The effects of sodium sulfite on Helicobacter pylori by establishing a hypoxic environment.

Helicobacter pylori (H. pylori) is an obligate microaerobion and does not survive in low oxygen. Sodium sulfite (SS) reacts and consume oxygen in solutions. The present study aimed to investigate the effects of SS on H. pylori. The effects of SS on oxygen concentrations in solutions and on H. pylori in vivo and in vitro were examined, and the mechanisms involved were explored. The results showed that SS decreased the oxygen concentration in water and artificial gastric juice. In Columbia blood agar and special peptone broth, SS concentration-dependently inhibited the proliferation of H. pylori ATCC43504 and Sydney strain-1 in Columbia blood agar or special peptone broth, and dose-dependently decreased the number of H. pylori in Mongolian gerbils and Kunming mouse infection models. The H. pylori was relapsed in 2 weeks withdrawal and the recurrence in the SS group was lower than that in the positive triple drug group. These effects were superior to positive triple drugs. After SS treatments, the cell membrane and cytoplasm structure of H. pylori were disrupted. SS-induced oxygen-free environment initially blocked aerobic respiration, triggered oxidative stress, disturbed energy production. In conclusion, SS consumes oxygen and creates an oxygen-free environment in which H. pylori does not survive. The present study provides a new strategy and perspective for the clinical treatment of H. pylori infectious disease.

GPR17 mediates ischemia-like neuronal injury via microglial activation.

GPR17 is a G (i)-coupled dual receptor, linked to P2Y and CysLT receptors stimulated by uracil nucleotides and cysteinyl leukotrienes, respectively. Recent evidence has demonstrated that GPR17 inhibition ameliorates the progression of cerebral ischemic injury by regulating neuronal death and microglial activation. The present study aimed to assess the detailed regulatory roles of this receptor in oxygen­glucose deprivation/recovery (OGD/R)­induced ischemia­like injury in vitro and explore the underlying mechanism. The results demonstrated that OGD/R induced ischemic neuronal injury and microglial activation, including enhanced phagocytosis and increased inflammatory cytokine release in neuron­glial mixed cultures of cortical cells. GPR17 upregulation during OGD/R was spatially and temporally correlated with neuronal injury and microglial activation. In addition, GPR17 knockdown inhibited OGD/R­induced responses in neuron­glial mixed cultures. GPR17 knockdown also attenuated cell injury induced by the agonist leukotriene D4 (LTD4) or uridine 5'­diphosphate (UDP) in neuron­glial mixed cultures. However, GPR17 knockdown did not affect OGD/R­induced ischemic neuronal injury in primary cultures of neurons. In primary astrocyte cultures, neither GPR17 nor OGD/R induced injury. By contrast, GPR17 knockdown ameliorated OGD/R­induced microglial activation, boosting phagocytosis and inflammatory cytokine release in primary microglia cultures. Finally, the results demonstrated that the conditioned medium of microglia pretreated with OGD/R induced neuronal death, and the neuronal injury was significantly inhibited by GPR17 knockdown. These findings suggested that GPR17 may mediate ischemia­like neuronal injury and microglial activation in vitro; however, the protective effects on ischemic neuronal injury might depend upon microglial activation. Whether GPR17 regulates neuronal injury mediated by oligodendrocyte linkage remains to be investigated.

Protective effects of paeonol on subacute/chronic brain injury during cerebral ischemia in rats.

Ischemic stroke is a highly complex pathological process that is divided into acute, subacute and chronic phases. Paeonol is a biologically active natural product with a variety of pharmacological effects, including those on neuronal activity. However, the effects of paeonol on subacute/chronic ischemic stroke have remained to be elucidated. The present study was designed to investigate the effects of paeonol against subacute and chronic cerebral ischemic injury and to explore the possible underlying mechanisms. Male adult Sprague Dawley rats were randomly divided into a sham group (treated with saline), a model group [subjected to middle cerebral artery occlusion (MCAO) and treated with saline] and a paeonol-treated group (MCAO + paeonol at 25 mg/kg). Behavioral impairment, infarct volume and ischemic/contralateral hemispheric ratios were assessed at 72 h and at 28 days after MCAO, respectively. Immunofluorescence was employed to determine the neuronal damage and glial responses after MCAO. Compared with the model group, paeonol treatment significantly attenuated behavioral impairment, ischemic infarct volume and moderate cerebral edema in the ischemic brain at 72 h, as well as brain atrophy at 28 days after reperfusion. Furthermore, paeonol treatment ameliorated neuronal damage in the ischemic core and boundary zone regions at 72 h after reperfusion and in the boundary zone at 28 days after reperfusion. In addition, paeonol treatment reduced the proliferation of astrocytes in the boundary zone, and inhibited microglial activation in the ischemic core and boundary zone regions at 72 h and 28 days after reperfusion. These results demonstrated the protective effects of paeonol against subacute/chronic cerebral ischemia, and the mechanism of action may include subacute/chronic microglial activation and astrocyte proliferation.

