Protective Effect of Long Term Administration of Gum Arabic

The action of key cell reinforcement catalysts assumes a basic part in the acceptance of hyperglycemia-related tissue harm [1]. Oxidative pressure initiated by the lopsidedness of oxidants/cancer prevention agents harm of organic macromolecules, including starches, proteins, lipids, and nucleic acids, cause aggravations in cell homeostasis and generation of other receptive atoms that reason more damage[2]. The significance of oxidative pressure and its association with the pathology of diabetes mellitus(DM) alongside related intricacies have been broadly investigated[1,3]. Past examinations announced that the generation of reactive oxygen species (ROS) in diabetes start the improvement of perpetual diabetic sores on blood vessels [4], retina[5], kidneys[6], and neurodegenerative diseases[7].

DM is a constant and most regular metabolic issue that has turned out to be pandemic in the twenty-first century [8]. Around 347 million individuals were influenced with DM in 2011 overall [9]. The World Wellbeing Association predicts that diabetes will be the seventh driving reason for death in2030. The oxidative worry in DM causes a few unfavorable consequences for the cell physiology [10]. It diminished glutathione (GSH) level in diabetes [11], diminished catalase activity[12], down managed renal SOD[13] and expanded heat shock protein 70 (HSP70) level in patients with type2 DM [14]. Oxidative pressure has been accounted for as a key factor at the beginning of pathogenesis and diabetic complexities [15]. Clinical and test ponders suggested that the liver might be affected by DM in the long haul [16– 18]. Histological highlights of fatty liver disease initiated by DM and non-alcoholic fatty liver disease (NAFLD) can't be recognized from ethanol-instigated hepatic steatosis [18].

Gum Arabic (GA) is a consumable, dried sticky exudate from Acaciaseyal and Acacia Senegal is rich with non-thick solvent fiber. It is usually utilized as a part of nourishment industry and pharmaceutical field as an emulsifier and additive [19]. In North Africa and the Middle East, it’s utilized as an oral cleanliness specialist by different groups for a few centuries [20]. GA is utilized as a part of Arabic society pharmaceutical to diminish both recurrence and need of hemodialysis in incessant renal failure patients [21]. It has solid cancer prevention agent properties and used to decrease the test nephrotoxicity against gentamicin [21], cisplatin [22] and to improve cardiotoxicity [23]. Be that as it may, the impacts of GA on oxidative worry in the liver of type I diabetic rats have not been accounted for. Regardless of whether GA can change oxidative related qualities articulation in the liver of type I diabetic rodent stays less clear.

Consequently, in the present examination, we utilized type I diabetic rodent model to explore our speculation that supplementation of GA in drinking water may secure liver by lessening oxidative harm, and the decrease of oxidative harm might be related with the balance of liver oxidative related qualities articulation.

The rationale of the study:

In the present study, we aim to use a diabetic rat model, to test whether adding GA to insulin therapy would ameliorate the diabetic-induced changes in liver function and whether the action is mediated by an effect on oxidative stress markers.

2. Material and Methods:

2.1 Animals. A total of 60 male Sprague-Dawley rats (age 5-6 weeks) weighing 200 ± 10 g were included in this study. Animals were housed in the animal house a controlled environment with a 12:12-hrs light/dark cycle. Animals were acclimatized for one week before the study and had free access to water and standard rat chow throughout the experimental period.

2.2.Animal groups: This study followed a randomized controlled animal experimental design. The 60 male Sprague-Dawley rats included in this study were divided into 6 groups (n = 10 each) as follows:

[A] Control groups:

1. Non- treated control (C): rats were injected intraperitoneally once with citrate buffer only.

2. Gum Arabic treated control (GAC): rats were injected intraperitoneally with buffer as “C” group and received 10% w/v of Gum Arabic in drinking water.

[B] Experimental groups:

1. None treated diabetic group (D): diabetic group neither treated with insulin nor GA.

2. Diabetic + insulin (DI): Rats received insulin only.

3. Diabetic + GA (DGA): rats received GA only after induction of diabetes.

4. Diabetic + insulin + GA (DIGA): rats received insulin and GA after induction of diabetes.

2.3 Induction of Diabetes Mellitus (DM):

Type I diabetes mellitus (DM) was induced as described by [24]. Briefly, a single intraperitoneal injection (i.p.) of streptozotocin STZ (65 mg/kg) in freshly prepared citrate buffer (0.1 M, pH 4.5) (Sigma Chemical Company, USA) was given after an overnight fasting. DM was verified by measuring blood glucose in surviving rats after 3 days, through tail/neck blood sampling. Rats with non-fasting blood glucose level of =20 mmol/L after 48 h of STZ injection were considered to be diabetic [25]. The experimental period was 12 weeks, a period which has been proven to induce detectable diabetic complications in the kidney [26].

2.4 Tissue preparation

The rats were anesthetized with pentobarbital sodium (60 mg/kg body weight, intraperitoneally). The liver was removed, cleaned of gross adventitial tissue, blotted dry and processed for biochemical measurements. Tissue was homogenized in 50 mM phosphate buffer (pH 7.4) using a Polytron homogenizer. The resultant supernatant was used for measurement of Thiobarbituric acid reactive substances (TBARS) and antioxidant enzymes. The TBARS levels were measured as an index of malondialdehyde production and then lipid peroxidation was assessed in the tissues by the method of Yagi [27] as previously described [28].

