Effect of Chitosan-Diosgenin Combination on Wound Healing.

The difficult-to-heal wounds continue to be a problem for modern medicine. Chitosan and diosgenin possess anti-inflammatory and antioxidant effects making them relevant substances for wound treatment. That is why this work aimed to study the effect of the combined application of chitosan and diosgenin on a mouse skin wound model. For the purpose, wounds (6 mm diameter) were made on mice's backs and were treated for 9 days with one of the following: 50% ethanol (control), polyethylene glycol (PEG) in 50% ethanol, chitosan and PEG in 50% ethanol (Chs), diosgenin and PEG in 50% ethanol (Dg) and chitosan, diosgenin and PEG in 50% ethanol (ChsDg). Before the first treatment and on the 3rd, 6th and 9th days, the wounds were photographed and their area was determined. On the 9th day, animals were euthanized and wounds' tissues were excised for histological analysis. In addition, the lipid peroxidation (LPO), protein oxidation (POx) and total glutathione (tGSH) levels were measured. The results showed that ChsDg had the most pronounced overall effect on wound area reduction, followed by Chs and PEG. Moreover, the application of ChsDg maintained high levels of tGSH in wound tissues, compared to other substances. It was shown that all tested substances, except ethanol, reduced POx comparable to intact skin levels. Therefore, the combined application of chitosan and diosgenin is a very promising and effective medication for wound healing.

Snail Mucus Protective Effect on Ethanol-Induced Gastric Ulcers in Mice.

Nowadays, an increased interest in natural compounds with preventive or therapeutic potential for various diseases has been observed. Given the involvement of oxidative stress in the pathogenesis of gastric ulcer (GU) and the wide range of bioactive compounds isolated from snails, this study aimed to investigate the protective effect of Cornu aspersum (Müller, 1774) mucus on ethanol-induced GUs. Male albino mice were divided into Control, Ethanol, Mucus + Ethanol and Mucus + Omeprazole treated groups. The GUs were induced by administration of 96% ethanol (10 mL/kg, per os). One hour before ulcer induction, the mice of Mucus + Ethanol group were pretreated with mucus (20 mg/kg, per os), and the mice of Mucus + Omeprazole group were pretreated with omeprazole (20 mg/kg, per os). Ethanol administration caused grave lesions of gastric mucosa and a significant decrease of glutathione (GSH) and superoxide dismutase (SOD), catalase, and glutathione reductase (GR) activities. In the animals with mucus or omeprazole pre-administration compared to the Ethanol group, the following were observed: only a small number of hemorrhagic fields, significantly reduced GU index with calculated 73% protection by mucus and 78% protection by omeprazole, and significant recovery of mucosal GSH and SOD and GR activities. In addition, the mucus inhibited Helicobacter pylori growth. Thus, the protective effect of C. aspersum mucus on both gastric mucosa and gastric antioxidant potential in ethanol-induced GU model suggests that it may serve as a good tool for prevention of this disease.

Chromatographic Profile and Redox-Modulating Capacity of Methanol Extract from Seeds of Ginkgo biloba L. Originating from Plovdiv Region in Bulgaria.

Oxidative stress underlies the pathogenesis of many diseases, which determines the interest in natural substances with antioxidant properties. Ginkgo biloba L. leaves are well known and widely used in the pharmaceutical industry, but the therapeutic properties of the seeds are less studied. This study aimed to identify the chromatographic profile and to evaluate the antioxidant properties of methanol extract from seeds of G. biloba (GBSE). In the GBSE, flavonoids and terpenes were found as terpenes predominated. The GBSE antioxidant capacity determined by 2,2 azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and 1-diphenyl-2-picrylhydrazyl (DPPH) methods were equal to 1.34% and 0.58% of the activity of reference substance Trolox, respectively. The results of the ferric reducing antioxidant power method showed that the effect of concentration 1 mg/mL (w/v) GBSE was equal to 7.418 mM FeSO4 used as a standard. The cupric reducing antioxidant capacity activity of the GBSE was found to be 215.39 µmol Trolox/g GBSE and is presented as Trolox equivalent. The metal chelation effect of 1 mg/mL (w/v) GBSE was equal to that obtained for 0.018 mM EDTA. In conclusion, GBSE showed a good ability to neutralize ABTS and DPPH radicals and could have a beneficial effect in pathological conditions with oxidative stress etiology.

