EFFECTS OF ARABIC GUM ON CARDIOMYOPATHY IN A RAT MODEL OF TYPE II DIABETES

EFFECTS OF ARABIC GUM ON CARDIOMYOPATHY IN A RAT MODEL OF TYPE II DIABETES

By

Al-Bayomi Fouda, Adel Abd El-Aziz, and Nageh Mabrouk*

Departments of Medical Physiology and Pharmacology*, Al-Azhar Faculty of Medicine

ABSTRACT

Background: Diabetes mellitus and the associated complications represent a global burden on human health and economics. Cardiovascular diseases are one of the leading causes of death in diabetic patients. Arabic gum (AG) is a natural compound that has a potent anti-inflammatory effect, antioxidant activity, and hypoglycemic effect.

Objective: Evaluation of the effects of Arabic gum on diabetic cardiomyopathy (DCM).

Material and Methods: Thirty-two adult male albino rats of a local strain were chosen as an animal model for this study, weighed 110 -130 g (average weight was 120 g). Diabetes mellitus type II was induced in experimental rats by using a high-fat diet (HFD) for two weeks followed by streptozotocin (STZ) injection. At the end of the experiment (8 weeks), serum was obtained for the determination of glucose, insulin, cardiac enzymes (lactate dehydrogenase,creatine kinase-MB fraction and AST), lipid profile (triglycerides and total cholesterol), and oxidative stress markers (Thiobarbituric acid-reactive substance and superoxide dismutase). ECG was recorded. Also, heart weight/body weight ratio (HW/BW) was calculated at the end of the experiment.

Results: Diabetic rats exhibited hyperglycemia, and hyperlipidemia accompanied by significant hypoinsulinemia. In addition, diabetic rats showed significantly increased HW/BW, serum CK-MB, AST, and lactate dehydrogenase. Oxidative stress marker increased, whereas antioxidant defenses significantly reduced in the diabetic heart. ECG also disturbed. Treatment with Arabic gum alleviated hyperglycemia, hyperlipidemia, and heart function markers. Arabic gum also minimized the oxidative stress and boosted antioxidant defenses in the heart of diabetic rats. HW/BW decreased and ECG ameliorated.

Conclusion: Arabic gum attenuated the development of DCM via amelioration of hyperglycemia/hyperlipidemia-mediated oxidative stress. Therefore, it might be worth considering the therapeutic potential of AG for human DCM.

Keywords: Hyperglycemia; Hyperlipidemia; Cardiomyopathy; Oxidative stress; Arabic gum, ECG and Diabetes mellitus.

INTRODUCTION

     Cardiovascular complications due to the long-term uncontrolled hyperglycemic condition are generally considered one of the leading causes of morbidity and mortality among diabetic patients, in both developed and developing countries (Abdul-Ghaniet al., 2019). Long-standing diabetes results in structural and functional alterations in the myocardium of diabetic patients leading to diabetic cardiomyopathy. Etiology of diabetic cardiomyopathy (DCM) is very complex with several interlinked mechanisms chargeable for damage to the heart in diabetic condition. Unfortunately, exact molecular mechanisms and the sequence of events leading to DCM are still elusive. Oxidative stress and inflammation have been implicated to play a central role in the progression of DCM (Huynh et al., 2014). Oxidative stress, in the diabetic heart, is a major contributing factor in the development and deterioration of DCM (Li et al., 2012). Diabetic complications are generally considered to be the result of oxidative stress. In addition, diabetic complications are interrelated with the inflammatory response and have been shown to be accelerated under a hyperglycemic state for the production of acute response factors in fat cells (Wang et al., 2015). Arabic gum is non-toxic, and used extensively in pharmaceutical preparations in folk medicine, and in most categories of processed foods as in candy products. It is indigestible to both humans and animals, not degraded in the intestine, but fermented in the colon to give short-chain fatty acids, leading to a large range of possible health benefits. It has a prebiotic effect because it causes significant increases in Bifidobacteria, Lactobacteria, and Bacteroides. It has anti-carcinogenic and anti-oxidant effect with a protective role against hepatic and cardiac toxicities (Elshama et al., 2014).