2.5 Evaluation of hepatic antioxidant enzyme activity

The Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH) commercial reagents were purchased from Sigma Chemical Company, USA. Liver tissues (1 g) were cut into small pieces and homogenized in ice-cold saline buffer (0.85%, pH 7.4) (1:9, wt/v) with an Ultra-Turrax (T8, IKA-labortechnik Staufen, Germany). Liver homogenates were centrifuged at 1000 g for 15 min at 4C, and the supernatants were collected. The supernatants were used for the assays of SOD, GPx, CAT, and GSH. The antioxidative status of the liver was estimated by measuring the level of different antioxidants in the liver. The activity of SOD was measured according to the method of [29]. The activities of GPx and CAT were measured by the methods described by [30], and [31] respectively. All assays were measured with the clinical chemistry assay kits according to the manufacturer’s recommended procedure. The antioxidant activity as a function of oxidative stress in hepatic tissue was determined according to the manufacturers information; catalase (abcam- ab83464 catalase assay kit- Colorimetric/Fluorometric), Superoxide dismutase (Cayman chemical- Superoxide Dismutase assay kit- item number. 706002), malondialdehyde (abcam- ab118970- Lipid Peroxidation (MDA) Assay kit - Colorimetric/ Fluorometric) and glutathione (SIGMA- ALDRICH- glutathione assay kit- catalog number. CS0260). The obtained results were analyzed by SPSS version 20. The mean values of the parameters of the two groups were analyzed using t-test.

2.6. RNA extraction and real-time PCR

About 100 mg of liver was ground in liquid N2, and a portion of about 50 mg was used for RNA extraction using TRIzol total RNA kit (Invitrogen, Biotechnology Co., Ltd., Carlsbad, CA, USA) according to the manufacturer’s instruction. Two approaches were taken to ensure that all the total RNA preparations are free of genomic DNA contamination. First, total RNAs were treated with 10 U DNase I (RNase Free, D2215, Takara, Japan) for 30 min at 37C, and purified according to the manufacturer’s protocol. Second, the primers for the reference gene (-actin) were designed to span an intron, so any genomic DNA contamination could be reported easily with an extra product in the melting curves for real-time PCR. Real-time PCR was performed in Mx3000P (Stratagene, USA) according to the previous publications [32,33]. Primers specific for SOD, and gGCL (Table 1) was synthesized by Geneary (Shanghai, China), and rat -actin was used as a reference gene for normalization purpose. The method of 2-Ct was used to analyze the real-time PCR data [34].The mRNA abundances were presented as the fold change relative to the average level of the control group.

2.7. Ethical clearance:

This study was conducted after academic and ethical approval from the faculty of medicine at the University of Al Neelain- Khartoum, Sudan.

2.8. Statistic analysis

Descriptive statistics were performed to check the normality and homogeneity of variances before using parametric analyses. Data were analyzed using SPSS statistical program. Values are expressed as frequency, percentage and mean ± SD. Testing significance was performed using 2 test and the one-way analysis of variance (ANOVA). P-values = 0.05 were considered statistically significant.

3. Results

3.1. Effect of GA on hepatic antioxidant enzymes activities

Key antioxidant enzymes including SOD, CAT, and GPx of rat liver were measured. The antioxidant activity as a function of oxidative stress in hepatic tissue in the six groups is presented in Figure 1 to 4. Values (mean±SD) obtained for the levels of reduced glutathione (GSH) in the livers of gum arabic treated group of rats were significantly different when compared to control group. Furthermore, there were also significantly different in rats with DM treated with Insulin and gum Arabic (Figure – 1). Diabetic rat group showed significant decreases in all antioxidant enzymes activities when compared to the control. Levels of total superoxide dismutase (SOD) in the livers of experimental groups of rats showed a diverse tendency wherein rats with diabetes mellitus did not show any significant increase in SOD upon treatment with gum arabic however the addition of insulin with gum Arabic to diabetic rats increased SOD very significantly (Figure – 3). Finally, the levels of catalase (CAT) showed a very significant increase in diabetic rats treated either with insulin and gum Arabic alone or in combination (Figure – 4). However, the treatment of GA significantly (P < 0.05) increased the antioxidant enzymes activities including SOD, CAT and GPx compared to that of control and diabetic groups.

3.2. Effect of GA on hepatic lipid peroxidation

Malondialdehyde (MDA) as a biomarker of oxidative stress is routinely used to evaluate the extent of lipid peroxidation. In the present study, we observed significant increases in hepatic MDA concentrations in diabetic group compared to the control. However, the treatment of GA significantly (P < 0.05) decreased MDA concentrations compared to that of diabetic rat group (Figure 2). In addition, the treatment of GA significantly increased liver GSH concentration compared to the control and diabetic rat groups. The levels of thiobarbituric reactive acid substances (GSH) in the livers of all experimental groups of rats showed a similar trend, however, their levels were significantly higher in diabetic rats treated with gum Arabic and insulin (Figure – 2).

3.3. Effect of GA on hepatic antioxidant genes expression

Q.PCR was used to measure antioxidant enzymes mRNA expression in the liver. Streptozotocin-induced diabetes caused a significant decrease in liver SOD mRNA expression. However, the treatment of GA significantly increased mRNA expression of hepatic SOD (Figure 4) compared to a diabetic group or those treated with insulin. Furthermore, mRNA significantly increased and reached on par with control in diabetic rats treated with insulin and GA. A similar trend was seen with gGLC mRNA expression in relation to -actin mRNA, wherein treatment of GA to diabetes-induced rats significantly increased its expression and it was much more significantly increased when GA treated in combination with insulin