| Backgroud:Myocardial infarction(MI) is a major cause of cardiovascular disease to death and disability.In our country, every year about millions of patients died of Acute myocardial infarction (AMI), the disease is regarded as "the first killer" to human health. Over the last30years, advanced coronary care and early reperfusion strategies have dramatically improved survival rates in patients suffering an acute myocardial infarction. However, this impressive success has resulted in a larger pool of patients who, having survived the acute infarction, are at risk of developing heart failure. Despite therapeutic advances, the risk of heart failure following myocardial infarction has remained high; in fact, some studies have suggested an increased incidence of postinfarction heart failure in recent decades that parallels the decreasing acute mortality rates. Development of heart failure following myocardial infarction is closely associated with profound alterations in cardiac geometry, function, and structure, also referred to as "ventricular remodeling."The molecular and cellular changes in the remodeling heart affect both the area of necrosis and the noninfarcted segments of the ventricle and manifest clinically as increased chamber dilation and sphericity, myocardial hypertrophy, and worsened cardiac function. Cardiac remodeling is linked to heart failure progression and is associated with poor prognosis in patients surviving a myocardial infarction. The extent of postinfarction remodeling is dependent on the size of the infarct and on the quality of cardiac repair. Cardiac repair is dependent on a superbly orchestrated inflammatory response that serves to clear the wound from dead cells and matrix debris, but also provides key molecular signals for activation of reparative cells. It is becoming increasingly apparent that timely repression and containment of inflammatory signals are needed to ensure optimal formation of a supportive scar in the infarcted area and to prevent development of adverse remodeling. Because even relatively subtle alterations in myocardial architecture, matrix compo sition, and cellular phenotype profoundly affect chamber geometry and ventricular function, defects, and aberrations in temporal and spatial regulation of the postinfarction inflammatory reaction may have catastrophic consequences. Excessive early inflammation may augment matrix degradation causing cardiac rupture. Prolongation of the inflammatory reaction may impair collagen deposition leading to formation of a scar with reduced tensile strength, thus increasing chamber dilation. Enhanced expression of proinflammatory mediators may activate proapoptotic pathways inducing further loss of cardiomyocytes. Finally defective containment of the inflammatory reaction may lead to extension of the inflammatory infiltrate into the noninfarcted myocardium enhancing fibrosis and worsening diastolic function.Because of the importance of inflammatory and reparative mechanisms in cardiac remodeling, defects in resolution of inflammation may be responsible for remodeling, injury, and heart failure in a large number of patients surviving a myocardial infarction. Emerging concepts on the significance of endogenous inhibitory pathways in cardiac repair may provide insight into the reasons for the failure of previous strategies targeting the inflammatory response in patients with myocardial infarction.Curcumin, the natural yellow pigment extracted from the rhizomes of the plant curcuma longa, has been demonstrated to exhibit a variety of potent beneficial effects, acting as an antioxidant, anti-inflammatory and anti-fibrotic. Curcumin attenuates plasma inflammatory cytokines surge and cardiomyocytic apoptosis following cardiac ischemia/reperfusion by inhibition of NF-kappaB activation. Curcumin can inhibit adverse ventricular remodeling caused by left ventricular pressure overload and improve left ventricular function.Curcumin can also improve cardiac function after myocardial infarction in rats. But the role of myocardial protective effect of curcumin is still not clear.Curcumin can regulate the expression of proinflammatory and anti-inflammatory cytokine, inhibit the NF-kappa B signaling pathway activation, removal of oxygen free radicals and inhibition of apoptosis, effectively reduce the level of interleukin1(IL-1), interleukin6(IL-6), interleukin12(IL-12), TNF alpha. At the same time can also induce up-regulation of transformed cell growth factor (TGF-beta) and interleukin10(IL-10). These results indicate that curcumin have the bidirectional immune regulation function, keeping the body’s inflammatory response in a low level of equilibrium, avoiding tissue damage caused by excessive inflammation.Based on the basis of inflammatory reaction is critical factor in ventricular remodeling after myocardial infarction, and curcumin is efficacious for inflammatory and cardiacvuacular disease, We infer that curcumin may through immuneregulation and inhibiting the inflammatory reaction after myocardial infarction, which play the role of anti-ventricular remodeling.In this study, we attempt to investigate the cardioprotection of curcumin after myocardial infarction. The study was divided into two parts as bellow. Part I was designed to explore the effects of curcumin on cardiac function and ventricular remodeling after myocardial infarction. Part II was to clarify curcumin could improving cardic function after myocardial infarction may be through immune regulation mechanism, and to finding a new thought of prevention for ventricular remodeling. Part I:Effects of curcumin on cardiac function in the