الفهرس 
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Abstract
Biochemical evaluation of the potential anti-endotoxin effects of curcumin, rutin and resveratrol in a rat model of endotoxemia were investigated.Sixty four male rats, 8-10 weeks old and average body weight 180-220 g were used in the experimental investigation of this study. Rats were housed in separated metal cages and kept at constant environmental and nutritional conditions throughout the period of experiment. The animals were fed on constant ration and water was supplied ad- libitum. Rats were left 15 days for acclimatization before the beginning of the experiment. Induction of Endotoxemia:Escherichia coli (serotype O55: B5) is a gram negative rod (bacillus) in the family Enterobacteriaceae and is chosen and used in this study to induce endotoxemia. Moreover, endotoxemia was induced in rats by a single dose of intraperitoneal (i.p) injection of LPS (E.coli, serotype O55:B5) at a dose level of (200mg/ kg b. wt).Antioxidants compounds:antioxidant compounds used in the present study were Curcumin: Curcumin was freshly prepared by dissolved in 7% of dimethyl sulfoxide ( DMSO) solution and administered orally at a dose of (100 mg/kg b.wt./day) for 21 days.Rutin: Rutin was dissolved within propylene glycol solution and administered orally to rats at a dose level of (200 mg/kg b.wt) once daily for 21 days.Resveratrol: Resveratrol was freshly prepared in 5% Ethanol and administered to rats at a dose level of (10 mg/kg b.wt/ i.p.) daily for 7 successive days.Design of the experimental work:Rats under study were randomly divided into five mean groups, placed in individual cages and classified as follows.Group I (control group): 12 Rats received no drugs, served as control non-treated for all experimental groups. This group was divided into two subgroups:Subgroup (a) 5 rats : sacrificed at the 8th day of the experiment , served as control normal for resveratrol protected group : Subgroup (b) 7 rats: sacrificed at the 22nd day of the experiment , served as control normal for rutin and curcumin protected group.Group (2): Endotoxin group: Included 15 rats injected intraperitoneally (i.p) with a single dose of LPS (200 mg/Kg b.wt).This group was divided into 2 subgroups:Subgroup (a) (5 rats):Considered as control group for comparison with RSV protected group. Received Endotoxin (200mg/kg b.wt. i.p.) at the 8th day of the experiment.Subgroup (b) (10 rats): Considered as control group for comparison with RTN and CUR protected groups. Rats received endotoxin (200mg/kg b.wt/ i.p.) at the 22th day of the experiment.Group III (Curcumin protected group): Comprised 15 rats received curcumin orally in a daily dose of 100 mg/Kg b.wt. for 3 weeks before endotoxemia indction. One hour after the last dose of curcumin administration, endotoxemia was induced in rats by a single i.p injection of LPS ( 200mg/ Kg b.wt).Group (IV) (Rutin protected group): Comprised 15 rats received rutin orally in a daily dose of 200 mg/kg/ b.wt. for 3 weeks before endotoxemia induction. One hour after the last dose of rutin administration, endotoxemia was induced in rats by a single intraperitoneally (i.p) injection of LPS (200 mg/ kg b.wt.).Group (V) (Resveratrol protected group): Comprised 7 rats, received resveratrol injected intraperitoneally (i.p) in a daily dose of 10 mg/kg/b.wt. for 7 days before endoxemia induction. one hour after the last dose of resveratrol administration, endoxemia was induced in rats by a single i.p injection of LPS ( 200 mg/ kg/b.wt).Sampling Random blood samples and tissue specimens (liver and brain) were collected from all animal groups (control and experimental groups) twice at one hour and three hours after endotoxemia induction. Blood Samples:1- Blood samples:Blood was collected from retro- orbital venous plexus of eyes in clean dry screw-capped tubes, then allowed to coagulate at room temperature for 30 minutes, and centrifuged at 3500 r.p.m for 15 minutes. The clean, clear-serum was separated by Pasteur pipette and received in dry sterile sample tube, processed directly for glucose determination, then, kept in a deep freeze at -20° C until used for subsequent biochemical analysis. All sera samples were analyzed for the following