Protective effects of melatonin and L-carnitine against methotrexate-induced toxicity in isolated rat hepatocytes.

The present study was designed to evaluate the possible protective effects of melatonin (MEL) and/or L-carnitine (L-CAR) against methotrexate (MTX)-induced toxicity in isolated rat hepatocytes. Hepatocytes were prepared using collagenase techniques of perfusion and digestion of rat liver. Trypan blue uptake, as well as, glutathione (GSH), lipid peroxidation (LPO), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-α) levels were measured. Caspase-3 activity was also assessed. Pre-incubation of hepatocytes with MEL (1 mM) and/or L-CAR (10 mM) 30 min prior to intoxication with MTX, significantly protected hepatocytes against toxicity. In addition, LPO, NO, TNF-α levels, and caspase-3 activity were decreased in comparison to the MTX-intoxicated group. Furthermore, the two drugs increased the MTX-depleted GSH level. MEL and L-CAR prevented MTX-induced hepatocytotoxicity, at least partly, by their antioxidative, antiinflammatory, and antiapoptotic effects. Further studies are recommended on the clinical pharmacologic and toxicologic effects of MEL and L-CAR in patients receiving MTX.

6-shogaol protects against diabetic nephropathy and cardiomyopathy via modulation of oxidative stress/NF-κB pathway.

Diabetes dramatically increases the risk of numerous heart and kidney troubles. Diabetic nephropathy (DN) and cardiomyopathy (DC) are major causes of death. The pathophysiology of DN/DC includes inflammatory and oxidative stress mechanisms. NF-κB is one of the transcription factor that mediates signal transduction processes. Nowadays, it is suggested that inhibition of NF-κB activation could delay the development of DN and DC. 6-shogaol was reported to modulate NF-κB besides its anti-oxidant and anti-inflammatory activities. Therefore, it is worth testing it against diabetic complications. Rats were divided to 4 groups: Normal control (NC), 6-shogaol (6S), diabetic control (DC), diabetic rats treated with 6-shogaol (DC+6S). BGL, BUN, serum creatinine, total urine protein, creatine kinase (CK), LDH, NO, TNF-α NF-κB were determined in serum. Heart and kidney tissues were isolated for GSH, MDA, SOD measurement and histopathology. NF-κB was estimated in kidney tissues using immunohistopathology and western blot techniques. Results showed that diabetic rats left untreated for 16 weeks showed kidney injury as evidenced from elevated BUN, serum creatinine, urine protein, TNF-α and NF-κB. Heart tissue damage was evidence from elevated CK, LDH. Diabetic rats simultaneously treated with 6-shogaol showed a protective effect on both kidney and heart as evidenced biochemically and histopathologically. Therefore, using 6-shogaol may be of value in protection against diabetic complications in kidney and heart of rats.

Irbesartan, an angiotensin II receptor antagonist, with selective PPAR-gamma-modulating activity improves function and structure of chemotherapy-damaged ovaries in rats.

Cyclophosphamide (CYP) is a chemotherapeutic agent with a potent ovarian toxic effect. CYP induces granulosa cell apoptosis and oxidative stress. Irbesartan (IRB) is a unique ARB with a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonistic activity. As PPAR-É£ activation exerts anti-inflammatory effects and reduces ROS production, IRB may further reduce inflammatory chemokine expression and suppress apoptotic cell death. Therefore, this study aimed to evaluate the effects of IRB on the development of CYP-induced ovarian damage. Rats were divided into four groups: control group, IRB group (100 mg/kg, orally), CYP group (100 mg/kg, i.p. single injection), and IRB+CYP group (IRB administered 9 days before and 6 days after CYP administration). Rats sacrificed on day 16 of experiment; estradiol (E2), FSH, and TNF-α levels were estimated in serum. Reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and caspase-3 activities, myeloperoxidase (MPO), and IL-10 levels were determined in ovarian tissues. Protein expressions of p53, caspase-3, Ki-67, and Rad-51 were estimated by immunohistochemical and Western blot techniques. CYP produced ovarian damage as indicated from the decline in serum E2; elevation in FSH; unbalance in tissue oxidative stress parameters; increase in MPO, TNF-α levels, caspase-3 activity/expression, p53, and Rad-51 expression; and decrease in IL-10 contents, without effect on Ki-67. On the other hand, IRB, significantly reduced the toxic effects of CYP as indicted from normalization of E2, FSH, oxidative stress, apoptotic, and inflammatory mediators. These data were further supported by histopathological studies. Thus, co-administration of IRB may be promising in alleviating the ovarian toxic effects of CYP.

