Pei-Xi Chen

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.

Curcumin protects PC12 cells against 1-methyl-4-phenylpyridinium ion-induced apoptosis by bcl-2-mitochondria-ROS-iNOS pathway

The aim of present study is to explore the cytoprotection of curcumin against 1-methyl-4-phenylpridinium ions (MPP+)-induced apoptosis and the molecular mechanisms underlying in PC12 cells. Our findings indicated that MPP+ significantly reduced the cell viability and induced apoptosis of PC12 cells. Curcumin protected PC12 cells against MPP+-induced cytotoxicity and apoptosis not only by inducing overexpression of Bcl-2, but also reducing the loss of mitochondrial membrane potential (MMP), an increase in intracellular reactive oxygen species (ROS) and overexpression of inducible nitric oxide synthase (iNOS). The selective iNOS inhibitor AG partly blocked MPP+-induced apoptosis of PC12 cells. The results of present study suggested that the cytoprotective effects of curcumin might be mediated, at least in part, by the Bcl-2-mitochondria-ROS-iNOS pathway. Because of its non-toxic property, curcumin could be further developed to treat the neurodegenerative diseases which are associated with oxidative stress, such as Parkinson’s disease (PD).

Hydrogen Sulfide Protects against Chemical Hypoxia-Induced Cytotoxicity and Inflammation in HaCaT Cells through Inhibition of ROS/NF-κB/COX-2 Pathway

Hydrogen sulfide (H2S) has been shown to protect against oxidative stress injury and inflammation in various hypoxia-induced insult models. However, it remains unknown whether H2S protects human skin keratinocytes (HaCaT cells) against chemical hypoxia-induced damage. In the current study, HaCaT cells were treated with cobalt chloride (CoCl2), a well known hypoxia mimetic agent, to establish a chemical hypoxia-induced cell injury model. Our findings showed that pretreatment of HaCaT cells with NaHS (a donor of H2S) for 30 min before exposure to CoCl2 for 24 h significantly attenuated CoCl2-induced injuries and inflammatory responses, evidenced by increases in cell viability and GSH level and decreases in ROS generation and secretions of IL-1β, IL-6 and IL-8. In addition, pretreatment with NaHS markedly reduced CoCl2-induced COX-2 overexpression and PGE2 secretion as well as intranuclear NF-κB p65 subunit accumulation (the central step of NF-κB activation). Similar to the protective effect of H2S, both NS-398 (a selective COX-2 inhibitor) and PDTC (a selective NF-κB inhibitor) depressed not only CoCl2-induced cytotoxicity, but also the secretions of IL-1β, IL-6 and IL-8. Importantly, PDTC obviously attenuated overexpression of COX-2 induced by CoCl2. Notably, NAC, a ROS scavenger, conferred a similar protective effect of H2S against CoCl2-induced insults and inflammatory responses. Taken together, the findings of the present study have demonstrated for the first time that H2S protects HaCaT cells against CoCl2-induced injuries and inflammatory responses through inhibition of ROS-activated NF-κB/COX-2 pathway.

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 Sulfide Protects against Chemical Hypoxia-Induced Injury by Inhibiting ROS-Activated ERK1/2 and p38MAPK Signaling Pathways in PC12 Cells