Role of Trace Elements for Oxidative Status and Quality of Human Sperm.

BACKGROUND: Oxidative stress affects sperm quality negatively. To maintain the pro/antioxidant balance, some metal ions (e.g. copper, zink, iron, selenium), which are co-factors of the antioxidant enzymes, are essential. However, iron and copper could act as prooxidants inducing oxidative damage of spermatozoa. AIMS: To reveal a possible correlation between the concentrations of some metal ions (iron, copper, zinc, and selenium) in human seminal plasma, oxidative stress, assessed by malondialdehyde and total glutathione levels, and semen quality, assessed by the parameters count, motility, and morphology. STUDY DESIGN: Descriptive study. METHODS: The semen analysis for volume, count, and motility was performed according to World Health Organization (2010) guidelines, using computer-assisted semen analysis. For the determination of spermatozoa morphology, a SpermBlue staining method was applied. Depending on their parameters, the sperm samples were categorized into normozoospermic, teratozoospermic, asthenoteratozoospermic, and oligoteratozoospermic. The seminal plasma content of iron, copper, zinc, and selenium was estimated by atomic absorption spectroscopy. The malondialdehyde and total glutathione levels were quantified spectrophotometrically. RESULTS: In the groups with poor sperm quality, the levels of Fe were higher, whereas those of Zn and Se were significantly lower than in the normozoospermic group. In all groups with poor sperm quality, increased levels of malondialdehyde and decreased glutathione levels were detected as evidence of oxidative stress occurrence. All these differences are most pronounced in the asthenoteratozoospermic group where values differ nearly twice as much compared to the normozoospermic group. The Fe concentration correlated positively with the malondialdehyde (r=0.666, p=0.018), whereas it showed a negative correlation with the level of total glutathione (r=-0.689, p=0.013). The total glutathione level correlated positively with the sperm motility (r=0.589, p=0.044). CONCLUSION: The elevated levels of Fe and the reduced Se levels are associated with sperm damage. The changes in the concentrations of the trace elements in human seminal plasma may be related to sperm quality since they are involved in the maintenance of the pro-/antioxidative balance in ejaculate.

Are nociceptin(1-13)NH2 and its structural analogue [ORN(9)]nociceptin(1-13)NH2 able to affect brain antioxidant status in control and kainic acid-treated rats?

In-vivo effects of nociceptin (N/OFQ(1-13)NH(2)) on the levels of lipid peroxidation and cell enzyme (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non-enzyme (glutathione) antioxidants in brain of control and kainic acid-treated rats were studied. N/OFQ(1-13)NH(2) effects were compared with those of its structural analogue [Orn(9)]N/OFQ(1-13)NH(2). Kainic acid (25 microg, i.c.v) increased the lipid peroxidation (4 and 24 h after kainic acid treatment) and decreased the glutathione level (1 h after kainic acid injection). We failed to find, any changes in antioxidant enzyme activities, independently of the time of kainic acid treatment. At the background of kainic acid-effects, N/OFQ(1-13)NH(2) and [Orn(9)] N/OFQ(1-13)NH(2), injected 30 min before kainic acid, had no effects on all parameters, tested in brain. In addition, the neuropeptides did not change the antioxidant status in brain of control animals. It might be concluded that N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2) have neither pro- nor anti-oxidant activity.

Effect of copper intoxication on rat liver proteasome activity: relationship with oxidative stress.

