| 1. Establishment and assessment of experimental type 2 diabetic cardiomyopathy rat modelObjective Diabetic cardiomyopathy (DC) is a specific and independent diabetic complication occurred in the absence of coronary artery disease or systemic hypertension. Clinical and epidemiological evidences have demonstrated the existence of diabetic cardiomyopathy in humans. Diabetic cardiomyopathy is diagnosed in diabetic patients in accordance with ventricular dysfunction in the absence of coronary atherosclerosis and hypertension and often occurs as an unknown asymptomatic heart disease, which counteracts clinical diagnosis and treatment. The major impediment of development of research in this area is the lack of appropriate animal models as well as the lack of standardized measures for phenotypes associated with diabetic cardiomyopathy. Importantly, animal model of diabetic cardiomyopathy should reflect the clinical features of patients and fulfill the definition of the disease before the onset of mechanism and medical studies. This study was to establish and evaluate a kind of experimental rat model of type 2 diabetic cardiomyopathy and assess the contribution of high fat-sucrose diet to the pathology.MethodsMale Wistar rats (150g-180g) were randomly divided into 3 groups, including the control group, high sucrose-high fat group (HSF) and high sucrose-high fat with low dose streptozotocin (STZ) group (HSF-STZ). Rats in the control group were fed with normal food, while rats in the HSF and HSF-STZ groups were fed with high sucrose-high fat diet (consisted of 20% sucrose,10% lard,2.5% cholesterol,1% bile salt and 67.5% normal food) for totally 12 weeks. Blood of all rats were collected after 12-hour fasting to measure fasting blood glucose (FBG), total cholesterol (TCH) and triglyceride (TG) before high sucrose-high fat diet and after 4 weeks of feeding (the fifth week). Then rats in HSF-STZ group were injected intraperitoneally (i.p.) singly with STZ (resolved in citric acid-sodium citrate buffer, pH=4.5) at the dose of 30 mg/kg after 6 weeks' high sucrose-high fat diet feeding. FBG were tested at 72 hours after injection and FBG≥7.77 mmol/L was considered as standard of the establishment of diabetes mellitus. Rats in control and HSF groups were i.p. with vehicle (citric acid-sodium citrate buffer, pH=4.5). At the end of experiment (the eleventh week), indexes of heart rate (HR), stroke volume (SV) and cardiac output (CO) of rats were detected by impedance plethysmography (IPG) method and left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), the maximum rate of myocardial contraction and the maximum rate of myocardial diastole (±dp/dtmax) were measured by left ventricular catheterization by MP150 polygraph physiological signal recorder to evaluate cardiac function. The whole heart weight (HW), left ventricular weight (LVW), and ratios of HW/body weight (HW/BW), LVW/BW, interventricular septal thickness (IST), left ventricular wall thickness (LVWT) and left ventricular collagen concentration were measured to assess cardiac structure. Then artery blood of rats were gathered to detected FBG, glycosylated hemoglobin (HbA1c), glycosylated serum protein (GSP), TCH, high density lipoprotein (HDL) and nonesterified fatty acid (NEFA).ResultsEffects of high sucrose-high fat on blood sugar, blood lipid levels in rats. It showed no significant differences of blood glucose and lipid levels between each groups at the beginning of the experiment. After 4-week intake of high sucrose-high fat diet, serum TCH and TG of rats in HSF group significantly increased by 26% and 34%, and in HSF-STZ group significantly increased by 16% and 58%, when compared with the control group(P<0.05), whereas FBG didn't increase significantly.Effects of high sucrose-high fat diet with STZ injection on lipid metabolism in rats. The blood lipid maintained high level after another 6 weeks of high sucrose-high fat diet intake. At same time, plasma NEFA of rats in HSF and HSF-STZ groups significantly increased by 131% and 100%, while HDL significantly reduced by 38% and 26%, respectively, when compared with the control group. TCH level of rats in HSF-STZ group was significantly increased by 44%, when compared with the control rats. The indexes of blood lipid indexes showed no significant difference between HSF-STZ-treated rats and HSF-treated rats.Effects of high sucrose-high fat diet with STZ injection on blood sugar in rats. Plasma FBG, HbA1c and GSP significantly increased by 233%,35% and 28% respectively in HSF-STZ treated rats, when compared with control