Rui-Xian Guo

Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance.

Compelling evidence has suggested that spinal glial cells were activated by chronic morphine treatment and involved in the development of morphine tolerance. However, the mechanisms of glial activation were still largely unknown in morphine tolerance. In present study, we investigated the role of p38 mitogen-activated protein kinase (p38 MAPK) in the spinal cord in the development of chronic morphine antinociceptive tolerance. We found that intrathecal administration of morphine (15 microg) daily for 7 consecutive days significantly induced an increase in number of phospho-p38 (p-p38) immunoreactive cells in the spinal cord compared with chronic saline or acute morphine treated rats. Double immunofluorescence staining revealed that p-p38 immunoreactivity was exclusively restricted in the activated spinal microglia, not in astrocytes or neurons. Repeated intrathecal administration of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (10 microg or 2 microg), a specific p38 inhibitor, 30 min before each morphine injection for 7 consecutive days significantly attenuated tolerance to morphine analgesia assessed by tail flick test. However, a single intrathecal administration of SB203580 (10 microg) did not antagonize the established tolerance to morphine analgesia. Taken together, these findings suggested that p38 MAPK activation in the spinal microglia was involved in the development of morphine antinociceptive tolerance. Inhibition of p38 MAPK by SB203580 in the spinal cord attenuated but not reversed the tolerance to morphine analgesia. The present study provides the first evidence that p38 activation in spinal microglia played an important role in the development of tolerance to morphine analgesia.

Protection of oxidative preconditioning against apoptosis induced by H2O2 in PC12 cells: mechanisms via MMP, ROS, and Bcl-2.

The present study is designed to investigate the effects of preconditioning with different doses of hydrogen peroxide (H2O2) on oxidative stress-induced apoptosis and the changes in mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) level, and expression of Bcl-2 during H2O2 preconditioning in rat pheochromocytoma (PC12) cells. It was shown that (1) H2O2 induced apoptosis in PC12 cells in a dose-dependent manner; (2) the preconditioning with 10 micromol L(-1) or 20 micromol L(-1) H2O2 can significantly protect PC12 cells against apoptosis induced by 50 or 100 micromol L(-1) H2O2, low (5 micromol L(-1)) and higher (30 micromol L(-1)) concentrations of H2O2 had no cytoprotections; (3) high concentration (100 micromol L(-1)) of H2O2 reduced MMP and expression of Bcl-2, and increased ROS level, but these effects were blocked by preconditioning with 10 micromol L(-1) H2O2; (4) the preconditioning with 10 micromol L(-1) H2O2 induced overexpression of Bcl-2. These results suggested that the preconditioning with low dose of H2O2 could protect the oxidative stress-induced PC12 cells apoptosis not only by preventing the reduction of MMP and expression of Bcl-2 as well as increase in ROS level, but also through overexpression of Bcl-2. It was indicated that overexpression of Bcl-2 may play a key role in the cytoprotection induced by preconditioning with low dose of H2O2 in PC12 cells.

Hydrogen sulphide protects H9c2 cells against chemical hypoxia-induced injury

1. The aim of the present study was to investigate the effect of hydrogen sulphide (H2S) on cobalt chloride (CoCl2)‐induced injury in H9c2 embryonic rat cardiac cells.

NMDA receptors are involved in upstream of the spinal JNK activation in morphine antinociceptive tolerance.

N-methyl-d-aspartate (NMDA) receptors and c-Jun N-terminal kinase (JNK) have been shown to be involved in morphine antinociceptive tolerance. However, whether chronic morphine-induced activation of the spinal JNK is NMDA receptor-dependent is unknown. The present study investigated the link between the spinal NMDA receptor NR2B subunit and the JNK activation during morphine antinociceptive tolerance in rats. Our results showed that chronic morphine treatment induced upregulation of the NR2B expression and activation of JNK in the spinal cord. Moreover, the increased NR2B-immunoreactivity (IR) and phosphorylated JNK-IR were observed mainly at the superficial dorsal horn laminae of the spinal cord; the spinal p-JNK was mainly expressed in astrocytes and NR2B in neurons. SP600125, a selective inhibitor of JNK, significantly attenuated morphine tolerance. MK-801, a noncompetitive NMDA receptor antagonist, not only suppressed morphine antinociceptive tolerance and the increase in NR2B, but also reduced the spinal JNK activation induced by chronic morphine treatment. These findings demonstrated for the first time that NMDA receptor-dependent activation of the spinal JNK contributes to morphine antinociceptive tolerance and that MK-801 attenuates morphine tolerance partly due to its inhibition on the spinal JNK activation.

