| BackgroundAcute myocardial infarction (MI) is a primary cause of mortality and mobidity, which becomes a severe health problem worldwide now. When acute MI occur, rapid restore coronary blood flow by either percutaneous coronary intervention or thrombolytic therapy is very important for salvaging myocardial tissue from inevitable necrosis and to decrease infarct size (IS), but paradoxically, reperfusion itself is frequently associated with an exacerbation of tissue injury and profound inflammatory response (named ischemia reperfusion injury). Moreover once a single organ expose to ischemia reperfusion may subequently result in inflammatory activation in other organs, eventually leading to multiorgan failure and greatly increases mortality and morbidity. Hence, that, to alleviate myocardial ischemia/reperfusion injury, is a very important stretagy in the management of acute myocardial ischemia.High mobility group box 1 (HMGB1), a ubiquitous and abundant nuclear protein, can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatiory signals. HMGBl is widely distributed in the liver, brain, spleen, lung, heart, kidney and lymphatic tissue. The first 40 peptides segments of B-box can induce the production of tumor necrotic factor-a (TNF-a) and the interleukin (IL)-6. As a pro-inflammation cytokine, HMGB1 takes an important role in many cardiovascular diseases, such as atherosclerosis, myocardial ischemia/reperfusion injuries, heart failure and myocardial infarction. Clinical studies show that circulating levels of HMGB1 correlates with the severety of coronary artery disease and may be a potential and independent predictor of cardiovascular mortality in patients with UA/NSTEMI. Extracellular HMGB1 not only represents an optimal "necrotic marker" selected by the innate immune system to recognize tissue damage and initiate reparative responses, but acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and promote I/R-induced myocardial injury as well.Recently, it has been reported that exogenous administration of HMGB1 after myocardial infarction or acute global ischemia/reperfusion leads to the recovery of left ventricular function through the regeneration of cardiomyocytes. But once the dose of HMGB1 increased, it can only inhibit inflammation reactions other than improves the recovery of left ventricular function. Furthermore, most of previous studies have given HMGB1 by ways of intracoronary injection or direct intra-myocardial injection in various animal models. However, the effects of intravenously infused HMGB1 on myocardial ischemic reperfusion injuries is less certain.To date, the effects and the underlying mechanisms of intravenous administration of HMGB1 on the ischemia reperfusion myocardium remain unclear. Therefore, the purpose of the present study was to investigate the changes of myocardial ischemia reperfusion injury and to elucidate the mechanism of effects of intravenous HMGB1 on ischemic reperfusion myocardium. We established an ischemic reperfusion model in vivo. The related inflammation cytokines, cardiomyocytes apoptosis, infact size of myocardium and related signal pathways were detected.The present study includes two parts:PART Ⅰ:Effects of intravenous HMGBlon myocardial ischemia reperfusion injury in rats.PART Ⅱ:Regulating mechnisms of PI3K/Akt signal pathway on the effects of HMGB1 on myocardial ischemia reperfusion injury in rats.PART I:EFFECTS OF INTRAVENOUS HMGB1 ON MYOCARDIAL ISCHEMIA REPERFUSION INJURY IN RATSObjectiveThe purpose of the present study was to investigate the cardioprotective effects of intravenous