A New Approach to Antivenom Preparation Using Chitosan Nanoparticles Containing EchisCarinatus Venom as A Novel Antigen Delivery System.

In recent years, use of biodegradable polymers based nanoparticles has received high interest in the development of vaccines delivery vehicles. The aim of study was to prepare chitosan nanoparticles (CS NPs) for loading Echis carinatus (EC) venom and evaluate their potential as an adjuvant and antigen delivery system on a pilot scale. CS NPs were prepared using ionic gelation method, and their characteristics were optimized. Venom-loaded CS NPs prepared under optimum conditions and traditional venom-loaded adjuvants were used to hyperimmunization of horse. Under optimum conditions, particle size, polydispersity index (PDI), and zeta potential of CS NPs were 127.9 ± 15 nm, 0.29, and +19.8 ± 1.92 mV, while those of venom-loaded CS NPs were 182.4 ± 20 nm, 0.35, +26.8 ± 1.98 mv, respectively. All CS NPs had integrated surface and good morphology. Optimum loading concentration of EC venom was 500 µg/mL. The loading capacity (LC) and loading efficiency (LE) were 87% and 94%, respectively, and release profile of venom-loaded CS NPs showed suitable correlation with Higuchi kinetics. Stability test showed good stability of the venom encapsulated in CS NPs. Furthermore, antivenom plasma obtained using the new antigen delivery system had significantly higher potency (P < 0.05) for neutralizing the venom than that obtained using conventional system. These results suggested that venom-loaded CS NPs could be a suitable alternative to conventional adjuvant for development antivenom.

The influence of NMDA receptor agonist and antagonist on morphine state-dependent memory of passive avoidance in mice.

The measurement of step-down latency in passive avoidance has been used to study memory in laboratory animals. The pre-training injection of 5 mg/kg morphine impaired memory, which was restored when 24 h later the same dose of the drug was administered. To explore the possible involvement of NMDA modulators on morphine-induced memory impairment, we have investigated the effects of intracerebroventricular (i.c.v.) administration of NMDA and the competitive NMDA antagonist, DL-AP5, on morphine-induced memory impairment or recall, on the test day. Morphine (5 mg/kg, s.c.) was administered 30 min before training to induce impairment of memory and 24 h later, 30 min before test to improve it. Pre-test administration of NMDA (0.00001, 0.0001 and 0.001 microg/mouse, i.c.v.) did not alter the retention latency compared to the saline-treated animals. But restored the memory impairment induced by pre-training morphine (5 mg/kg, s.c.). Pre-test administration of DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) by itself decreased the retention latencies. The same doses of DL-AP5 increased pre-training morphine-induced memory impairment. Co-administration of NMDA (0.0001 and 0.001 microg/mouse, i.c.v.) and morphine (5 mg/kg, s.c.) on the test day increased morphine memory improvement. Conversely, DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) inhibited morphine-induced memory recall. It is concluded that NMDA receptors may be involved, at least in part, in morphine state-dependent learning in mice.

Cholecystokinin and GABA interaction in the dorsal hippocampus of rats in the elevated plus-maze test of anxiety.

In the present study, we have investigated the effects and interaction of CCK and GABAergic systems in the dorsal hippocampus of rats using the elevated plus-maze test of anxiety. Bilateral injection of different doses of CCK(8s) (0.01, 0.05 and 0.1 microg/rat) into the dorsal hippocampus (intra-CA1) decreased percentage of open arm time (%OAT) and open arm entries (%OAE) that are representative of anxiogenic-like behavior. The bilateral injection of three doses of LY225910, a selective CCK2 receptor antagonist (0.01, 0.1 and 0.5 microg/rat) produced significant anxiolytic behavior. Although muscimol (GABA(A+)) (0.1, 0.5 and 1 microg/rat, intra-CA1) produced dose dependent increase in %OAT and a slight increase in %OAE, bicuculline (GABA(A-)), (1, 2 and 4 microg/rat, intra-CA1) failed to change the anxiety profile. Both muscimol (0.1 microg/rat) and bicuculline (1 microg/rat), when co-administered with LY225910, reversed the effect of latter drug on anxiety but when co-administered with CCK8s (0.05 microg/rat) showed no effect on anxiety profile. In conclusion, it seems that both CCK and GABAergic systems not only play a part in the modulation of anxiety in the dorsal hippocampus of rats but also have demonstrated a complex interaction as well.

Neuro-protective effects of cerium and yttrium oxide nanoparticles on high glucose-induced oxidative stress and apoptosis in undifferentiated PC12 cells.