Renoprotective effects of montelukast, a cysteinyl leukotriene receptor antagonist, against methotrexate-induced kidney damage in rats.

Methotrexate (MTX) is a cytotoxic chemotherapeutic agent used for treatment of several cancers. Nephrotoxicity, an adverse side effect of high-dose MTX, is attributed to abnormal production of reactive oxygen species (ROS), inflammatory mediators, and neutrophil infiltration. Montelukast (MON) is a cysteinyl leukotriene receptor antagonist. Recently, it has gained a considerable interest as a ROS scavenger and inflammatory modulator. In this study, we investigated the effect of MON against MTX-induced nephrotoxicity. Rats were divided into four groups: control group, MON group (10 mg/kg, orally), MTX group (20 mg/kg, i.p., single injection), and MON + MTX group (MON was administered 5 days before and 5 days after MTX administration). At the end of the experiment, serum was collected for analysis of blood urea nitrogen (BUN) and creatinine. Glutathione (GSH), lipid peroxides (malondialdehyde), tumor necrosis factor alpha (TNF-α) levels, superoxide dismutase, myeloperoxidase activities, and nuclear factor kappa beta (NF-κB) protein expression were determined in renal tissues. In addition, kidney tissues were examined histopathologically and immunohistochemically for NF-κB. MTX administration produced acute renal damage as indicated from severe elevation in BUN and serum creatinine. The role of oxidative stress and inflammatory mechanisms in MTX-induced nephrotoxicity was evidenced from the unbalance in tissue oxidative parameters, increased TNF-α levels, and NF-κB expression in renal tissues. On the other hand, MON significantly reduced the toxic effects of MTX as indicted from normalization of kidney-specific parameters, oxidative stress, and inflammatory mediators. This data was further supported by histopathological studies. Thus, co-administration of MON may be promising in alleviating the systemic side effects of MTX.

Accelerated wound healing and anti-inflammatory effects of physically cross linked polyvinyl alcohol-chitosan hydrogel containing honey bee venom in diabetic rats.

Diabetes is one of the leading causes of impaired wound healing. The objective of this study was to develop a bee venom-loaded wound dressing with an enhanced healing and anti-inflammatory effects to be examined in diabetic rats. Different preparations of polyvinyl alcohol (PVA), chitosan (Chit) hydrogel matrix-based wound dressing containing bee venom (BV) were developed using freeze-thawing method. The mechanical properties such as gel fraction, swelling ratio, tensile strength, percentage of elongation and surface pH were determined. The pharmacological activities including wound healing and anti-inflammatory effects in addition to primary skin irritation and microbial penetration tests were evaluated. Moreover, hydroxyproline, glutathione and IL-6 levels were measured in the wound tissues of diabetic rats. The bee venom-loaded wound dressing composed of 10 % PVA, 0.6 % Chit and 4 % BV was more swellable, flexible and elastic than other formulations. Pharmacologically, the bee venom-loaded wound dressing that has the same previous composition showed accelerated healing of wounds made in diabetic rats compared to the control. Moreover, this bee venom-loaded wound dressing exhibited anti-inflammatory effect that is comparable to that of diclofenac gel, the standard anti-inflammatory drug. Simultaneously, wound tissues covered with this preparation displayed higher hydroxyproline and glutathione levels and lower IL-6 levels compared to control. Thus, the bee venom-loaded hydrogel composed of 10 % PVA, 0.6 % Chit and 4 % BV is a promising wound dressing with excellent forming and enhanced wound healing as well as anti-inflammatory activities.

Cardioprotective effects of nicorandil, a mitochondrial potassium channel opener against doxorubicin-induced cardiotoxicity in rats.