mice model of myocardial infarctionObjective:To explore the effects of lycopene on cardiac function and ventricular emodeling after myocardial infarction.Method:Selecting male C57BL/6with SPF level, ligating the left coronary artery a id resulting in the myocardial infarction. Dividing those falling into4groups: Model control group, Curcumin group with high dosage, low dosage, Enalapril group; plus Sham operation group. A total of5groups:Curcumin group with low dosage:50mg/kg·d, intraperitoneal injection; Curcumin group with high d ssage:100mg/kg·d, intraperitoneal injection; Enalapril group:enalapril7.5mg/kg.1,and the same amount of solvent, intraperitoneal injection; Sham operation gro up and Model control groups:the same amount of solvent of curcumin, intra peritoneal injection. Continuous administration for4weeks. Using echocardiogr iphy to checkout cardiac function on4weeks after operation. Then all mice were killed, the heart mass index(HMI) was calculated and the myocardial tiss aes were sampled and then pathologically observed through HE staining techni c and Masson trichrome staining technic. Testing the protein of myocardial tiss ae collagen I, collagen Ⅲ through immunohistochemical methods; observing th e effect of Curcumin on the apoptosis of cardiomyocyte after myocardial infarc tion by TUNEL staining.Statistical analysis:All statistical anlyses were performed with SPSS13.0software. All values are presented as mean±SD. Statistical significance was determined using one-way ANOVA, multiple comparisons using Fisher’s LSD post hoc tests. When the variance of arrhythmia, the use of robust estimation Welch, then the use of T3compared methods. Significance was taken as P<0.05.Result: 1. Results of cardiac ultrasonographyCompared with the sham group, model group mice had significantly increased LVEDd, LVESd values and declined EF, FS values (P<0.01),Curcumin group with high dosage, low dosageg and Enalapril group can had significantly declined LVEDd, LVESd values and increased EF、FS values (P<0.01,)compared with the model group, but there was no significant difference between Curcumin group with high dosage and Enalapril group. These are both better than Curcumin group with low dosage.2. The Result of heart weight/body weight constitutions in experiment miceCompared with the sham group, model control group mice had significantly increased heart weight and HMI significantly(P<0.01); Compared with the model group, heart weight and HMI decreased significantly in curcumin group with high dosage, low dosage and Enalapril group mice, the difference was significant (P<0.01), but there was no significant difference between them(P>0.05).3. Result of HE stainingSham operation group:the pigmentation of myocardium is even and in neat band, the cell boundary is clear, indicating normal myocardial morphology. Model control group:thickening, elongation, disordered arrangement and widened interspace of myocardial fibers, the cell nucleus is big and dark stained indicating the infiltration of inflammatory cells. The junctional zone of curcumin group with high dosage, low dosage and Enalapril shows mild myocardial cell hypertrophy and hyperplasia with spotted fibroplasias, clear and neat myocardial cells display around the non infarct zone, less inflamed cell infiltration compared with the Model control group and curcumin group with high dosage is better than group with high dosage.4. Result of Masson trichrome stainingCompared with MI model group, the myocardial fibrosis area of curcumin group with high dosage, low dosage and enalapril is reduced, the difference was statistically significant (P<0.01); Compared with enalapril group, fibrosis area of curcumin with low dosage increases, the difference was statistically significant (P<0.01), fibrosis area of curcumin with high dosage group is no statistical difference (P=0.771).5. Expression results of Immunohistochemical detection of myocardial collagen type I, III collagen proteinSham operation group:no significant expression of collagen,no stromal fibrosis; Model control group:obvious fibrosis, showing significant expression of collagen; Curcumin group with high dosage, low dosage and Enalapril group:a small amount of collagen fiber, obviously much less than that in Model control group, but the occurrence is still inevitable. The content of myocardium collagen and the ratio of myocardial collagen type I to III collagen protein in model control group were obviously higher than the sham operation group(P<0.01); the content of myocardium collagen and the ratio of myocardial collagen type I to III collagen protein in Curcumin group with high dosage, low dosage and Enalapril group were lower than which in model control group(P<0.01); the ratio of myocardial collagen type I to III collagen protein in Cucumin group with high dosage, low dosage and Enalapril group was no significant (P>0.05). And Curcumin with high dosage is better than Curcumin with low dosage.6. Effect of curcumin on cardiomyocyte apoptosis in myocardial infarction miceAfter3days of MI model operation and drug intervention, TUNEL kit was used to detecting cardiomyocyte apoptosis in border of infarted myocardium. Compared with model control group, curcumin group with high dosage, low dosage and enalapril can reduce cardiomyocyte apoptosis, the difference was statistically significant (P<0.01); Compared with Enalapril group and Curcumin group with low dosage, Curcumin group with low dosage increased cardiomyocyte apoptosis, the difference was statistically significant(P<0.01);the cardiomyocyte apoptosis index of Curcumin group with high dosage and enalapril was no