rats. However, values of blood sugar did not significantly increased in the HSF group, when compared with the control group.Effects of high sucrose-high fat diet with STZ injection on cardiac function in rats. The high sucrose-high fat diet plus STZ treated rats showed the same hemodynamic characteristics of diabetic cardiomyopathy. Levels of LVSP, SV and CO significantly reduced by 15%,26% and 23%, while LVEDP and -dp/dtmax significantly increased by 44% and 29%, when compared with the control rats. Meanwhile, LVSP significantly decreased by 12%, and LVEDP significantly increased by 43% in the HSF-treated rats, when compared with the control rats. However, changes of±dp/dtmax, SV and CO did not get any statistical differences in HSF rats, when compared with the control rats. Interestingly, SV significantly decreased in HSF-STZ treated rats, when compared with HSF-treated rats.Effects of high sucrose-high fat diet with STZ injection on cardiac structure in rats. Ratios of HW/BW and LVW/BW,IST and LVWT parameters, and collagen concentration significantly increased by 15%,10%,80%,33% and 18% in the HSF-STZ rats, when compared with the control rats. It also showed significant correlations between cardiac function and structure parameters.ConclusionIn summary, experimental type 2 diabetic cardiomyopathy rat model can be established by high sucrose-high fat diet plus single low dosage of STZ injection. Diabetic cardiomyopathy rat model could be assessed efficiently and simply by synthetic indexes including glucose and lipid metabolism as well as heart function. The rat model demonstrated hyperglycemia, hyperlipidemia, and cardiac dysfunction especially diastolic dysfunction, which were similar to clinical symptoms.2. Berberine attenuates cardiac dysfunction in experimental type 2 diabetic cardiomyopathy rat modelObjectiveBerberine, an isoquinoline alkaloid, derived from medical herbs including Berberis, Hydrastis canadensis, Coptis chinensis Franch. and Cortex Phellodendri Chinensis, has antibacterial and anti-inflammatory activities. Recent studies demonstrated the reduction of blood sugar and lipids, increase in insulin sensitivity in type 1 and typer 2 diabetic animals, antiarrhythmia, as well as inhibition of cardiac hypertrophy with berberine treatment. Clinical researches have indicated that berberine can improve metabolic dysfunction and decrease ventricular premature complexes in the patients with dyslipidemia and congestive heart failure. Up to now, however, little attention has been focus on the role of berberine for treating diabetic cardiomyopathy. Hence our experiments were aimed to explore the effects of berberine on cardiac dysfunction and metabolic disorders in the experimental type 2 diabetic cardiomyopathy rat model induced by high sucrose-fat diet and streptozotocin.MethodsAnimal treatment. Rat model of experimental type 2 diabetic cardiomyopathy was induced as described in experiment 1. To speak simply, the control rats were fed with normal food and administrated with vehicle for totally 12 weeks. Rat model of experimental type 2 diabetic cardiomyopathy were fed with high sucrose-high fat diet (consisted of 20% sucrose,10% lard,2.5% cholesterol,1% bile salt and 66.5% normal food) for 6 weeks, then i.p. injection with streptozotocin at the dose of 30 mg/kg following a 12 h fast at the seventh week, and fed with special food as before for another 6 weeks. Rat model were daily intragastrically with relative medication including berberine (7.5 mg/kg,15 mg/kg and 30 mg/kg), Metformin (140 mg/kg), Rosiglitazone (2 mg/kg), and Captopril (45 mg/kg) at 72 h after STZ i.p. injection for totally consecutive 6 weeks.Plasma profiles assessment. Whole blood samples were obtained from the right carotid artery of rats and collected in fresh vials containing anticoagulant, and plasma samples were prepared by centrifuging the whole blood for 10 min at 2000 g. Levels of fasting blood glucose, glycated hemoglobin, fructosamine, glycosylated serum protein, total cholesterol and triglyceride in plasma samples were determined using ultraviolet spectrophtometric method according to the manufacturer's protocol.Cardiac function, biomarker and histology assessment. At the thirteenth week, rats were anesthetized with pentobarbital sodium (35 mg/kg, i.p.) following a 12 h fast. Stroke volume and cardiac output were detected by means of non-invasive impedance plethysmography. On completion of the cardiac output measurements, a catheter was positioned in the left ventricle via the right carotid artery for measurement of left ventricular systolic