and then subjectedto simulated anoxia for2h, followed by4h of reoxygenation. The activity of cells, LDHrelease in the culture medium and the expression of JAK2, p-JAK2, STAT3and p-STAT3in each group were measured.3. Further, the effect of JAK2/STAT3pathway inhibitor AG490on the anti-IRI of Cur inisolated rat hearts was explored. Meanwhile, the effect of AG490on the indicators ofmitochondrial oxidative stress and molecules in apoptosis pathways was also studied. Thehearts were randomly divided into4groups:①IR;②Cur+IR;③Cur+AG490+IR;④AG490+IR. After being perfused steadily, the isolated hearts were perfused with KHbuffer containing1μM Cur (with/without1μM AG490) for5min and then subjected toischemia for45min, followed by60min of reperfusion. The cardiac hemodynamicparameter LVDP was recorded. The LDH release in the coronary effluent, the myocardialinfarct size and the apoptosis rate of cardiomyocytes were measured at the same time. Themyocardial mitochondria was isolated and extracted for measuring a series of indicators ofmitochondrial oxidative stress, including superoxide dismutase (SOD), methanedicarboxylic aldehyde (MDA), H2O2, redox potential (Eh), succinate dehydrogenase (SDH)and cytochrome c oxidase (COX). Then, the samples were analyzed for the expression ofJAK2, p-JAK2, STAT3, p-STAT3, Bcl2and Bax by Western Blot.4. Finally, the effect of blocking JAK2/STAT3pathway by JAK2siRNA on theanti-SIRI of Cur in cardiomyocytes was explored. Meanwhile, the effect of JAK2siRNAon the indicators of mitochondrial oxidative stress and molecules in apoptosis pathwayswas also studied. The cells were randomly divided into4groups:①Control siRNA+SIR;②Control siRNA+Cur+SIR;③JAK2siRNA+Cur+SIR;④JAK2siRNA+SIR. After beingtransfected with JAK2siRNA, the cardiomyocytes were incubated with the transfectionmixture for24h, later incubated in10μM Cur for2h, and then subjected to simulated anoxia for2h, followed by4h of reoxygenation. The activity of cells, LDH release in theculture medium and the apoptosis rate of cardiomyocytes in each group were detected.The myocardial mitochondria was isolated and extracted for detecting a series ofindicators of mitochondrial oxidative stress, including SOD, MDA, SDH and COX. Then,the samples were analyzed for the expression of JAK2, p-JAK2, STAT3, p-STAT3, Bcl2and Bax by Western Blot.Part II: Exploring whether SIRT1pathway is involved in the protective effect ofCur’s fighting against myocardial IRI1. Firstly, the effect of Cur on the related molecules of SIRT1pathway on isolated SD rathearts and neonatal rat cardiomyocytes which were subjected to IRI was explored.2. Then, the effect of SIRT1pathway inhibitor Sirtinol on the anti-IRI of Cur in isolatedrat hearts was explored. Meanwhile, the effect of Sirtinol on the indicators ofmitochondrial oxidative stress and molecules in apoptosis pathways was also studied.After being perfused steadily, the hearts were randomly divided into4groups:①IR;②Cur+IR;③Cur+Sirtinol+IR;④Sirtinol+IR. The isolated hearts were perfused with KHbuffer containing1μM Cur (with/without3.75μM Sirtinol) for5min and then subjected toischemia for45min, followed by60min of reperfusion. The cardiac hemodynamicparameter LVDP was recorded. The LDH release in the coronary effluent, the myocardialinfarct size and the apoptosis rate of cardiomyocytes were detected at the same time. Themyocardial mitochondria was isolated and extracted for detecting a series of indicators ofmitochondrial oxidative stress, including SOD, MDA, H2O2, Eh, SDH and COX. Then, thesamples were analyzed for the expression of SIRT1, acetylated-FOXO1(Ac-FOXO1),Bcl2and Bax by Western Blot.3. Further, he effect of blocking SIRT1pathway by