     The present work aimed to detect the effects of Arabic gum on diabetic cardiomyopathy in a rat model of type II diabetes.

MATERIALS AND METHODS

Animals:

     Thirty-two adult male albino rats of a local strain were chosen as an animal model for this study. They kept in suitable cages (20x32x20 cm for every four rats) at room temperature, with the natural light/dark cycle. They weighed 110 -130 g (average weight was 120 g). They were fed on a standard food in addition to bread and green vegetables with free water supply.   They were kept for 10 days for the adaptation to the new environments before the start of the experiment.

Chemicals:

     Streptozotocin (STZ) was purchased from Sigma-Aldrich Co., St Louis, USA. Arabic gum was obtained from the local market, Cairo, Egypt.

Study design:

Rats were divided into four equal groups:

GroupI (Normal control group): provided with standard animal pellet and water.

Group II (Arabic gum -treated normal group): Rats were offered 10% AG in drinking water for 8 weeks.

Group III (Diabetic control group).

Group IV (Arabic gum-treated diabetic group): Arabic gum was offered in 10% concentration in drinking water for 8 weeks.

Induction of diabetes:

     Type II DM was experimentally induced using HFD/STZ model as described by (Skovso, 2014). Briefly, feeding with a high-fat diet (HFD) for two weeks followed by single i.p. injection of Streptozotocin (35 mg/kg body weight). Streptozotocin was dissolved in citrate buffer (pH 4.5).At day 5, following STZ administration, the level of blood glucose was measured by collecting whole blood from the tail vein. Rats that had a blood glucose level of >200 mg/ dl were considered diabetic. The blood glucose level was measured using Accu-Chek glucometer (Roche, Germany).

Preparation of Arabic gum:

     Crude AG was obtained as spherical tears which were milled and sieved to obtain a fine pure powder. Ten grams from the powder was dissolved into 100 ml warm water and given to the animals orally (Abd-Allah et al., 2002). The treatments were initiated on the fifth day after STZ exposure.

ECG recording:

     One day prior to euthanasia, rats were anesthetized with ketamine (100) mg/kg i.p), and ECGs were recorded through needle electrodes inserted under the skin of the animals in lead II position using Biopac MP 35 data acquisition system (Biopac system, Canada).

Biochemical parameters in blood:

     At the end of the experiment, rats were fasted for 12 h, and samples of blood were obtained from retro-orbital venous plexus by using a heparinized capillary tube (about 0.75 – 1.0 mm internal diameter) inserted in the medial canthus. The collected blood samples were kept in centrifuge tubes until coagulated, then set centrifuged at 5000 rotations per minute for 15 minutes to separate the serum. Serum was sucked out into Eppendorf tubes and stored frozen at -20oC till used for the determination of blood glucose level (Kaplan, 1984), Insulin level (Chevenne et al., 1994), cardiac enzymes (lactate dehydrogenase “LDH” - Pesce, 1984, Creatine kinase-MB fraction “CK-MB” - Lee and Goldman, 1986) and A spartate transaminase “AST”-Reitman and Frankel, 1957), Lipid profile (triglycerides “TG ”- Fossati and Prencipe, 1982) and total cholesterol “TC” - Allain et al., 1974).

Heart weight/body weight:

     Prior to euthanasia,the rat was weighed. After euthanasia, the heart was isolated, dried and weighed, and heart weight/body weight was calculated (Neha and Lubna, 2014).

Biochemical estimation in heart homogenate: The isolated heart tissue was homogenized in 50 mM phosphate buffer (pH 7.4) using hand-held tissue homogenizer. The resultant supernatant was used for measurement of antioxidant enzyme (SOD) and Cardiac malondialdehyde (MDA) which were measured as thiobarbituric acid reactive substances (TBARS) by the colorimetric technique (Gupta et al., 2015).

Statistical analysis:

     The data were presented as mean ± SD. SPSS software version 25 (IBM Co., USA) was used for the statistical analysis to perform ANOVA followed by post-hoc-test (LSD), where P≤0.05 was considered to indicate a statistically significant difference.

RESULTS

     In the DM group, STZ-induced diabetic rats exhibited a markedly higher HW/BW ratio when compared with the control and AG-treated groups, respectively. No statistically significant difference in the HW/BW was observed between the AG-treated control group and the normal control group (Table 1).

