Cinobufotalin Induces Ferroptosis to Suppress Lung Cancer Cell Growth by lncRNA LINC00597/hsa-miR-367-3p/TFRC Pathway via Resibufogenin.

BACKGROUND: Lung cancer is the leading cause of cancer-associated death and the first most diagnosed cancer in the world. More than 2 million new cases are diagnosed and 1.6 million people die due to lung cancer every year. It is urgent to explore novel drugs and approaches for lung cancer treatment. Cinobufotalin is a TCM isolated from dried toad venom, which has been used to treat lung cancer. However, the precise mechanism remains unclear. OBJECTIVE: This study was to investigate the mechanism of cinobufotalin treated in lung cancer. METHODS: Cell growth was identified by Cell Counting Kit-8 (CCK-8) assay. Besides, ferroptosis of lung cancer cells was determined by intracellular iron content, lactate dehydrogenase (LDH) release and mitochondrial membrane potential. Moreover, RNA levels and proteins were detected by quantitative reverse transcription-PCR (qRT-PCR) and Western blot (WB), respectively. In addition, the regulatory effect of hsa-miR-367-3p on TFRC was confirmed by luciferase reporter assay. RESULTS: This study indicated that cinobufotalin suppressed lung cancer cell growth through resibufogenin. Besides, cinobufotalin induced ferroptosis in lung cancer cells through resibufogenin. Moreover, cinobufotalin increased lncRNA LINC00597 level, whereas it downregulated hsa-miR-367-3p expression in lung cancer cells via resibufogenin. In addition, ferroptosis inducer transferrin receptor (TFRC) was the target of hsa-miR-367-3p, and lncRNA LINC00597 upregulates TFRC expression through sponging hsa-miR-367-3p in lung cancer cells. CONCLUSION: In summary, this study indicated that cinobufotalin induced ferroptosis to suppress lung cancer cell growth by lncRNA LINC00597/hsa-miR-367-3p/TFRC pathway via resibufogenin might provide novel therapeutic targets for lung cancer therapy.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs.

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.

Surface-Enhanced Raman Scattering Based on Controllable-Layer Graphene Shells Directly Synthesized on Cu Nanoparticles for Molecular Detection.

Graphene shells with a controllable number of layers were directly synthesized on Cu nanoparticles (CuNPs) by chemical vapor deposition (CVD) to fabricate a graphene-encapsulated CuNPs (G/CuNPs) hybrid system for surface-enhanced Raman scattering (SERS). The enhanced Raman spectra of adenosine and rhodamine 6G (R6G) showed that the G/CuNPs hybrid system can strongly suppress background fluorescence and increase signal-to-noise ratio. In four different types of SERS systems, the G/CuNPs hybrid system exhibits more efficient SERS than a transferred graphene/CuNPs hybrid system and pure CuNPs and graphene substrates. The minimum detectable concentrations of adenosine and R6G by the G/CuNPs hybrid system can be as low as 10(-8) and 10(-10) M, respectively. The excellent linear relationship between Raman intensity and analyte concentration can be used for molecular detection. The graphene shell can also effectively prevent surface oxidation of Cu nanoparticles after exposure to ambient air and thus endow the hybrid system with a long lifetime. This work provides a basis for the fabrication of novel SERS substrates.

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.

AIMS: 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. METHODS: Morphine tolerance was induced by intrathecal (i.t.) administration of morphine (15 µg) daily for seven consecutive days. The analgesia effect was assessed by hot-water tail-flick test. Expression of proteins was detected by Western blot and immunohistochemistry assay. RESULTS: Chronic morphine markedly increased the expression of spinal Cx43. Gap26, a specific Cx43 mimic peptide, attenuated not only morphine antinociceptive tolerance, but also the up-regulation of spinal Cx43 expression, the activation of astrocytes, and N-methyl-D-aspartic acid (NMDA) receptors (NR1 and NR2B subunits), as well as the decreased GLT-1 expression induced by chronic morphine. MK-801, a noncompetitive NMDA receptors antagonist, suppressed the chronic morphine-induced spinal Cx43 up-regulation, astrocytes activation and decline of GLT-1 expression. CONCLUSIONS: The spinal astrocytic Cx43 contributes to the development of morphine antinociceptive tolerance by activating astrocytes and NMDA receptors, and inhibiting GLT-1 expression. We also demonstrate that the role of interaction between the spinal astrocytic Cx43 and neuronal NMDA receptors is important in morphine tolerant rats.

