Journal of Advanced Pharmacy Education and Research

Introduction  

Diabetes mellitus (DM) is a metabolic disorder with a variety of etiologies [1, 2]; it is characterized by chronic hyperglycemia and disturbance in protein, fat, and carbohydrate, metabolism due to a lack of insulin excretion/or insulin action. DM has become a public health threat around the globe [3-5]; it is also a major health issue in the Kingdom of Saudi Arabia. The progression of diabetic hepatosclerosis is triggered by oxidative stress. It is caused by an excess of reactive oxygen/nitrogen radicals, as well as a reduction in endogenous antioxidant systems, which leads to the development of degenerative diseases [6, 7]. Although there are several anti-diabetes drugs that regulate hyperglycemia, treatment options that target DM sequelae, including oxidative stress and dyslipidemia, are limited. Therefore, the focus of the current research is to explore new factors and strategies that would curb the spread of the diabetes epidemic and its fatal consequences. A combination of anti-diabetic drugs supplied with other chemicals has emerged as an interesting strategy for managing hyperglycemia and other DM disorders. Curcumin (CUR) is a South Asian spice and is a highly pleiotropic molecule that is a widely-known antioxidant [7] and has been demonstrated to have anti-bacterial, [8] anti-inflammatory, anticancer [9], antiatherosclerotic, antimicrobial [10], wound healing [11], and other pharmacological actions. Moreover, it has the ability to scavenge reactive oxygen species (ROS), including alkoxy radicals, hydroxyl radicals, and superoxide radicals, directly in the living cells [12], completely degrades superoxide through mimicking superoxide dismutase [13, 14], and acts as a protective barrier in conditions when high levels of peroxynitrite and hydrogen peroxide molecular oxidants are produced inside the cells [15].

Zinc, an essential trace element, has been found to improve glucose absorption and transport in numerous tissues while suppressing pancreatic insulin release and raising pancreatic insulin content [16]. Evans' research [17] has also revealed that zinc supplementation has a beneficial influence on the progression of age-related macular degeneration; individuals who took antioxidants and zinc supplements were less likely to lose visual acuity. Furthermore, studies have found promising results when curcumin is used in combination with anti-diabetic drugs [18] or other phytochemicals [19] to regulate glycemia and reduce other diabetic problems.

The aim of this research is to study how CUR combined with ZnCl2 affects the levels of oxidative stress biomarkers and serum lipid profile; and how the combination exerts defensive actions against morphological changes in liver tissue in STZ-induced diabetic rats. (Figure 1) This would benefit in determining the role of the combination, as well as alleviating the deteriorating effects of diabetes worldwide.

Materials and Methods

Chemicals and experimental procedures

High purity analytical grade chemical reagents were procured from Sigma, Merck, Aldrich. All kits were manufactured by Biosystems (Alcobendas, Madrid, Spain), Sigma (St. Louis, MO, USA), and Biodiagnostic (Cairo, Egypt). Seventy-two Albino Wister male rats, in the weight range of 200g to 205g, were purchased from Animal House, King Faisal University. Rats were kept in an individual cage in a room at a controlled temperature of 25°C, 50% humidity, 12 h of light and dark cycle with screened bottoms. These rats were fed for 10 days with the basal diet containing corn starch (70%), casein (10%), corn seed oil (10%), cellulose (5%), a mixture of salt (4%), and vitamins (1%). Then, rats were weighed and separated into six groups, with 12 rats in every group. The assigned 6 diet groups were as follows: G1 signified the negative control (NC), G2 designated the positive control (PC) in which rats were injected by STZ (60 mg/kg BW), G3 enclosed the rats that received STZ (60 mg/kg BW) + CUR (100 mg/kg BW), in G4 rats were treated with STZ (60 mg/kg BW) + CUR (200 mg/kg BW), rats in G5 were treated with [STZ (60 mg/kg BW) + CUR (100 mg/kg BW)+ ZnCl2 (15 mg/kg BW)] and those in G6 were treated with [STZ (60 mg/kg BW) + CUR (200 mg/kg BW) + ZnCl2 (15 mg/kg BW)]. Decapitation was used to kill all the rats at the end of the experiment (21 days). Following Schermer [20], blood samples were obtained from the orbital plexus using heparinized capillary glass tubes. First, a dry clean centrifuge tube was utilized to place each blood sample and then centrifuged at 4°C with the revolution of 1500g for 30 min to acquire the blood serum. An approval (lRB-2018-10-216) from the University was obtained to perform the study.

Biochemical analysis

ALT, AST, glucose, insulin, and lipid profile assay

ALT, AST activities [21], Glucose [22], Insulin [23], Total cholesterol [24], Triglycerides [25] and HDL-C and LDL-C [26]  concentrations were estimated by using standard kits.

Thiobarbituric acid (TBA) assay

Lipid peroxidation was estimated according to Hommouda et al.’s method [27] and upon the TBA reactivity principle. Briefly, 2.5mls of 10% trichloracetic acid (TCA) and 0.5ml of plasma were mixed in test tubes. After 15 minutes of incubation at 90°C and then cooling via a cold water bath, the mixture was centrifuged for 10 minutes at 3000 rpm. Then 2mls of the supernatant was added to 1ml of 0.675% TBA. The tubes were capped and incubated for 15 minutes at 90°C and then allowed to cool down at room temperature. The optical density was measured at 532nm by a spectrophotometer (Schimadzu, AA6800, Tokyo, Japan).