parameters.Tumor Necrosis Factor (TNF-α) , Interleukin – 6( IL-6), Sialic acid (SA), L-Malondialdehyde (L-MDA) and Nitric oxide (NO). Tissue Samples:a- Liver samples:After finishing blood samples, rats of each group were sacrificed using highly sterilized scissors and liver samples were directly excised and it was wrapped in aluminum foil and put immediately in liquid nitrogen container which used forPCR analysis. All liver samples were used for determination of gene expression of Cytochrome P450 2E1 (CYP2E1).b- Brain samples:The skull was opened carefully and the brain was quickly removed, cleaned by rinsing with ice-cold isotonic saline, cleared off blood, and immediately transferred into ice-cold isotonic saline for a second time, then blotted between 2 filter papers. The brain tissue samples were quickly frozen in a deep freeze at -20 °C for consequent biochemical analysis. Brain tissue samples were analyzed for determination of the following biochemical parameters: L-malondialdehyde (L-MDA), nitric oxide (NO), antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione Peroxidase (GPx), reduced glutathione (GSH), monoamine oxidase (MAO) and myeloperoxidase (MPO). Also, brain tissue Lipid extraction were performed with 5ml of isopropanol and the aliquots were used for determination of total cholesterol and phospholipids.The obtained results summarized the followings: 1- Serum tumor necrosis factor-alpha (TNF-α) and interleukin - 6( IL-6):The mean value of serum TNF- α and IL-6 concentrations significantly increased at one hour and 3 hours after endotoxin injection in rats when compared with control group.Curcumin and resveratrol pretreatment endotoxin groups significantly decreased serum TNF- α and IL-6 concentrations at one hour and 3 hours after endotoxin injection as compared with endotoxin group. However, rutin pretreatment endotoxin group non significantly decreased serum TNF- α and IL-6 concentrations at one and three hours after endotoxin treatment as compared with endotoxin group .2- Serum Sialic acid.Serum sialic acid concentration was significantly decreased in endotoxin injected rats after one hour and three hours when compared with control group.A significant increase in serum sialic acid concentration was observed in curcumin and resveratrol pretreatment endotoxin treated rats after one hour and 3 hours.Rutin pretreatment endotoxin group non significantly decrease serum sialic acid concentration at one hour and non significantly increased sialic acid level at 3 hours after endotoxin injection in rats when compared with endotoxin group.3- Serum L-malondialdehyde (L-MDA):Endotoxin injection in rats significantly increased serum L-MDA concentration at one and 3 hours after injection in comparison with control group.A significant decrease in the value of serum L- MDA concentration was observed in curcumin, rutin and resveratrol pretreatment endotoxin group at one and 3 hours after endotoxin injection when compared with endotoxin treated rats only.4- Serum nitric oxide(NO): A significant decrease in serum nitric oxide concentration was observed in endotoxin injected rats after one and 3 hours in comparison with control group. Pretreatment with curcumin, rutin and resveratrol significantly increased serum NO concentration at one and 3 hours after endotoxin injection when compared with endotoxin group only. 5- Brain tissue L-malondialdehyde (L- MDA):The mean value of brain tissue L-MDA concentration was significantly increased in endotoxin injected rats after one and 3 hours in comparison with control group. Pretreatment with curcumin, rutin and resveratrol significantly decreased brain L- MDA concentrations at one and 3 hours after endotoxin injection when compared with endotoxin treated rats group. 