high mobility group box 1 (HMGB1) (in different doses) on (1) serum myocardial inflammatory and necrosis biomarkers levels of interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and cardiac troponin I (c-TnI), (2) myocardial oxidative stress products levels of malondialdehyde (MDA)and activity of superoxide dismutase (SOD) and (3) myocardial infarction size (IS) via construction of acute myocardial ischemia/reperfusion (I/R) models in rats. Thus, to study the cardioprotective effects of intravenous HMGB1 in different doses on I/R myocardium in rats following acute myocardial ischemia.MethodsMale Wistar rats (weighs 250g-300g) were chosen in the present study and were randomly divided into five groups that includes:sham group (n=15), I/R group (n =15), HMGB50 (HMGB150 ng/kg, i.v., n=15) group, HMGB100 (HMGB1100 ng/kg, i.v., n=15,) group and HMGB200 (HMGB1200 ng/kg, i.v., n=15) group. Left anterior descending coronaries were occlused for 30 min and reperfused for 4h in each I/R and HMGB groups. HMGB1, in different doses, were solved in 0.5 ml normal saline, and was given via tail vein 10 min before reperfusion, respectively. LAD was not ligated while was just placed suture beneth the LAD artery in the sham group.After reperfusion for 4h, the following steps were taken:1. Serum IL-6, TNF-a and cTNI levels and myocardial MDA and SOD levels were measured by an enzyme link immunosorb assay2. Measurements of myocardial sizes by 2,3,5-Triphenyltetrazolium chloride evans blue staining methods (TUNEL).3. Pathological measurements of myocardial tissue in each groups (by haematoxylin and eosin staining).4. Investigation of apoptosis of myocardiocytes by terminal deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) methods.Results1. As shown in figure 1, figure 1 (a) shows normal sinus cardiac rhythms. It, ST segment elevated after LAD ligated for 30 min, suggests that the I/R model was constructed successfully. Ventricular arrhythmias, namely ischemia reperfusion arrhythmias, was found after LAD was reperfused (as shown in figure 1(c)).8 rats (53.3%) suffered from ventricular premature beats and 2 with ventricular fibrillation. When treated with HMGB1, ventricular arrhythmia rates were decreased (p>0.05) (7 rats (46.7%) in HMGB 50 group,6 rats (40.0%) in HMGB 100 group and 6 rats (40.0%) in HMGB 200 group, respectively). It suggests that treatment with HMGBl can reduce ventricular arrhythmias in rats undergone I/R (p>0.05).2. As shown in table 1 (a-e) and figure(2-6), serum levels of IL-6 (377.46 ± 16.86/166.87 ± 15.45 pg/ml, p<0.01), TNF-a (77.23 ± 1.01/16.68 ± 2.57 pg/ml, p <0.01) and cTnI (78.46 ± 8.60/0.13 ± 0.12 ng/ml, p<0.01) and myocardial MDA (8.59 ± 0.71/1.48 ± 0.17 nmol/mg, p<0.01) contents were very higher in the I/R group than that in the sham group. Myocardial contents of SOD was significantly decreased in the I/R group (71.33 ± 5.81 u/mg) than that in the sham group (140.77 ± 10.55 u/mg,p<0.01).Serum levels of IL-6 in HMGB50 (296.97 ± 24.08/377.46 ± 16.86 pg/ml, p <0.01), HMGB100 (271.05±16.94/377.46 ± 16.86 pg/ml, p<0.01) and HMGB200 (227.62 ± 16.22/377.46 ± 16.86 pg/ml, p<0.01) groups were greatly reduced than that in the I/R group in a dose dependant manner.Serum levels of TNF-a in HMGB50 (58.62 ± 5.83/77.23 ± 1.01 pg/ml, p<0.01), HMGB100 (52.69±6.74/77.23 ± 1.01 pg/ml, p<0.01) and HMGB200 (45.70± 8.71 /77.23 ±1.01 pg/ml, p<0.01) groups were greatly reduced than that in the I/R group in a dose dependant manner.Serum levels of cTnI in HMGB50 (66.26 ± 6.87/78.46 ± 8.60ng/ml, p<0.01), HMGB100 (58.74 ± 7.05/78.46 ± 8.60 