Effect And Mechanism On The Oxidative Stress And Cardiac Remodeling Of Diabetic Rats From Carvedilol

Background and objectiveDiabetes is the largest morbidity of patients with heart failure and adversely affects outcomes of cardiovascular diseases. Diabetic mortality from heart diseases accounts for two part in all reasons. The characteristics of diabetic heart failure are faster deteriorating, high mortality and difficult to be diagnosed early. Thus, we considered an ideal treatment were to inhibit the pathway towards the heart failure in early period. It has been a consensus there were excessive activity of sympathetic nerve system, renin-angiotensin system, oxidative stress, accumulation of extracellular matrix, all of which accelerated cardiac remodeling and disfunction. Unfortunately, the symptoms often lacked in early period.Carvedilol is a nonselective agentadrenoceptor blocker. It was demonstrated that carvedilol has antioxidant, anti-proliferation, anti-apoptosis and anti-inflammatory properties, which therapeutic dose, moreover, had no negative effect on the blood sugar and lipids. Therefore, we deduced carvedilol could have positive effect on the diabetic hearts. Our objective is to investigate the effect of carvedilol on the balance of oxidant and antioxidant systems, cardiac remodeling in the early diabetic rat model, and study its possible mechanisms.Methods1. Induction of diabetes and treatment protocolsMale Sprague-Dawley rats weighing 200-220g were injected i.p. with streptozotocin (60mg /kg body weight) after 12 hour fasting. Diabetes was verified 72h later by measuring tail vein blood glucose, and the rats with a blood glucose level ≥16.7mmo/l were considered the diabetes groups. Rats were divided into the following groups: (1) untreated control rats (C, n = 9);(2)rats treated with carvedilol (CS, 1mg/kg/day, orally) (n=8);(3)rats treated with carvedilol (CL, 10mg/kg/day, orally) (n = 9);(4)untreated diabetic rats (D, n=9);(5) diabetic rats treated with carvedilol (DS, 1mg/kg/day, orally) (n=9);(6) diabetic rats treated with carvedilol (DL, 10mg/kg/day, orally) (n = 9).2. Echocardiography and blood dynamicsUnder half the anesthetic dose 10% hydrated chloral (200mg/kg), two-dimensional echocardiography (Acuson 128 XP/10) was performed 5th weeks after treatment. Anteroposterior diameter (D) and short-axis area (A) of left ventricle (LV) at the papillary muscle level were measured at end diastole (ED) and end systole (ES). Left ventricular enject fraction (EF), fractional shortening fraction (FS%), the intraventricular septum thickness and Left ventricular posterior wall (LVPW) thickness at the end of systolic or diastolic term were measured. Rats were anesthetized with 10% hydrated chloral (350mg/kg), then the right carotid arterywas cannulated with a BD Angiocatheter (20GA 1.1 mm * 48 mm, Italy) for recording of arterial pressure. The aortic catheter was then advanced into the left ventricle for recording left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), maximal rate of rise/fall left ventricle pressure development and decline (+/-dP/dtmax), and heart rate (HR).3. Ratio of ventricular weight to body weight (VW/BW)Rats heart were sheared along atrioventricular interval, then discarded atrium and connective tissue. The ventricles were quantified, and the ratio (VW/BW) was calculated.4. Heart tissue disposal and biochemical procedureOne third near the apex of heart was harvested and stored at -80°C, medial heart was dipped in the formalin, and the rest was homogenized. The protein content of the supernatant was determined using the Lowry method. Malondialdehyde (MDA) levels were estimated by the thiobarbituric acid (TBA) method. Total (Cu-Zn and Mn) superoxide dismutase (SOD) activity was estimated by the xanthine-xanthine oxidase method. Catalase (CAT) activity was estimated by the H2O2 method. The blood plasma was centrifuged, and then the total cholesterol was measired by the oxidase- peroxide method.5. Pathological staining and result judgementAfter embedded in paraffin, heart was formed into sections (5 um thick), then which were stained with hematoxylin and eosin, Masson's trichrome as well as immunohistochemistry staining. Fibrosis was quantitated using trichrome-stained sections. Green staining collagen fibers were quantitated as a measure of fibrosis. The cardiac collagen volume fraction (CVF) and the per-invascular collagen area (PVCA) were calculated.6. Reversal transcription polymerase chain reaction (RT-PCR)Cardiac tissue was pulverized to powder. Total RNA was extracted using Trizol. The primers were desighed by the Primer 3, and then synthesized by Sangon Co. The one-step RT reaction was amplified using TaqDNA polymerase and primers to BCL-2> TGF-pimRNA. GAPDH was as a reference. Half-quantity of different genes mRNA expression were gained by the image scan and analysis after electrophoresis.Results1. After 5 weeks, animals injected with STZ had lost weight, higher total cholesterol and blood sugar in plasma compared with normal controls, and carvedilol treatment alleviated the weight loss significantly in diabetes without change of blood sugar and total cholesterol. There was no significant change in healthy rats treated with carvedilol.2. VW/BW in the 5th diabetic rats was elevated significantly, increased to 1.4 times. Treatment with small dosage carvedilol reduced VW/BW near to the normal level, and treatment with large dosage carvedilol depressed this ratio further.3. Cardiac function: EF, FS%, IVS%, LVPS% and LVDd decreased in untreated diabetes (D) compared with C. EF, FS%, IVS%, LVPS% were elevated by administration of carvedilol, whereas LVDs was decreased significantly. LVDP, and ±dP/dtmax were significantly lower in D, and LVEDP was substantially elevated compared with normal control rats (P<0.01). ±dP/dtmax in diabetic rats treated with small and large dosage of carvedilol were significantly elevated, whereas LVEDP values were lowered in DL (P<0.05vs. D). HR in untreated diabetic rats was significantly decreased (P<0.05). Large dosage of carvedilol treatment reduced HR further (P>0.05 vs. D). There was reduced HR in healthy control treated with carvedilol, and this fall was dose-related.4. The level of MDA in the heart was increased markedly in untreated diabetic rats compared with the rats in the control group (P<0.0001). Treatment with carvedilol reduced the level of MDA in both healthy groups and diabetic groups. SOD and GSH-Px activities were lower in diabetic untreated rats significantly (P<0.0001 vs. C). Treatment with carvedilol increased activities of CAT, SOD and GSH-Px in healthy control and diabetic ratssignificantly (P<0.05 vs. C). Moreover, large dosage of carvedilol administration enhanced this rise (P<0.05 vs. C).5. HE and MASSON staining: In the myocardium of diabetic rats we observed disorganized cardiac muscle, fibrosis along interstitial cells and perivascular collegen were markedly increased in diabetic rats. There were no obvious stenosis and atherosclerotic plaque in coronary vascular. Moreover, both the CVF and PVCA were increased significantly. Carvedilol reduced collagen positive interstitial cells and perivascular fibrosis, the CVF and PVCA were also reduced markedly.6. Increased expression of TGF-pimRNA, reduced expression of BCL-2 mRNA were observed in untreated diabetic rats. The expression BCL-2 mRNA was significantly upregulated and expression of TGF-p1mRNA were downregulated in carvedilol-treated diabetic groups compared with untreated groups.In conclusionThe 5th STZ-induced diabetic rats not only displayed systolic and diastolic disfunction, but also cardiac remodeling and oxidative-antioxidative turbulence. 5 weeks administration with carvedilol improved diabetic cardiac function, increased antioxidant ability, and inhibited cardiac remodeling. Moreover, these positive effects were related to the upregulation of the BCL-2 mRNA, downregulation of TGF—pimRNA due to carvedilol.