Hydrogen sulfide (H2S) has been proposed as a novel neuromodulator and neuroprotective agent. Cobalt chloride (CoCl2) is a well-known hypoxia mimetic agent. We have demonstrated that H2S protects against CoCl2-induced injuries in PC12 cells. However, whether the members of mitogen-activated protein kinases (MAPK), in particular, extracellular signal-regulated kinase1/2(ERK1/2) and p38MAPK are involved in the neuroprotection of H2S against chemical hypoxia-induced injuries of PC12 cells is not understood. We observed that CoCl2 induced expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α), decreased cystathionine-β synthase (CBS, a synthase of H2S) expression, and increased generation of reactive oxygen species (ROS), leading to injuries of the cells, evidenced by decrease in cell viability, dissipation of mitochondrial membrane potential (MMP) , caspase-3 activation and apoptosis, which were attenuated by pretreatment with NaHS (a donor of H2S) or N-acetyl-L cystein (NAC), a ROS scavenger. CoCl2 rapidly activated ERK1/2, p38MAPK and C-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 or p38MAPK or JNK with kinase inhibitors (U0126 or SB203580 or SP600125, respectively) or genetic silencing of ERK1/2 or p38MAPK by RNAi (Si-ERK1/2 or Si-p38MAPK) significantly prevented CoCl2-induced injuries. Pretreatment with NaHS or NAC inhibited not only CoCl2-induced ROS production, but also phosphorylation of ERK1/2 and p38MAPK. Thus, we demonstrated that a concurrent activation of ERK1/2, p38MAPK and JNK participates in CoCl2-induced injuries and that H2S protects PC12 cells against chemical hypoxia-induced injuries by inhibition of ROS-activated ERK1/2 and p38MAPK pathways. Our results suggest that inhibitors of ERK1/2, p38MAPK and JNK or antioxidants may be useful for preventing and treating hypoxia-induced neuronal injury.

Inhibition of neuronal nitric oxide synthase antagonizes morphine antinociceptive tolerance by decreasing activation of p38 MAPK in the spinal microglia

We have demonstrated that the activation of p38 mitogen-activated protein kinase (MAPK) in the spinal microglia played an essential role in the development of morphine antinociceptive tolerance. The aim of this study was to investigate whether inhibition of neuronal nitric oxide synthase (nNOS) attenuated tolerance to morphine analgesia by modulating p38 activation in the spinal microglia. It was shown that the selective inhibitor of nNOS, 7-NINA (7-Nitroindazole, sodium salt) (25 microg, i.t.) attenuated not only the development of morphine antinociceptive tolerance, but also the activation of p38 MAPK in the spinal microglia induced by chronic intrathecal administration of morphine. Our results suggest that neuronal NO signals to microglia, leading to the upregulation of microglial phospho-p38 MAPK. Such p38 MAPK activation in microglia is consistent with a potential role in the development of morphine antinociceptive tolerance. We demonstrated for the first time that the inhibition of nNOS attenuated morphine antinociceptive tolerance by reducing p38 MAPK activation in the spinal microglia.

Exogenous Hydrogen Sulfide Protects against Doxorubicin-Induced Inflammation and Cytotoxicity by Inhibiting p38MAPK/NFκB Pathway in H9c2 Cardiac Cells

Background/Aim:We have demonstrated that exogenous hydrogen sulfide (H2S) protects H9c2 cardiac cells against the doxorubicin (DOX)-induced injuries by inhibiting p38 mitogen-activated protein kinase (MAPK) pathway and that the p38 MAPK/nuclear factor-κB (NF-κB) pathway is involved in the DOX-induced inflammatory response and cytotoxicity. The present study attempts to test the hypothesis that exogenous H2S might protect cardiomyocytes against the DOX-induced inflammation and cytotoxicity through inhibiting p38 MAPK/NF-κB pathway. Methods: H9c2 cardiac cells were exposed to 5µM DOX for 24 h to establish a model of DOX cardiotoxicity. The cells were pretreated with NaHS( a donor of H2S) or other drugs before exposure to DOX. Cell viability was analyzed by cell counter kit 8 ( CCK-8), The expression of NF-κB p65 and inducible nitric oxide synthase (iNOS) was detected by Western blot assay. The levels of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-a (TNF-a) were tested by enzyme-linked immunosorbent assay (ELISA). Results: Our findings demonstrated that pretreatment of H9c2 cardiac cells with NaHS for 30 min before exposure to DOX markedly ameliorated the DOX-induced phosphorylation and nuclear translocation of NF-κB p65 subunit. Importantly, the pretreatment with NaHS significantly attenuated the p38 MAPK/NF-κB pathway-mediated inflammatory responses induced by DOX, as evidenced by decreases in the levels of IL-1ß, IL-6 and TNF-a. In addition, application of NaHS or IL-1ß receptor antagonist (IL-1Ra) or PDTC (an inhibitor of NF-κB) attenuated the DOX-induced expression of iNOS and production of nitric oxide (NO), respectively. Furthermore, IL-1Ra also dramatically reduced the DOX-induced cytotoxicity and phosphorylation of NF-κB p65. The pretreatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS) prior to exposure to DOX depressed the phosphorylation of NF-κB p65 induced by DOX. Conclusion: The present study has demonstrated the new mechanistic evidence that exogenous H2S attenuates the DOX-induced inflammation and cytotoxicity by inhibiting p38 MAPK/NF-κB pathway in H9c2 cardiac cells. We also provide novel data that the interaction between NF-κB pathway and IL-1ß is important in the induction of DOX-induced inflammation and cytotoxicity in H9c2 cardiac cells.