     The serum glucose and insulin level showed insignificant changes in the AG-treated control group when compared to the normal control group. In the DM group, STZ-induced diabetic rats exhibited markedly higher blood glucose and lower insulin level when compared with the control group. However, in the AG-treated diabetic group, the serum glucose level showed a significant decrease, while insulin level significantly increased as compared with the diabetic group (Table 1).

Table (1): Effects of treatment with Arabic gum on Blood glucose (mg/dl), insulin(μIU/ml) and HW/BW(mg/g) in the various experimental groups (Mean±SD)

P* vs. control group; P†vs. DM group. AG, Arabic gum; HW/BW, heart weight to body weight ratio; SD, standard deviation.

Figure (1): Effect of Arabic gum on cholesterol & triglyceride (mean ± SD). P* vs. control group; P† vs. DM group (n=8 per group); AG, Arabic gum; DM, diabetes mellitus.

Figure (2): Effect of Arabic gum on CK-MB, LDH and AST (mean ± SD). P* vs. control group; P† vs. DM group (n=8 per group); AG, Arabic gum; DM, diabetes mellitus.

     ECG recordings of AG-treated control rats revealed insignificant changes in heart rate, QRS, and QT interval as compared to normal control rats. ECG of diabetic rats revealed a significantly slower heart rate and prolonged both QRS, and QT interval comparing to normal control rats. However, after adding Arabic gums to diabetic rats, heart rate, QRS, and QT interval were close to values of the normal control group (Table 2).

Table (2): Effects of treatment with Arabic gum on ECG parameters in the various experimental groups (Mean±SD)

P*<vs. control group; P†vs. DM group. AG, Arabic gum; ms, millisecond.

     The untreated DM rats in the present study showed higher cardiac MDA and lower SOD activity. By contrast, the administration of AG to diabetic rats for 8 weeks exhibited a significant amelioration of both MDA and SOD. Normal rats treated with AG showed insignificant changes in the cardiac levels of MDA and SOD as compared with control rats (Table 3).

Table (3): Effects of treatment with Arabic gum on Cardiac MDA (nmol/ g tissue) and SOD (U/g tissue) in the various experimental groups (Mean±SD)

P* vs. control group; P†vs. DM group. AG, Arabic gum; MDA, malondialdehyde; SOD, Super oxide dismutase.

DISCUSSION

     Diabetic cardiomyopathy is defined by the existence of abnormal myocardial structure and performance in the absence of other cardiac risk factors, such as coronary artery disease, hypertension, and significant valvular disease, in individuals with diabetes mellitus (Jia et al., 2018). Streptozotocin (STZ) type II diabetic rat model was used to investigate the protective effect of Arabic gum against diabetic cardiomyopathy. STZ induced diabetes was associated with myocardial injury in rats (Al-Rasheed et al., 2016).

     In the present study, STZ diabetic rats showed a notable increase in HW/BW when compared with the control group. Diabetic rats treated with AG for 8 weeks reduced the level of HW/BW. Normal rats, received AG for 8 weeks, showed insignificant changes in HW/BW.Weight loss is a complication of diabetesdue to an increase ofmuscle protein catabolism, glycogenolysis lipolysis, gluconeogenesis, and polyuria which result in muscle wasting and loss of body weight (Bolla et al., 2015).

     The present study was in agreement with Jia et al. (2018) who reported that hyperglycemia in diabetic patients induces cardiac hypertrophy through cardiac insulin resistance and metabolic disorders that increase mitochondria dysfunction, oxidative stress, advanced glycation end products (AGEs), impairment of mitochondria Ca²⁺ handling, inflammation, activation of renin-angiotensin-aldosterone system (RAAS), autonomic neuropathy, endoplasmic reticulum stress, cardiomyocyte death, as well as microvascular dysfunction.