Oral administration of ampelopsin protects against acute brain injury in rats following focal cerebral ischemia.

Ampelopsin (AMP) is isolated from the Chinese medicinal herb Ampelopsis grossedentata (Hand-Mazz) and has been associated with numerous biological and pharmacological activities. However, it is not clear whether AMP has a direct protective effect on cerebral ischemia reperfusion injury. Therefore, the present study investigated its role in acute brain injury following focal cerebral ischemia in rats. The current study induced transient focal cerebral ischemia by performing middle cerebral artery occlusion (MCAO) for 60 min, followed by 24 h of reperfusion. Rats were exposed to 40, 80 and 160 mg/kg AMP by oral administration 30 min prior to MCAO and the cysteinyl leukotriene receptor 1-antagonist, pranlukast (0.1 mg/kg, i.p.) was used as a positive control. Neurological deficit scores were observed and an inclined board test was used to assess behavioral dysfunction. The coronal slices were stained with 3,5-triphenyltetrazolium chloride to determine the infarct volume and brain edema. Neuronal morphology was assessed in brain sections stained with cresyl violet and degenerating neurons were identified using Fluoro-Jade B staining. Blood-brain barrier permeability was determined with immunoglobulin (Ig)G immunohistochemistry. Interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) in serum and cerebrospinal fluid were measured using ELISA kits. AMP at 80 and 160 mg/kg attenuated neurological deficits, reduced infarct volume, brain edema, IgG exudation and neuron degeneration and loss. Similar to pranlukast, AMP also inhibited the MCAO-induced IL-1ß and TNF-α release. Thus, AMP has a neuroprotective effect on acute brain injury following focal cerebral ischemia in rats at an effective oral dose of 80-160 mg/kg. The results of the current study indicate a therapeutic role for AMP in the treatment of ischemic stroke.

NAMPT inhibitor protects ischemic neuronal injury in rat brain via anti-neuroinflammation.

Nicotinamide phosphoribosyltransferase (NAMPT) is an important neuroprotective factor in cerebral ischemia, and it has been reported that NAMPT inhibitors can aggravate neuronal injury in the acute phase. However, because it is a cytokine, NAMPT participates in many inflammatory diseases in the peripheral system, and its inhibitors have therapeutic effects. Following cerebral ischemia, the peripheral and resident inflammatory and immune cells produce many pro-inflammatory mediators in the ischemic area, which induce neuroinflammation and impair the brain. However, the effects of NAMPT inhibitors in the neuroinflammation after ischemic brain injury remain unknown. Here, we found that FK866, a potent NAMPT inhibitor, decreased the level of TNF-α, NAMPT and IL-6 in the ischemic brain tissue one day after middle-cerebral-artery occlusion and reperfusion (MCAO/R), improved neurological dysfunction, decreased infarct volume and neuronal loss, and inhibited microgliosis and astrogliosis 14days after MCAO/R. The expression of NAMPT protein was induced in Iba1-positive microglia/macrophages in the ischemia core 14days after MCAO/R. In vitro studies show that oxygen-glucose deprivation and recovery (OGD/R) activate microglia. Activated microglia increased the activity of NF-κB, increased the mRNA synthesis of TNF-α, NAMPT and IL-6, and increased the secretion of TNF-α, NAMPT and IL-6. On the other hand, NAMPT can act synergistically with other cytokines and activate microglia. FK866 strongly inhibited these changes and alleviated OGD/R-induced activation of microglia. As such, NAMPT is a crucial determinant of cellular inflammation after cerebral ischemia. NAMPT inhibitors are novel compounds to protect neuronal injury from ischemia via anti-inflammatory effects.

Nicotinamide phosphoribosyltransferase inhibitor APO866 induces C6 glioblastoma cell death via autophagy.

APO866 is a potent inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), and inhibits nicotinamide adenine dinucleotide (NAD) synthesis. Our previous study showed that APO866 inhibits the proliferation of C6 glioblastoma cells, but failed to induce apoptosis. Since APO866 inhibits cellular metabolism and such metabolic stress is closely related with autophagy, thus we determined whether APO866 can induce autophagy in C6 glioblastoma cells and whether the autophagy induced by APO866 is pro-death or pro-survival. Using LC3 immunofluorescence imaging and transmission electron microscopy detection, we found that APO866 at 1-100 nM induced autophagy in C6 glioblastoma cells. APO866 at 1 nM mainly induced initial autophagic vacuoles. Whereas APO866 at 100 nM induced degrading autophagic vacuoles, as well as induced nuclei malformation and mitochondria swelling. In addition, APO866 concentration-dependently decreased the cell viability of C6 glioblastoma cells, and this effect was attenuated by autophagy inhibitors, including 3-methyladenine and LY294002. APO866 concentration-dependently decreased intracellular NAD level. Interestingly, APO866 at 1 nM slightly decreased intracellular NAD level, but dramatically increased autophagy-positive cells. The dramatical cell viability decreasing required the decreasing of intracellular NAD level to a very low threshold. Thus, our results indicated that APO866 induced pro-death autophagy in C6 glioblastoma cells by decreasing intracellular NAD, and low concentration of APO866 can be used as an autophagy inducer in autophagic-death sensitive glioblastoma.