ng/ml, p<0.01)and HMGB200 (52.40 ± 6.70 /78.46 ± 8.60 ng/ml, p<0.01) groups were greatly reduced than that in the I/R group in a dose dependant manner.Myocardial contents of MDA in HMGB50 (6.81 ± 1.43/8.59 ± 0.71 nmol/mg, p <0.01), HMGB100 (5.50 ± 0.88/8.59 ± 0.71 nmol/mg, p<0.01) and HMGB200 (3.88 ± 1.12/8.59 ± 0.71 nmol/mg, p<0.01) groups were greatly reduced than that in the I/R group in a dose dependant manner.Myocardial content of SOD was significantly increased in the HMB50 (83.47 ± 5.75 u/mg), HMGB100 (92.55 ± 6.01 u/mg) and HMGB200 (117.26 ± 10.31 u/mg) groups than that in the I/R group (71.33 ± 5.81 u/mg, all p<0.01) in a dose dependant manner.3. As shown in figure 7, the normal myocardial tissue dyed blue, the ischemic damaged myocardium stained brick like red, while infarcted myocardium dyed white.As shown in table 1(f) and figure(8), myocardial size in the HMGB50 ((45.80 ± 4.37)%, p<0.01), HMGB100 ((40.60 ± 5.04)%, p<0.01) and HMGB200 ((34.10 ± 5.99)%, p<0.01) groups were significantly reduced than that in the I/R group ((56.30 ± 4.47)%). The higher the HMGB1 doses, the smaller the myocardial sizes were.4. As shown in figure 9, I/R group, histological examination showed focal myocardial damage with uneven staining. Morphological changes in affected cardiomyocytes primarily comprised different degrees of swelling, necrosis, myocytolysis, myofibrillar loss. Myocardial fibers were broken and arranged irregularly. The myocardial infarction foci were infiltrated with a large number of inflammatory cells such as neutrophils. But in the HMGB groups, myocardial fibers arranged relatively regular. Infiltration of a large number of neutrophils can be found.5. As shown in figure (10-11) and in table 1(g), myocardiocytes apoptosis rate is significantly higher in the I/R group ((29.09 ± 2.41)%, p<0.01) than that in the sham group ((5.43 ± 1.58)%, p<0.01).Myocardiocytes apoptosis rate in the HMGB50 ((24.90 ± 4.43)%, p<0.01), HMGB100 ((16.04 ± 1.99)%, p<0.01) and HMGB200 ((13.39 ± 1.64)%, p<0.01) groups were significantly decreased than that in the I/R group ((29.09 ±2.41)%). HMGB1 decreased myocardiocytes apoptosis rates in a dose dependant manner.Conclusion1. Myocardial ischemia reperfusion injury manifests severe myocardiocytes necrosis, irregular arrangement of myocardial fibers, increased serum levels of inflammatory and myocardial necrosis biomarkers and oxidative stress substances.2. HMGB1 may attenuate myocardial ischemia reperfusion injury by inhibiting myocardial inflammation responses and release of myocardial enzyme, reducing infarct size and inhibiting myocardiocytes apoptosis. HMGB1 may exert its cardioprotective effects by inhibiting the expression of inflammatory cytokines and myocytes apoptosis.Myocardiocytes apoptosis rate in the HMGB50 ((24.90 ± 4.43)%, p<0.01), HMGB100 ((16.04 ± 1.99)%, p<0.01) and HMGB200 ((13.39 ± 1.64)%, p<0.01) groups were significantly decreased than that in the I/R group ((29.09 ±2.41)%). HMGB1 decreased myocardiocytes apoptosis rates in a dose dependant manner.Conclusion1. Myocardial ischemia reperfusion injury manifests severe myocardiocytes necrosis, irregular arrangement of myocardial fibers, increased serum levels of inflammatory and myocardial necrosis biomarkers and oxidative stress substances.2. HMGB1 may attenuate myocardial ischemia reperfusion injury by inhibiting myocardial inflammation responses and release of myocardial enzyme, reducing infarct size and inhibiting myocardiocytes apoptosis. HMGB1 may exert its cardioprotective effects by inhibiting the expression of inflammatory cytokines and