SIRT1siRNA on the anti-SIRI ofCur in cardiomyocytes was explored. Meanwhile, observe the effect of SIRT1siRNA onthe indicators of mitochondrial oxidative stress and molecules in apoptosis pathways wasstudied. The cells were randomly divided into4groups:①Control siRNA+SIR;②ControlsiRNA+Cur+SIR;③SIRT1siRNA+Cur+SIR;④SIRT1siRNA+SIR. After beingtransfected with SIRT1siRNA, the cardiomyocytes were incubated with the transfection mixture for24h, later incubated in10μM Cur for2h, and then subjected to simulatedanoxia for2h, followed by4h of reoxygenation. The activity of cells, LDH release in thecoronary effluent and theapoptosis rate of cardiomyocytes in each group were detected.The myocardial mitochondria was isolated and extracted for detecting a series ofindicators of mitochondrial oxidative stress, including SOD, MDA, SDH and COX. Then,the samples were analyzed for the expression of SIRT1, Ac-FOXO1, Bcl2and Bax byWestern Blot.4. Finally, the effect of SIRT1pathway inhibitor Sirtinol on the anti-IRI of Cur in vivorat hearts was explored. Meanwhile, the effect of Sirtinol on the indicators ofmitochondrial oxidative stress and molecules in apoptosis pathways was also been studied.The rats were randomly divided as follows:①IR;②Cur+IR;③Cur+Sirtinol+IR;④Sirtinol+IR. The SD rats (220-250g) were given200mg/kg Cur by intragastricadministration10days before operation. Sirtinol was administered2mg/kg byintraperitoneal injection every other day,5times in total. The control group rats weretreated with saline once a day. The myocardial infarct size, serum creatine kinase (CK)and LDH levels, indicators of mitochondrial oxidative stress (SOD and MDA) and theexpression of SIRT1, Ac-FOXO1, Bcl2and Bax were detected.Part III: Exploring the upstream and downstream relationship of JAK2/STAT3andSIRT1pathway in normal cardiomyocytesand in the process of Cur’s fighting againstmyocardial IRITo block JAK2/STAT3pathway by JAK2siRNA and STAT3siRNA or block SIRT1pathway by SIRT1siRNA in normal cardiomyocytes or in the process of Cur’s fightingagainst myocardial IRI. Then the effect on the expression of relevant molecules in eachother pathway and the cross-talking or upstream and downstream relationship wasexplored.Results:Part I: Exploring the effect of Cur on myocardial IRI and whehter JAK2/STAT3pathway is involved in the protective effect of Cur1. Firstly, we explored the protective effect of Cur against IRI and the effect of Cur on the expression of related molecules in JAK2/STAT3pathway. Studies in isolated heartsrevealed that different concentrations (0.25μM,0.5μM,1μM) of Cur had concreteprotective effect in isolated hearts subjected to IRI, which was manifested by theimprovement of cardiac hemodynamic parameters (HR, LVDP,+dP/dtmaxand CF)(vs. IRgroup, P<0.01or P<0.05), the reduction of myocardial infarct size and LDH release in thecoronary effluent (vs. IR group, P<0.01), all of which were concentration-dependent.These results accorded with the experiments in the cardiomyocyte model, i.e. differentconcentrations (1μM,5μM,10μM) of Cur significantly increased the activity ofcardiomyocytes subjected to SIRI and decreased the level of LDH release into the culturemedium (vs. SIR group, P<0.01). Studies in the isolated hearts and cardiomyocytes bothfound that, Cur treatment significantly increased the expression of p-JAK2and p-STAT3(vs. IR or SIR group, P<0.01).2. Then, studies in isolated hearts revealed that Cur significantly increased the LVDP,decreased the myocardial infarct size, decreased the LDH release in the coronary effluentand decreased the apoptosis rate of cardiomyocytes (vs. IR group, P<0.01). However, theprotective effect of Cur was reversed by AG490(vs. Cur+IR