The Molecular Effect Mechanism Of Myocardial Protection On Resveratrol Against Stress-induced Injury In Mice

BackgroundStress is the non-specific response of the body to the various positive andnegative non-lethal stimuli caused by enviroment factors. Moderate stressresponse in the body can create a protective effect, but overactive stress responseoften result in various injuries in many organs of body. Heart is the hub of thebody’s cardiovascular system,and thus, it is one of the major stress-inducedtarget organs. With the development of social economy, direct hazards renderedby lethal trauma perse have been substantially reduced, whereas secondary organfailure induced by non-lethal trauma (natural disaster, traffic accidents, the familymember died, etc.) cannot be ignored. In a recent study that non-lethal traumamice model was used to simulate the stress-induced myocardial injury of humanheart caused by natural disasters, researchers found that secondary heart failureoccurred in post-traumatic mice after non-lethal trauma and cardiomyocyteapoptosis was also significantly increased. The increasing cardiomyocyteapoptosis can induce cardiac dysfunction even directly cause death. Therefore, itis imperative to develop the methods for preventing and alleviatingstress-induced injury of heart caused by various stimulating factors.Resveratrol, a natural polyphenol compound, has a variety of biological effect and plays an important role in preventing and treating heart disease,atherosclerosis, malignant neoplasm related to aging, neurodegenerativedisease,etc. Since the rapid progress in recent years, the myocardial protection ofresveratrol has become a hotspot field. An evidence showed that cardioprotectioneffects afforded by resveratrol are due to alleviation of myocardial ischemia/reperfusion injury caused by activating endogenous AMP. Whether resveratrolcan reduce stress-induced myocardial injury or not, and the molecularmechanisms of cardioprotection of resveratrol remain elusive.STAT3is a signal transducer and activator of transcription protein and ispresent in high abundance in various cells and tissues. STAT3plays an importantrole in multiple physiological functions especially in regulating cell growth,differentiation, apoptosis. Studies have suggested that STAT3has closely relatedto inflammation, cancer and immune response. Activation of STAT3signalpathway promote the anti-apoptosis of cardiocytes. Survivin is a member of theinhibitor of apoptosis family and is expressed highly in many tumors, fetal tissue,hematopoietic stem cell, endometrium, villus and decidua. Survivin is so far thestrongest found apoptosis inhibitory factor. Studies showed that the expression ofsurvivin was significantly increased during cardiocytes suffering ischemicdamage and played a role in inhibiting apoptosis, promoting wound healing andinducing angiogenesis. Furthermore, activation of STAT3can upregulate theexpression of survivin in breast cancer cell. Therefore,we hypothesized that themolecular mechanisms of resveratrol alleviate stress-induced myocardial injurycaused by non-lethal trauma might be the activation of STAT3signal pathwayand then the expression of survivin increased. In the present study, we aimed totest this hypothesis and further to elucidate the molecular mechanisms for thecardioprotection of resveratrol against stress-induced injury. Our study mightshed light to cardiovascular protection function of resveratrol and further provideexperimental basis for the mechanisms underlying stress-induced secondarycardiac injury. Objective1. To investigate the effects of stress-induced injury caused by non-lethal traumaon mice myocardium (cardiac function and myocardical apoptosis).2. To ascertain the cardioprotection of resveratrol against stress-induced cardiacinjury caused by non-lethal trauma.3. To observe the molecular mechanisms of cardioprotection of resveratrolagainst stress-induced cardiac injury.Methods1. Resveratrol concentration gradient experiment: Resveratrol was dissolved bycyclodextrin. According to the injection concentration of resveratrol, fortyhealthy adult male Kunming mice were randomly divied into4groups(n=10): group control, group5μM resveratrol, group10μM resveratrol, group20μM resveratrol. Group control mice were injected (p.i)0.2mlcyclodextrin per day, and other three group mice were injected (p.i)equivalent concentration of resveratrol. After1week, the techniques of2D,M-mode and Doppler echocardiography were applied to evaluate the cardiacfunctions of mice.2. Non-lethal truama mice model: Adult male Kunming mice (20-25g) werefully anesthetized with1%inhalation isoflurane and placed in a Noble-Collipdrum apparatus. Whole-body non-lethal trauma was induced by a total of5minutes at a rate of40rpm. Sham traumatized mice were subjected to thesame revolution but the animals were taped on the inner wall of drum, thusavoiding traumatic