Ganoderma atrum polysaccharide attenuates oxidative stress induced by d-galactose in mouse brain

AimsGanoderma atrum polysaccharide (PSG-1), the main constituent of G. atrum, has been reported to attenuate oxidative stress in vitro. The aim of this study was to investigate whether PSG-1 has a protective effect on the brain against oxidative stress induced by d-galactose (D-gal) in vivo.Main methodsMice were intraperitoneally (i.p.) injected with D-gal (100 mg/kg body weight) once daily for 10 weeks. From the seventh week, D-gal-treated mice received PSG-1 (50, 100, or 150 mg/kg body weight) once daily for the last 4 weeks. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GSH-Rd), and the contents of glutathione (GSH), glutathione disulfide (GSSG) and malondialdehyde (MDA) in the brain were measured using different biochemical methods to evaluate the changes of the antioxidant ability in the PSG-1 treated mice. Apoptosis, reactive oxygen species (ROS) and calcium levels were determined by flow cytometry.Key findingsAdministration of PSG-1 significantly reduced apoptosis in the mouse brain in a dose-dependent manner. PSG-1-evoked reduction of apoptosis was associated with the decrease of MDA and GSSG contents, and the increase of SOD, CAT, GPx and GSH-Rd activities, and GSH contents. PSG-1 treatment was also found to attenuate ROS production and calcium accumulation.SignificancePSG-1 has a potential to be used as a novel therapeutic agent for the protection of aging brain tissue against oxidative damage by modifying the redox system and maintaining calcium homeostasis.     

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.Main methodsMice were divided into sham, I/R, Rb1 + I/R (Rb1 postconditioning, 20 mg/kg, i.p. after ischemia), sham + L-NAME, I/R + L-NAME, and Rb1 + I/R + L-NAME groups using 60 min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p < 0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p < 0.05), while it dramatically reduced the concentration of MDA (p < 0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p < 0.05).SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.     

Prevention of diabetic nephropathy in rats through enhanced renal antioxidative capacity by inhibition of the proteasome

AimsOxidative stress may play an important role in the pathogenesis of diabetic nephropathy (DN). Recent studies have shown that the ubiquitin–proteasome pathway (UPP) and oxidative stress have interaction. We aimed to investigate whether inhibiting the proteasome has a preventive effect on DN through suppression of renal oxidative stress.Main methodsMale Sprague–Dawley rats were randomly divided into three groups: a normal control (NC) group, a streptozotocin-induced DN model group, and a DN plus MG132 (10 μg/kg) treatment group.Key findingsIncreased 24-h urinary protein excretion rate (UPER) and renal pathological changes were all improved after MG132 administration. Furthermore, enhanced renal 26S proteasome activity and concentration in DN rats were effectively reduced after MG132 administration. Increased p47phox and nitrotyrosine (NT) expressions in kidneys of DN rats were decreased after MG132 treatment. Renal mRNA and protein expressions of NF-E2 related factor 2 (Nrf2) were up-regulated by MG132 in comparison to DN alone. Decreased renal mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) in DN rats was heightened after MG132 intervention. Depressed activities of renal SOD, CAT and GPx in DN rats were also improved by MG132 treatment. Increased renal nuclear factor κB (NF-κB) activity was inhibited after MG132 administration in DN rats at the end of 12 weeks.SignificanceOur present data suggest that inhibition of the proteasome by low-dose MG132 has a preventive effect on DN development and progression in rats through the up-regulation of antioxidant genes.     

Exogenous intravascular nitric oxide enhances ventricular function after hemodilution with plasma expander