OBJECTIVE: Oxidative stress has been recognized as the major factor for the development of diabetes and its complications. Cerium oxide and Yttrium oxide nanoparticles are known as free radicals scavengers. The aim of this study was to investigate the protective effect of CeO2 and Y2O3 on oxidative stress induced by high glucose in undifferentiated rat pheochromocytoma (PC12) cells. METHODS: In this study, undifferentiated PC12 cells were exposed to high glucose (25 mg/ml, 24 hours) and the protective effects of CeO2 and Y2O3 nanoparticles were evaluated. The viability of undifferentiated PC12 cells was determined by MTT assay. The levels of reactive oxygen species (ROS) were measured using 2,7-dichlorodihydrofluorescein diacetate (DCF). The expression levels of pro-apoptotic Bax, anti-apoptotic Bcl-2 and caspase3 proteins were also detected by western blotting. Total antioxidant power (TAP), total thiol molecules (TTM) and lipid peroxidation (LPO) were also evaluated. RESULTS: CeO2 and Y2O3 increased survival of undifferentiated PC12 cells exposed to high glucose-induced oxidative stress. CeO2 and Y2O3 pre-treatment decreased ROS production, LPO, Bax and caspase-3 proteins expression. Both nanoparticles have also increased the TTM and Bcl-2 protein expression. DISCUSSION: These findings suggest that CeO2 and Y2O3 protect the undifferentiated PC12 cells against the oxidative stress and apoptosis induced by high glucose.

Potentiation of anandamide effects in mesenteric beds isolated from bile duct-ligated rats: role of nitric oxide.

Changes in vascular responsiveness are proposed as the basis for some of the cardiovascular complications in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and evidence of nitric oxide (NO) overproduction. On the other hand, it is well known that anandamide, an endogenous cannabinoid ligand, causes hypotension and a decrease in systemic vascular resistance. In the present study, the possible role of the cannabinoid system in cholestasis-induced mesenteric vascular bed responsiveness was investigated. Mesenteric arteries of bile duct-ligated and sham-operated rats receiving daily administrations of saline were used for evaluating phenylephrine or anandamide dose-response, acute effects of N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), a non-selective inhibitor of NO synthase (NOS), or naltrexone, an opioid receptors antagonist (1 microM). The other groups of bile duct-ligated and sham-operated rats received daily intraperitoneal administration of L-NAME (20 mg/kg/day), aminoguanidine, a selective inducible NOS (iNOS) inhibitor (150 mg/kg/day) or naltrexone (10 mg/kg/day). After 7 days, the superior mesenteric artery was cannulated and the mesenteric vascular bed was perfused according to the McGregor method. Anandamide-induced relaxation was significantly potentiated in mesenteric vascular beds of bile duct-ligated rats. Chronic treatment of bile duct-ligated animals with L-NAME and aminoguanidine blocked this hyperresponsiveness while the hyperresponsiveness was potentiated at large doses of anandamide on chronic treatment of these animals with naltrexone. Although acute L-NAME treatment of mesenteric beds completely blocked the anandamide-induced vasorelaxation in sham-operated rats, this vasorelaxation still was present in bile duct-ligated animals. Anandamide-induced vasorelaxation remained unaffected after acute naltrexone treatment of mesenteric beds in both bile duct-ligated and sham-operated rats. Our results indicate that (1) there is enhanced anandamide-induced vasorelaxation in cholestatic rats, probably due to a defect in cannabinoid or vanilloid receptors and (2) NO overproduction may be involved in cholestasis-induced vascular hyperresponsiveness.

Modulation of cholestasis-induced antinociception by CCK receptor agonists and antagonists.

Acute cholestasis is associated with increased activity of the endogenous opioid system. Agonists and antagonists of cholecystokinin (CCK) receptors are known to modulate opioid-induced antinociception. In the present study, the effect of the CCK receptor agonist caerulein and the antagonist proglumide on antinociception induced during acute cholestasis was investigated in rats using the tail-flick test. A significant increase in nociception threshold was observed in bile duct ligated (BDL) rats compared to sham-operated controls that was maximum on day 7 after the operation and decreased thereafter. Proglumide (40 mg/kg, i.p.) did not affect nociception in unoperated and sham-operated animals, but exerted a significant potentiation of antinociception in cholestatic rats in a way similar to its potentiation effect on unoperated morphine-treated (2 mg/kg, s.c.) animals. Caerulein (0.005, 0.001, 0.01 and 0.02 mg/kg, s.c.), which did not change nociception per se or in sham-operated animals, also significantly potentiated the antinociception in BDL rats as well as in morphine-treated unoperated controls. Caerulein-induced potentiation of antinociception in BDL animals was completely reversed by proglumide pretreatment. Our findings show that, in cholestatic animals, modulation of nociception by the CCK system is different from normal subjects and resembles the state observable in morphine-administered subjects.