pressure, left ventricular end diastolic pressure, the maximum rate of myocardial contraction (+dp/dtmax) and the maximum rate of myocardial diastole (-dp/dtmax). Data were collected using MP150 systems. After that, rat hearts and left ventricles were obtained and weighted to calculate the ratios of heart weight and left ventricular weight to the body weight, respectively.The homogenate of heart tissues were prepared in the physiological saline (1:9) and centrifuged for 10 min at 2000 g. The myocardial nonesterified free fatty acids were measured by the biochemical method.Hydroxyproline concentration was determined in heart tissue by alkaline hydrolysis method. Tissue samples were hydrolyzed in 2 mol/L sodium hydroxide at 100℃for 1 h. Chloramine-T (0.05 mol/L) was used to oxidize for 10 min at room temperature (pH=6.0-6.8). Then Ehrlich's reagent was added to each sample and the samples were mixed and incubated at 65℃for 15 min. The absorbance of samples were read at 550nm using a spectrophotometer to determine the content of hydroxyproline. Left ventricular collagen content was estimated from its hydroxyproline concentration, with multiplying its value by 7.46, since the imino acid represents 13.4% of collagen. Levels of myocardial fatty acid transport protein-1, fatty acid transport proteins and fatty acidβ-oxidase were assessed by ELISA.Then equator annulus of left ventricles were collected and placed in 10% buffered formalin. Sections (4μm) were cut and stained with hematoxylin and esosin (H&E). The left ventricular wall thickness and interventricular septum thickness were measured by Image-ProPlus 5.0 image analysis software (USA) on the H&E slices microscopically.Cardiac PPAR a, PPAR y and GLUT4 mRNA expression assessment. Tissue samples obtained from left ventricles were rapidly frozen in liquid nitrogen and stored at -70℃prior to quantitative real-time (RT)-polymerase chain reaction (PCR) analysis. Total RNAs were isolated using TRIzol reagent according to the manufacturer's protocol, and then reverse transcribed to synthesize cDNA. The RT primers were designed by Prime 5.0 software.Total Realtime PCR reaction system was performed in Rotor-Gene 3000 Realtime PCR instrument, as previously described. To allow for comparisons between samples and groups, quantities of all targets in test samples were normalized to the constitutive housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH).Cardiac PPARα, PPAR y and GLUT4 protein expression assessment. Total GLUT4, PPARa and PPARy protein expression in the heart homogenate extracts were determined by Western blot as described previously. GAPDH was probed as an internal loading control. Western blot band density analysis was made using ImageJ. Total GLUT4, PPARa and PPARy proteins were shown in arbitrary units.Statistical analysis. All data were presented as mean±SEM and analyzed by one-way analysis of variance (ANOVA). Multiple group comparisons were made with least significant difference's (LSD) post hoc test by SPSS 17.0. Statistical significant difference was defined as a value of P< 0.05.ResultsEffects of berberine on general state, blood sugar and lipid levels in experimental type 2 diabetic cardiomyopathy rat model. The body weight of DC rat model significantly decreased at day 5 after i.p. with STZ and kept low level until the end of experiment, while the food and water intake significantly increased by 16% and 84%, when compared with the control rats. Berberine 30 mg/kg treatment could prevent the body weight reduction. In the rat model of diabetic cardiomyopathy, plasma fast blood glucose, glycated hemoglobin, glycosylated serum protein, fructosamine, total cholesterol, triglyceride and low density lipoprotein cholesterol significantly increased by 422%,40%,73%,86%, 205%,70% and 188% respectively, when compared with the control group. Berberine 30 mg/kg treatment significantly decreased plasma FBG, HbA1c, GSP, FMN, TCH, TG and LDL by 69%,40%,46%,40%,42%,42% and 40%, respectively, when compared with the drug-untreated rat model of DC. Metformin 140 mg/kg treatment significantly decreased FBG, HbA1c, GSP, FMN and TG levels by 67%,34%,42%,34% and 41% respectively in DC rat model. Rosiglitazone 2 mg/kg treatment could reduce plasma FBG, HbA1c, TG and LDL by 36%,24%,40% and 37%, when compared with DC rat model.Effects of berberine on cardiac function in experimental type 2 diabetic cardiomyopathy rat model. The rats treated with high fat-high sucrose plus STZ showed the same hemodynamic characteristics of diabetic cardiomyopathy. Cardiac output, left ventricular