الفهرس 
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Abstract
Diabetes mellitus (DM) is a serious worldwide medical and social problem. DM is characterized by persistent hyperglycemia with impaired metabolism of glucose, lipids and protein resulting from malfunction in insulin secretion and/or insulin action. Untreated or poorly controlled hyperglycemia can cause many complications that lead to decrease in the quality of life and increase in the rate of mortality.
The aim of the present study is to investigate the effect of phenolic acids, gallic acid and p-coumaric acid, on the impaired glucose tolerance and various biochemical, molecular, histopathological alterations associated with diabetes and to suggest the possible mechanism(s) of action of the tested agents.
Adult male albino rats (Rattus norvegicus) weighing about 130±10 g were used in the present study. For acclimatization and excluding any intercurrent infection, rats were kept under observation for two weeks before the onset of the experiment. Type 2 diabetes was induced in overnight fasted rats by single intraperitoneal (i.p.) injection of streptozotocin (65 mg/kg b.wt.), freshly dissolved in cold citrate buffer (pH 4.5), after 15 min of i.p. injection of nicotinamide (120 mg/kg b.wt.) prepared in normal physiological saline. Seven days after streptozotocin injection, rats were deprived of food (8˗10 hours), blood samples were taken from lateral tail vein after 2 hours of oral glucose administration (3 g/kg b.wt.) and plasma glucose concentration was measured. Rats with a 2-hour plasma glucose level ranging from 200˗300 mg/dl were considered mildly diabetic and included in the experiment.
The experimental animals were randomly allocated into four groups as follows; group I served as normal control, group II served as diabetic control, group III acted as diabetic rats administered gallic acid (20 mg/kg b.wt.) and group IV acted as diabetic rats administered p-coumaric acid (40 mg/kg b.wt.).
At the end of the sixth week of treatment, oral glucose tolerance test (OGTT) was performed for all experimental groups, rats were then sacrificed and two blood samples were collected from each rat. The first blood sample was collected into a tube containing EDTA and immediately preserved in the refrigerator for subsequent analysis of blood glycosylated hemoglobin (HbA1c) and complete blood count (CBC). The second blood sample was allowed to coagulate then centrifuged to separate serum that used for quantitative determination of glucose, insulin, adiponectin, TNF-α, lipid profile, protein profile, ALT, AST, urea and uric acid.
Livers were excised quickly after dissection of the sacrificed animals and perfused with ice-cold saline then homogenized. The homogenate was centrifuged and the supernatant was used for biochemical analysis including liver glycogen content, MDA, NO, SOD, CAT, GPx, GST, GSH and total thiols. Moreover, visceral adipose tissue samples were taken for determination of mRNA expression levels of PPARγ using real time-PCR. Hippocampus was isolated from one half of brain and used for determination of protein expression levels of Bax and Bcl-2 using Western blotting. The remnant tissue of the brain half was homogenized, centrifuged and used for determination of MDA, NO, SOD, CAT, GPx, GST, GSH and total thiols in addition to levels of monoamines (EP, NE, DA and 5-HT). Pancreas and other half of brain were fixed in 10% neutral buffered formalin for paraffin section preparation, staining and microscopic examination to detect histopathological alterations.
In addition to the in vivo study, the present investigation was extended to include in vitro and in situ studies such as insulin release from isolated islets in vitro, peripheral glucose uptake by rat’s diaphragm in vitro, alpha amylase inhibitory activity in vitro and intestinal glucose absorption in situ.
The obtained data from the above mentioned investigations revealed the following:
The treatment of diabetic rats with gallic acid or p-coumaric acid showed protection against body weight loss.
Oral glucose tolerance curve of diabetic control group was elevated and the treatment with either gallic acid or p-coumaric acid significantly ameliorated the impaired glucose tolerance.
Blood HbA1c level was markedly increased in diabetic rats and was potentially decreased as a result of administration of the tested agents.
Serum insulin level decreased in diabetic rats and increased as a consequence of administration of the tested agents.
Histological examination of pancreatic islets of diabetic rats revealed a marked degeneration of cells, represented by necrosis and vacuolations, and decrease in number of β-cells and intensity of insulin granules. Treatment with gallic acid or p-coumaric acid reversed these histopathological alterations.
Regarding liver glycogen content, it enormously depressed in diabetic rats and increased as a result of treatment with the tested agents.