     Wang et al. )2014) observed that decreased heat shock protein20 (Hsp₂₀) in chronic diabetic hearts may be a primary factor causing the development of cardiac hypertrophy as Hsp₂₀-mediated protection against diabetes-induced cardiac injury. Heat shock protein 20 engineered exosomes have a beneficial role in the regulation of cardiomyocyte exosome secretion and restoration of hyperglycemia-induced cardiac dysfunction (Wang et al., 2016). Also, Tan et al., )2011) found that hyperglycemia and hyperlipidemia provoke the production of pro-inflammatory cytokines as TNF-α which implicated in cardiac hypertrophy and dysfunction. In the present study, diabetic rats treated by AG for 8 weeks showed reduction in HW/BW, This may be due to the ability of AG to ameliorate the expression of HSP₂₀ in cardiomyocyte in diabetic rats or due to its anti-inflammatory properties which decrease TNF-α production.

     The present study showed an insignificant change in HW/BW in healthy rats receiving AG for 8 weeks. A similar result was obtained by Tageldinet al., )2006) who found that weight gain significantly depressed in rabbits that consumed Arabic gum in the first week of the experiment, but weight gain was recovered thereafter. This result was in contrast with the result of (Babiker et al., 2012) who concluded that ingestion of Arabic gum caused a significant reduction in body mass index on adult females. The difference may be due to the hormonal difference as that done on female, while the present study was done on male rats.

     It was evident from the results of the present work that, STZ-induced diabetic rats, exhibited significant hyperglycemia and hypoinsulinemia level than their levels in the control group. This could be due tothe selective uptake of STZ into β cells via glucose transporter GLUT2 and destroying β-cells through damaging the nuclear DNA. Non-β endocrine cells in pancreatic islets remain intact after STZ injection, indicating selective properties of STZ to beta cell (Goudet al., 2013).The increased blood glucose level can undergo autoxidation and no enzymatic reaction, forming a glycated product, which forms oxidants with the generation of reactive oxygen species (ROS). The increased ROS causes further damage to the β-cells because these cells have low intrinsic oxidant enzymes. Thus, the prooxidant-antioxidant balance is impaired (Abdel Aziz et al., 2013).

     Although the concentration levels of plasma glucose in the DM + AG group did not reduce to the same extent as that observed in the control group, the levels significantly decreased compared with the level in the DM group, while oral administration of AG for 8 weeks did not affect glucose or insulin levels in normal rats. The present study was in agreement with Pal et al., )2013) who revealed that Arabic gum exerted a significant hypoglycemic effect by initiating the release of insulin from pancreatic beta cells through removing free radicals and lipid peroxidation repression. Nasir et al., )2010) reported that AG causes down regulating of sodium glucosetransporter1 (SGLT1), causing delay the intestinal glucose transport in diabetic mice treated with AG. On the other hand, the down-regulation of SGLT1 leads to the enhancement of hunger-related hormones including leptin, cholecystokinin, and glucagon-like peptide 1 (Gorboulev et al., 2012). These hormones decrease hunger by improving post-meal satiety through many mechanisms.

     The result of the current study disagreed with the result of Yasir et al., )2012) who reported that AG has a hypoglycemic effect in normal control rats. There is no conflict between the present study and that study as the study examined only the acute effect within few hours of administration of the AG in normal control animals, and it suggested that the plant stimulates insulin secretion. This effect seemed to be counteracted by the regulatory mechanisms in case of chronic administration, which was observed in our study.

     The hyperlipidemia observed in STZ-induced diabetic rats could be due to oxidative stress secondary to persistent hyperglycemia (Kumar et al., 2013), or due to insulin resistance as insulin resistance correlates with hyperglycemia, and alteration in lipid metabolism (Stahlman et al., 2013).

     The present study was in harmony with Almohaimeed et al., (2018) who reported that a decrease in triglyceride and cholesterol level in diabetic rats which obtained oral administration of Arabic gum.

     Numerous mechanisms have been reported to explain the hypocholesterolemic effect of AG. Some studies suggested that AG increase the viscosity ofthe intestinal content and therefore, interfere with intestinal lipid absorption (Longdet et al., 2018). Another mechanism suggested that AG act by disrupting the enterohepatic circulation of bile acids, consequential to increased bile acid excretion, and subsequently reduces plasma cholesterol concentrations, in addition to inducing increased numbers of lipoprotein receptors in the liver and decreased plasma cholesterol concentration (Brockman et al., 2014).

