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Our previous studies confirm that exogenous reduced nicotinamide adenine dinucleotide phosphate (NADPH) exerts a neuroprotective effect in animal models of ischemic stroke, and its primary mechanism is related to anti-oxidative stress and improved energy metabolism. However, it is unknown whether nicotinamide adenine dinucleotide (NADH) also plays a neuroprotective role and whether NADPH is superior to NADH against ischemic stroke? In this study we compared the efficacy of NADH, NADPH, and edaravone in ameliorating brain injury and metabolic stress in ischemic stroke. Transient middle cerebral artery occlusion/reperfusion (t-MCAO/R) mouse model and in vitro oxygen glucose deprivation/reoxygenation (OGD/R) model were established. The mice were intravenously administered the optimal dose of NADPH (7.5 mg/kg), NADH (22.5 mg/kg), or edaravone (3 mg/kg) immediately after reperfusion. We showed that the overall efficacy of NADPH in ameliorating ischemic injury was superior to NADH and edaravone. NADPH had a longer therapeutic time window (within 5 h) after reperfusion than NADH and edaravone (within 2 h) for ischemic stroke. In addition, NADPH and edaravone were better in alleviating the brain atrophy, while NADH and NADPH were better in increasing the long-term survival rate. NADPH showed stronger antioxidant effects than NADH and edaravone; but NADH was the best in terms of maintaining energy metabolism. Taken together, this study demonstrates that NADPH exerts better neuroprotective effects against ischemic stroke than NADH and edaravone.

Crotoxin (CrTX) is the primary toxin in South American rattlesnake (Crotalus durissus terrificus) venom, and exhibits antitumor and other pharmacological actions in vivo and in vitro. Here, we investigated the molecular mechanisms of the antitumor action of CrTX in human lung carcinoma cells in vitro.Human lung squamous carcinoma SK-MES-1 cells were tested. The cytotoxicity of CrTX was evaluated in both MTT and colony formation assays. Cell cycle was investigated with flow cytometry. Cell apoptosis was studied with Hoechst 33258 and Annexin V-FITC staining. The levels of relevant proteins were analyzed using Western blot assays.CrTX (25, 50, 100 μmol/L) inhibited the growth and colony formation of SK-MES-1 cells in dose- and time-dependent manners. CrTX increased the proportion of S phase cells and dose-dependently induced cell apoptosis, accompanied by down-regulating the expression of proliferating cell nuclear antigen (PCNA), and increasing the level of cleaved caspase-3. Furthermore, CrTX dose-dependently increased the expression of autophagy-related proteins LC3-II and beclin 1, and decreased the level of p62 in the cells. Moreover, CrTX (50 μmol/L) significantly increased p38MAPK phosphorylation in the cells. Pretreatment of the cells with SB203580, a specific inhibitor of p38MAPK, blocked the inhibition of CrTX on cell proliferation, as well as CrTX-induced apoptosis and cleaved caspase-3 expression.The p38MAPK signaling pathway mediates CrTX-induced apoptosis and autophagy of human lung carcinoma SK-MES-1 cells in vitro.

To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover,SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex.ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.

To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.Treatment of A549 cells with CrTX (25-200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC(50)=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G(1) arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.

Cobratoxin (CTX), the long-chain alpha-neurotoxin from Thailand cobra venom, has been demonstrated to have analgesic action in rodent pain models. The present study evaluated the anti-inflammatory and anti-nociceptive effects of CTX on adjuvant arthritis (AA) in rats.Arthritis was induced by injection of complete Freund's adjuvant (CFA) in rats. Paw swelling and hyperalgesia of AA rats were measured at various times after CFA administration. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-2 (IL-2) and interleukin-10 (IL-10) levels in serum were determined with ELISA. Histopathological changes in synoviocytes were examined under a microscope. Involvement of the cholinergic system in the effects of CTX was examined by pretreatment of animals with the alpha(7) nicotinic receptor (alpha(7)-nAChR) antagonist methyllycaconitine (MLA).CFA induced marked paw swelling and reduced thresholds of mechanical and cold-induced paw withdrawal. The levels of TNF-alpha, IL-1 and IL-2 in the serum of AA rats were increased, whereas the level of IL-10 was decreased. Histopathological examination of synoviocytes showed pronounced inflammation and accumulation of collagen. The administration of CTX (17.0 microg/kg, ip) significantly reduced paw swelling and mechanical and thermal hyperalgesia. CTX also reduced the production of TNF-alpha, IL-1, and IL-2 but increased the production of IL-10 and altered pathohistological changes. The analgesic and anti-inflammatory efficacy of CTX was significantly reduced by MLA (3 mg/kg, sc).These results indicate that CTX has a beneficial effect on CFA-induced arthritis by modulating the production of inflammatory cytokines. alpha(7)-nAChR appears to mediate the anti-nociceptive and anti-inflammatory actions of CTX.

