Protective Effects And Mechanisms Of Curcumin On Cardiomyopathy In Type2Diabetes

Backgroup:Diabetes mellitus (DM) has been recognized as a major health problem. The number of diabetic patients worldwide is increasing very fast and expected to reach439millions by2030. The leading cause of death in diabetic patients is cardiovascular disease, which are responsible for the three quarters of the deaths among this population. Diabetic cardiomyopathy (DCM), as an independent diabetic cardiac complication, is characterized by the myocardial dysfunction in the absence of coronary artery disease, hypertension, or valvular heart disease. It is associated with both type1and type2DM and presents as by early-onset diastolic and late-onset systolic dysfunction. DCM can cause diastolic and systolic dysfunction damage, eventually lead to myocardial ischemia and heart failure. The mortality of DCM is very high along with the progressed cardiac function deterioration. The development of DCM has been poorly understood and the mechanisms underlying has not been completely elucidated. Previous studies demonstrated that hyperglycemia, lipid accumulation, excessive generation of reactive oxygen species, cardiac inflammation, accumulation of cardiac fibrosis, and apoptosis are involved in the pathophysiology of DCM. So to explore deeply the mechanism of DCM, and seek an effective new drug to block the pathway of DCM development is important.Curcumin, the yellow curry pigment isolated from turmeric powder, main chain formula for C21H20O6, unsaturated aliphatic and aromatic groups, is a kind of natural active substances. Turmeric has bee used to treat DM in ancient Chinese and India. Modern pharmacological studies have shown that curcumin have many effects such as antioxidant, scavenging free radicals, anti-inflammatory, anti-tumor, anti-microbial, and so on. Research within the past two decades has revealed that curcumin can reverse insulin resistance, hyperglycemia, hyperlipidemia, and other symptoms linked to obesity and obesity-related metabolic diseases. Curcumin has been reported to exhibit protective effects against diabetic nephropathy. However, little has been known about the effect of curcumin on DCM.Objectives:In the present study, type2DM model was induced by low dose streptozoticin (STZ) combined with high energy intake on rats. We aim to investigate the potential effects of curcumin on alterations of cardiac function and morphology in diabetic rats, and the associated mechanisms as well.Methods:There are seventy-five male wistar rats. Ten normal rats in control group fed with basal diet. Sixty-five rats were fed with high fat diet which was prepared by adding20%sucrose (w/w),20%lard (w/w) into basal diet. After8weeks of the high fat diet, overnight-fasted rats were intraperitoneally injected with a single dose of streptozotocin (STZ,40mg/kg, in0.1mol/L citrate buffer, pH4.5). Control rats were administered an equivalent volume of citrate buffer. Blood glucose levels were measured72h and one week after STZ injection. Diabetic rats were diagnosed by a sustained glucose levels≥11.6mmol/L. One week after STZ injection, diabetic rats were randomly divided into three groups:DCM group: untreated diabetic rats; DCM+Cur100mg/kg group:diabetic rats treated with100mg-kg-1·d-1curcumin; DCM+Cur200mg/kg group:diabetic rats treated with200mg-kg·d-1curcumin. Curcumin was treated for16weeks, animals were placed under light anesthesia with pentobarbital sodium. Cardiac function was evaluated by serial echocardiography. The blood was taken by abdominal aorta. The rat heart was separated, the heart and body of every group was weighted. Serum triglyceride (TG), total cholesterol (TC) and myocardial enzymes were determined by auto-biochemical analysis system.The rat heart was homogenated and centrifuged for detecting superoxide dismutase (SOD) activity with xanthine oxidase, glutathione peroxidase (GSH-Px) activity with chromatometry and malondialdehyde (MDA) level with thio-barbituric Bcid method respectively. Inflammatory cytokines such as interleukin-1beta (IL-1β)、tumor necrosis factor-alpha (TNF-a) and cardiac troponin I (cTnI) in serum were determined by enzyme-linked immunosorbent assay (ELISA). HE and masson’s trichrome stain were used to observe the microstructure changes and myocardial fibrosis in the heart tissue. Electron microscopy was used for observation of ultrastructural changes of the heart. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method was used to detect cardiomyocytes apoptosis. Advanced glycation end products (AGEs), Bcl-2, Bax and caspase-3were detected by immunohistochemistry. Caspase-3activity in heart was quantified according to Caspase-3activity assay kit. RacI activity in heart tissue was measured by GST-pull down method. The NADP+and NADPH ratio was determined using EnzyChromTMNADP+/NADPH assay kit. Expression of AGE receptor (RAGE) mRNA in rat heart was detected by RT-PCR. Expression of RAGE, Bcl-2, Bax, gp91phox, p47phox, Protein kinase B (Akt) and glycogen synthase kinase-3beta (GSK-3β) proteins in rat heart were measured by Western blot.Results:1. The metabolic characteristics of the experimental animals analyzed in this study are shown:In DCM group, untreated diabetic rats had markedly lower body weight and higher blood glucose levels compared with the control group (P<0.05). Moreover, heart-to-body weight ratio (HW/BW) in DM group exhibited higher than control group (P<0.05). Compared with the DM group,200mg/kg curcumin-treated group gained significantly higher body weight and lower HW/BW (P<0.05). Furthermore, all doses of curcumin could significantly decrease blood glucose levels in diabetic rats (P<0.05). Whereas,200mg/kg curcumin-treated group preserved blood glucose levels within normal range. In the basal fasting state, DCM group showed significantly higher TG and TC than control group (P<0.05), and TG was markedly decreased in diabetic rats treated with200mg/kg curcumin (P<0.05vs DCM group). However, no significant differences in TC were observed between DCM group and curcumin-treated group. 