Hydrogen sulfide protects H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of endoplasmic reticulum stress

The roles of hydrogen sulfide (H2S) and endoplasmic reticulum (ER) stress in doxorubicin (DOX)-induced cardiotoxicity are still unclear. This study aimed to dissect the hypothesis that H2S could protect H9c2 cells against DOX-induced cardiotoxicity by inhibiting ER stress. Our results showed that exposure of H9c2 cells to DOX significantly inhibited the expression and activity of cystathionine-γ-lyase (CSE), a synthetase of H2S, accompanied by the decreased cell viability and the increased reactive oxygen species (ROS) accumulation. In addition, exposure of cells to H2O2 (an exogenous ROS) mimicked the inhibitory effect of DOX on the expression and activity of CSE. Pretreatment with N-acetyl-l-cysteine (NAC) (a ROS scavenger) attenuated intracellular ROS accumulation, cytotoxicity, and the inhibition of expression and activity of CSE induced by DOX. Notably, the ER stress-related proteins, including glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were obviously upregulated in DOX-treated H9c2 cells. Pretreatment with sodium hydrosulfide (NaHS, a H2S donor) before DOX exposure markedly suppressed DOX-induced overexpressions of GRP78 and CHOP, cytotoxicity and oxidative stress. In conclusion, we have demonstrated that ROS-mediated inhibition of CSE is involved in DOX-induced cytotoxicity in H9c2 cells, and that exogenous H2S can confer protection against DOX-induced cardiotoxicity partly through inhibition of ER stress.

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.

Activation of the p38 MAPK/NF-κB pathway contributes to doxorubicin-induced inflammation and cytotoxicity in H9c2 cardiac cells.

A number of studies have demonstrated that inflammation plays a role in doxorubicin (DOX)-induced cardiotoxicity. However, the molecular mechanism by which DOX induces cardiac inflammation has yet to be fully elucidated. The present study aimed to investigate the role of the p38 mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) pathway in DOX-induced inflammation and cytotoxicity. The results of our study demonstrated that the exposure of H9c2 cardiac cells to DOX reduced cell viability and stimulated an inflammatory response, as demonstrated by an increase in the levels of interleukin-1β (IL-1β) and IL-6, as well as tumor necrosis factor-α (TNF-α) production. Notably, DOX exposure induced the overexpression of phosphorylated p38 MAPK and phosphorylation of the NF-κB p65 subunit, which was markedly inhibited by SB203580, a specific inhibitor of p38 MAPK. The inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-κB, significantly ameliorated DOX-induced inflammation, leading to a decrease in the levels of IL-1β and IL-6, as well as TNF-α production in H9c2 cells. The pretreatment of H9c2 cells with either SB203580 or PDTC before exposure to DOX significantly attenuated DOX-induced cytotoxicity. In conclusion, our study provides novel data demonstrating that the p38 MAPK/NF-κB pathway is important in the induction of DOX-induced inflammation and cytotoxicity in H9c2 cardiac myocytes.