Evaluation of Cardioprotective Effects of Genistein against Diabetes-induced Cardiac Dysfunction in Rats

Purpose: To investigate the possible cardioprotective effects and potential pharmacological mechanism of genistein. Methods: Six-week old ZDF and lean rats were randomized into 4 groups (8 rats/group), including group 1 (control lean rats); group 2 (lean rats treated with genistein, 2.5 mg/kg); group 3 (control ZDF rats); and group 4 (ZDF treated with genistein). Two groups (2 and 4) were treated with genistein for 12 weeks, and cardiac functions and metabolic alterations were determined. Macrophage/monocyte chemo-attractant protein-1 (MCP-1), vascular cellular adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) secretion and their messenger RNA transcription level also were observed. Results: Genistein attenuated diabetes-induced cardiac dysfunction and pathological alterations, by improving glucose tolerance and insulin resistance; facilitating Akt activation and glucose utilization, and attenuating oxidative stress and interrelated MAP kinase and NF- κB signalling pathways. In addition, genistein treatment markedly reduced diabetic-induced MCP-1 (83.33 %), VCAM-1 (74.66 %) and ICAM-1 (71.42 %) secretion and mRNA transcription in ZDF rats. Conclusion: The results demonstrate the putative effects of genistein against cardiovascular dysfunction by improving glucose homeostasis, attenuating oxidative stress and reduced diabetic-induced endothelial dysfunction in ZDF rats. Thus, genistein is a potential candidate for the prevention of cardiovascular diseases.


INTRODUCTION
The American Heart Association considers diabetes to be one of the seven major controllable risk factors for cardiovascular disease. Diabetic induced cardiovascular disease was characterized in ZDF rats by increased oxidative stress, apoptosis, fibrosis, inflammation, activation of MAP kinases and NF-κB signalling and diminished Akt phosphorylation, along with decreased glucose transport, increased cardiac lipid accumulation, and ultimately diastolic dysfunction [1,2]. The most prevalent cardiovascular complications of diabetes, characterized by both systolic and diastolic dysfunction, due to reduced contractility, prolonged relaxation and decreased compliance, are diabetic cardiomyopathy [3] and atherosclerosis [4]. Endothelial dysfunction (ED) is an early event in atherosclerosis and plays a pivotal role in the atherogenesis process [5].
The emerging experimental data suggest that cardiac insulin resistance and metabolic perturbations largely contribute to the development of cardiac dysfunction. The diminished glucose utilization and increased fatty acid oxidation in diabetic heart leads to lipid accumulation in myocardium [6]. An inflammatory response has already been shown to have an important role in the pathogenesis of myocardial ischemia-reperfusion injury [7]. Endothelial impairment can be detected in the early stage of atherosclerosis and serves as one of the leading mechanisms for HHcy-induced vascular dysfunction [8,9].
Genistein, a major isoflavone abundant in soy, has been shown its potent vascular protective function [10,11]. Genistein could decrease ox-LDL-induced ICAM-1 in human vascular endothelial cells [12], attenuate hyperglycemiainduced MCP-1 expression in human aortic endothelial cells [13], and reduce lipopolysaccharide (LPS)-induced ICAM-1 and ICAM-1 in human endothelial cells [14]. However, the mechanisms have not been clarified. In the present study, we have demonstrated the cardio protective effect of genistein and its possible mechanism involved in the protection of cardiac dysfunction.

EXPERIMENTAL Experimental animals
Male ZDF (Zucker diabetic fatty) rats and age matched Zucker lean rats were housed in a temperature-controlled room under a 12/12 h light/dark cycle, with access to water and diet ad libitum. The 6 weeks old ZDF and lean rats were randomized into 4 groups (8 rats/group) such as group 1 -control lean rats; group 2-lean rats treated with genistein (2.5 mg/kg); group 3control ZDF rats; group 4 -ZDF treated with genistein. Genistein was dissolved in olive oil (total concentration 1 %) and administered 1 g daily for 12 weeks. Body weight and fasting glucose levels were measured weekly. Before sacrificing, an oral glucose tolerance test (GTT) was performed. The animals were maintained in accordance with the Chinese legislation guidelines and the international guidelines on the protection, care and handling of laboratory animals and all the protocols were approved by the Ethical Committee of Third Affiliated Hospital of Harbin Medical University, China (approval ref. no. CLG/PSMG-GH/2014/007983).

Blood chemistry and metabolic analysis
Serum samples were collected from rats that were fasted overnight and insulin levels determined (Insulin ELISA Kit from Millipore). For the GTT, D-glucose (2 g/kg body weight, Sigma Aldrich) was orally administrated after overnight fasting. Blood glucose was measured before, and after 30, 60 and 120 min of glucose uptake, using a commercially available glucometer (Bayer, IN). The area under the glucose curve (AUC) from 0-30, 0-60 and 0-120 min was calculated using the trapezoidal method [15]. Insulin sensitivity of individual animals was evaluated using the previously validated homeostasis model assessment (HOMA) index [16]. Plasma total cholesterol was measured using the polychromatic (452, 540, 700 nm) endpoint technique. The triglycerides (TG) and high-density lipoprotein (HDL) were measured using a dichromatic (510/700 nm; 600/700 nm) end-point technique.