Aim: Heat shock proteins (HSPs) are important regulators of cellular survival and exert neuroprotective effects against cerebral ischemia. Both prostaglandin El (PGE1) and lithium have been reported to protect neurons against ischemic injury. The present study was undertaken to examine if lithium could potentiate the neuroprotection of PGE1 against cerebral ischemia, and if the synergetic effects take place at the level of HSPs. Methods: Brain ischemia was induced by a permanent middle cerebral artery occlusion (pMCAO) in rats. Rats were pretreated with subcutaneous injection of lithium for 2 d and a single intravenous administration of PGE1 immediately after ischemic insult. Cerebrocortical blood flow of each group was closely monitored prior to onset of ischemia, 5 min, 15 min, 30 min and 60 min after surgical operation. Body temperature was measured before, 5 min, 2 h and 24 h after the onset of pMCAO. The infarct volume, brain edema and motor behavior deficits were analyzed 24 h after ischemic insult. Cytoprotective HSP70 and heme oxygenase-1 (HO-1) in the striatum of the ipsilateral hemisphere were detected by immunoblotting. Brain sections from the striatum of the ipsilateral hemisphere were double-labeled with the anti-HSP70 antibody and 4,6-diamidino-2-phenylindole (DAPI). Results: Treatment with PGE1 (8 and 16 μg/kg, iv) or lithium (0.5 mEq/kg, sc) alone reduced infarct volume, neurological deficits and brain edema induced by focal cerebral ischemia in rats. Moreover, a greater neuroprotection was observed when PGE1 and lithium were given together. Co-administration of PGE1 and lithium significantly upregulated cytoprotective HSP70 and HO-1 protein levels. Conclusion: Lithium and PGE1 may exert synergistic effects in treatment of cerebral ischemia and thus may have potential clinical value for the treatment of stroke.

Aim: To investigate the effects of lithium (Li) and prostaglandin A 1 (PGA1) on the expression of heat shock factor 1 (HSF-1), heat shock proteins (HSP), and apoptosis protease activating factor-1 (Apaf-1) induced by permanent focal ischemia in rats. Methods: The rats were pretreated with a subcutaneous (sc) injection of Li for 2 d or a single intracerebral ventricle (icv) administration of PGA 1 for 15 min before ischemic insult, or a combination of Li (sc, 1 mEq/kg, 2 d) and PGA 1 (icv, 15 min prior to ischemic insult). Brain ischemia was induced by the permanent middle cerebral artery occlusion (pMCAO). Twenty-four hours after the occlusion, the expression of HSF-1, HSP, andApaf-1 in the ischemic striatum were examined with Western blot analysis. Results: The expression of HSF-1, heme oxygenase-1 (HO-1), HSP90α, and Apaf-1 were significantly increased, but the expression of HSP90β was significantly decreased 24 h after the pMCAO. PGA1 and Li and their combination significantly enhanced the ischemia-induced elevation in the levels of HSF-1, HO-1, and HSP90α, and recovered HSP90β expression, but decreased Apaf-1 levels in the ischemic striatum. Conclusion: The present study demonstrates that PGA1 and Li have synergistic effects on the enhancement of the expression of HSP, suggesting that the synergistic effects of PGA1 and Li in the rat model of permanent focal cerebral ischemia may be mediated by the enhancement expression of HSP expression and the downregulation of Apaf-1. Our studies suggest that combined PGA1 and Li may have potential clinical value for the treatment of stroke.