Doxorubicin is a chemotherapeutic drug used to treat solid and haematopoietic tumours. Its use is limited by a major side effect of cardiotoxicity. It was reported that doxorubicin-induced cardiotoxicity is mediated through oxidative stress coupled with impaired NO bioavailability and NF-κB activation. Nicorandil, a mitochondrial ATP-dependent potassium (KATP ) channel opener, was reported to be cardioprotective on ischaemic myocardium. However, the effect of nicorandil against doxorubicin-induced cardiotoxicity has not yet been clarified. Accordingly, six groups of rats were used. The first three groups were injected with vehicle, nicorandil (3 mg/kg) orally and doxorubicin (a single intraperitoneal injection of 20 mg/kg), respectively. Group four was treated with nicorandil, whereas group five was treated with glibenclamide and then nicorandil starting 2 days before doxorubicin and continued for five consecutive days. Group six was treated with glibenclamide alone. At the end of the experiment, the rats were killed. Cardiac enzyme indexes were measured in serum. Heart tissues were processed for determination of nitrite/nitrate, NF-κB protein expression, glutathione (GSH), lipid peroxide (TBARS) levels and superoxide production. In addition to body-weight reduction, doxorubicin produced cardiotoxicity as indicated from the increase in lactate dehydrogenase (LDH), creatine kinase (CK) activities, TBARS, superoxide production, NF-κB expression and caspase-3 activity. Moreover, doxorubicin decreased GSH and nitrite/nitrate levels. Histopathological examination of doxorubicin-treated hearts revealed degenerative changes. On the other hand, nicorandil protected cardiac tissues against doxorubicin cardiotoxicity as demonstrated from normalization of cardiac biochemical and oxidative stress parameters and amelioration of histopathological changes. Glibenclamide, a blocker of the KATP channel, reversed most of the cardiac effects of nicorandil.

Hepatoprotective effect of the selective mineralocorticoid receptor antagonist, eplerenone against carbon tetrachloride-induced liver injury in rats.

BACKGROUND: Eplerenone is a selective mineralocorticoid receptor (MR) antagonist, and its potential protective role in cardiovascular injury has been reported by several studies. However, whether and how this drug can ameliorate hepatic injury in rats is unknown. MATERIAL AND METHODS: The present study was conducted to investigate effect of eplerenone against liver injury induced by carbon tetrachloride (CCl(4)) in rats. The biochemical liver function tests and oxidative stress parameters including malondialdehyde (MDA), reactive oxygen species (ROS), in addition to the reduced glutathione (GSH) levels were evaluated. Moreover, serum tumor necrotic factors (TNF-α) level and histopathological changes were examined. RESULTS: Our results show that pre-treatment with eplerenone (4 mg/kg per day for 4 weeks) revealed attenuation in serum activities of alanine aminotransferase (ALT), aspartate aminotransferase, (AST), alkaline phosphatase (ALP) and bilirubin levels that were enhanced by CCl(4). Further, pre-treatment with eplerenone inhibited the elevated hepatic MDA content and restored hepatic GSH to its normal level. The enhanced hepatic ROS production in CCl(4)-treated group was markedly decreased by eplerenone administration. Eplerenone pre-treatment significantly attenuated the inflammatory responses caused by CCl(4) as evident by the decreased serum TNF-α level. Histopathological studies showed that eplerenone alleviated the liver damage and reduced the lesions caused by CCl(4). CONCLUSION: Collectively, the present study provides a proof to hepatoprotective actions of eplerenone via reducing oxidative stress and inflammatory responses in CCl(4)-induced liver damage in rat model.

Gastroprotective effect of rutin against indomethacin-induced ulcers in rats.