The Effect of Pentoxifylline on bcl-2 Gene Expression Changes in Hippocampus after Ischemia-Reperfusion in Wistar Rats by a Quatitative RT-PCR Method.

Ischemia-reperfusion injury is the tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines. Twenty-four male Wistar rats (250-300 g body wt) were used in this study. The animals were divided into four groups of 6 rats each: I: Control group that was subjected to ischemia-reperfusion, II: Ischemia-reperfusion group that was subjected to all surgical procedures, III: Drug group that received pentoxifylline (200, 400 and 600 mg/kg) 60 min before and after ischemia and IV: Vehicle group that received saline. Seventy two h after ischemia-reperfusion, the hippocampus was taken for studying the changes in bcl-2 gene expression. We used quantitative real-time PCR for the detection of bcl-2 gene expression in ischemia and drug groups and then compared them to normal samples. The results showed the gene dosage ratio of 0.66 and 1.5 for ischemia group and the drug groups, respectively. The results also showed the bcl-2 gene expression declined in ischemia group as compared to the drug group. Furthermore, we observed a significant difference in the bcl-2 gene expression between ischemia and drug groups. These findings are consistent with anti-apoptotic properties of bcl-2 gene. Furthermore this method provides a powerful tool for the investigators to study brain ischemia and respond to the treatment drugs with anti-apoptotic agents.

The involvement of nitric oxide in the anti-seizure effect of acute atorvastatin treatment in mice.

OBJECTIVES: In addition to reducing the serum level of cholesterol, statins have various pleiotropic effects such as increasing nitric oxide and reducing oxidative stress, neuroinflammation, and neurotoxicity. Increasing evidence indicates that statins are neuroprotective in several conditions, including stroke, cerebral ischemia and traumatic brain injury. However, only a few studies have investigated whether statins modulate seizure activity. METHODS: In the current study, we investigated whether acute treatment with atorvastatin alters the seizures induced by electroshock or pentylenetetrazole in mice. We also evaluated whether nitrergic system is involved in the effects of acute atorvastatin on seizure. RESULTS: The results of the present study demonstrate that acute atorvastatin (10 and 20 mg/kg) treatment decreased the incidence of tonic seizure and death in electroshock-induced seizure model. We also showed that acute atorvastatin (10 mg/kg) treatment increased the clonic seizure threshold in pentylenetetrazole-seizure model. Acute L-NAME (5 mg/kg), a non-selective inhibitor of nitric oxide synthase, or aminoguanidine (100 mg/kg), a selective inhibitor of inducible nitric oxide synthase, administration prevented the anti-convulsant effect of atorvastatin in electroshock-induced seizure model. We also demonstrated that acute L-NAME (5 mg/kg) or aminoguanidine (100 mg/kg) administration decreased the enhanced clonic latency of clonic seizure threshold induced by atorvastatin in mice in pentylenetetrazole model. DISCUSSION: In conclusion, anti-convulsant effect of atorvastatin was demonstrated in two seizure models of electroshock and intraperitoneal pentylenetetrazole. Nitric oxide release, probably through inducible nitric oxide synthase at least in part is responsible for this effect.

Anti-mitogenic and apoptotic effects of 5-HT1B receptor antagonist on HT29 colorectal cancer cell line.

PURPOSE: There is lack of evidence about impact of 5-HT receptors on colorectal cancers. The current study was designed to investigate the role of serotonin and its receptors in colorectal cancer cell line and tissues. METHODS: In cell cultures, we investigated the effects of 5-HT and 5-HT(1A,1B,1D) agonists and antagonists on proliferation of HT29 cells. We also tested apoptosis for the receptor antagonists. The expression of 5-HT1(A,B,D) receptor subtypes was examined by immunohistochemistry and western blotting. RESULTS: Our data indicated that 5-HT(1B) receptor was fully expressed in HT29 cell line and tumor tissues. MTT proliferation assay also revealed that serotonin and CP93129 dihydrochloride, a selective 5-HT(1B) receptor agonist, stimulated growth of HT29 cells. Further, SB224289 hydrochloride (that is a selective 5-HT(1B) receptor antagonist) had anti-proliferative and apoptotic effects on HT29 cells. CONCLUSIONS: The findings of this study provide evidence for the potential role of 5-HT(1B) receptor in colorectal cancer. Further investigation is required to explore the effect of receptor antagonists on the prevention, prognosis and treatment of patients with colorectal cancer.