AimsThis study evaluated the hypothesis that exogenous nitric oxide (NO) supplementation during acute hemodilution with plasma expander (PE) provides beneficial effects on cardiac function.Main methodsAcute hemodilution in golden Syrian hamsters was induced by a 40% of blood volume exchange with dextran 70 kDa. Intravascular NO supplementation after hemodilution was accomplished with a NO donor, diethylenetriamine NONOate (DETA NONOate). The test group was treated with DETA NONOate, while the control group received only vehicle. Left ventricular cardiac function was studied using pressure–volume measurements obtained with a miniaturized conductance catheter.Key findingsCardiac output increased to 122 ± 5% and 107 ± 1% of the baseline in the group treated with NO donor and the vehicle group, respectively. Stroke work per stroke volume (SW/SV) after hemodilution reduced to 90% of the baseline and the NO donor significantly reduced SW/SV compared to the vehicle. The minimum rate of pressure change (dP/dt_min) was significantly lower in animals treated with the NO donor compared to vehicle treated animals. Systemic vascular resistance (SVR) decreased to 62 ± 5% of the baseline in the NO donor group whereas the vehicle group SVR decreased to 83 ± 5% of the baseline. Using intravital microscopy analysis of microvessel in the dorsal skinfold window chamber, we established that the NO donor group induced significant vasodilation compared to the vehicle group.SignificanceNO supplementation in an acute hemodilution with PE has beneficial effects on cardiac performance. However, the NO supplementation effects with a NO donor are dose-independent and short-lasting.     

Effect of green tea extract (catechins) in reducing oxidative stress seen in patients of pulmonary tuberculosis on DOTS Cat I regimen

Background and AimThe role played by free radicals in pathogenesis of pulmonary tuberculosis and treatment mediated toxicity is well established. Hence, the present study was undertaken to assess the effect of crude green tea catechin in reducing the oxidative stress seen in patients of AFB positive pulmonary tuberculosis.MethodsA total of 200 newly diagnosed cases of AFB positive pulmonary tuberculosis, who received CAT I regimen were enrolled consecutively from DOTS center. Out of 200 patients, 100 randomly selected patients received catechin (500 μg) with antitubercular treatment (ATT) (cases) and 100 received starch (500 μg) with ATT (control). Oxidative stress level in blood samples of cases and controls as compared at the time of enrollment and after one and four months of treatment. Oxidative stress was measured in terms of free radicals (lipid peroxidation, nitric oxide), enzymatic antioxidant (catalase, superoxide dismutase, glutathione peroxidase) and non enzymatic antioxidant (total thiol, reduced glutathione) levels.ResultsThe results showed significant difference in all the parameters among cases and controls. A significant decrease (p≤0.001) in LPO level was observed in cases as compare to controls during the follow up while the level of NO was significantly increased (p≤0.001) in cases as compare to controls. Significant decrease (p≤0.001) in catalase and GPx level was observed in cases as compare to controls while SOD levels significantly rose (p≤0.001) in cases as compared to controls. Significant decrease (p≤0.001) in SH level was observed in cases as compared to controls while the level of GSH was significantly increased (p≤0.001) .ConclusionThese findings suggest that crude catechin extract can play a definite role as adjuvant therapy in management of oxidative stress seen in pulmonary tuberculosis patients. More detailed studies are needed to document use of catechin in reducing the frequency and severity of side effects of treatment.     

Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats

AimsThe present study aims to elucidate the role of oxidative stress in erectile dysfunction associated with aging and to investigate the effect of treatment with vitamin E in this respect.Main methodsRats were divided into four groups: young (3-month-old), aged rats (18-month-old), aged rats given 80 IU of vitamin E/rat/day for 21-days, aged rats given 5 mg/kg of sildenafil/day for 21-days. Intracavernosal pressure/mean arterial pressure (ICP/MAP), nitric oxide production, TBARS, GSH levels and SOD activity in corpus cavernosum were measured.Key findingsSignificant decrease in ICP/MAP was observed in aged rats at both low and high frequency of stimulation. Significant increase in ICP/MAP was observed in aged rats treated with vitamin E over the range of 0.8 to 5 Hz but young control values were not restored. Percentage potentiation of ICP/MAP than aged group at 0.8 Hz was 326 ± 41.3% and 897 ± 72.2% for vitamin E and sildenafil respectively. Decreased levels of NO₂/NO₃ and SOD activity in the penile tissue observed with aging were elevated back to control by either vitamin E or sildenafil. Penile concentration of TBARS was 20.86 ± 0.83 for aged rats vs. 11.39 ± 0.79 nmol/g tissue for young controls. Both vitamin E and sildenafil reduced penile TBARS in aged rats.SignificanceThis study proves that antioxidant therapy with vitamin E ameliorates the age-associated erectile dysfunction. Sildenafil may exert some antioxidant properties which add to the advantages of its long-term use. The effect of combinations of low-dose sildenafil and vitamin E on age-associated erectile dysfunction merits to be studied.     