Real-time RT-PCR
The total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. cDNA was synthesized with Revertaid First Stand cDNA Synthesis Kit (Fermentas). Real-time RT-PCR using Taq Man fluorescence methods were performed for quantitative analysis of mRNA. The products were amplified using Light Cycler II PCR machine (Roche Diagnostics, Basel, Switzerland) in the reaction mixture (20 ml) containing 2 ml of Light Cycler-Fast Start DNA Master Taqman (Roche, Tucson, AZ) and 0.5 mmol/L each primer. The primers specific for oxidative stress genes such as; SOD1, SOD2, Bcl2, Bax and Caspase-9; and primer specific for inflammatory marker such as; MCP-1, VCAM-1, ICAM-1, HO-1 and β-actin were designed from Integrated DNA Technologies and synthesized from invitrogen (Life Technologies). By using Light Cycler Relative Quantification Software, the amount of target gene expression was calculated as a ratio of a target transcript relative to β-actin (a housekeeping gene) in the same sample.

Echocardiography
Transthoracic echocardiography was performed in anesthetized rats using HP sonos 5500 (Hewlett-Packard) with a 12-MHz imaging transducer. Left ventricle (LV) wall thickness, diameter, systolic and diastolic function were analyzed as described previously [17], by an experienced sonographer who was blinded to treatment.

Statistical analysis
All the data were analyzed using SPSS13.0 software (SPSS Inc, Chicago, IL, USA) and each experiment was performed at least three times, all values were represented as mean ± SD; n=8. Comparisons between groups were tested by One-Way ANOVA analysis; p < 0.05 was considered statistically significant

Genistein mediated changes in body weight
At 6 weeks of age, the ZDF rats had a higher body weight (BW) than the lean rats (252 ± 3 g vs 204 ± 5 g). The lean control rats gained BW steadily from 6 to 12 weeks, whereas the ZDF rats gained BW between 9 to 21 weeks, and there was no further gain from 21 to 25 weeks. Their BW was comparable to the lean rats at 8 weeks (333 ± 7 g in ZDF vs 350 ± 6 g in the lean group) and lower at 12 weeks (340 ± 8 g in ZDF vs 320 ± 6 g in the lean group, p < 0.05). The effect of genistein on body weight was observed following 2 weeks of treatment. Rats receiving genistein for 2 weeks, significantly inhibited body weight gain in the ZDF rats. However, the untreated ZDF rats significantly gained BW through the experimental period (Table I).

Genistein improve glucose homeostasis and cardiac glucose metabolism
Fasting blood glucose levels were significantly elevated throughout the entire experimental period, in the untreated-ZDF rats (Table I), along with increased glucose tolerance (GTT), AUC-GTT and HOMA-IR index at the age of 12 weeks (Fig 1, 2A), suggesting insulin resistance in ZDF rats. Genistein lowered the fasting glucose level and improved insulin resistance, as evidenced by the decreased GTT, AUCGTT and HOMA-IR index. The beneficial effect of RAR and RXR ligands on glucose metabolism was observed as early as 1 to 2 weeks following treatment ( Fig  2B), Genistein significantly lowered the blood glucose level (after 2 weeks of treatment: 260 ± 12 mg/dL in ZDF+Genistein vs 478 ± 12 mg/dL in ZDF, p < 0.05) and HOMA-IR index (Fig 2C) after 2 weeks of treatment. Hyperinsulinemia was observed at age 6 weeks in ZDF rats (6.08 ± 0.75 ng/dL in ZDF vs 0.44 ± 0.03 ng/dL in lean, p < 0.05). At age 12 weeks, the serum insulin level in the ZDF rats was decreased to a level comparable with the lean rats (0.71 ± 0.07 ng/dL in ZDF vs 0.76 ± 0.09 ng/dL in lean, (Fig. 2). This observation was accompanied by a significantly increased HOMA-IR index and decreased HOMA-β% index, suggesting that impaired β cell function may contribute to the decreased insulin level and progression of the diabetic process. At the age of 12 weeks, serum insulin was maintained at a higher level in the genistein treated ZDF rats (p < 0.05, vs ZDF), along with a decreased HOMAIR index and increased HOMA-β% index, (Fig. 2D), suggesting that genistein treatment has beneficial effects on improving insulin resistance and β cell function.

Genistein inhibit oxidative stress, apoptosis and activation of MAP kinases in ZDF rat hearts
The results showed a significantly increased intracellular ROS generation (Fig. 3) and decreased expression of SOD1 and SOD2 in Figure 3A were observed in the ZDF rat's hearts. Increased apoptosis was also observed in the ZDF rat's hearts,    (Fig. 3B, C). Genistein inhibited ROS generation and the activated MAP kinases and apoptotic signaling, as well as the percent of TUNEL-positive myocytes (10 and 2.3 %, respectively, p < 0.05, vs ZDF). These data suggest that genistein inhibits apoptosis of cardiomyocyte, by reducing oxidative stress and associated MAP kinase pathways.