     The normal rats received AG for 8 weeks showed insignificant changes in serum lipids. This finding was supported by the results of Hegazy et al.,( 2013) who reported that AG did not change the lipid profile, in the normal control rats. On the other hand, these findings were contradicted with those of Yasir et al., (2012) who reported that AG has both hypoglycemic and hypolipidemic effect in normal rats as prevotellaruminicola-like bacterium was the predominant organism that is most likely responsible for fermentation of AG to propionate. Propionate could limit the induction of key enzymes of cholesterol metabolism hence lowers cholesterol levels. That difference may be due to the duration of administration of the AG as they administrated AG for only three weeks, while in the present study the extract of AG was administrated for 8 weeks.

     Diabetes-associated cardiac injury in the current study was evidenced by the elevated circulating levels of CK-MB, LDH, and AST. Creatin kinase-MB is a well-known sensitive marker of cardiomyocyte damage and a positive correlation between the myofibrillar disintegration and increased serum levels of CK-MB has been reported (Upaganlawar and Balaraman, 2010). In support of these findings, Al-Rasheed et al., (2016) demonstrated that serum CK-MB and AST elevated in experimental–induced DCM in rats. Interestingly, treatment with AG significantly ameliorated the circulating CK-MB, LDH and AST levels, and prevented hyperglycemia-induced cardiomyocyte injury, demonstrating its cardioprotective efficacy. The present study was in line with Nemmar et al., (2019) who reported that Arabic gum ameliorates cardiotoxicity induced by water- pipe smoke exposure in mice by a mechanism which involves activation of the Nrf2 (nuclear factor-like 2) signaling pathway.Nrf₂ is a critical transcription factor which plays a major role in activating antioxidant enzymes to respond to oxidative stress (Barancik et al., 2016).

     Regarding electrocardiographic (ECG) parameters, HR, QRS duration and QT interval are valid predictors of heart disease and fatal ventricular arrhythmia. The prevalence of a prolonged QT interval is higher in people with diabetes as compared to non-diabetic individuals. Moreover, QRS duration and R-wave amplitude (RWA) are early indicators of evolving cardiovascular disease and increased cardiovascular risk (Nakos et al., 2018).

     In the present study, the STZ induced diabetic rats showed a decrease in HR than control rats. This result was in agreement with Mostarda et al., (2013) who found that streptozotocin-induced diabetes produces bradycardia through autonomic neural dysfunction and sinoatrial node impairment.

     Various studies proved the beneficial role of bioflavonoid in improving cardiac function/ electrophysiology in diabetic animals (Annapurna et al., 2009). In the present study, AG improves cardiac function in diabetic rats by normalizing cardiac electrophysiology as it contains bioflavonoid.

     The untreated DM rats in the present study showed a significant increase in the cardiac MDA, and the activity of SOD significantly diminished. By contrast, the administration of AG to diabetic rats for 8 weeks exhibited a significant amelioration of both MDA and SOD. Normal rats treated with AG showed insignificant changes in the cardiac levels of MDA and SOD.

     In the present study, STZ-induced diabetic rats showed reductions in activities of SOD with increments in MDA that could reflect oxidative pressure. This might be a mirror to diminished antioxidant defense potentialas hyperglycemia and hyperlipidemias are associated with increased production of ROS (Tiwari et al., 2013). Arabic gum ameliorated both MDA and SOD due to its containing four antioxidant minerals, i.e. copper, iron, manganese, and zinc, or due to its positive effect on the expression of the antioxidant enzymes (Kong et al., 2014), or due to the  ability of AG to  reduce the expression of  HSP70 mRNA in the heart of diabetic rat. Heat-shock protein 70 (HSP70), a stress-induced protein is proposed to play a protective role against oxidative stress (Ahmed et al., 2015). Ali, (2004) reported that Arabic gum did not significantly affect lipid peroxidation (LP), and superoxide dismutase (SOD) in the kidneys and liver of healthy rats.

CONCLUSION

         AG attenuated cardiomyopathy via amelioration of hyperglycemia and attenuation of oxidative stress, inflammation, and consequent apoptotic cell death. Thus, AG possesses a therapeutic potential for the treatment and/or prevention of DCM.

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