Copper/zinc and copper/selenium ratios,and oxidative stress as biochemical markers in recurrent aphthous stomatitis

ProjectRecurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent, painful oral aphthae, and oxidative stress presumably contributes to its pathogenesis. The aim of this study is to scrutinize the relationship between oxidative stress and serum trace elements (copper, Cu; zinc, Zn; selenium, Se), and to evaluate the ratios of Cu/Zn and Cu/Se in this disorder.ProcedurePatients with RAS (n = 33) and age- and sex-matched healthy control subjects (n = 30) were enrolled in this study. Malondialdehyde (MDA) concentrations in plasma and the activities of superoxide dismutase (SOD1; CuZnSOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocyte were determined as spectrophotometric. Also, the levels of Se, Zn and Cu in serum were determined on flame and furnace atomic absorption spectrophotometer using Zeeman background correction.Results and conclusionsOxidative stress was confirmed by the significant elevation in plasma MDA, and by the significant decrease in CAT, SOD1, and GPx (p < 0.05). When compared to controls, Zn and Se levels were significantly lower in patients, whereas Cu levels was higher in RAS patients than those in controls (p < 0.05). In addition, the correlation results of this study were firstly shown that there were significant and positive correlations between Se–CAT, Se–GPx, and Cu–MDA parameters, but negative correlations between Se–Cu, Se–MDA, Cu–CAT, Cu–SOD1 and Cu–GPx parameters in RAS patients. Furthermore, the ratios of Cu/Zn and Cu/Se were significantly higher in the patients than the control subjects (p < 0.05). Our results indicated that lipid peroxidation associated with the imbalance of the trace elements seems to play a crucial role in the pathogenesis of RAS. Furthermore, the serum Cu/Zn and Cu/Se ratios may be used as biochemical markers in these patients.     

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

AimTo investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta.MethodsThe relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10^?6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10^?5 M), NG-nitro-l-arginine methyl ester (L-NAME, 10^?4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10^?7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10^?5 M) and methyl-β-cyclodextrin (10^?2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10^?5 M).ResultsThe extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca²⁺/calmodulin and Na⁺/Ca²⁺ exchanger (NCX), and partially blunted by the caveolae disassembly.ConclusionsThese results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca²⁺ concentration and activating the Ca²⁺/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.     

The characteristics of therapeutic effect of pinocembrin in transient global brain ischemia/reperfusion rats

AimsThe therapeutic effect of pinocembrin, together with the therapeutic time window, in the transient global cerebral ischemia/reperfusion (I/R) rats was investigated.Main methodsAdult male Sprague–Dawley rats were subjected to global cerebral ischemia for 20 min by four-vessel occlusion. Pinocembrin (1 and 5 mg/kg) was administrated intravenously 30 min before ischemia and 30 min, 2 h, 6 h after reperfusion, respectively. Neurological scores, brain edema and histological examination by Nissl staining were employed to assess the neuronal injury after ischemia and the neuroprotection by pinocembrin. The activities of superoxide dismutase (SOD), myeloperoxidase (MPO) and the content of malondialdehyde (MDA) in brain tissue were tested by colorimetric assays. Alterations of neurotransmitters were determined by a high performance liquid chromatography–electrochemical method.Key findingsPinocembrin significantly ameliorated neurological deficits and brain edema, and alleviated the degree of hippocampal neuronal loss at 24 h after global cerebral I/R with a broad therapeutic time window. It was found that treatment with pinocembrin reduced the compensatory increase of SOD activity and decreased the MDA level and MPO activity in a dose-dependent manner. The metabolic balance between excitatory and inhibitory amino acids was modulated by pinocembrin treatment.SignificanceThese findings suggest that pinocembrin provides neuroprotection against global cerebral ischemic injury with a wide therapeutic time window, which may be attributed to its antioxidative, antiinflammatory and antiexcitotoxic effects.     