significant (P=0.356).Conclusion:1. The left anterior descending coronary artery is subjected to ligation to establishing model in mice with myocardial infarction. If the AMI model mice was successfully made, the local myocardial would be color and electrocardiogram (ECG) changed. The tissue morphology, cardiac function and collagen expression detection have been showed to detected the pathological changes of the model exists ventricular remodeling in mice. 2. The treatments used in Curcumin can improve cardiac function of the mice, reduce myocardial fibrosis, maintain cardiomyocyte’ normal morphology, inhibit the cardiomyocyte apoptosis in myocardial infarction mice. Curcumin with high dosage same effect as Enalapril.3. Curcumin might be one of Effective medicine for anti-ventricular remodeling after myocardial infarction. Curcumin with low and high dosage both improve cardiac function and ameliorate ventricular remodeling after myocardial infarction in mice, and the effects of curcumin with high dosage is better than the group with low dosage, the protect effective has a certain dosage-response relationship. Part Ⅱ:Effects of curcumin on inflammatory response in the mice model of myocardial infarctionObjective:Inflammatory response is the basic mechanism of the development of chronic heart failure. The activation of NF-kappa B signaling pathway can promote inflammation and cardiomyocyte apoptosis. Studies have reported that curcumin can inhibit the activation of TLR4and the NF-kappa B signaling pathway, so we speculated that curcumin may inhibit the inflammatory response through suppression the expression of TLR4in and the activation of NF-kappa B signaling pathways in infracted myocardium, thereby effectively reversing ventricular remodeling and improving the cardiac function after myocardial infarction. The purpose of this experimental research is to observe influence of curcumin on the infiltration of dendritic cells in the edge area of infracted myocardium and the expression of inflammatory cytokine IL-1β, tumor necrosis factor a, IL-6and IL-10, and the expression of TLR4and the signal pathway downstream of NF-kappa B in infarcted heart.Methods:Selecting male C57BL/6with SPF level, ligating the left coronary artery and resulting in the myocardial infarction. Dividing those falling into2groups:Model control group, Curcumin group; plus Sham operation group. A total of3groups: Curcumin group with high dosage:100mg/kg-d, intraperitoneal injection; Sham operation group and Model control groups:the same amount of solvent of curcumin, intraperitoneal injection. And those groups will be divided into4subgroups:the group of3days,7days,14days and28days after the MI model operation and intervention. Immunofluorescence staining was observed to dendritic cells infiltration. The expression of TLR4and iNOS, and NF-κB pathway activation in ischemic zone surrounding infarted myocardium were detected by western blot analysis. The relative mRNA expression of TLR, the inflammatory cytokine IL-1β, tumor necrosis factor a, IL-6and IL-10were detected by quantitative real-time PCR. Statistical analysis:All statistical anlyses were performed with SPSS13.0software. All values are presented as mean±SD. Statistical significance was determined using one-way ANOVA, multiple comparisons using Fisher’s LSD post hoc tests. When the variance of arrhythmia, the use of robust estimation Welch, then the use of T3compared methods. Significance was taken as P<0.05.Results:1. The influence of curcumin on the infiltration of dendritic cells in the border of infarcted myocardiumImmunofluorescence analysis of paraffin section is used to detect the expression of CD11c+dendritic cells. Results show that:the infiltration of CD11c+dendritic cells into infarcted myocardium3days, and reached its peak7days after MI in MI control group was significantly higher than in sham group(P<0.05), and returned to normal level on day28after MI. The infiltration of dendritic cells could be reduced by curcumin (P<0.05).2. The influence of curcumin on the expression of inflammatory cytokines in infarcted heartReal time PCR was used to test the mRNA expression of proinflammatory factor IL-1beta, tumor necrosis factor a, IL-6and anti-inflammation factor IL-10. Results show that:IL-1β, tumor necrosis factor a, IL-6, which are proinflammatory, increased in3days after MI and gradually deseased thereafter. The expression of those proinflammatory factor in MI control group was significantly higher than in sham group; The relative mRNA expression of proinflammatory factor could be reduced by curcumin compared with MI control group (P<0.05).3. The influence of curcumin on the expression of iNOS in infarcted heartiNOS was upregulated in the ischemic myocardium and attenuated by curcumin campared with mice in MI control group(P<0.05).4. The influence of curcumin on the expression of TLR4and activation of NF-κB signaling pathway in infarcted heartPhosphorylation and degradation of IκBα and phosphorylation of NF-κB p65 were induced with mice after MI but were significantly suppressed in crucumin group. Curcumin attenuate TLR4expression and inhibite NF-κB signaling pathway activation campared with mice in MI control group(P<0.05).Conclusion:Curcumin reduce the infiltration of dendritic cells in border of infracted myocardium and the expression of TLR4, proinflammatory factor, iNOS in infracted myocardium. Curcumin reduce inflammatory response after MI through inhibiting activation of NF-κB signaling pathway, which could be the mechanism of cardioprotective effects of curcumin. |
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