Superoxide dismutase (SOD) assay

The SOD activity of erythrocytes was determined in the hemolysates via commercially available kits from Randox Laboratory, Crumlin, Ireland. Hemolysis of erythrocytes was done by using ice-cold deionized water followed by strong vortexing. SOD activity was determined through the superoxide radicals that are generated from xanthine and xanthine oxidase, which in turn react with 2-(4- iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to produce a red formazan dye. The degree of inhibition of such a reaction is correlated to SOD activity [28].

Catalase (CAT) assay

CAT activity was determined via Aebi method [29], which employs a UV spectrophotometer at 240nm. To determine the ceruloplasmin activity in serum, 5ml of phenylenediamine substrate was added to 2 tubes (test and curve tubes). Then, 1ml sodium azide (NaN3) solution was added only into the curve tube while 100μls of serum was added to both tubes. Both tubes were mixed and kept for 15 minutes at 37°C. Finally, 1ml of NaN3 was added to the test tube only, and a spectrophotometer was used to measure the OD at 546nm.

Glutathione (GSH) assay

Concentrations of serum GSH were measured according to Beutler et al. method [30].

Statistical analysis

Data were analyzed by comparing the results of various treatment groups to the results of control groups. All data were presented as mean±SD. ANOVA with posthoc least significant difference test (LSD) at (p 0.05) were integrated and used to examine significant differences between values.

Histopathology

 Liver specimens were fixed in 10% formalin, paraffinized, sectioned (4µm thickness), and stained using hematoxylin and eosin dyes. Light microscopy was used to demonstrate hepatic pathological alterations such as degeneration, atrophy, cell distortion, and necrosis, as well as the effectiveness of micronutrients in alleviating these pathological aspects [31].

Results and Discussion

Table 1 shows the effect of CUR+ZnCl₂ on serum glucose during 21 days in STZ-induced diabetes in rats. The level of serum glucose increased significantly (p≤0.05) in PC group (STZ-treated); and reduced significantly (p≤0.05) in CUR (100 & 200 mg/kg b.w) and CUR coupled with ZnCl₂ (15 mg/kg b.w) treated groups, concerning the PC.

Results for 12 rats in every group are shown as mean±SD; statistical significance at p≤0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

The levels of insulin were given in Table 2. The results demonstrated that the level of serum insulin was reduced significantly (p≤0.05) in PC when compared with NC. Administration of CUR (100 & 200 mg/kg b.w) and CUR combined with ZnCl₂ (15 mg/kg b.w) led to a marked increase (p≤0.05) in insulin level when compared to PC. The treatment of 200 mg/kg b.w CUR + ZnCl₂ 15mg/kg b.w gave results near to the NC after 21 days.

The results of TC, TG, HDL-C, and LDL-C were given in Table 3. The data indicated that serum levels of TC, TG, and LDL-C were significantly (p≤0.05) elevated in PC; however, HDL-C was significantly (p≤0.05) reduced compared to NC and other treated groups. A significant (p≤0.05) inhibition in TC, TG, and LDL-C levels was observed in CUR (100 & 200 mg/kg b.w) and CUR combined with ZnCl₂ treated groups while HDL-C levels were elevated, compared to PC. Moreover, the group that was treated with CUR 200 + ZnCl₂ 15mg/kg b.w, gave the best results.

Results for 12 rats in every group are shown as mean ± SD with statistical significance at p ≤ 0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

Table 3 shows the effect of CUR (100 & 200 mg/kg b.w) and CUR + ZnCl₂ 15mg/kg b.w on serum AST and ALT enzymes. ALT and AST activities significantly (p≤0.05) increased in PC group compared with NC and other treated groups. The CUR and CUR + ZnCl₂ treated groups showed a lower compared to PC, especially at CUR 200 + ZnCl₂ 15mg/kg b.w treated group.

STZ exposure significantly (p≤0.05) elevated the levels of SOD, TBA, and CAT, while GSH was significantly (p≤0.05) decreased as compared with the NC group. Administration of CUR (100 & 200 mg/kg b.w) and CUR combined with ZnCl₂ 15mg/kg b.w significantly (p≤0.05) lowered SOD, TBA and CAT levels (Table 4). CUR 200 + ZnCl₂ 15mg/kg b.w treated group improved the levels of GSH and SOD close to NC.

Results for 12 rats in every group are shown as mean±SD with statistical significance at p≤0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

Results for 12 rats in every group are shown as mean±SD with statistical significance at p ≤ 0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

Histopathological investigations revealed that the diabetic liver had a high degree of hydropic degeneration accompanied by coagulative necrosis (Figure 2). Treatment with CUR (100 & 200 mg/kg b.w) and their combination with ZnCl₂ 15mg/kg b.w showing improvement appeared in the hepatocytes, Kupffer cells nuclei, and blood sinusoids (Figure 2).