group, P<0.01). Cur alsoeffectively increased the levels of SOD, Eh, SDH and COX in myocardial mitochondriasubjected to IRI and significantly decreased mitochondrial MDA and H2O2(vs. IR group,P<0.01). However, AG490reversed the effect of Cur fighting against mitochondrialoxidative injury (vs. Cur+SIR group, P<0.01). Moreover, Cur significantly increased theexpression of p-JAK2, p-STAT3and the anti-apoptotic protein Bcl2and decreased theexpression of Bax after IRI (vs. IR group, P<0.01). These effect of Cur was also reversedby AG490(vs. Cur+IR group, P<0.01).3. Further, Studies in the cardiomyocytes turned out accordant with studies in isolatedhearts. Cur treatment significantly increased the activity of cardiomyocytes subjected toSIRI, decreased the level of LDH release into the culture medium and decreased theapoptosis rate of cardiomyocytes (vs. Control siRNA+SIR group, P<0.01). However, theprotective effect of Cur was reversed by JAK2siRNA (vs. Control siRNA+Cur+SIRgroup, P<0.01). Cur also effectively elevated the levels of SOD, SDH and COX in myocardial mitochondria subjected to SIRI and significantly decreased mitochondrialMDA (vs. Control siRNA+SIR group, P<0.01). However, JAK2siRNA reversed the effectof Cur fighting against mitochondrial oxidative injury (vs. Control siRNA+Cur+SIR group,P<0.01). Moreover, Cur significantly increased the expression of p-JAK2, p-STAT3andthe anti-apoptotic protein Bcl2and decreased the expression of Bax after SIRI (vs. ControlsiRNA+SIR group, P<0.01). These effect of Cur was also reversed by JAK2siRNA (vs.Control siRNA+Cur+SIR group, P<0.01).Part II: Study on SIRT1pathway involved in the protective effect of Cur’s fightingagainst myocardial IRI1. Studies in isolated hearts or cardiomyocytes revealed Cur significantly increased theexpression of SIRT1compared to the IRI or SIRI group (P<0.01).2. Studis in isolated hearts revealed that Cur significantly increased LVDP after themyocardial IRI and decreased myocardial infarct size, the LDH release in the coronaryeffluent and the apoptosis rate of cardiomyocytes (vs. IR group, P<0.01). However, theprotective effect of Cur was reversed by Sirtinol (vs. Cur+IR group, P<0.01). Cur alsoeffectively elevated the level of SOD, Eh, SDH and COX in myocardial mitochondriasubjected to IRI and significantly reduced mitochondrial MDA and H2O2(vs. IR group,P<0.01). However, Sirtinol reversed the effect of Cur fighting against mitochondrialoxidative injury (vs. Cur+IR group, P<0.01). Moreover, Cur significantly increased theexpression of SIRT1and Bcl2, and reduced the expression of Ac-FOXO1and Bax afterIRI (vs. IR group, P<0.01). The effect of Cur was also reversed by Sirtinol (vs. Cur+IRgroup, P<0.01).3. Studies in the cardiomyocytes turned out accordant with study on isolated hearts. Curtreatment significantly increased the activity of cardiomyocytes subjected to SIRI anddecressed the level of LDH release into the culture medium and the apoptosis rate ofcardiomyocytes (vs. Control siRNA+SIR group, P<0.01). However, the protective effectof Cur was reversed by SIRT1siRNA (vs. Control siRNA+Cur+SIR group, P<0.01). Curalso effectively elevated the level of SOD, SDH and COX in myocardial mitochondriasubjected to SIRI and significantly reduced mitochondrial MDA (vs. Control siRNA+SIR group, P<0.01). However, SIRT1siRNA reversed the effect of Cur fighting againstmitochondrial oxidative injury (vs. Control siRNA+Cur+SIR group, P<0.01). Moreover,Cur significantly increased the expression of SIRT1and Bcl2, and decreased theexpression of Ac-FOXO1and Bax after SIRI (vs. Control siRNA+SIR group, P<0.01).The effect of Cur was also reversed by SIRT1siRNA (vs. Control siRNA+Cur+SIR