Several reports have indicated that indomethacin-induced gastropathy is mediated through generation of free radicals, neutrophil infiltration and disturbance in nitric oxide production. Rutin is a potent antioxidant flavonoid. Recently, rutin was reported to inhibit neutrophil infiltration and to modulate nitric oxide production in gastric mucosa. Therefore, the aim of this study was to investigate the protective effect of rutin against indomethacin-induced gastric injury. Accordingly, four groups of rats were used. The first three groups were injected orally with vehicle, rutin (200 mg/kg) and indomethacin (48 mg/kg) respectively. The fourth group was injected with rutin 1 hr before indomethacin. Animals were killed after 6 hr of indomethacin administration. Gastric juice acidity and gastric injury were evaluated directly. Moreover, the activities of myeloperoxidase, superoxide dismutase and the contents of reduced glutathione, thiobarbituric acid reactive substance and total nitrite/nitrate (as a marker of nitric oxide production) were determined in mucosal tissues. Indomethacin increased gastric ulcer index, gastric myeloperoxidase activity, gastric acidity and thiobarbituric acid reactive substance contents compared with control. On the other hand, indomethacin decreased glutathione, nitrite/nitrate contents and superoxide dismutase activity. Histopathological examination of the stomachs of indomethacin-treated rats revealed degenerative changes in gastric tissues. Pre-treatment with rutin protected gastric tissues against indomethacin-induced gastropathy as demonstrated from reduction in the ulcer index, attenuation of histopathological changes and amelioration of the altered oxidative stress and biochemical parameters. These results indicate that rutin has a protective effect against indomethacin-induced gastropathy probably through inhibiting neutrophil infiltration, suppression of oxidative stress generation and replenishing nitrite/nitrate levels regardless of gastric acidity.

Green tea ameliorates renal oxidative damage induced by gentamicin in rats.

Recent studies indicate that free radicals are important mediators of renal damage induced by gentamicin (GM), an aminoglycoside antibiotic widely used in treating severe gram-negative infections. Green tea extract (GTE) was reported to have antioxidant and free radical scavenging activities. Therefore, the aim of this work was to investigate the possible protective effect of GTE against gentamicin-induced nephrotoxicity. For this purpose, rats were divided into four groups. Group-1 (control) received normal saline. Group-2 received GTE (300 mg/kg/d, orally). Group-3 received gentamicin (80 mg/kg/d, intraperitoneally). Group-4 was injected with GTE plus gentamicin simultaneously. Daily urinary total protein levels were estimated to assess kidney dysfunction. The rats were sacrificed on the seventh day and kidneys were collected for histopathological studies. Blood urea nitrogen (BUN) and creatinine levels were measured in the blood. Moreover, glutathione (GSH), lipid peroxide expressed as thiobarbituric acid reactive substance (TBARS) levels, superoxide dismutase (SOD) and catalase (CAT) activities were determined in renal tissues. GM produced elevation in urinary total protein, BUN, serum creatinine and TBARS levels. On the other hand, GM reduced the GSH level and SOD, CAT activities. The simultaneous administration of GTE plus gentamicin protected kidney tissues against nephrotoxic effect of gentamicin as evidenced from amelioration of histopathological alterations and normalization of kidney biochemical parameters.

Role of ursodeoxycholic acid in prevention of hepatotoxicity caused by amoxicillin-clavulanic acid in rats.

Incidence of hepatotoxicity caused by the broad spectrum antibiotic combination amoxicillin-clavulanic acid (Co-amoxyclav) has been increasingly recognized and the mechanism of this toxicity remains undefined. On the other hand, Ursodeoxycholic acid (UDCA) has been suggested as efficient antioxidant therapy in various liver diseases. Therefore, the present study was designed to elucidate the possible role of oxidative stress in hepatotoxicity induced by Co-amoxyclav and the putative protective role of UDCA in rats. Effects of amoxicillin (Amox; 50 mg/kg, orally, 21 d) or clavulanic acid (Clav; 10 mg/kg, orally, 21 d) and their combined administration on the biochemical liver parameters, reduced glutathione (GSH), lipid peroxidation measured as hepatic malondialdehyde (MDA) levels. In addition, myeloperoxidase (MPO) activity and reactive oxygen species (ROS) production in liver homogenate were also evaluated. On the other hand, the protective effects of pretreatment with UDCA (20 mg/kg, orally, 21 d) on these parameters were also evaluated. Our results show that pretreatment with UDCA reduced the liver parameters that were enhanced by single or combined administration of Amox and/or Clav such as serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and serum bilirubin levels. Moreover, pretreatment with UDCA normalized the GSH level and inhibited the elevation in hepatic MDA concentration. The enhanced MPO activity and ROS production in liver homogenate of rats treated with Clav or Co-amoxyclav were also normalized by UDCA pretreatment. In conclusion, the present data suggest that UDCA acts as effective hepatoprotective agent against liver dysfunction caused by Co-amoxyclav and this effect is related to its antioxidant properties.