Novel insights into the role of HSP90 in cytoprotection of H2S against chemical hypoxia-induced injury in H9c2 cardiac myocytes

The present study evaluated potential mechanisms of hydrogen sulfide (H2S)-mediated cardioprotection using an in vitro chemical hypoxia-induced injury model. We have demonstrated that H2S protects H9c2 cardiomyoblasts (H9c2) against chemical hypoxia-induced injuries by suppressing oxidative stress and preserving mitochondrial function. The aim of this study was to investigate the role of heat shock protein 90 (HSP90) in cardioprotection of H2S in H9c2 cells. The findings of the present study showed that cobalt chloride (CoCl2), a chemical hypoxia agent, significantly enhanced the expression of HSP90 and that 17-allylamino-17-demethoxy geldanamycin (17-AAG), a selective inhibitor of HSP90, aggravated concentration-dependent cytotoxicity induced by CoCl2. Exogenous administration of NaHS (a donor of H2S) augmented not only HSP90 expression under normal conditions, but also CoCl2-induced overexpression of HSP90. Pre-treatment with 17-AAG significantly blocked the cardioprotection of H2S against CoCl2-induced injuries, leading to increases in cytotoxicity and apoptotic cells. Furthermore, pre-treatment with 17-AAG also antagonized the inhibitory effects of NaHS on overproduction of reactive oxygen species (ROS), a loss of mitochondrial membrane potential (MMP) and ATP depletion induced by CoCl2. In conclusion, these results demonstrate that the increased expression of HSP90 may be one of the endogenous defensive mechanisms for resisting chemical hypoxia-induced injury in H9c2 cells. We also provide novel evidence that HSP90 mediates the cardioprotection of H2S against CoCl2-induced injuries by its antioxidant effect and preservation of mitochondrial function in H9c2 cells.

Spinal MCP-1 Contributes to the Development of Morphine Antinociceptive Tolerance in Rats

Background:The chemokine monocyte chemoattractant protein-1 (MCP-1) has been shown to contribute to neuropathic pain. However, whether MCP-1 is involved in the development of morphine antinociceptive tolerance is incompletely understood. Methods:Morphine antinociceptive tolerance was induced by intrathecal administration of 15 &mgr;g of morphine daily for 7 days. Immunohistochemistry was used to test the changes in the morphology of spinal MCP-1 immunoreactivity and OX-42-IR. The role of MCP-1 in morphine antinociceptive tolerance is explored by hot-water tail-flick test. Results:Our findings showed that intrathecal chronic morphine exposure obviously increased MCP-1 immunoreactivity in the spinal cord. Moreover, the increased MCP-1 immunoreactivity was observed mainly in the spinal neurons. Intrathecal injections of MCP-1-neutralizing antibody significantly reduced the development of morphine antinociceptive tolerance, suggesting that spinal neuronal MCP-1 contributes to tolerance to morphine antinociception. Treatment with MCP-1-neutralizing antibody also reduced the spinal microglial activation induced by chronic morphine treatment. Conclusions:This study revealed for the first time that spinal neuronal MCP-1 is a key mediator of the spinal microglial activation and that spinal MCP-1 is involved in morphine antinociceptive tolerance. Inhibition of MCP-1 may provide a new therapy for morphine tolerance management.

Cyclooxygenase mediates cardioprotection of angiotensin-(1-7) against ischemia/reperfusion-induced injury through the inhibition of oxidative stress

Angiotensin (Ang)-(1-7) exhibits cardioprotective effects in myocardial ischemia reperfusion (I/‌R)-induced injury. However, the roles of oxidation and cyclooxygenase (COX) in the cardioprotection of Ang-(1-7) remain unclear. This study was conducted to investigate whether oxidation and COX were involved in the cardioprotection of Ang-(1-7) against I/‌R-induced injury in isolated rat hearts. The hearts were subjected to 15 min regional ischemia followed by 30 min reperfusion. Myocardial I/‌R treatment induced significant cardiac dysfunction, including ventricular arrhythmia (VA) and a reduction of left ventricular systolic pressure (LVSP), cardiomyocyte apoptosis and oxidative stress, manifesting as an increase in malondialdehyde (MDA) production and a decrease in superoxide dismutase (SOD) activity. Pretreatment of the hearts with 1.0 nmol/‌l Ang-(1-7) for 30 min prior to ischemia considerably attenuated I/‌R-induced VA, apoptosis and MDA production, and enhanced LVSP and SOD activity. These cardioprotective effects of Ang-(1-7) were antagonized by the intraperitoneal injection of 5 mg/‌kg body weight indomethacin (IDM, a COX inhibitor), presenting as an enhancement of VA, apoptosis and MDA production as well as a reduction of LVSP and SOD activity. In conclusion, COX mediated Ang-(1-7)-induced cardioprotection via its antioxidative mechanism.