Copper toxicity is associated with formation of reactive oxygen species, which are capable to oxidize proteins. The selective removal of the latter by the 20S proteasome is considered an essential part of the cell antioxidant defense system. The aim of the present study was to investigate whether peptidase activities of rat liver proteasomes were affected by chronic (40 mg CuSO(4)/rat/daily with the drinking water for 2 weeks) and acute (20 mg/kg CuSO(4), s.c.) copper treatment. To evaluate the role of proteasome, its inhibitor MG132 was also used. The degree of copper-induced oxidative stress (OS), established by measuring lipid peroxidation, protein oxidation, and cellular glutathione level, as well as activities of antioxidant enzymes--catalase, superoxide dismutase, and gultathionine peroxidase, depended on the mode of copper administration. Chronic copper administration (mild oxidative stress) did not affect proteasome activities, whereas acute copper treatment (severe oxidative stress) caused a decline in chymotryptic- and tryptic-like activities. The treatment of copper-loaded animals with MG132 did not change copper-induced alterations in the tested indices, except an additional increase in protein oxidation and inhibition of glutathionine peroxidase activity. The results suggested that the in vivo copper-induced oxidative stress was associated with changes in the catalytic activity of proteasome.

Effects of proteasome inhibitor, MG132, on proteasome activity and oxidative status of rat liver.

In vivo effects of N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal (MG132) on chymotryptic-like (ChT-L), tryptic-like, and post-glutamyl peptide hydrolytic-like proteasome activities, protein oxidation, lipid peroxidation (LP), glutathione (GSH) level, as well as on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione-reductase) in the rat liver were studied. The possibility of MG132 provoking the formation of free oxygen radicals was also assayed in primary hepatocytes. The following results were obtained: (1) In vivo, MG132 did not change the spontaneous LP, but increased Fe-induced LP and the amount of oxidized proteins; it decreased the GSH level in liver. From the proteasome activities studied in liver cytosol only ChT-L activity was significantly decreased after MG132 administration. Furthermore, MG132 increased antioxidant enzyme activities of SOD, CAT, and GSH-Px. (2) In vitro, MG132 increased free radical oxygen species in hepatocytes; this effect disappeared in the presence of CAT or mannitol. In conclusion, since nowadays proteasome inhibitors are entering into the swing of laboratory and clinical practice, the present data could provide useful information for MG132 action. Consequently, future in vivo experiments with MG132 could highlight the possibility of its use at different pathological conditions.

Effect of MG132 on proteasome activity and prooxidant/antioxidant status of rat liver subjected to ischemia/reperfusion injury.

AIM: Previous studies have shown that proteasome inhibitors exerted protective effects against ischemia/reperfusion injury (IRI) of brain, heart, kidney and intestine. The aim of the present study was to investigate: (i) whether the proteasome inhibitor MG132 protects rat liver against IRI; and (ii) whether MG132 modulates prooxidant/antioxidant status of rat liver subjected to warm IRI. METHODS: The left lateral and medial lobes (approximately 70% of the total liver volume) of livers of male Wistar rats were subjected to 30-min ischemia followed by 60-min reperfusion. Lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured in the plasma. Proteasome chymotryptic-like (ChT-L) activity, levels of thiobarbituric acid-reactive substances (TBARS), protein carbonyls (PC) and glutathione (GSH), as well as superoxidase dismutase (SOD), catalase (CAT), glutathionine peroxidase and glutathionine reductase activities were measured in liver fractions. RESULTS: Thirty-min ischemia followed by 60-min reperfusion increased liver TBARS and PC, CAT and SOD activities, but decreased GSH level. Ischemia/reperfusion-induced oxidative stress was exacerbated in mitochondria, indicating that these organelles are the preferential target of IRI. Plasma LDH and AST levels were decreased by MG132 during both ischemia and reperfusion, while ALT values were decreased only after 30 min of reperfusion. MG132 did not significantly affect liver TBARS and GSH levels, but it increased PC and decreased ChT-L activity; the activities of CAT and SOD were also decreased. CONCLUSIONS: MG132 exerts a protective effect during the early phase of reperfusion and it modulates prooxidant/antioxidant status of rat liver subjected to warm IRI.

In vivo effects of CB1 receptor ligands on lipid peroxidation and antioxidant defense systems in the rat brain of healthy and ethanol-treated rats.