To study the effects of the caspase-1 inhibitor Ac-YVAD-CHO on quinolinic acid (QA)-induced apoptosis. Rats were pre-treated with intrastriatal infusion of Ac-YVAD-CHO (2-8 μg) before intrastriatal injection of QA (60 nmol). Striatal total proteins, genomic DNA, and nuclear proteins were isolated. The effects of Ac-YVAD-CHO on QA-induced caspase-1 activity, Internucleosomal DNA fragmentation, IκB-α degradation, NF-κB, and AP-1 activation, and increases in p53 protein levels were measured with enzyme assays, agarose gel electrophoresis, electrophoresis mobility shift assays, and Western blot analysis. Pre-treatment with Ac-YVAD-CHO inhibited QA-induced Internucleosomal DNA fragmentation. Ac-YVAD-CHO inhibited QA-induced increases in caspase-1 activity and p53 protein levels, but had no effect on QA-induced IκB-α degradation, NF-κB or AP-1 activation. Caspase-1 is involved in QA-induced p53 upregulation but not IκB-α degradation. Inhibition of caspase-1 attenuates QA-induced apoptosis in rat striatum.

To examine the effects of a mixed formulation composed of prostaglandin E1 and lithium (PGE1+Li mixture) on brain damage after cerebral ischemia. The effects of the mixture on protein expression of heat shock proteins (HSPs), p53, and Bcl-2 were also determined.Brain ischemia was induced with a permanent middle cerebral artery occlusion (pMCAO) in rats. Rats were treated with a single intravenous administration of PGE1, lithium or a PGE1+Li mixture immediately after the ischemic insult. The infarct volume and motor behavior deficits were analyzed 24 h after the ischemic insult. The protein levels of HSP70, glucose-regulated protein 78 (GRP78), HSP60, Bcl-2, and p53 in the striatum of the ipsilateral hemisphere were examined using immunoblotting.The mixture (PGE1 22.6 nmol/kg+Li 0.5 mmol/kg) reduced infarct volume and neurological deficits induced by focal cerebral ischemia. Moreover, the mixture had a greater neuroprotective effect against cerebral ischemia compared with PGE1 or lithium alone. The mixture was effective even if it was administered 3 h after ischemia. PGE1+Li also significantly upregulated cytoprotective HSP70, GRP78, HSP60, and Bcl-2 protein levels, while decreasing p53 expression.These results demonstrated a PGE1+Li mixture with a therapeutic window of up to 3 h for clinical treatment of cerebral ischemia. The PGE1+Li mixture potentially exerts a protective effect after stroke through the induction of HSPs and Bcl-2 proteins.

Heat shock proteins (HSPs) are important regulators of cellular survival and exert neuroprotective effects against cerebral ischemia. Both prostaglandin E1 (PGE1) and lithium have been reported to protect neurons against ischemic injury. The present study was undertaken to examine if lithium could potentiate the neuroprotection of PGE1 against cerebral ischemia, and if the synergetic effects take place at the level of HSPs.Brain ischemia was induced by a permanent middle cerebral artery occlusion (pMCAO) in rats. Rats were pretreated with subcutaneous injection of lithium for 2 d and a single intravenous administration of PGE1 immediately after ischemic insult. Cerebrocortical blood flow of each group was closely monitored prior to onset of ischemia, 5 min, 15 min, 30 min and 60 min after surgical operation. Body temperature was measured before, 5 min, 2 h and 24 h after the onset of pMCAO. The infarct volume, brain edema and motor behavior deficits were analyzed 24 h after ischemic insult. Cytoprotective HSP70 and heme oxygenase-1 (HO-1) in the striatum of the ipsilateral hemisphere were detected by immunoblotting. Brain sections from the striatum of the ipsilateral hemisphere were double-labeled with the anti-HSP70 antibody and 4,6-diamidino-2-phenylindole (DAPI).Treatment with PGE1 (8 and 16 microg/kg, iv) or lithium (0.5 mEq/kg, sc) alone reduced infarct volume, neurological deficits and brain edema induced by focal cerebral ischemia in rats. Moreover, a greater neuroprotection was observed when PGE1 and lithium were given together. Co-administration of PGE1 and lithium significantly upregulated cytoprotective HSP70 and HO-1 protein levels.Lithium and PGE1 may exert synergistic effects in treatment of cerebral ischemia and thus may have potential clinical value for the treatment of stroke.