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-α (TNF-α) 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-α. 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.

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-1ß (IL-1ß) 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-1ß 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.

Interaction between ROS and p38MAPK contributes to chemical hypoxia-induced injuries in PC12 cells.

The present study investigated whether there is an interaction between reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK) during chemical hypoxia-induced injury in PC12 cells. The results of the present study showed that cobalt chloride (CoCl2), a chemical hypoxia agent, markedly induced ROS generation and phosphorylation of p38MAPK, as well as neuronal injuries. N-acetylcysteine (NAC), a ROS scavenger, blocked CoCl2-induced phosphorylation of p38MAPK. In addition, SB203580, an inhibitor of p38MAPK attenuated not only CoCl2-induced activation of p38MAPK, but also ROS production. These results suggest that ROS and p38MAPK are capable of interacting positively during chemical hypoxia. Furthermore, NAC and SB203580 markedly prevented CoCl2-induced cytotoxicity, apoptosis and a loss of mitochondrial membrane potential. Taken together, our findings suggest that the positive interaction between CoCl2 induction of ROS and p38MAPK activation may play a significant role in CoCl2-induced neuronal injuries. We provide new insights into the mechanisms responsible for CoCl2-induced injuries in PC12 cells.

Inhibition of ROS-activated ERK1/2 pathway contributes to the protection of H2S against chemical hypoxia-induced injury in H9c2 cells.

Hydrogen sulfide (H(2)S) has been shown to exert cardioprotective effects. However, the roles of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in H(2)S-induced cardioprotection have not been completely elucidated. In this study, cobalt chloride (CoCl(2)), a chemical hypoxia mimetic agent, was applied to treat H9c2 cells to establish a chemical hypoxia-induced cardiomyocyte injury model. The results showed that pretreatment with NaHS (a donor of H(2)S) before exposure to CoCl(2) attenuated the decreased cell viability, the increased apoptosis rate, the loss of mitochondrial membrane potential (ΔΨm), and the intracellular accumulation of reactive oxygen species (ROS) in H9c2 cells. Exposure of H9c2 cells to CoCl(2) or hydrogen peroxide (H(2)O(2)) upregulated expression of phosphorylated (p) ERK1/2, which was reduced by pretreatment with NaHS or N-acetyl-L-cysteine, a ROS scavenger. More importantly, U0126, a selective inhibitor of ERK1/2, mimicked the above cytoprotection of H(2)S against CoCl(2)-induced injury in H9c2 cells. In conclusion, these results indicate that H(2)S protects H9c2 cells against chemical hypoxia-induced injury partially by inhibiting ROS-mediated activation of ERK1/2.

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

The roles of hydrogen sulfide (H(2)S) and endoplasmic reticulum (ER) stress in doxorubicin (DOX)-induced cardiotoxicity are still unclear. This study aimed to dissect the hypothesis that H(2)S 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 H(2)S, accompanied by the decreased cell viability and the increased reactive oxygen species (ROS) accumulation. In addition, exposure of cells to H(2)O(2) (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 H(2)S 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 H(2)S can confer protection against DOX-induced cardiotoxicity partly through inhibition of ER stress.

Hydrogen sulfide protects against chemical hypoxia-induced injury by inhibiting ROS-activated ERK1/2 and p38MAPK signaling pathways in PC12 cells.