injury.3. Determination of cardiac function by high resolution small-animal ultrasonicimaging system: According to the instruction manual of Vevo770, mice werefully anesthetized with1%inhalation isoflurane and placed in a constanttemperature (37℃) cushion, then its limbs were connected with4electrodes.Heart rate, left ventricular end diastolic volume (LVEDV), ejection fraction(LVEF), fractional shortening (LVFS) and E/A value, E peak decelerationtime (EDT) were obtained using high resolution small-animal ultrasonic imaging system. All kinds of contraction and relaxation indexes wereautomatically calculated by Visual Sonics Vevo770TMsoftware.4. Determination of myocardial apoptosis: Myocardial apoptosis wasdetermined by terminal deoxynucleotidyltransferase-mediated dUTP nickend labeling staining (TUNEL) and caspase-3activity assay.5. Expression of STAT3protein, survivin and p-STAT3level assay: Expressionof STAT3protein, p-STAT3level and survivin were determined by Westernblotting.Results1. Mice injected resveratrol showed increased value of left ventricular ejectionfraction (LVEF) as compared to those in control group (0μM Res)(P <0.05).However, there is no dose-dependent relationship between LVEF andresveratrol. LVEF of mice in10μM Res group was highest compared to theother3groups (P <0.01), and LVEF of mice in20μM Res group was lowerthan5μM Res group. Given that LVEF is in general not subject to the otherfactors interference, it can be used to reflect cardiac systolic function. Ourresveratrol concentration gradient experiment results showed that10μM ofresveratrol exerted an optimum myocardial protection efficacy.2. Compaired with control group, the cardiac function of mice in Trauma,trauma+Res and trauma+Res+WP1066group were impaired after trauma(P<0.01vs control).①The returned blood volume of mice in Trauma,Trauma+Res and Trauma+Res+WP1066were significantly decreased,and compared to pro-trauma, LVEDV of mice in3trauma group decreasedby44.6％(P <0.001),25.6％(P=0.009),39.3％(P <0.001), respectively;②Under stimuli situation, cardiac function of mice in Trauma, Trauma+Resand Trauma+Res+WP1066group have an obvious compensated change.Compared to pro-trauma, LVEF and LVFS of post-traumatic mice in thesegroups were decreased by14.7％(P <0.001) and25.3％(P <0.001),9.4％(P=0.007) and15.8％(P=0.016) and12.3％(P=0.006) and23.7％(P= 0.003);③Compared to pro-trauma, E/A and EDT of post-traumatic mice inthese groups were decreased by36.3％(P=0.001) and28.8％(P=0.01),25.6％(P=0.005) and20.9％(P=0.009),32.5％(P=0.001) and27.3％(P=0.014).3. Apoptosis index of cardiocytes of mice in Trauma, Trauma+Res andTrauma+Res+WP1066group showed significantlu increased as comparedto those in control group [(37.17±2.28)%vs (3.98±1.32)%，(8.72±1.59)%vs (3.98±1.32)%，(28.93±1.95)%vs (3.98±1.32)%，P <0.01], andactivation of caspase-3was also increased (P <0.01).4. Stress-induced injury of myocardium was significantly alleviated byresveratrol preconditioning.①The cardiac function decline of mice inTrauma+Res group was much lower than those in Trauma and Trauma+Res+WP1066group(P=0.005);②LVEF of mice in Trauma+Res group wassignificantly higher than those in other groups either pro-or post-trauma (P=0.007and P=0.008). whereas, LVFS exhibited no significantly differencebefore trauma compared to other2groups (P=0.909), but the decline ofLVFS was much lower than those in Trauma and Trauma+Res+WP1066group after trauma (P=0.048);③The values of E/A and EDT of mice inTrauma+Res were decreased lower than those in Trauma and Trauma+Res+WP1066group, but there was no significant;④Compared with Traumaand Trauma+Res+WP1066group, apoptosis index of cardiocytes of micein Trauma+Res group exhibited a significant decrease[(8.72±1.59)%vs(37.17±2.28)%，(8.72±1.59)%vs (28.93±1.95)%，P <0.01], andactivation of caspase-3was also increased (P <0.01).5. Expression of STAT3in cardiocytes of mice was significantly increased aftertrauma(P <0.05vs. control), but phosphorylation level of STAT3andexpression of survivin had not obviously ascending trend.6. After trauma, expression of STAT3in cardiocytes of mice was increased which triggered by resveratrol pre-conditioning(P <0.05vs. control).Meanwhile, mice in the former group had increased phosphorylation level ofSTAT3(P <0.001vs. control) and higher expression of survivin(P <0.01vs.control).7. STAT3inhibitor (WP1066) can inhibit cardioprotection of resveratrolthrough regulating expression of STAT3, phosphorylation level of STAT3and expression of survivin（P <0.05vs.Trauma+Res）.Conclusions1. Non-lethal trauma can reduce cardiac function decrease and cardiocyteapopptosis and in turn lead to stress-induced cardiac injury in mice.2. Resveratrol preconditioning can significantly alleviate stress-induced cardiacinjury in mice caused by non-lethal trauma and exert protective effects onheart.3. Resveratrol alleviate stress-induced cardiac injury in mice throughSTAT3/Survivin pathway.