systolic pressure and +dp/dtmax in DC rat model significantly decreased by 36%,18% and 45%, while left ventricular end diastolic pressure and -dp/dtmax significantly increase by 44% and 38%, when compared with normal rats. Berberine 30 mg/kg could significantly increase CO, LVSP, and +dp/dtmax by 60%,14% and 81%, and decrease LVEDP and-dp/dtmax by 80% and 55%, while Berberine 15 mg/kg only increased LVSP significantly, when compared with the drug-untreated DC rat model. Metformin 140 mg/kg treatment significantly increased CO and +dp/dtmax by 71% and 60%, while Captoprile 45 mg/kg treatment could increase CO, LVSP,+dp/dtmax by 54%,15% and 77%, and decrease -dp/dt max by 52%, when compared with drug-untreated DC rat model.Effects of berberine on cardiac structure in experimental type 2 diabetic cardiomyopathy rat model. When rats treated with high fat-high sucrose food and STZ, the ratios of heart weight and left ventricular weight to the body weight, interventricular septum thickness, left ventricular thickness, and myocardial collagen content were significantly increased by 11%,10%,78%,33% and 22%. Berberine 30 mg/kg treatment decreased HW/BW, LVW/BW,IST, LVWT and collagen content by 6%,9%,20%,46% and 32% respectively in the DC rats, when compared with the drug-untreated rat model. Metformin, Rosiglitazone as well as Captopril treatment could significantly decrease IST by 26%,47% and 40% in the DC rat model.Effects of berberine on cardiac structure in experimental type 2 diabetic cardiomyopathy rat model. Levels of myocardial nonesterified free fatty acid and enzymes involved in fatty acids transport and oxidation were measured. Myocardial nonesterified fatty acid in the DC rat model was significantly increased by 69%, while fatty acid transport protein-1, fatty acid transport proteins and fatty acidβ-oxidase were decreased by 68%,22% and 47%, respectively, when compared with the normal rats. Berberine at the dosage of 7.5, 15 and 30 mg/kg and Metformin 140 mg/kg treatment significant reduced myocardial NEFA by 34%,27% and 25% respectively, when compared with the rat model of DC. Meanwhile, Berberine 30 mg/kg treatment could significantly increase FATP-1, FATPs, and FA-β-oxidase by 159%,56% and 91%, when compared with the drug-untreated rat model. Cardiac PPAR a, PPAR y and GLUT4 mRNA expression. Genes involved in cardiac glucose and lipid metabolism was evaluated by quantitative real-time PCR. As expected, cardiac mRNA expression of PPAR y and GLUT4 mRNA expression reduced by 38% and 34%, while PPAR a mRNA gene expression increased by 80% in the DC rat model, when compared with normal rats. Berberine 30 mg/kg treatment increase PPAR y and GLUT4 mRNA expression by 50% and 32%, and decreased PPAR a mRNA levels by 54% in the DC rat model, when compared with drug-untreated rat model.Cardiac PPARα, PPAR y and GLUT4 protein expression. Protein expression of PPAR y and GLUT4 reduced by 40% and 68% respectively in the DC rat model, when compared with control rats. Berberine treatment significantly increased PPAR y and GLUT4 protein expression by 83% and 133% in the DC rat model, when compared with the drug-untreated rat model. However, berberine at the dosage of 30 mg/kg did not show any detectable changes in the cardiac PPAR a protein expression.ConclusionPhenotypes such as hyperglycemia, hypercholesterolemia, cardiac dysfunction and left ventricular hypertrophy, cardiac lipid accumulation, which mimics to the diabetic cardiomyopathy, were found in the rat model induced by high fat-high sucrose food plus streptozotocin treatment. Berberine treatment could effectively recover the diastolic and systolic dysfunction, inhibit the cardiac left ventricular hypertrophy, lower plasma sugar and lipids levels in the DC rat model. It could also alleviate cardiac lipid accumulation, increase intracellular fatty acid transport proteins and fatty acid beta-oxidase, and impressively increase mRNA and protein expression of PPAR y and GLUT4 and repress PPAR a gene expression, indicating a protective effect of berberine on experimental diabetic cardiomyopathy.3. Inhibitory differentiation effect of berberine on 3T3-L1 fibroblastsObjectiveTo study the effect of berberine on preadipocyte differentiation, lipid accumulation and adipokines secretory in the murine 3T3-L1 cell line.MethodsCell culture and treatments. Murine 3T3-L1 preadipocyte were grown on FALCON 6-well plates in a 5% CO2 atmosphere at 37℃and maintained in low glucose Dullbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum,100 U/mL penicillin, and 100μg/mL streptomycin. The 2-day