Hydrogen sulfide (H(2)S) has been proposed as a novel neuromodulator and neuroprotective agent. Cobalt chloride (CoCl(2)) is a well-known hypoxia mimetic agent. We have demonstrated that H(2)S protects against CoCl(2)-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 H(2)S against chemical hypoxia-induced injuries of PC12 cells is not understood. We observed that CoCl(2) induced expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α), decreased cystathionine-ß synthase (CBS, a synthase of H(2)S) 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 H(2)S) or N-acetyl-L cystein (NAC), a ROS scavenger. CoCl(2) 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 CoCl(2)-induced injuries. Pretreatment with NaHS or NAC inhibited not only CoCl(2)-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 CoCl(2)-induced injuries and that H(2)S 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.

Hydrogen sulfide protects against chemical hypoxia-induced cytotoxicity and inflammation in HaCaT cells through inhibition of ROS/NF-κB/COX-2 pathway.

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

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.

Heat shock protein 90 mediates cytoprotection by H2S against chemical hypoxia-induced injury in PC12 cells.

1. Increasing evidence indicates that hydrogen sulphide (H2S) may serve as an important biological cytoprotective agent. Heat shock protein (Hsp) 90 can attenuate stress-induced injury. However, whether Hsp90 mediates the cytoprotective effect of H2S against chemical hypoxia-induced injury in PC12 cells is not known. 2. In the present study, CoCl2 (a chemical hypoxia mimetic) was used to treat PC12 cells to create a model of chemical hypoxia. To explore the role of Hsp90 in the cytoprotection afforded by H2S against chemical hypoxia-induced injury, 2 µmol/L 17-allylaminogeldanamycin (17-AAG), a selective inhibitor of Hsp90, was administered for 30 min prior to preconditioning with 400 µmol/L NaHS, followed by chemical hypoxia. 3. Cobalt chloride reduced cell viability (by 52.7 ± 1.5%), increased PC12 cell apoptosis (by 42.1 ± 1.5%), induced reactive oxygen species (ROS) by 3.79% compared with control and induced the dissipation of mitochondrial membrane potential (MMP) by 2.56% compared with control. 4. Pretreatment of PC12 cells with 100-400 µmol/L sodium hydrosulphide (NaHS), an H2S donor, for 3 h prior to exposure to 600 µmol/L CoCl2 provided significant, concentration-dependant protection to PC12 cells against CoCl2-induced cytotoxicity. Specifically, pretreatment of PC12 cells with 400 µmol/L NaHS decreased apoptosis to 16.77 ± 1.77% and blocked the CoCl2-induced increase in ROS production and loss of MMP. 5. At 400 µmol/L, NaHS upregulated Hsp90 in a time-dependant manner (over the period 0-180 min). In addition to its effects on Hsp90 expression, NaHS pretreatment of PC12 cells augmented the overexpression of Hsp90 induced by 600 µmol/L CoCl2 by 1.38-fold (P < 0.01). 6. Treatment of PC12 cells with 2 µmol/L 17-AAG for 30 min prior to NaHS pretreatment blocked the overexpression of Hsp90 induced by NaHS preconditioning, as evidenced by decreased cell viability (by 54.2 + 1.2%; P < 0.01), increased PC12 cell apoptosis (by 36.6 ± 1.2%; P < 0.01) and increasing ROS production. 7. The findings of the present study provide novel evidence that Hsp90 mediates H2S-induced neuroprotection against chemical hypoxia-induced injury via anti-oxidant and anti-apoptotic effects.

[Reactive oxygen species scavenger protects cardiac cells against injuries induced by chemical hypoxia].