myocytes apoptosis.PART II REGULATING MECHNISMS OF PI3K/Akt SIGNAL PATHWAY ON THE EFFECTS OF HMGB1 ON MYOCARDIAL ISCHEMIA REPERFUSION INJURY IN RATSObjectiveThe purpose of the present study was to construct a rat myocardial ischermia reperfusion (I/R) model, and to investigate the effects of intravenous high mobility group box 1 (HMGB1) on (1) serum inflammatory and necrosis biomarkers levels of interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and cardiac troponin I (c-Tnl), (2) myocardial oxidative stress substances levels of malondialdehyde (MDA) and superoxide dismutase (SOD) and (3) myocardial infarction size (IS) in I/R rats. Meanwhile, wortmannin, a PI3K inhibitor, was used to explore whether or not the PI3K/Akt signal pathway play a regulating effects in the process of cardioprotection of HMGB1 on myocardial ischemia reperfusion injury in rats.MethodsMale wistar rats (250g-300g) were chosen in the present study and were randomly divided into five groups includes:sham group (n=20), I/R group (n=20), I/R+ HMGB200 group (n=20), I/R+Wortmannin group (n=20), and I/R+HMGB200+ ortmannin group (n=20). I/R+HMGB200 group:LADs were ligated for 30 min and reperfused for 4h. HMGB200(200ng/kg) was dissolved in 0.5 ml normal saline and injected via tail vein 10 min before reperfusion and 0.5 ml normal saline injected via tail vein 15 min before reperfusion. I/R+wortmannin group:LADs were ligated for 30 min and reperfused for 4h. Wortmannin (20μg/kg, dissolved in 0.5 ml normal saline) and normal saline 0.5 ml were injected via tail vein 15 min and 10 min before reperfusion, respectively. I/R+HMGB200+wortmannin group:LADs were ligated for 30 min and reperfused for 4h. HMGB200 (200μg/kg) and wortmannin (20μg/kg) were dissolved in 0.5 ml normal saline, respectively and injected via tail vein 10 min and 15 min before reperfusion, respectively. While in the sham group, suture was just placed beneth the LAD artery without ligation of the artery.After reperfusion lasts for 4h:1. Serum levels of IL-6, TNF-a and cTn1 were measured by an enzyme linked immunosorbed assay methods. And myocardial MDA and SOD content were also determined.2. Detection of myocardial infarction size by 2,3,5-Triphenyltetrazolium chloride evans blue staining methods.3. Measurement of myocardial pathologic changes (by haematoxylin and eosin staining methods).4. Investigation of apoptosis rates of myocardiocytes by Terminal-deoxynucleoitidyl transferase mediated nick end labeling.5. Detecting of myocardial expression of p-Akt、HIF-1 a and VEGF protein.After reperfusion for 4h,4 rats in each group were continuously raised for 4 weeks and for the following detections.1. Detection of heart function by echocardiography.2. Detection of severity of myocardial fibrosis by Masson staining.Results1. As shown in table 1 (a-e) and figure(1-5), serum levels of IL-6、TNF-α、cTnl and myocardial MDA contents were significantly increased in the I/R (377.46 ± 16.86 pg/ml,77.23±1.0 pg/ml,78.46 ± 8.60 ng/ml and 8.59 ± 0.71 μmol/L), I/R+HMGB (227.62 ± 16.62 pg/ml,45.70 ± 8.71 pg/ml,52.40 ± 6.70 ng/ml and 3.88 ± 1.12 nmol/mg), I/R+wortmannin (363.45±11.53 pg/ml,74.32±1.42 pg/ml, 73.53±4.53 ng/ml and 7.04±0.53 nmol/mg)and I/R+HMGB200+wortmannin (363.28±11.42 pg/ml,76.35±3.35 pg/ml,77.58±6.24 ng/ml and 9.042 ± 0.42 nmol/mg) groups than that in the sham group (166.87 ±15.45 pg/ml,16.68 ± 2.57 pg/ml,0.13 ± 0.12 ng/ml and 1.48 ± 0.17 nmol/mg), respectively (all p<0.01).Serum levels of IL-6、TNF-α、cTnI and myocardial MDA content were significantly increased in the I/R+wortmannin (363.45±11.53 