In vivo experiments were conducted to study the effects of N-(piperidin-l-yl)-5-(4-chlorophenyl)-1-(2,4-cochlo-rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; a potent and selective CB(1)-receptor antagonist) and ara-chidonyl-2-chloroethylamide (ACEA; a selective CB(1)-receptor agonist) on spontaneous lipid peroxidation, glutathione (GSH) level and activities of antioxidant enzymes in rat tissues. Single doses of SR141716A(3 mg/kg, ip) and ACEA(10 mg/kg, ip) had no effect on all indices, studied in the brain, except for a decrease in GSH level by 10 mg/kg of SR141716A. The effects of repeated administration of the CB(1)-receptor ligands (3 mg/kg, ip, once daily for 2 days) on the above indices in the brain and liver of control and ethanol-treated animals were also studied. Two weeks after ethanol exposure, the rats lost weight (by 41%), which correlated with their decreased water and food consumption (by 52% and 33%, respectively). The time of ethanol action was not sufficient to change the biochemical parameters in the brain, except for the lipid peroxidation. However, a decrease in GSH level and superoxide dismutase activity, as well as an increase in lipid peroxidation and glucose-6-phosphate dehydrogenase activity were registered in the liver. The repeated administration of CB(1) receptor ligands restored some of ethanol-induced changes. The present results suggested lack of pro-oxidant activity and potential antioxidant ability of the studied CB(1) receptor ligands, which might contribute to their beneficial effects.

A peptide mimic of selectin ligands abolishes in vivo inflammation but has no effect on the rat heart allograft survival1.

Acute heart allograft rejection is characterized by leukocyte infiltration and myocyte damage, key elements in the histological grading of rejection. The induction of selectins and their ligands on the graft postcapillary venular endothelium increases leukocyte tethering to, rolling on, and extravasation through the endothelium into graft parenchyma. We have characterized peptide mimicking selectin ligands by screening phage peptide libraries using anti-Lewis A antibodies and E-selectin as target molecules. The effect of this selectin- binding peptide, IELLQAR, on the prevention of inflammation and tissue damage and on the prolongation of graft survival in inbred DA (RT1a) rat heart allografts transplanted to WF (RT1v) recipients was tested. Bovine serum albumin (0.1%, solvent), VTSIAQA (control peptide), or IELLQAR were either continuously infused into the peritoneum via osmotic mini pumps or injected twice daily IV. Treatment with bovine serum albumin and VTSIAQA did not alter the number of graft infiltrating leukocytes or the histological grade of acute rejection, all scored as grade 4. On the contrary, the selectin binding peptide, IELLQAR, dose-dependently reduced inflammation and at the highest dose (6.0 mg/kg per day) eliminated the majority of graft infiltrating leukocytes, reduced the histological grade from 4 to 1B, but had no effect on graft survival. These data indicate that the intensity of inflammation related to the allograft rejection does not correlate to the graft survival.

Elimination of vascular fibrointimal hyperplasia by somatostatin receptor 1,4-selective agonist.

The somatostatin analogs octreotide and lanreotide, selective to receptor subtypes 2 and 5, failed clinical efficacy for the prevention of restenosis after percutaneous transluminal angioplasty. These findings might have been the result of targeting a wrong subset of receptors. In rat arteries, subtypes 1 and 4 are expressed 3-4 times more prominently than 2 and 5, and subtype 1 is the nearly exclusive subtype in atherosclerotic human vessels. Here, we demonstrate that daily s.c. injections (50-500 microg/kg/d) of CH275 (DesAA1,2,5(D-W8,IAmp9)Somatostatine-14), selective to subtypes 1 and 4, dose-dependently inhibited intimal hyperplasia 14 days after rat carotid denudation injury (for intimal area P=0.0002 across the dose range). CH275 was more effective than somatostatin-14 (equal affinity to all five subtypes, P=0.03), or octreotide (selective to subtypes 2 and 5, P=0.098). When rats were given the peptides for 14 days with end-point at 28 days, CH275 still significantly inhibited intimal area expansion. Both CH275 and octreotide inhibited the outgrowth of cells from postinjury aortic tissue punch-explants and the distance migrated in vitro, but not cell replication, which indicated that the effects of somatostatin analogs were directed on the migration of intimal cell progenitors rather than on their proliferation.