OBJECTIVE: To investigate the protective effect of reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), against H9c2 cardiomyocytes from injuries induced by chemical hypoxia. METHODS: H9c2 cells were treated with cobalt chloride (CoCl2), a chemical hypoxia-mimetic agent, to establish the chemical hypoxia-induced cardiomyocyte injury model. NAC was added into the cell medium 60 min prior to CoCl2 exposure. The cell viability was evaluated using cell counter kit (CCK-8), and the intercellular ROS level was measured by 2', 7'- dichlorfluorescein-diacetate (DCFH-DA) staining and photofluorography. Mitochondrial membrane potential (MMP) of the cells was observed by Rhodamine123 (Rh123) staining and photofluorography, and the ratio of GSSG/ (GSSG+GSH) was calculated according to detection results of the GSSG kit. RESULTS: Exposure of H9c2 cardiomyocytes to 600 micromol/L CoCl2 for 36 h resulted in significantly reduced cell viability. Pretreatment with NAC at the concentrations ranging from 500 to 2000 micromol/L 60 min before CoCl2 exposure dose-dependently inhibited CoCl2-induced H9c2 cell injuries, and obviously increased the cell viability. NAC at 2000 micromol/L obviously inhibited the oxidative stress induced by CoCl2, decreased the ratio of GSSG/(GSSG+GSH), increased ROS level, and antagonized CoCl2-induced inhibition on MMP. CONCLUSION: NAC offers obvious protective effect on H9c2 cardiomyocytes against injuries induced by chemical hypoxia by decreasing in the ratio of GSSG/(GSSG+GSH) and ROS level and ameliorating MMP.

Nuclear factor-kappaB mediates cytoprotection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells.

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

Inducible nitric oxide synthase and cyclooxgenase-2 mediate protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells.

The induction of inducible nitric oxide synthase (iNOS) in response to different stress is associated with simultaneous induction of cyclooxygenase-2 (COX-2) in various cell types. Both iNOS and COX-2 have been reported to mediate the late phase of cardioprotection induced by different preconditioning. However, whether both iNOS and COX-2 are mediators in the neuroprotection induced by preconditioning with hydrogen peroxide (H(2)O(2)) at low concentration is unknown. In this study, using the neurosecretory cell line-PC12 cells to set up the model of neuroprotection of preconditioning with H(2)O(2) against apoptosis, we first investigate what changes in expression of iNOS and COX-2 appear during H(2)O(2) preconditioning, then determine if both iNOS inhibitor and COX-2 inhibitor interfere with the neuroprotection elicited by preconditioning with H(2)O(2). We found that preconditioning with H(2)O(2) at 10 microM significantly protected PC12 cells against apoptosis induced by lethal H(2)O(2) (50 microM) and increased the expression of iNOS and COX-2 and that selective iNOS inhibitor, aminoguanidine (AG) and COX-2 inhibitor, NS-398 obviously blocked the protective effects induced by preconditioning with 10 microM H(2)O(2). The results of this study suggest that both iNOS and COX-2 are mediators of the neuroprotection induced by preconditioning with oxidative stress (H(2)O(2) at low concentration) in PC12 cells.

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 peroxide preconditioning protects PC12 cells against apoptosis induced by oxidative stress.

Oxidative stress can induce significant cell death by apoptosis. We explore whether prior exposure to H2O2 (H2O2 preconditioning) protects PC12 cells against the apoptotic consequences of subsequent oxidative damages and what role the ATP-sensitive potassium (K(ATP)) channels play in the preconditioning protection. PC12 cells were preconditioned with 90 min exposure to H2O2 at 10 micromol/L, followed by 24-h recovery and subsequent exposures to different concentrations (20, 30, 50 and 100 micromol/L) of H2O2 for 24 h respectively. We used PI staining flow cytometry (FCM) to observe the apoptosis of PC12 cells. It was shown that 24-h exposures to H2O2 at 20, 30, 50 and 100 micromol/L respectively induced substantial cell apoptosis, which was greatly prevented in the preconditioning cells, indicating that H2O2 preconditioning protected PC12 cells against apoptosis induced by H2O2. Administration of pinacidil (10 micromol/L), an K(ATP) channel activator, significantly attenuated the apoptosis of PC12 cells induced by H2O2 at 30 and 50 micromol/L for 24 h respectively. Glybenclamide (10 micromol/L), a K(ATP) channel inhibitor, significantly suppressed or abolished the protective effects caused by the pinacidil but not by H2O2 preconditioning. However, when both H2O2 preconditioning and pinacidil were co-applied, their protection against the apoptosis of PC12 cells was much stronger than that of the individual one of them. These results suggest that H2O2 preconditioning protects PC12 cells against apoptosis and that the activation of K(ATP) channels is not involved in, but synergetically enhances adaptive protection of H2O2 preconditioning.