Hesperidin alleviates doxorubicin-induced cardiotoxicity in rats.

BACKGROUND: Doxorubicin (DOX) is a potent chemotherapeutic agent used in the treatment of several tumors but its cardiac toxicity prevents its use at a maximum dose, representing an important problem. Increased reactive oxygen species (ROS) and imbalance in nitric oxide (NO) production have been implicated in the cardiotoxicity of doxorubicin. Hesperidin is a citrus bioflavonoid that possesses a potent antioxidant and NO modulating activities. OBJECTIVES: Therefore, the aim of this study was to investigate the possible protective role of hesperidin against doxorubicin-induced cardiac toxicity. METHODS: Four groups of animals were used in this study. First group served as a control and injected with the vehicle. Second group was given 200 mg/kg of hesperidin orally for seven consecutive days. The third group was injected with a single dose (20 mg/kg) of doxorubicin intraperitoneally and was sacrificed after 48 h. The fourth group was treated with hesperidin for seven days but on day five, 1-hour after hesperidin treatment, rats were injected with the single dose of doxorubicin. On day seven, the rats were scarified by decapitation. Blood was collected and processed for determination of serum lactate dehydrogenase (LDH), creatine kinase (CK) and NO. The hearts were removed and processed for both histopathological examination and determination of oxidative stress parameters like reduced glutathione (GSH), lipid peroxide (TBARS) levels and superoxide dismutase (SOD) activity. RESULTS: Our results showed that doxorubicin produced severe cardiotoxicity as indicated from increase in serum LDH, CK activities and NO level. Histopathological examination of DOX-treated rats revealed degenerative changes in heart tissues. The significant decrease in GSH levels, SOD activity and increase in TBARS levels, indicated that DOX-induced cardiotoxicity was mediated through ROS generation. On the other hand, pretreatment of rats with hesperidin protected cardiac tissues against the cardiotoxic effects of doxorubicin as evidenced from amelioration of histopathological changes and normalization of cardiac biochemical parameters. CONCLUSION: Hesperidin may have a protective effect against DOX-induced cardiotoxicity.

Protein kinase C-alpha mediates TNF release process in RBL-2H3 mast cells.

1 To clarify the mechanism of mast cell TNF secretion, especially its release process after being produced, we utilized an antiallergic drug, azelastine (4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4-yl)-1-(2H)- phthalazinone), which has been reported to inhibit TNF release without affecting its production in ionomycin-stimulated RBL-2H3 cells. 2 Such inhibition was associated with the suppression of an ionomycin-induced increase in membrane-associated PKC activity rather than the suppression of Ca2+ influx, suggesting that PKC might be involved in TNF release process. 3 To see whether conventional PKC family (cPKCs) are involved, we investigated the effects of a selective cPKC inhibitor (Gö6976) and an activator (thymeleatoxin) on TNF release by adding them 1 h after cell stimulation. By this time, TNF mRNA expression had reached its maximum. Gö6976 markedly inhibited TNF release, whereas thymeleatoxin enhanced it, showing a key role of cPKC in TNF post-transcriptional process, possibly its releasing step. 4 To determine which subtype of cPKCs could be affected by azelastine, Western blotting and live imaging by confocal microscopy were conducted to detect the translocation of endogenous cPKC (alpha, betaI and betaII) and transfected GFP-tagged cPKC, respectively. Both methods clearly demonstrated that 1 microM azelastine selectively inhibits ionomycin-triggered translocation of (alpha)PKC without acting on betaI or betaIIPKC. 5 In antigen-stimulated cells, such a low concentration of azelastine did not affect either (alpha)PKC translocation or TNF release, suggesting a functional link between (alpha)PKC and the TNF-releasing step. 6 These results suggest that (alpha)PKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment of (alpha)PKC in the pathway activated by ionomycin in RBL-2H3 cells.