Antihyperglycemic, Antidyslipidemic and Antifibrotic effect of EGCG in STZ - High Fat Diet Induced DCM rats

 

K. Pramila*, A. Julius

¹Research Scholar, Bharathiyar University, Coimbatore. Sree Balaji Dental College and Hospital,

Bharat Institute of Higher Education and Research (BIHER), Pallikaranai, Chennai – 600100.

²Professor and Head, Department of Biochemistry, Sree Balaji Dental College and Hospital,

Bharat Institute of Higher Education and Research (BIHER), Pallikaranai, Chennai – 600100.

 

ABSTRACT:

This study shows the potential effect of EGCG in DCM induced rats by exerting anti- hyperglycemic, anti-hyperlipidemic and anti-fibrotic effects. Hence EGCG a natural occurring compound have important clinical implications in terms of prevention and progression of diabetic cardiomyopathy.

 

 

 

INTRODUCTION:

In 1972, diabetic cardiomyopathy (DCM) was first defined by Rubler (Rubler et al), which was characterized by the myocardial dysfunction in absence of hypertension and coronary artery disease (CAD). Diabetic cardiomyopathy was associated with both type 1 and type 2 diabetes mellitus (Boudina and Abel). Hyperglycemia, hyperlipidemia and inflammation were the three main metabolic abnormalities potentiates the severity of diabetic complications through stimulation of reactive oxygen species (Aneja et al). In addition, hyperglycemia stimulate overexpression of transforming growth factor – 1 by cardiac fibroblasts resulting in a condition of cardiac fibrosis (Mizushige et al). Impaired glucose and fatty acid metabolic pathways were also contributing to the development of DCM, in which diabetic heart was characterized by increased fatty acid uptake and oxidation with decreased glucose uptake and oxidation, results in reduced myocardial high energy reserves and contractile dysfunction. (Brownlee M; Liu et al). So very effective treatment was necessary to prevent the pathogenesis and progression of cardiomyopathy to reduce diabetes related mortality.

 

 

Green tea was made from Camellia sinensis leaves, originated from China. The leaves of the tea plant produce more catechins than any other plant, (Mak J. C.) (-)-epigallocatechin gallate (EGCG), was the most abundant and powerful catechin in controlling high glucose and lipid in diabetic heart (Suzuki et al). Our work aimed to investigate the ability of EGCG in emphasizing the biochemical parameters in STZ induced high fat diet albino rats.   

 

MATERIALS AND METHODS:

1.     Induction of Diabetic cardiomyopathy (DCM) in Experimental rats:

Experimental rats were induced diabetes by feeding with high fat diet (HFD) during the whole experimental period, whereas the rats consuming basal diet (BD), served as a control group. HFD was given for 60 days. On 60^th day rats fed with HFD were intraperitoneally injected with STZ - streptozotocin at the dose of 35 mg/kg body weight for just one time. Blood glucose levels were measured 72 h after STZ injection using glucometer by tail vein puncture blood sampling. If the blood glucose level reaches 16.7 mmol/L or 300 mg/dl the rats were considered to have DCM and used for this study.  DCM rats were treated with EGCG (100 mg/kg body weight for 28 days.  All the animals were provided with food and water. The histopathological study was conducted using H and E stain method.

 

2.       Experimental groups:

Group - 1   Control rats,

Group - 2   Diabetic cardiomyopathy – DCM rats,

Group - 3   EGCG treated DCM rats and

Group - 4   Drug control rats. 

 

3.     Biochemical parameters:

i.      Determination of blood glucose and HbA1c:

Fasting blood glucose levels were measured by one touch ultra-glucometer. Invitro estimation of HbA1c in serum was quantitatively done using ELISA kit works under the principle of Sandwich – ELISA method.

 

ii.    Lipid and lipoprotein profile:

Lipid profile includes Total cholesterol (TC), triglycerides (TG), and high density lipoprotein (HDL) which were examined before the rats were sacrificed.  LDL cholesterol was calculated according to the Fried Wald formula (LDL = TC – (VLDL+HDL) and (VLDL = TG ̸ 5). The values of TC, TG, low density lipoprotein (LDL), very low density lipoprotein (VLDL) and HDL were expressed as mg/dl.

 

The results were expressed as mean ± standard deviation (SD). Differences between groups were analysed by one-way analysis of variance (ANOVA) using the SPSS Software package. Post hoc testing was performed for inter-group comparison using the least significant difference (LSD) test; significance at p values < 0.05 has been given in tables and figures.