Effect of 593C > T GPx1 SNP alone and in synergy with 47C > T SOD2 SNP on the outcome of critically ill patients

During critical illness and sepsis there is severe antioxidant depletion, and this scenario raises the critical ill patient’s mortality risk. Glutathione peroxidase (GPx) is one of the first endogenous antioxidant defense enzymes, and it works cooperatively with superoxide dismutase (SOD) and catalase (CAT) to detoxify free radicals from the cellular environment. Genetic studies are important to understand the complexity of human oxidative stress and how the organism responds to an extreme situation such as critically care conditions. Previous studies with a GPx1 single nucleotide polymorphism (593C > T SNP; rs1050450; protein variant in GPx1: Pro198Leu) showed 593T carriers and 593TT homozygotes present higher risk to develop different diseases. We assessed the relationship of the genotype distribution of GPx1 SNP in critically ill patients with their conditions (organ dysfunction, sepsis, and septic shock) and their outcome. We monitored 626 critically ill patients daily from the ICU (intensive care unit) admission to their discharge from hospital, or death. Our study revealed a significant association between 593TT GPx1 genotype and mortality; the mortality rate was higher in homozygous 593TT GPx1 (N = 94) when compared with the group of subjects with genotypes 593CT or 593CC GPx1 (N = 532) (52% vs. 38%, P = 0.009; OR = 1.79; 95% CI = 1.13–2.85). Evaluating the subgroup of 293 ICU patients with sepsis, a pooled analysis including two genetic variants GPx1 and SOD2 (47C > T SNP, rs4880; protein variant in MnSOD: Ala–9Val) showed a significant difference in relation to progression to septic shock. The frequency of septic shock among septic patients with 593T GPx1 and 47C SOD2 alleles (N = 122) was higher when compared with septic patients carrying other settings of genotypes (N = 174) (78% vs. 66%; P = 0.028; OR = 1.81; 95% CI = 1.03–3.18). Accepting the previously reported functional effects of these two SNPs on GPx1 and SOD2 gene expressions and, consequently, on GPx1 and MnSOD enzyme activities, we believe our results may be considered as an important contribution for the understanding of oxidative imbalance during the critical ill.     

Is preconditioning by oxytocin administration mediated by iNOS and/or mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart?

AimsOxytocin (OXT) pretreatment protects the heart during ischemia–reperfusion injury by activating ATP-dependent potassium (K_ATP) channels. The aim of the current study was to elucidate the roles of nitric oxide synthaseNOS and myocardial biochemistry in the cardioprotective effects of OXT and ischemic preconditioning (IPC).Main methodsMale New Zealand White anesthetized rabbits (13 groups) were subjected to 30 min of occlusion of the left coronary artery and 120 min of reperfusion with or without IPC.Key findingsIPC (1 cycle), OXT (0.03 μg/kg, i.p.) or IPC + OXT yield significant infarct size reductions (21.8 ± 1.5%, 20.5 ± 1.2% and 19.4 ± 1.4%, respectively, versus 38.9 ± 3.5% in the S-CONT group; P < 0.01) and antiarrhythmic effects, including VF (0%, 0% and 0%, versus 50% in S-CONT group; P < 0.05) sustained VT (13%, 13% and 13%, versus 100% in S-CONT group; P < 0.005) and other arrhythmias (25%, 13% and 25%, versus 100% in S-CONT group; P < 0.005, P < 0.01 and P < 0.005, respectively). Atosiban (ATO, a selective OXT receptor antagonist), 5-HD and l-NAME (a nonspecific NOS inhibitor) abolished the beneficial effects of IPC and OXT, suggesting that the benefits are achieved via selective activation of OXT receptors, mitochondrial K_ATP channels and NO. An iNOS inhibitor (1400 W) blocked the beneficial effects of IPC but not OXT. The IPC, OXT, IPC + OXT and 1400 W + OXT interventions significantly preserved ATP levels in the heart.SignificanceThis study demonstrates similarities between acute OXT pretreatment and IPC in terms of infarct size reduction, antiarrhythmic activity, and metabolic status.     