A Novel Role of Spinal Astrocytic Connexin 43: Mediating Morphine Antinociceptive Tolerance by Activation of NMDA Receptors and Inhibition of Glutamate Transporter‐1 in Rats

Connexin 43 (Cx43) has been reported to be involved in neuropathic pain, but whether it contributes to morphine antinociceptive tolerance remains unknown. The present study investigated the role of spinal Cx43 in the development of morphine tolerance and its mechanisms in rats.

NUCLEAR FACTOR‐κB MEDIATES CYTOPROTECTION OF HYDROGEN PEROXIDE PRECONDITIONING AGAINST APOPTOSIS INDUCED BY OXIDATIVE STRESS IN PC12 CELLS

1 Cytoprotection by H2O2 preconditioning against oxidative stress‐induced apoptosis of PC12 cells has been demonstrated previously. In the present study, we investigated the effects of H2O2 preconditioning on nuclear factor (NF)‐κB activation and the role of NF‐κB in the adaptive cytoprotection of H2O2 preconditioning in PC12 cells. 2 The PC12 cells were preconditioned with 100 µmol/L H2O2 for 90 min, followed by 24 h recovery and subsequent exposure to 300 µmol/L H2O2 for a further 12 h. 3 The results showed that preconditioning with 100 µmol/L H2O2 upregulated NF‐κB expression and enhanced its nuclear translocation and DNA binding activity. In addition to its own effects on NF‐κB expression, H2O2 preconditioning also promoted the overexpression of NF‐κB induced by a lethal concentration of H2O2 (300 µmol/L). 4 N‐Tosyl‐l‐phenylalanine chloromethyl ketone (TPCK; 20 µmol/L), an inhibitor of NF‐κB, was administered 20 min before preconditioning with 100 µmol/L H2O2. At this concenteration, TPCK blocked the overexpression of NF‐κB induced by H2O2 preconditioning, accompanied by attenuation of H2O2 preconditioning‐induced cytoprotection. The inhibition of NF‐κB by TPCK enhanced caspase 3 activity induced by 300 µmol/L H2O2. 5. The findings of the present study provide novel evidence for the effects of preconditioning with H2O2 on constitutive activation of NF‐κB, which contributes to the adaptive cytoprotection of H2O2 preconditioning against PC12 cells apoptosis.

Protective effects of early hypoxic post-conditioning in cultured cortical neurons

Primary objective: Recent evidence suggests that delayed hypoxic post-conditioning is neuroprotective. The aim of the present study was to test whether early post-conditioning applied immediately after hypoxia could protect cultured neurons from hypoxia/reoxygenation (H/R)-induced injuries. Methods: Primary cortical neuronal culture depleted of microglia was exposed to H/R. Post-conditioning started immediately after hypoxia and consisted of three cycles of 15-minutes of reoxygenation and 15-minutes of hypoxia. Cell viability assay was performed using Cell Counting Kit-8 (CCK-8). Apoptosis was evaluated by Hoechst 33258 staining, FITC-Annexin V/PI double staining and Western blot assay (testing the cleaved caspase-3 expression). Reactive oxygen species (ROS), intracellular Ca2+ and mitochondrial membrane potential (MMP) were examined using confocal laser-scanning microscopy. Main results: H/R significantly reduced cell viability and increased neuronal apoptosis and necrosis. Furthermore, the expression of cleaved caspase-3, ROS production and intracellular Ca2+ were increased. MMP was attenuated. Injuries induced by H/R were substantially attenuated by early hypoxic post-conditioning. Changes in cleaved caspase-3 expression, ROS production, intracellular Ca2+ level and MMP in response to H/R were significantly decreased by the post-conditioning. Conclusions: The findings demonstrated that early hypoxic post-conditioning could protect neurons against H/R-induced injuries independent of microglial cells, possibly by inhibiting ROS over-production and intracellular Ca2+ accumulation and maintaining MMP.