 

RESULT:

Effect of EGCG in body weight changes in each experimental groups:

Table – 1 shows that there were no differences in initial weight among group 1 to 4.  In group 2 and 3, initial weight differs due to induction of high fat diet in DCM rats and EGCG treated DCM rats. Rats of DCM group shows increased body weight when compared to the control rats (p<0.05). DCM rats when treated with EGCG shows decreased body weight when compared to group 2. From these results it shows clearly that EGCG treatment reduced the body weight in DCM rats.

 

Table - 1

	0 days (Base line)	90 days
Gp - 1 Control	150 ± 3.6	190 ± 3.6
Gp - 2 DCM	147.9 ± 4.0	299.9 ± 4.0
Gp -3 DCM + EGCG	151.9 ± 3.8	269.9 ± 3.8
Gp - 4 EGCG	144.9 ± 3.7	179 ± 3.6

Effect of EGCG on body weight in control and DCM rats. Gp 1 – control rats, Gp 2 – DCM rats, Gp 3 – EGCG treated DCM rats and Gp 4 – only EGCG treated rats. Values are expressed as the mean ± SEM for six animals in each group. Values are statistically significant at the level p < 0.05.

 

Blood parameters:

Effect of EGCG on blood glucose and lipid profile levels in diabetic cardiomyopathy rats:

Table 2 shows the effect of EGCG on serum lipid profile of various experimental groups. Levels of serum lipid profile in the present study depicted that a significant (p<0.05) increase in the levels of Total cholesterol (TC), Triglycerides (TG), Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL) with significant decrease in High Density Lipoprotein (HDL) was observed in group 2 alone when compared to the control rats. In EGCG treated DCM rats, the levels of Triglycerides (TG), Total cholesterol (TC), Low Density Lipoprotein (LDL) were significantly lower than those in the DCM group indicating that EGCG have the ability in controlling the levels of lipids.

 

Outcome of EGCG on blood glucose and HbA1c:

Graph 1 and 2 shows the effect of EGCG on blood glucose and HbA1c levels of four different experimental groups. Assessment of blood glucose and HbA1c in the present study depicted that a significant (p<0.05) increase in the levels was observed in group 2 alone when compared to the control rats. In EGCG treated DCM rats, both the levels were significantly lower than those in the DCM group 2 indicating that EGCG have the ability in controlling the levels of blood glucose and HbA1c.

 

 

Table - 2

	GP 1 Control n=6	GP 2 DCM n=6	GP 3EGCG + DCM n=6	Gp 4 EG CG n=6
Total cholesterol (mg/dl)	50.11 ±2.26	138.90±6.2	63.01±2.8	52.16±2.3
Triglycerides (mg/dl)	63.01±2.8	126.41±5.6	70.40±3.1	64.89±2.8
LDL (mg/dl)	19.39±0.87	100.01±4.51	30.01±1.34	21.9±0.99
VLDL (mg/dl)	13.9±0.63	25.9±1.17	16.9±0.76	12.9±0.58
HDL (mg/dl)	22.9±1.03	12.9±0.58	18.9±0.85	20.9±0.94

Effect of EGCG on lipid profile in control and DCM rats. Gp 1 – control rats, Gp 2 – DCM rats, Gp 3 – EGCG treated DCM rats and Gp 4 – only EGCG treated rats. Values are expressed as the mean ± SEM for six animals in each group. Values are statistically significant at the level p < 0.05.

 

Graph – 1 and 2:

Gp 1- Control Rats; Gp 2- DCM rats; Gp 3- DCM + EGCG treated rats; Gp 4- EGCG alone treated rats.  Each value represents mean ± SEM for six rats in each group. Values are statistically significant at the level of p< 0.05, where ‘a’ - compared with Gp 1, ‘b’ –compared with Gp 2.

 

 

Histopathological changes:

Figure – 1 shows microscopic investigation of H and E stained heart tissue reveals the myocardial architecture in all 4 groups of experiment. In the control and EGCG treated rats (group 1 and 4) demonstrated normal endocardium, myocardium and normal distribution of vascular endomysium between cardiac cells. In DCM rats (group 2), clearly shows the presence of increased fibrosis tissue and thickness of the myocardium of LV, indicating early inflammation. In group 3, DCM rat treated with EGCG shows marked decrease in thickness of LV myocardium and also reduced inflammatory cellular infiltration when it was compared with the control rats.

 

Gp-1                                                                        Gp-2

 

Gp-3                                                        Gp-4

Figure -1

Effect of EGCG on heart histoarchitecture of high fat diet and streptozotocin induced diabetic cardiomyopathy (DCM) in wistar rats. (H and E 40X). Gp 1 – Control Rats, Gp 2 – DCM Rats, Gp 3 – EGCG treated DCM Rats and Gp 4 – Only EGCG treated Rats.