Y25130 hydrochloride, a selective 5HT3 receptor antagonist has potent antimitogenic and apoptotic effect on HT29 colorectal cancer cell line.

Serotonin (5-hydroxytryptamine, 5-HT) is known to be a mitogenic factor in several malignancies. It elicits its mitogenic effect through a wide range of 5-HT receptor subtypes and several internal cellular transcription pathways. According to wide distribution of 5-HT3 and 5-HT4 receptors in the gastrointestinal tract, the main aim of this study was to investigate the effect of these receptor agonists and antagonists in a colorectal cell line. In cell culture, we investigated the effects of 5-HT, 5-HT3 and 5-HT4 receptor agonists and antagonists on proliferation of HT29 cells. We also tested apoptosis for the receptor antagonists with TUNEL apoptosis test. In addition, we assayed effects of 5-HT receptor antagonists on cell cycle kinetics with flow cytometery. Proliferation assay revealed that phenylbiguanide (a 5-HT3 receptor selective agonist) increased proliferation of HT29 cells significantly and Y25130 hydrochloride (a 5-HT3 receptor antagonist) had the opposite effect; but for 5-HT4 receptor antagonist, these effects were not significant. In addition, potent apoptotic and cell cycle arresting effect was found for a selective 5-HT3 receptor antagonist but we have not seen any significant effect for the 5-HT4 receptor antagonist. The findings of this study provide strong evidence for the potential role of 5-HT3 receptors in colorectal cancer.

Study of 5HT3 and HT4 receptor expression in HT29 cell line and human colon adenocarcinoma tissues.

BACKGROUND: Serotonin (5HT) has been shown to be a mitogenic factor in several carcinomas. Its mitogenic effect is elicited through a wide range of 5HT receptor subtypes. In this study, the effects of 5HT, 5HT3 (1-phenylbiguanide hydrochloride) and 5HT4 (cisapride) agonists in promoting the growth of the HT29 cell line and the growth-inhibition effect of the 5HT3 receptor antagonist (Y-25130 hydrochloride) and 5HT4 receptor antagonist (RS 23597-190) were investigated. The expressions of 5HT3 and 5HT4 receptors in human colon cancer tissues and the HT29 cell line were studied. METHODS: The growth-promoting and growth-inhibition effects of 5-HT, 5HT3 and 5HT4 agonists and antagonists on the HT29 cell line were studied using MTT assay. Receptor expression has been demonstrated by western blotting. RESULTS: The results showed that 5HT, 5HT3, and 5HT4 agonists caused significant proliferation of HT29 cells. 5HT3 and 5HT4 receptor antagonists had an inhibitory effect on the growth of these cells. Western blot analysis gave bands from colon tissue extracts and the HT29 cell line. CONCLUSION: The results indicate which 5HT3 and 5HT4 receptors are significantly expressed in both colon cancer tissue and the HT29 cell line. Expression for the 5HT3 receptor is more potent. Furthermore, 5HT plays a mitogenic role in colon cancer cells and antagonists of 5HT3, and 5HT4 receptors can inhibit cancer cell growth.

Involvement of NMDA receptors in morphine state-dependent learning in mice.

In the present study, the effects of intracerebroventricular (i.c.v.) injection of NMDA receptor agonist and antagonist on impairment of memory formation and the state-dependent learning by morphine have been investigated in mice. Pretraining administration of morphine (5 mg/kg; s.c.) decreased the learning of one-trial passive avoidance task. Pretest administration of morphine (5 mg/kg) induced state-dependent learning acquired under pretraining morphine influence. Pretest administration of NMDA receptor agonist, L-glutamate (0.00001 and 0.0001 and 0.001 microg/mouse, i.c.v.) following pretraining saline treatment did not affect retention. Amnesia induced by pretraining morphine was significantly reversed by pretest administration of L-glutamate (0.0001 and 0.001 microg/mouse, i.c.v.). Pretest administration of noncompetitive NMDA receptor antagonist, MK-801 (0.5, 1, and 2 microg/mouse, i.c.v.) significantly impaired memory formation. Amnesia induced by pretraining morphine was increased by pretest administration of MK-801 (2 microg/mouse, i.c.v.). Pretest coadministration of L-glutamate (0.0001 and 0.001 microg/mouse, i.c.v.) or MK-801 (0.5, 1, and 2 microg/mouse, i.c.v.) with morphine (5 mg/kg, s.c.) increased and decreased morphine state-dependent learning, respectively. The results suggest that NMDA receptors are involved in morphine state-dependent learning in mice.