Baicalin upregulates the genetic expression of antioxidant enzymes in Type-2 diabetic Goto-Kakizaki rats

AimThe primary purpose of this study was to characterize and investigate the antioxidant and anti-diabetic activities of the flavonoid baicalin in type 2 diabetic Goto-Kakizaki rats.Main methodsFour groups of Goto-Kakizaki rats (n = 6) were subjected to the following oral treatments for 30 days: (1) metformin — 500 mg/kg (2) baicalin — 120 mg/kg (3) metformin 500 mg/kg and baicalin — 120 mg/kg (4) vehicle treated diabetic controls receiving distilled water. The plasma glucose, triglyceride, total cholesterol, lipid peroxide and protein carbonyl contents were measured on a weekly basis. Following the completion of the treatment, the rats were sacrificed and their blood, heart, pancreatic and hepatic tissues were collected for analysis. The antioxidant enzyme activities as well as their expression were quantified using Western Blot, microarray and RT-PCR.Key findingsThe respective analyses showed that the baicalin- and the metformin and baicalin-treated groups had statistically significant increases (p < 0.05) in the activity and expression of the antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) compared with vehicle- and metformin-treated groups. Further complementing the antioxidant enzyme activity increases, the oxidative stress markers of plasma lipid peroxide and protein carbonyl contents were reduced in these groups as well. These treatment groups also had reduced plasma total cholesterol and triglyceride levels compared with vehicle-treated and metformin-treated groups (p < 0.05).SignificanceBaicalin was an efficient antioxidant in reducing hyperglycemia-induced oxidative stress through the increased expression of antioxidant enzyme activities. It was also an efficient anti-hypertriglyceridemic as well as anti-hypercholesterolemic agent compared with metformin.     

Counteraction of early circulatory derangement by administration of low dose steroid treatment at the onset of established endotoxemic shock is not directly mediated by TNF-α and IL-6

BackgroundOnce a septic condition is progressing, administration of steroids in the pro-inflammatory phase of septic shock ought to yield maximal effect on the subsequent, devastating inflammatory response. Recently, a retrospective study showed that early initiation of corticosteroid therapy improved survival in septic shock. We aimed to prospectively evaluate effects of early administrated hydrocortisone therapy on physiologic variables in a porcine model of septic shock.ExperimentEight anesthetized pigs were given a continuous infusion of endotoxin during this 6 h prospective, randomized, parallel-grouped placebo-controlled experimental study. At the onset of endotoxemic shock, defined as the moment when the mean pulmonary arterial pressure reached the double baseline value, the pigs were either given a single intravenous dose of hydrocortisone (5 mg kg^?1) or the corresponding volume of saline.ResultsMean arterial pressure and systemic vascular resistance index were significantly higher (both p < 0.05), and heart rate was significantly lower (p < 0.05), in the endotoxin + hydrocortisone group as compared to the endotoxin + saline group. Body temperature and blood hemoglobin levels increased significantly in the endotoxin + saline group (both p < 0.05). Urinary hydrocortisone increased significantly in both groups (p < 0.05). There were no significant differences in the plasma levels of TNF-alpha, IL-6 or nitrite/nitrate between the groups.ConclusionEarly treatment with hydrocortisone ameliorates some endotoxin mediated circulatory derangements, fever response and microvascular outflow. Our results suggest that these effects are not directly mediated by the pro-inflammatory cytokines TNF-alpha or IL-6, nor by NO.     

Involvement of NO-guanylyl cyclase pathway for the depression of spinal monosynaptic reflex by Mesobuthus tamulus venom in neonatal rat in vitro