pg/ml,74.32±1.42pg/ml, 73.53±4.53 ng/ml and 7.04±0.53 nmol/mg) and I/R+HMGB200+wortmannin (363.28±11.42 pg/ml,76.35±3.35pg/ml,77.58±6.24ng/ml and 9.042±0.42 nmol/mg) than that in the I/R+HMGB200 group (227.62 ± 16.62 pg/ml,45.70±8.71 pg/ml, 52.40 ± 6.70 ng/ml and 3.88 ± 1.12 nmol/mg) (all p<0.01).On the contrary, myocardial SOD contents in the I/R ((71.33 ± 5.81) u/mg), I/R+HMGB 200((117.26 ± 10.31) u/mg), I/R+wortmannin ((68.43 ± 3.55) u/mg) and I/R+HMGB200+wortmannin ((75.28 ± 7.53) u/mg) groups were significantly higher than that in the sham group ((140.77 ± 10.55) u/mg) (all p<0.01).2. Comparison of infarct size:as shown in figure(6-7) and table 1(f), as compared with the I/R ((56.30 ± 4.47)%), I/R+ wortmannin ((55.35 ± 5.25)%) and the I/R+HMGB200+wortmannin ((53.55 ± 3.25)%) groups, infarct size in the HMGB200 treated group ((34.10 ± 5.99)%) was significantly decreased (all p<0.01). No great difference was found between the I/R group and the I/R+HMGB200+wortmannin group.3. Histopathological changes of myocardial tissue (H&E staining,400x). As shown in figure 8, myocardial fibers arranged regularly with clear boundaries in the sham group. There was no inflammatory infiltration. Histological examination showed focal myocardial damage with uneven staining in the I/R group. Morphological changes in affected cardiomyocytes primarily comprised different degrees of swelling, necrosis, myocytolysis, myofibrillar loss. Myocardial fibers were broken and arranged irregularly. The myocardial infarction foci were infiltrated with a large number of neutrophils. Myocardial fibers arranged relatively regular in the I/R+HMGB200 group. Infiltration of a large number of neutrophils can be found. Myocardial fibers were broken and dissolved, and stripes disappeared in the I/R+wortmannin group. A large number of inflammatory cells infiltrated around myocardial infarction foci and capillaries. Myocardial fibers arranged irregularly and parts of them were dissolved and broken in the I/R+HMGB200+wotmannin group. The number of myocardiocytes reduced and the intermuscular spaces were widened. Myocardial infarction foci and capillaries are surrounded by infiltration of inflammatory cells.4. Results of myocardiocytes apoptosis rates in each groups. As shown in figure (9-10) and table 1(g), myocardiocytes apoptosis rates in the I/R ((29.09 ± 2.41)%), I/R+HMGB200 ((13.39± 1.64)%), I/R+wortmannin ((25.64±3.53%) and I/R+HMGB200+wortmannin ((29.40 ± 3.28)%) were significantly higher than that in the sham group ((5.43 ± 1.58)%) (all p<0.01). Myocardiocytes apoptosis rates in the I/R+HMGB200 ((13.39 ± 1.6)%) was greatly decreased than that in the I/R ((29.09 ±2.41)%) group (p<0.01).Myocardiocytes apoptosis rates in the I/R+wortmannin ((25.64 ± 3.53%) and I/R+HMGB200+wortmannin ((27.13±3.53)%) were significantly increased than that in the I/R+HMGB200 ((13.39 ± 1.64)%) (all p<0.01).5. figure11 Comparison of myocardial expression of p-Akt> HIF-la and VEGF protein. As shown in figure 4, as compared with the sham group, myocardial expression of p-Akt=、HIF-1α and VEGF protein in the I/R, I/R+HMGB200, I/R+ wortmannin and I/R+HMGB200+wortmannin groups were increased significantly (all p<0.05). As compared with the I/R group, myocardial expression of p-Akt, HIF-1α and VEGF protein in the I/R+HMGB200 group were significantly increased (all p <0.05). But no great diffirence was found of myocardial expression of p-Akt、HIF-1α and VEGF protein among the I/R group, I/R+ wortmannin and I/R+HMGB200+wortmannin groups. The myocardial expression of p-Akt、HIF-1α and VEGF protein in the I/R+wortmannin