[Antioxidative effects of cysteinyl leukotriene receptor antagonists montelukast and HAMI 3379 on ischemic injury in rat cortical neurons in vitro].

OBJECTIVE: To investigate the antioxidative effects of two cysteinyl leukotriene receptors antagonists (CysLT1R and CysLT2R) montelukast and HAMI 3379 on ischemic injury of rat cortical neurons in vitro. METHODS: Cultured rat cortical neurons were pretreated with CysLT1R antagonist montelukast and CysLT2R antagonist HAMI 3379, and then exposed to oxygen-glucose deprivation/recovery (OGD/R）or H2O2. Reactive oxygen species (ROS) mitochondrial membrane potential (MMP) depolarization, neuronal viability and lactate dehydrogenase (LDH) release were determined. Meanwhile, RNA interference was used to inhibit the expression of CysLT1R and CysLT2R，and the effects were observed. RESULTS: ROS production in neurons was significantly increased after 1 h OGD, which reached the peak at 30 min and lasted for 1.5 h after recovery. Montelukast and HAMI 3379 at 0.01-1µmol/L moderately decreased OGD/R-induced ROS production (P<0.05). Montelukast mildly attenuated OGD/R-induced MMP depolarization (P<0.05)，but HAMI 3379 had no effect. H2O2 reduced neuronal viability and increased LDH release, namely inducing neuronal injury. Montelukast and HAMI 3379 at 0.1-1µmol/L moderately attenuated H2O2-induced neuronal injury (P<0.05). However, both CysLT1R siRNA and CysLT2R shRNA did not significantly affect the responses mentioned above. CONCLUSION: In ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.

HAMI 3379, a CysLT2 receptor antagonist, attenuates ischemia-like neuronal injury by inhibiting microglial activation.

The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonist HAMI 3379 [3-({[(1S,3S)-3- carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy)benzoic acid] in comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1ß and tumor necrosis factor-α) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R- and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.

Cerebral ischemia is exacerbated by extracellular nicotinamide phosphoribosyltransferase via a non-enzymatic mechanism.

Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT) is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO) and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD) induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.

Aggravated inflammation and increased expression of cysteinyl leukotriene receptors in the brain after focal cerebral ischemia in AQP4-deficient mice.

OBJECTIVE: Aquaporin-4 (AQP4), the main water channel protein in the brain, plays a critical role in water homeostasis and brain edema. Here, we investigated its role in the inflammatory responses after focal cerebral ischemia. METHODS: In AQP4-knockout (KO) and wild-type mice, focal cerebral ischemia was induced by 30 min of middle cerebral arterial occlusion (MCAO). Ischemic neuronal injury and cellular inflammatory responses, as well as the expression and localization of cysteinyl leukotriene CysLT(2) and CysLT(1) receptors, were determined at 24 and 72 h after MCAO. RESULTS: AQP4-KO mice showed more neuronal loss, more severe microglial activation and neutrophil infiltration, but less astrocyte proliferation in the brain after MCAO than wild-type mice. In addition, the protein levels of both CysLT(1) and CysLT(2) receptors were up-regulated in the ischemic brain, and the up-regulation was more pronounced in AQP4-KO mice. The CysLT(1) and CysLT(2) receptors were primarily localized in neurons, microglia and neutrophils; those localized in microglia and neutrophils were enhanced in AQP4-KO mice. CONCLUSION: AQP4 may play an inhibitory role in postischemic inflammation.

Intracerebroventricular injection of HAMI 3379, a selective cysteinyl leukotriene receptor 2 antagonist, protects against acute brain injury after focal cerebral ischemia in rats.

Cysteinyl leukotrienes (CysLTs) induce inflammatory responses by activating their receptors, CysLT(1)R and CysLT(2)R. We recently reported that CysLT(2)R is involved in neuronal injury, astrocytosis and microgliosis after focal cerebral ischemia in rats. Here, we determined whether HAMI 3379, a selective CysLT(2)R antagonist, protects against acute brain injury after focal cerebral ischemia in rats. We induced transient focal cerebral ischemia by 30 min of middle cerebral artery occlusion (MCAO), followed by 24h of reperfusion. HAMI 3379 (1, 10 or 100 ng) was injected intracerebroventricularly (i.c.v.) 30 min before MCAO, and the CysLT(1)R antagonist pranlukast (0.1mg/kg, i.p.) was used as a positive control. HAMI 3379 at 10 and 100 ng (but not at 1 ng) attenuated the neurological deficits, and reduced infarct volume, brain edema, IgG exudation, neuronal degeneration and neuronal loss. This protective effect was similar to that of pranlukast. Thus, HAMI 3339 at 10-100 ng i.c.v. is neuroprotective against acute brain injury after focal cerebral ischemia in rats. These findings suggest therapeutic potential for CysLT(2)R antagonists in the treatment of ischemic stroke.