AimsThe present study was undertaken to evaluate the role of nitric oxide (NO) in Mesobuthus tamulus (MBT) venom-induced depression of spinal reflexes.Main methodsExperiments were performed on isolated hemisected spinal cords from 4 to 6 day old rats. Stimulation of a dorsal root with supramaximal strength evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root.Key findingsSuperfusion of MBT venom (0.3 μg/ml) depressed the spinal reflexes in a time-dependent manner and the maximum depression was seen at 10 min (MSR by 63%; PSR by 79%). The time to produce 50% depression (T-50) of MSR and PSR was 7.7 ± 1.3 and 5.7 ± 0.5 min, respectively. Pretreatment with bicuculline (1 μM; GABA_A receptor antagonist) or strychnine (1 μM; glycine_A receptor antagonist) did not block the venom-induced depression of spinal reflexes. However, Nω-nitro-L-arginine methyl ester (L-NAME, 100 or 300 μM; NO synthase inhibitor) or hemoglobin (Hb, 100 μM; NO scavenger) antagonized the venom-induced depression of MSR. Further, soluble guanylyl cylase inhibitors (1 H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ; 1 μM or methylene blue, 100 μM) also antagonized the venom-induced depression of MSR but not PSR. Nitrite concentration (indicator of NO activity) of the cords exposed to venom (0.3 μg/ml) was not different from the control group.SignificanceThe results indicate that venom-induced depression of MSR is mediated via NO-guanylyl cyclase pathway without involving GABAergic or glycinergic system.     

Combined effect of bone marrow derived mesenchymal stem cells and nitric oxide inducer on injured gastric mucosa in a rat model

AimTo study the effect of intravenous injection of bone marrow mesenchymal stem cells (BMMSCs), alone and combined with NO inducer in gastric ulcer healing in a rat model.MethodsRats were divided into controls, gastric ulcer, gastric ulcer receiving mesenchymal stem cells (MSCs), gastric ulcer receiving NO inducer (l-Arginine), gastric ulcer receiving MSCs plus NO inducer (l-Arginine) groups. MSCs were given in a dose of (10⁶cells) by intravenous injection. l-Arginine was given 300 mg/kg body weight intraperitoneally. 24 h and 7 days after BMMSCs and NO inducer injection, VEGF, PGE, TNF-α were assessed by ELISA. Gene expression of HGF, caspase-3, eNOS and BAX/Bcl-2 in gastric tissues were studied by real time PCR. Histopathology staining of gastric tissues was performed.ResultsInjection of MSCs or NO inducer or both to the gastric ulcer group significantly decreased caspase-3 and BAX genes expression (apoptotic factors) and increased Bcl-2 gene expression (anti-apoptotic factor) compared to that of the gastric ulcer group after both 24 h and 7 days with more significant results in the gastric group received both MSCs and NO inducer. HGF gene expression was significantly increased in the groups injected with MSCs or NO inducer or both compared with the corresponding gastric ulcer group (p < 0.05, p < 0.05 & p < 0.001 respectively). There was a significant decrease in the mean PGE2 and TNF-α levels in the gastric ulcer group receiving MSCs, the gastric ulcer group receiving NO and the gastric ulcer group receiving both MSCs and  NO compared to the gastric ulcer group after both 24 h and 7 days. Histopathological examination of gastric tissue of groups that received stem cells or NO alone, showed mucosal regenerative changes with increased thickness together with reduced inflammatory cellular infiltrate in the submucosa and decreased congestion. There was complete restoration in gastric mucosa in the group that received both stem cells and NO.ConclusionAdministration of MSCs, NO, or MSCs plus NO may exert a therapeutic effect on the mucosal lesion in gastric ulcer through their anti-inflammatory, angiogenic and antiapoptotic actions.     

A targeted high-efficiency angiogenesis strategy as therapy for myocardial infarction

AimsThe aim of this study was to prove that an intramyocardial injection of a mixture of low-dose human growth factor (HGF) plasmid and microbubbles (MB) in combination with insonation was an effective therapy for myocardial infarction.Main methodsTwenty dogs with myocardial infarction were divided into 4 groups: (1) HGF, MB and ultrasound (HGF-US/MB), (2) HGF and US (HGF-US), (3) HGF alone and (4) surgery alone (control). In the HGF-US/MB group, HGF plasmid DNA (500 μg) mixed with 0.5 ml of MB solution was injected 5 min after coronary occlusion followed by insonation. With the exception of the control group, the other dogs were divided into two groups, one treated with the HGF gene and insonation and the other with the HGF gene only.Key findingsCompared to the HGF group, infarct size decreased from 32% ± 7% (control) to 23% ± 5% in the HGF-US/MB group 28 d later (P < 0.05). Capillary density increased from 21.7 ± 4.2/mm² (control) to 114.3 ± 28.9/mm² in the HGF-US/MB group (P < 0.01). Compared to the HGF group, there was a 14% decrease in the ratio of left ventricle weight/body weight and a 25% decrease in hydroxyproline content. We also observed a 29% and 20% decrease in collagen volume fraction of type I and type III collagen, respectively in the HGF-US/MB group.SignificanceIntramyocardial injection of HGF and MB in combination with insonation enhances neovascularization and reduces ventricular remodeling and infarct size.     