DISCUSSION:

The present study showed that the STZ induced diabetic rats exhibited enhanced cardiac dysfunction, cardiac fibrosis when they were subjected to high fat diet, therefore, these adverse effects were normalised by EGCG administration.

 

This study showed the protective effects of EGCG in treating STZ induced HFD diabetic cardiomyopathy rats. The experimental model was treated with high fat diet which played an important role in the development of DCM. Mild dose of STZ (30mg/kg) injection was given to induce diabetic condition. Diabetic rat model showed DCM condition has exhibited progressive aggravation of cardiac function, which includes increased level of triglycerides, total cholesterol, low density and very low density lipoprotein (LDL/VLDL) and decreased level of high density lipoprotein. Changes in left ventricular mass was also observed and increased cardiac fibrosis with myocardial cell degeneration procured.

 

The effectiveness of EGCG (100mg/kg) in a high fat diet to wistar rats showed remarkable antihyperglycemic and antidyslipidemic activity. In line with these reports, EGCG treated DCM rats showed the significant reduction in blood glucose, HbA1c and lipid profile when compared with diabetic induced DCM rat models. From these results it was evidenced the therapeutic potential of EGCG against the progression of DCM was confirmed.

 

Cardiac inflammation and fibrosis have been recognised in DCM condition. The histological findings displayed the focal areas of myocardial degeneration and some patches of fibrous tissue in the heart section of HFD and STZ induced diabetic rats which affect both functional and structural perturbations in DM damaged cardiac tissue. According to Boudina and Abel, left ventricular hypertrophy and fibrosis were associated with progression of DCM.

 

A plethora of literature has identified EGCG as a cardio protective molecule, particularly through its antifibrotic and anti-inflammatory activities (Sheng et al).  In the present investigation, when compared to DCM rats, EGCG treated DCM model shows the beneficial effect on post infarction fibrotic healing by reducing the density of scar tissue in the infarcted area and regulates cardiac fibroblast proliferation and differentiation, thereby reversing cardiac fibrosis in vivo condition. The histological study reports that EGCG have the ability to protect the heart against diabetes induced cardiac dysfunction through its dual ability by decreasing inflammation and by controlling the glucose and lipid levels.

 

CONCLUSION:

To conclude, the present study shows that EGCG exerts protective effect against DM induced cardiomyopathies in STZ induced HFD DCM animal models. In addition, EGCG possess strong antihyperglycemic and antihyperlipidemic effects and attenuated cardiac dysfunction and reduced severity of histopathological lesions in DM damaged cardiac tissue. Thus, EGCG might be considered as a novel therapeutic target in preventing DCM condition.   

 

REFERENCE:

1.      Rubler S, Dlugash J, Yüceoğlu YZ, Kumral T, Branwood AW, Grishman A. New type of cardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol 1972; 30: 595-602.

2.      Boudina S, Abel ED. Diabetic cardiomyopathy revised. Circulation 2007; 115: 3213-23.

3.      Aneja A, Tang WH, Bansilal S, Garcia MJ, Farkouh ME. Diabetic cardiomyopathy: insights into pathogenesis, diagnostic challenges, and therapeutic options. Am J Med 2008; 121: 748-57.

4.      Mizushige K, Yao L, Noma T, Kiyomoto H, Yu Y, Hosomi N, et al.Alteration in left ventricular diastolic filling and accumulation of myocardial collagen at insulin-resistant prediabetic stage of a type II diabetic rat model. Circulation 2000; 101: 899-907.

5.      Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001; 414:813–20.

6.      Liu TS, Pei YH, Peng YP, Chen J, Jiang SS and Gong JB 2014 Oscillating high glucose enhances oxidative stress and apoptosis in human coronary artery endothelial cells. Journal of Endocrinological Investigation 37 645–651.

7.      Mak J. C. (2012). Potential role of green tea catechins in various disease therapies: progress and promise. Physiol. 39, 265–273.

8.      Suzuki J., Ogawa M., Futamatsu H., Kosuge H., Sagesaka Y. M., Isobe M. (2007). Tea catechins improve left ventricular dysfunction, suppress myocardial inflammation and fibrosis, and alter cytokine expression in rat autoimmune myocarditis. Eur. J. Heart Fail. 9, 152–159.

9.      Sheng R., Gu Z. L., Xie M. L., Zhou W. X., Guo C. Y. (2009). EGCG inhibits proliferation of cardiac fibroblasts in rats with cardiac hypertrophy. Planta Med. 75, 113–120.

 

 

 

 

 

Received on 04.01.2019           Modified on 25.02.2019

Accepted on 06.03.2019         © RJPT All right reserved