In light of the antinociceptive activity of the short-chain neurotoxin, cobrotoxin, and other acetylcholine antagonists, the antinociceptive activity and mechanisms of cobratoxin (CTX), a long-chain postsynaptic alpha-neurotoxin, was investigated in rodent pain models.CTX was administered intraperitoneally (30, 45, 68 microg/kg), intra-cerebral ventricularly (4.5 microg/kg) or microinjected into periaqueductal gray (PAG; 4.5 microg/kg). The antinociceptive action was tested using the hot-plate and acetic acid writhing tests in mice and rats. The involvement of the cholinergic system and opioid system in CTX-induced analgesia was examined by pretreatment of animals with atropine (0.5 mg/kg, im; or 10 mg/kg, ip) or naloxone (1 and 5 mg/kg, ip). The effect of CTX on motor activity was tested using the Animex test.CTX exhibited a dose-dependent analgesic action in mice as determined by both the hot-plate and acetic acid writhing tests. The peak effect of analgesia was seen 3 h after administration. In the mouse acetic acid writhing test, the intra-cerebral ventricular administration of CTX at 4.5 microg/kg (1/12th of a systemic dose) produced marked analgesic effects. Microinjection of CTX (4.5 microg/kg) into the PAG region did not elicit an analgesic action in rats in the hot-plate test. Atropine at 0.5 mg/kg (im) and naloxone at 1 and 5 mg/kg (ip) both failed to block the analgesic effects of CTX, but atropine at 10 mg/kg (ip) did antagonize the analgesia mediated by CTX in the mouse acetic acid writhing test. Acetylsalicylic acid (300 mg/kg) did not enhance the analgesic effects of CTX. At the highest effective dose of 68 microg/kg the neurotoxin did not change the spontaneous mobility of mice.CTX has analgesic effects, which are mediated in the central nervous system though not through the PAG. The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CTX.

To investigate the involvement of L-type Ca(2+) channels in antinociceptive action induced by the 5-HT precursor, 5-hydroxytryptophan (5-HTP).Female Kunming mice were treated with either 5-HTP (20-80 mg/kg, ip) alone, or the combination of 5-HTP and fluoxetine (2-8 mg/kg, ip), pargyline (15-60 mg/kg, ip), nimodipine (2.5-10 mg/kg, ip), nifedipine (2.5-10 mg/kg, ip), verapamil (2.5-10 mg/kg, ip), CaCl(2) (5-20 mmol/L, icv), or EGTA (0.5-3 mmol/L, icv) prior to the hot-plate test (55 degree, hind-paw licking latency). In addition, locomotor activity in mice treated with 5-HTP alone was measured using an ambulometer with five activity boxes.Ip injection of 5-HTP alone had no influence on the spontaneous locomotor activity, whereas dose-dependently increased the latency to licking hind-paw in the hot-plate test in mice. The inhibitory effects of 5-HTP on nociceptive response were significantly enhanced by fluoxetine in the mouse hot-plate test. At a sub-effective dose, pargyline could cause a leftward shift in the dose-response curve of 5-HTP-induced antinociception. Co-administration with 5-HTP and nimodipine, nifedipine, or verapamil obviously potentiated the antinociceptive effects elicited by 5-HTP. Interestingly, 5-HTP-induced antinociception was antagonized by CaCl(2) and enhanced by EGTA injected icv in the mouse hot-plate test.These findings suggest that systemic administration of 5-HTP may yield the antinociceptive effects, which are related to Ca(2+) influx from extracellular fluid through L-type Ca(2+) channels.