Beta blocker metoprolol protects against contractile dysfunction in rats after coronary microembolization by regulating expression of myocardial inflammatory cytokines

AimsTo examine the effects of metoprolol on expression of myocardial inflammatory cytokines and myocardial function in rats following coronary microembolization (CME).Main methodsMale rats were randomly assigned to receive either sham-operation (control group), CME plus saline (CME group), or CME plus metoprolol (metoprolol group). CME was induced by injecting 3000 polyethylene microspheres (42 μm) into the left ventricle during a 10-second occlusion of the ascending aorta. Metoprolol (2.5 mg/kg) or saline was administered as three intravenous bolus injections after CME. At 3 h, 6 h, 12 h, 24 h and 4 weeks after CME, myocardial function was measured with echocardiography; and the mRNA and protein levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and interleukin 1-β (IL-1β) were determined.Key findingsInduced CME led to markedly higher mRNA and protein levels of TNF-α, IL-1β and IL-10 at 3, 6, 12, and 24 h, as well as reduced left ventricular function, compared to the control group. Metoprolol administration reduced TNF-α and IL-1β levels, but increased IL-10 levels at 3, 6, 12, and 24 h compared to the CME group. Moreover, metoprolol treatment resulted in significantly improved left ventricular function at 12 h, 24 h and 4 weeks, but afforded no cardiac protection at 3 h and 12 h, compared to the CME group.SignificanceHigher levels of TNF-α and IL-1β in rats following CME are associated with the development of myocardial contractile dysfunction. Metoprolol-conferred protection against progressive contractile dysfunction following CME may be mediated by its anti-inflammation potential.     

Estrogen dependence of the renal vasodilatory effect of nicotine in rats: role of α7 nicotinic cholinergic receptor/eNOS signaling

AimsWe recently reported that acute exposure to nicotine vasodilates the renal vasculature of male rats via facilitation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether this effect of nicotine is sexually dimorphic and the role of estrogen in modulating the nicotine effect.Main methodsNicotine-evoked vasodilation was evaluated in phenylephrine-preconstricted perfused kidneys obtained from male, proestrus female, ovariectomized (OVX) and estrogen-replaced OVX (OVXE₂) rats.Key findingsNicotine infusion (5 × 10^? 5, 1 × 10^? 4, and 5 × 10^? 4 M) produced greater concentration-dependent reductions in the renal perfusion pressure (RPP) in an isolated kidney from proestrus females than from males. Inhibition of NOS by N^G-nitro-l-arginine abolished the nicotine-evoked reduction in RPP and abolished the gender difference in the nicotine effect. Nicotine vasodilation was also attenuated in kidneys isolated from OVX and diestrus rats, models characterized by reduced estrogen levels. Further, estrogen or l-arginine supplementation in OVX rats largely restored the renal vasodilatory response to nicotine. Estrogen receptor blockade by tamoxifen abrogated the enhanced nicotine-evoked vasodilation elicited by E₂ in OVX rats. The nitrite/nitrate levels and protein expressions of eNOS and α₇ nicotinic cholinergic receptor (α₇ nAChRs) were significantly higher in renal tissues of OVXE₂ compared with OVX rats, suggesting a facilitatory effect for E₂ on α₇ nAChRs/eNOS signaling.SignificanceEstrogen-dependent facilitation of NOS signaling mediates the enhanced vasodilator capacity of nicotine in the renal vasculature of female rats. Preliminary evidence also suggests a potential role for α₇ nAChRs in this estrogen-dependent phenomenon.