group,P<0.01).4. Studies in vivo found that Cur treatment significantly reduced the myocardial infarctsize and serum CK and LDH levels (vs. IR group, P<0.01). However, the protective effectof Cur was reversed by Sirtinol (vs. Cur+IR group, P<0.01). Cur also effectively elevatedthe level of SDH and COX in myocardial mitochondria subjected to IRI (vs. IR group,P<0.01). However, Sirtinol reversed the effect of Cur fighting against mitochondrialoxidative injury (vs. Cur+IR group, P<0.01). Moreover, Cur significantly increased theexpression of SIRT1and Bcl2, and reduced the expression of Ac-FOXO1and Bax afterIRI (vs. IR group, P<0.01). The effect of Cur was also reversed by Sirtinol (vs. Cur+IRgroup, P<0.01).Part III: Exploring the upstream and downstream relationship of JAK2/STAT3andSIRT1pathway in normal cardiomyocytes and in the process of Cur’s fightingagainst myocardial IRI1. Studies in the normal myocardial cell models found that JAK2siRNA, STAT3siRNAand SIRT1siRNA had no significant influence on the activity of normal cardiomyocytes(vs. Control siRNA group, P>0.05). The results also showed that JAK2siRNA and STAT3siRNA effectively inhibited not only JAK2/STAT3pathway concomitant with thedown-regulated expression of p-JAK2, JAK2, p-STAT3and STAT3(vs. Control siRNAgroup, P<0.01), but also SIRT1pathway concomitant with the down-regulated expressionof SIRT1and up-regulated expression of Ac-FOXO1(vs. Control siRNA group, P<0.01).SIRT1siRNA effectively inhibited SIRT1pathway concomitant with the down-regulatedexpression of SIRT1and up-regulated expression of Ac-FOXO1(vs. Control siRNAgroup, P<0.01), it also had significant effect on the JAK2/STAT3pathway relatedmolecules (decreased expression of p-JAK2and p-STAT3)(vs. Control siRNA group, P<0.01). The above results indicated that JAK2/STAT3pathway crosstalks with SIRT1patheway in normal cardiomyocytes.2. Studies in the model of Cur’s fighting against myocardial SIRI revealed that JAK2siRNA, STAT3siRNA and SIRT1siRNA all reversed the protective effect of Cur (vs.Control siRNA+Cur+SIR group, P<0.01), which was accordant with the results of Part IIand Part III. Moreover, Cur significantly increased the expression of p-JAK2, p-STAT3and SIRT1(vs. Control siRNA+SIR group, P<0.01). JAK2siRNA and STAT3siRNAreversed not only Cur-activated JAK2/STAT3pathway concomitant with thedown-regulated expression of p-JAK2and p-STAT3(vs. Control siRNA+Cur+SIR group,P<0.01), but also Cur-activated SIRT1pathway concomitant with the down-regulatedexpression of SIRT1and up-regulated expression of Ac-FOXO1(vs. ControlsiRNA+Cur+SIR group, P<0.01). SIRT1siRNA reversed Cur-activated SIRT1pathwayconcomitant with the down-regulated expression of SIRT1and up-regulated expression ofAc-FOXO1(vs. Control siRNA+Cur+SIR group, P<0.01), it also had significant effect onJAK2/STAT3pathway (decreased expression of p-JAK2and p-STAT3)(vs. ControlsiRNA+Cur+SIR group, P<0.01). The above indicated that JAK2/STAT3pathwaycrosstalks with SIRT1-FOXO1pathway in the process of Cur’s fighting againstmyocardial SIRI.Conclusions:1. Cur has definite effect that fight against myocardial IRI.2. Cur alleviates IRI-induced mitochondrial oxidative stress insults and cellular apoptosisthrough the activation of JAK2/STAT3pathway, and further exerts protective effectagainst myocardial IRI.3. Cur alleviates IRI-induced mitochondrial oxidative stress insults and cellular apoptosisthrough the activation of SIRT1pathway, and further exerts protective effect againstmyocardial IRI.4. In the cardioprotection of Cur, JAK2/STAT3pathway crosstalks with SIRT1-FOXO1pathway to fight against myocardial IRI. |
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