postconfluent 3T3-L1 cells (designed as Day 0) were incubated with 10% FBS/high glucose DMEM and antibiotics,500μM 3-isobutyl-1-methylxanthine, 1μM dexamethasone, and 5μg/ml insulin for 3 days (Day 0-2). Then the cells were incubated for 2 days in 10% FBS/HG-DMEM with insulin (Day 3-4), and, thereafter incubated in 10% FBS/HG-DMEM that was changed once every 2 days (Day 5-9). Cells receiving berberine chloride were given 10% FBS/HG-DMEM and insulin containing a final concentrantion of 5,10 and 20μM berberine in DMSO at Day 3 and Day4, then medium changed to only 10% FBS/HG-DMEM with berberine for last 5 days (Day 5-9).Oil-Red-O staining. After differentiation (Day 9), cells were stained with Oil-Red-O to detect droplets in adipocytes. Cells were washed 3 times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min and then stained with 1.8 mg/ml Oil-Red-O in 60% isopropanol for 30 min. Cells were washed with 60% isopropanol until colorless and observed under a microscope in PBS. Stained oil droplets in the cells were dissolved by Nonidet-P40 (4%, v/v, diluted in isopropanol) with gentle agitation for 5 min. Supernatant was measured with a spectrophotometer at 500 nm.Reverse Transcription-Polymerase Chain Reaction. Total RNA was extracted from cultured 3T3-L1 adipocyte (Day 8) and preadipocyte by using QIAGEN RNeasy Mini Kit. Complementary DNA was generated from 2μg of total RNA and synthesized by using High Capacity cDNA Reverse Transcription Kit. The PCR conditions were as follows:for glyceraldehydes-3-phosphoate dehydrogenase (GAPDH) and adiponectin,25 cycles of 95℃for 30s,55℃for 30s,72℃for 30s; for resistin,27 cycles of 95℃for 30s,55℃for 30s,72℃for 30s; for peroxisome proliferator-activated receptor-y (PPAR y), adipocyte fatty acid binding protein (aP2), fatty acid synthase (FAS), angiotensinogen (AGT), monocyte chemoattractant protein 1 (MCP-1),28 cycles of 95℃for 30s,55℃for 30s,72℃for 30s; for CCAAT/enhancer binding protein (3 (C/EBP P), plasminogen activator inhibitor-1 (PAI-1),30 cycles of 95℃for 30s,55℃for 30s,72℃for 30s; for C/EBP a and leptin,31 cycles of 95℃for 30s,55℃for 30s,72℃for 30s. To allow for comparisons between samples and groups, quantities of all targets in test samples were normalized to GAPDH.ResultsInhibitory effects of berberine on triglyceride content and lipid accumulation in 3T3-L1 cells. Berberine at the dosage of 5μM,10μM and 20μM significantly decreased triglyceride content (26%,37%, and 43%, respectively) and lipid accumulation of 3T3-L1 adipocyte in a dose-dependent manner.Effects of berberine on transcription factors during differentiation process in 3T3-L1 cells. Berberine 10μM and 20μM significantly decreased C/EBP a (21% and 47%) and PPAR y mRNA (21% and 55%) gene expression in 3T3-L1 adipocyte in a dose dependent manner. Berberine at a dose of 20μM significantly reduced C/EBPβmRNA gene expression by 16%, whereas 5μM significantly increased C/EBP (3 mRNA gene expression by 21%.Effects of berberine on expression of genes involved in lipid metabolism. Berberine at dosage of 10 and 20μM significantly decreased aP2 mRNA expression (18% and 40%). Berberine 20μM significantly reduced FAS mRNA expression by 18%, however, berberine 5μM significantly increased FAS gene expression by 15%.Effects of berberine on adipokines (adiponectin, leptin, resistin, MCP-1, PAI-1 AGT) in 3T3-L1 cells. Berberine 5μM,10μM and 20μM significantly decreased adiponectin (9%,46% and 85%), leptin (33%,75% and 100%) and resistin (13%,63% and 82%) mRNA expression of 3T3-L1 cells in a dose dependent manner. Berberine 10μM and 20μM significantly decreased AGT mRNA expression (15% and 17%). Berberine in dose of 20μM caused significant reduction of PAI-1 (12%) and MCP-1 (28%) mRNA expression, whereas 5μM significantly increased PAI-1 (10%) and MCP-1 (26%) mRNA expression in 3T3-L1 adipocyte.ConclusionBerberine inhibited triglyceride and lipid accumulation in 3T3-L1 adipocyte. Berberine could inhibit differentiation of 3T3-L1 fibroblast via reducing transcription factors such as C/EBPβ, C/EBP a and PPAR y and their downstream genes aP2 and FAS. In term of inhibitory effect on differentiation in 3T3-L1 cells of berberine, adipokines including adiponectin, leptin, resistin, and AGT also showed significant decrease with berberine treatment, which might indicate anti-obesity action as well as cardiovascular protective effect of berberine. However, the reduction effect of berberine on MCP-1 and PAI-1 mRNA expression might be independent of differentiation inhibitory action.
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