6- Brain tissue nitric oxide (NO):A significant decrease in brain tissue NO concentration was reported in endotoxin injected rats after one and three hours in comparison with control group. On the other hand, a significant increase in brain tissue NO concentration was observed in curcumin, rutin and resveratrol pretreatment endotoxin injected rats at one and 3 hours in comparison with endotoxin group only.7- Brain tissue Myeloperoxidase:Intraperitoneal injection of endotoxin to normal rats significantly decreased brain tissue myeloperoxidase activity at one hour and 3 hours after injection as compared with control group. A significant increase in brain tissue myeloperoxidase activity was observed in curcumin, rutin and resveratrol pretreatment endotoxin group at one hours and 3 hours after endotoxin injection when compared with endotoxin treated rats only. 8- Brain tissue Monoamine Oxidase (MAO):Intraperitoneal injection of endotoxin to normal rats caused significant decrease in the value of brain tissue MAO activity after one and 3 hours in comparison with control group. Pretreatment with curcumin, rutin, and resveartol caused significant increase in brain tissue MAO activity at one hour and 3 hours after endotoxin injection in comparison with endotoxin injected rats only .9- Brain tissue Catalase (CAT):Intraperitoneal injection of endotoxin to normal rats significantly decreased brain tissue CAT activity after one and 3 hours in comparison with control group. A significant increase in brain tissue CAT activity was reported in curcumin, rutin and resveratrol pretreatment group at one and 3 hours after endotoxin injection when compared with endotoxin treated rats group only.10-Brain tissue Superoxide Dismutase (SOD): The mean value of brain tissue SOD activity was non significantly decreased in endotoxin treated rats at one hour. This decrease become significant after 3 hours when compared with control group. Pretreatment with curcumin, rutin and resveratrol exhibited a non-significant increase in brain tissue SOD activity at one hour after endotoxin injection. This increase becomes significant at 3 hours after injection when compared with endotoxin treated rats only. 11- Brain tissue Reduced Glutathione (GSH):A significant increase in brain tissue GSH concentration was observed in endotoxin injected rats after one hour and 3 hours when compared with control group. Pretreatment with curcumin, rutin and resveratrol significantly decreased brain tissue GSH level at one hour and 3 hours after endotoxin injection when compared with endotoxin injected rats group only. 12- Brain tissue Glutathione peroxidase (GPX):Endotoxin treatment significantly decreased brain tissue GPX activity in rats after one and 3 hours in comparison with control group. Pretreatment with curcumin, rutin and resveratrol significantly reversed endotoxin induced decrease in brain GPX activity after one and 3 hours when compared with endotoxin injected rats.13- Brain tissue total Cholesterol:Brain tissue total cholesterol concentration was significantly increased in endotoxin injected rats after one hour and 3 hours when compared with control group. Pretreatment with curcumin, rutin and resveratrol caused a significant decrease in brain tissue total cholesterol concentration at one hour and 3 hours after endotoxin injection when compared with endotoxin injected rats group only. 14- Brain tissue Phospholipids:Intraperitoneal injection of endotoxin to normal rats significantly increased the value of brain tissue phospholipid concentration after one and 3 hours in comparison with control group. A significant decrease in brain tissue phospholipid concentration was observed in curcumin, rutin, and resveratrol pretreatment endotoxin group at one hour and 3 hours after injection when compared with endotoxin treated rats only.15- Polymerase chain reaction product of Cytochrome p450 2E1 gene expression in liver tissue:Intraperitoneal injection of endotoxin to normal rats markedly increased cytochrome p450 2E1 gene expression in the liver tissue after 3 hours as compared with rats after one hour in comparison with control group.Pretreatment with curcumin showed a significant down regulation of cytochrome p450 2E1 at one and 3 hours after endotoxin injection when compared with endotoxin treated rats only. Pretreatment with resveratol showed inhibition of cytochrome p450 2E1 gene expression in rat liver tissue at one and 3 hours after endotoxin injection in comparison with endotoxin group only. However, pretreatment with rutin showed up regulation and slightly higher of cytochrome p450 2E1 gene expression in liver tissue at one and 3 hours after endotoxin injection as compared with endotoxin injected rats only.