2. Cardiac performance parameters derived from echocardiography are shown:DCM group had a slower heart rate (HR) than control group, while200mg/kg curcumin-treated group caused a marked increase in HR compared to DM group (P<0.05). Left ventricular inner diameter include left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD), left ventricular posterior wall thickness (LWPWT) and interventricular septal diastolic wall thickness (IVSD) exhibited higher in DCM group than control group (P<0.05). left ventricular fractional shortening (FS) and ejection fraction (EF), the index of left ventricular systolic function, were significantly decreased in DCM group (P<0.05). Curcumin-treated group showed lower Left ventricular inner diameter, LWPWT and IVSD than that of DCM group (P<0.05), which indicated that curcumin could inhibit interventricular septal hypertrophy. And compared with DCM group, Curcumin-treated group increased the left ventricular FS and EF (P<0.05).3. Myocardial enzymes activities and cTnI level are shown:As compared with control group, the myocardial enzymes of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and aspartate amino transferase (AST) and the level of cTnI were significantly elevated in the DCM group (P<0.05). Curcumin protected diabetic rats against cardiac injury, which was evidenced by decreased myocardial enzymes and cTnI level in curcumin-treated group, it showed a good dose dependent (P<0.05).4. Inflammatory factors results are shown:IL-1β and TNF-a were also increased in DCM group compared to control group (P<0.05). By contrast, lower levels of IL-1β and TNF-a were found in200mg/kg curcumin-treated group compared to DCM group.5. Oxidative stress results are shown:Compared with control group, there was increased accumulation of lipid peroxides with concordant increase content of MDA and decrease activitives of SOD and GSH-Px in hearts of DCM group rats (P<0.05). Diabetic rats treatment with200mg/kg curcumin markedly inhibited MDA content and up-regulated the SOD and GSH-Px activitives, compared with the DCM group, it has significant difference (P<0.05). 6. The myocardial structural are shown:Well organized, typical symmetric myofibrils were seen in control rats.In contrast, perinuclear vacuolization, destruction and loss of myofibrils were evident in DCM group rat heart. Curcumin treatment normalized alterations in myofilaments.7. Masson’s trichrome are shown:Myocardium was stain with red and fibrosis was stain with blue. Obvious fibrosis in the heart was shown in DCM group rats, with a no uniform pattern, as well as destroyed and disorganized collagen network structure in the interstitial and perivascular areas. However, the fibrotic changes in the heart were significantly mitigated when diabetic rats treated with curcumin.8. Ultrastructural results are shown:Well organized, typical symmetric myofibrils comprised of Z lines with sarcomeres, packed mitochondria beside the fibers over sarcomere units, swollen mitochondria and a number of glycogen lysis were evident in DCM group rats. Curcumin treatment normalized alterations in myofilaments, Z-lines and mitochondria, along with reduced lysis of glycogen in diabetic rats.9. Cardiomyocytes apoptosis results are shown:Apoptotic index (AI) of DCM group were much higher than control group (P<0.05). And compared with DCM group, curcumin treatment significantly decreased AI (P<0.05). Enhanced of Bax expression and activity of caspase-3but reduced expression of Bcl-2was presented in DCM group in contrast to control group. Remarkably, diabetic rats treated with200mg/kg curcumin could significantly up-regulate Bcl-2expression and down-regulate Bax expression, caspase-3activity.10. AGEs and RAGE expression results are shown:In the present study, AGEs formation was more prominent in DCM group rats (P<0.05). In addition, there was marked increase in RAGE mRNA expression and RAGE protein level in DCM group compared to control group (P<0.05). Curcumin treatment hampered the diabetes-induced AGEs accumulation and RAGE expression (P<0.05). 11. NADPH oxidase in heart tissue results are shown:The ratio of NADP+/NADPH and Racl activity were significantly higher in DCM group than those in control group. Compared with DCM group, the ratio of NADP+/NADPH and Racl activity in100or200mg/kg curcumin-treated group were significantly lower than those of DCM group (P<0.05). Moreover, increased expression of NADPH subunits gp91phox and p47phox were found in DCM group compared to control group, which were significantly reduced in200mg/kg curcumin-treated groups (P<0.05).12. Akt/GSK-3β signaling pathway results are shown:Akt phosphorylation was significantly hampered in the DCM group compared with control group. In addition, there was marked activation of GSK-30in the DCM group. In contrast,100or200mg/kg curcumin induced a significant increase in phosphorylation of Akt and GSK-3β in myocardium (P<0.05). There was no significant difference for total Akt and GSK-3β levels in all groups (P>0.05). Conclusions:Taken together, type2DM model was induced by low dose streptozoticin (STZ) combined with high energy intake on rats. These results suggest that curcumin may have great therapeutic potential in the treatment of DCM, and perhaps other cardiovascular disorders, by attenuating fibrosis, oxidative stress, inflammation and cell death. Furthermore, Akt/GSK-3β signaling pathway may be involved in mediating these effects.