Concerning in vitro studies, gallic acid and p-coumaric acid caused a marked increase in glucose-induced insulin secretion from isolated islets and increase of glucose uptake by diaphragm indicating insulinotropic and insulin mimetic actions of both agents. Furthermore, both treatments produced a marked α-amylase inhibitory activity at all concentrations in a dose dependent manner.
Intestinal glucose absorption in situ was observed to be decreased in a dose dependent manner as a result of the presence of gallic acid and p-coumaric acid in the perfusion solution.
Serum adiponectin in diabetic rats was obviously decreased and the oral treatment with gallic acid or p-coumaric acid caused profound increase in its level. In contrast, serum TNF-α level exhibited an opposite behavioral pattern as it increased in diabetic rats and decreased after treatments.
Real time-PCR showed that mRNA expression levels of PPARγ significantly decreased in diabetic control rats and increased in diabetic rats treated with the tested agents.
It was observed that diabetic rats showed marked elevations of serum triglycerides, total cholesterol, LDL-cholesterol and vLDL-cholesterol, while HDL-cholesterol level was detectably lowered. Treatment of diabetic rats with either gallic acid or p-coumaric acid had profound ameliorative effects on the disrupted lipid profile.
Cardiovascular risk index 1 and 2 were increased in diabetic rats as compared to normal ones. Treatment with both tested agents produced remarkable amelioration of these altered parameters.
The antiatherogenic index showed an opposite behavioral pattern; it was significant decreased in diabetic rats. Treatment with either gallic acid or p-coumaric acid alleviated the altered antiatherogenic index.
In view of protein profile, serum total protein, albumin, globulin were decreased in diabetic rats and markedly increased following administration of tested agents.
The activities of liver function enzymes, ALT and AST, were significantly increased in diabetic rats and decreased by treatment with both agents.
Serum urea and uric acid concentrations were increased in diabetic rats and the oral treatment with gallic acid or p-coumaric acid caused marked decrease of these parameters.
The levels of MDA and NO in both liver and brain were increased in diabetic rats, while they were decreased following treatment with gallic acid or p-coumaric acid, indicating the free radical scavenging activities.
The activities of SOD, CAT, GPx and GST as well as the levels of total thiols and GSH were decreased in liver and brain of diabetic rats and increased as a result of treatment with both agents.
Western blotting analysis showed that Bax protein expression level in hippocampus of diabetic control revealed a significant increase as compared to normal control and exhibited a significant decrease upon oral administration of gallic acid or p-coumaric acid. Bcl-2 protein expression level exhibited an opposite behavioral pattern; they were decreased in diabetic rats and profoundly increased upon treatment with both agents.
Concerning the histological examination of hippocampal sections, diabetic rats exhibited marked alterations in the normal structure of hippocampus in the form of disorganization, cell loss and decrease in number of pyramidal cells resulting from apoptotic cell death. Daily treatment with gallic acid or p-coumaric acid caused improvement in the histological architecture in the form of increase in the number of surviving pyramidal cells and marked decrease of apoptosis.
The obtained data indicated that diabetic rats exhibited significantly high brain levels of monoamines (EP, NE, DA and 5-HT) as compared to normal control. Gallic acid or p-coumaric acid oral administration markedly ameliorated the levels of brain monoamines in diabetic rats.
The hematological study showed decrease in the levels of RBCs, Hb, HCT, MCV, MCH and MCHC in diabetic rats indicating an anemic condition. Moreover, platelets count was decreased in diabetic rats. The results also revealed marked alterations in the total and differential leukocyte count of diabetic rats, especially an increase of WBCs count, neutrophils and monocytes. The treatment of diabetic rats with gallic acid or p-coumaric acid reversed these alterations.
In conclusion, the present investigation revealed that gallic acid and p-coumaric acid have potent antihyperglycemic activity which may be mediated via several mechanisms including inhibition of carbohydrate digestion and glucose absorption in the intestine, stimulation of insulin release from the pancreatic β-cells, enhancing glucose uptake in muscles and improving insulin action as evidenced by in vivo, in vitro and in situ studies. Both agents also exerted powerful anti-hyperlipidemic, cardiovascular protective, hepatoprotective and renoprotective effects in diabetic rats. In addition, the tested agents exhibited neuroprotective effects in the diabetic brain through scavenging free radicals and improving antioxidant status, anti-inflammatory and anti-apoptotic activities. All hematological alterations were also improved by the tested agents.
Hence, our results suggest that gallic acid and p-coumaric acid may be considered as promising candidates for treatment of diabetic patients, pending further investigations in order to trace out their antidiabetic efficacies and exact mechanistic pathways.