Effect of genistein on left ventricular structural and functional changes
There was no difference observed in heart rate between the groups (Table I). Increased heart weight (HW), HW/BW ratio and HW/TL (tibia length) ratio were observed in untreated-ZDF rats (p < 0.05, vs lean control). Due to changes in the body weight between groups, we used the HW/TL ratio, as an index, to compare the effect of genistein on cardiac hypertrophy. Genistein significantly inhibited the increased HW/TL in ZDF rats (29 ± 0.49 mg/mm in ZDF + Am vs 33.4 ± 0.86 mg/mm in ZDF, p < 0.05). A significantly increased thickness of the systolic posterior wall of the LV (LVPWs), along with decreased systolic inner diameter of the LV was observed in untreated ZDF rats at 12 weeks of age (Fig 5A), representing a concentric cardiac hypertrophy, which was not observed in genistein treated ZDF rats. The increased HW/TL and LVPWs were in line with the increased expression of the hypertrophic markers ANP and BNP (Fig 5B).
There was no significant decrease in LV ejection fraction (LVEF) and fractional shortening (FS) in ZDF rats, suggesting no significant systolic dysfunction in ZDF rats, at 12 weeks of age. However, a significantly decreased E/A ratio, and increased IVRT and DT were observed (data not shown) in ZDF rats, at 8 and 12 weeks of age (p < 0.05, vs lean rats), indicating that diastolic function was impaired.

DISCUSSION
The present study demonstrated that genistein ameliorated diabetes-induced cardiac dysfunction and other pathological alterations. Genistein, a major isoflavone abundant in soy, has been shown its potent cardiovascular protective function [10,11]. The beneficial effects of genistein may be associated with the mechanisms like improvement of the impaired cardiac insulin metabolic signalling by reducing systemic hyperglycemia and insulin resistance; enhancement of cardiac glucose uptake/utilization and reducing glucotoxityinduced oxidative stress; inhibition of MAP kinases activation and amelioration of cardiac inflammatory responses.
In the present study, hyperinsulinemia, hyperglycemia, hyperlipidemia and insulin resistance were observed in ZDF rats. Diabetic induced cardiac dysfunction as evidenced by diastolic dysfunction with hypertrophy, fibrosis, inflammation and apoptosis developed in ZDF rats.
Our results showed significantly increased HOMA-IR index and decreased HOMA-β% index in ZDF rats, suggesting that impaired β cell function may contribute to the decreased insulin level and development of the diabetes. This observation was consistent with previous studies, which showed a decreased serum insulin level in aged ZDF rats [18,19]. A number of factors could contribute to the pathological effects observed. The hyperglycemia and hyperlipidemia-induced intracellular generation of ROS can act as signal transduction molecules to activate various signalling pathways, which ultimately lead to an inflammation, a cell apoptosis and fibrosis [20,21]. We observed significantly increased oxidative stress, apoptosis, inflammatory responses and activation of MAP kinases in ZDF rats. However, genistein administration have prevented these effects may be due to the antioxidation and anti-angiogenesis effects as well as estrogenic activities of genistein [22]. Increased ROS (reactive oxygen species) formation activates MAP kinase-signalling pathways and promotes cardiomyocyte apoptosis [23,24]. Our data further confirmed that diabetes-induced oxidative stress plays vital role in regulation of the development of diabetic complications.
Insulin resistance reduces phosphorylation of Akt pathway, which is involved in regulation of genes that control glucose and lipid homeostasis and other cellular processes like cellular hypertrophy, protein translation, nitric oxide generation, and apoptosis [25,26]. The mechanisms involved in cardio protective function of genistein are obscure. Besides its non-specific tyrosine kinase inhibitory role, genistein could exert vascular protective action through many other signaling pathways, such as cAMP/protein kinase A and PI3 K/AKT pathways [27].
Furthermore, we analyzed the cardiac inflammatory response, where the increased mRNA level of MCP-1, VCAM-1 and ICAM-1 found in untreated ZDF rats. However, genistein significantly inhibited diabetic-induced MCP-1, VCAM-1 and ICAM-1 expression. This is consistent with the previous report that genistein increased heme oxygenase (HO) activity, where HO system is tightly involved in the inhibitory activities of genistein against inflammation [28]. This part of our study showed that the down regulation of the expression levels of MCP-1, VCAM-1 and ICAM-1 was achieved by genistein mediated induction of HO-1.

CONCLUSION
Altogether, the findings reveal the protective effect of genistein on cardiovascular dysfunction by maintaining glucose homeostasis and cardiac glucose metabolism, inhibiting oxidative stress, apoptosis and activation of MAP kinases and prevention of inflammatory response. Thus, administration of genistein against cardiovascular diseases may be a positive approach to the development of a potential drug.