and I/R+HMGB200+wortmannin groups were significantly decreased than that in the I/R+HMGB group(all p<0.05).6.Changes of heart function parameters measured by echocardiography.As shown in figure(12-15) and table1 (h-k),as compared with the sham group,Left ventricular internal diameter at end-systole(LVIDs)(5.55±1.13 mm vs 4.11±0.85 mm,p<0.01), left ventricular internal diameter at end -diastole (LVIDd)(6.61±0.85 mm vs 4.04± 0.28 mm,p<0.01),left ventricular end diastolic volume(LVEDV)(0.71±0.16 ml vs 0.41±0.02 ml,p<0.01),left ventricular end systolic volume(LVESV)(0.50±0.22 ml vs 0.28±0.07 ml,p<0.05)and left ventricular mass(LV mass)(1.43±0.11 g vs 1.13 ±0.08 g,p<0.01)in the I/R group were significantly increased.But left ventricular ejection fraction(LVEF)(46.68±3.92%vs 89.90±7.13%,p<0.01)and fractional shortening(FS)(37.90±13.07% vs 77.09±5.64%,p<0.01)were decreased significantly in the I/R group than those in the sham group(all p<0.01).Compared with the I/R group,LVIDs(4.48±0.60 mm vs 5.55±1.13 mm,p <0.01),LVIDd(5.07±0.64 mm vs 6.61±0.85 mm,p<0.05),LVEDV(0.54±0.10 ml vs 0.71±0.16 ml,p<0.01),LVESV(0.29±0.12 ml vs 0.50±0.22 ml,p<0.05)and LV Mass(1.20±0.11 g vs 1.43±0.11 g,p<0.01)in the I/R+ HMGB200 group decreased significantly,while LVEF(77.39±4.98% vs 46.68±3.92%,p<0.01)and FS(59.71±13.40% vs 37.90±13.07%,p<0.01)increased greatly.Compared with the I/R+ HMGB200 group,LVIDs(4.48±0.60 mm vs 6.28± 0.64 and 5.89±0.59 mm,all p<0.01).LVIDd(5.07±0.64 mm vs 6.73±0.78 mm and 6.21±1.0 mm,p<0.01.p<0.05).LVEDV(0.54±0.10 ml vs 0.78±0.06 ml and 0.72±0.12 ml,p<0.01).LVESV(0.29±0.12 ml vs 0.45±0.07 ml and 0.43±0.11 ml, p<0.05)and LV Mass(1.20±0.11 g vs 1.50±1.17 g and 1.41±0.44 g,all p<0.01)in the I/R+ wortmannin group and the I/R+HMGB200+wortmannin were increased significantly.LVEF(77.39±4.98% vs 42.88±6.36% and 45.87±6.22%, p<0.01) and FS(59.71±13.40% vs 25.02±3.49% and 39.70±8.43%,p<0.01)in the I/R+ HMGB200 group were very higher than those in the I/R+ wortmannin group and in the I/R+HMGB200+wortmannin group.7.Detection of myocardial fibrosis by Masson staining.As shown in figure (16-17)and table 1(1),as compared with the sham group,myocardial CVF in the I/R group was increased significantly(1.29±0.41% vs 35.31±3.92％,p<0.01)four weeks after ischemia reperfusion. Myocardial CVF was decreased significantly in the I/R+HMGB200 group (10.94±3.54%) than those in the I/R+wortmannin group (34.10±2.76%,p<0.01) and the I/R+HMGB200+wortmannin group (29.02±2.01%, p <0.01).Conclusions1. Myocardial ischemic reperfusion injury is manifested by myocardial necrosis, increasement of serum myocardial injury biomarkers such as IL-6 and TNF-a, myocardial fibers were broken and arranged irregularly, myocardial fibrosis (myocardial remodeling), decreament of myocardial expression of p-Akt, HIF-la and VEGF protein and reducing of heart function.2. Intravenous HMGB1 can effectively attenuates myocardial ischemic reperfusion injury. HMGB1 may exert its cardioprotective effects by reducing myocardial infarction size, inhibition of apoptosis rates of myocardiocytes, decreament of biomarkers of myocardial damagement, increased myocardial espression of p-Akt, HIF-la and VEGF protein, inhibition of myocardial fibrosis.3. After the inhibition of the PI3K/Akt signal pathway with wortmannin, the cardioprotective effects of intravenous HMGB1 was cancelled. It suggests that the activation of PI3K/Akt signal pathway was involved in the regulation of cardioprotective effects of HMGB1 on myocardial ischemic reperfusion injury in rats. |
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