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Recently, a novel mechanism known as 'programmed necrosis' or necroptosis has been shown to be another important mechanism of cell death in the heart. In this study, we investigated the role of necroptosis in high glucose (HG)-induced injury and inflammation, as well as the underlying mechanisms. In particular, we focused on the interaction between necroptosis and reactive oxygen species (ROS) in H9c2 cardiac cells. Our results demonstrated that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) markedly enhanced the expression level of receptor-interacting protein 3 (RIP3), a kinase which promotes necroptosis. Importantly, co-treatment of the cells with 100 µM necrostatin-1 (a specific inhibitor of necroptosis) and HG for 24 h attenuated not only the increased expression level of RIP3, but also the HG-induced injury and inflammation, as evidenced by an increase in cell viability, a decrease in ROS generation, the attenuation of the dissipation of mitochondrial membrane potential and a decrese in the secretion levels of inflammatory cytokines, i.e., interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Furthermore, treatment of the cells with 1 mM N-acetyl‑L‑cysteine (a scavenger of ROS) for 60 min prior to exposure to HG significantly reduced the HG-induced increase in the RIP3 expression level, as well as the injury and inflammatory response described above. Taken together, the findings of this study clearly demonstrate a novel damage mechanism involving the positive interaction between necroptosis and ROS attributing to HG-induced injury and inflammation in H9c2 cardiac cells.

It has been reported that exogenous hydrogen sulfide (H2S) protects against high glucose (HG)-induced cardiac injury and has a modulatory effect on heat shock protein (HSP) and Akt, which play a cardioprotective role. In this study, we examined whether the HSP90/Akt pathway contributes to the protective effects of exogenous H2S against HG-induced injury to H9c2 cardiac cells. Our results revealed that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) for 1 to 24 h decreased the expression of HSP90 and markedly reduced the expression level of phosphorylated (p)-Akt in a time-dependent manner. Co-exposure of the cells to HG and geldanamycin (GA; an inhibitor of HSP90) aggravated the inhibition of the p-Akt expression level by HG. Of note, treatment of the cells with 400 µM NaHS (a donor of H2S) for 30 min prior to exposure to HG significantly attenuated the HG-induced decrease in the expression levels of both HSP90 and p-Akt, along with inhibition of HG-induced cell injury, as indicated by the increase in cell viability and superoxide dismutase (SOD) activity, and by a decrease in the number of apoptotic cells, reactive oxygen species (ROS) generation, as well as by the decreased dissipation of mitochondrial membrance potential (MMP). Importantly, treatment of the cells with GA or LY294002 (an inhibitor of Akt) prior to exposure to NaHS and HG considerably blocked the cardioprotective effects of NaHS against the HG-induced injury mentioned above. On the whole, the findings of this study demonstrate that the inhibition of the HSP90/Akt pathway may be an important mechanism responsible for HG-induced cardiomyocyte injury. We also provide novel evidence that exogenous H2S protects H9c2 cells against HG-induced injury by activating the HSP90/Akt pathway.

Tolerance to hypoxia can be induced by reducing oxygen consumption. Dexmedetomidine (DEX) decreases locomotor activity and induces bradycardia and hypothermia in mice. The present study examined the hypothesis that DEX improves hypoxia tolerance in mice. Adult mice received an intraperitoneal injection of 1, 5, 10, 20, 40, 80, 160 or 320 µg/kg DEX, 20 mg/kg propranolol or saline. Acute hypoxic conditions were induced by placing the mice in a limited enclosed container with soda lime. Core body temperature (CBT) and heart rate (HR) were measured prior to and 30 min after drug administration. Survival time was monitored in the sealed container. Survival times (mean ± standard deviation) of mice in the saline, 1, 5, 10, 20, 40, 80, 160 and 320 µg/kg DEX, and the 20 mg/kg propranolol groups were 22.4±1.1, 23.4±1.1, 26.0±0.9, 36.9±5.2, 42.4±2.9, 43.2±2.3, 58.2±4.2, 80.5±4.0, 79.2±6.0, and 38.2±2.8 min, respectively. Pretreatment with propranolol and 10, 20, 40, 80, 160 or 320 µg/kg DEX, but not 1 or 5 µg/kg, significantly prolonged survival time compared with saline‑injected mice (P

Dexmedetomidine has been reported to provide neuroprotection against hypoxia-induced damage. However, the underlying mechanisms remain unclear. We examined whether dexmedetomidine's neuroprotective effects were mediated by the NF-κB/COX-2 pathways. Adult male C57BL/6 mice were subjected to a 30-min hypoxic treatment followed by recovery to normal conditions. They received dexmedetomidine (16 or 160 μg/kg) or 25 mg/kg atipamezole, an α2-adrenoreceptor antagonist, intraperitoneally before exposure to hypoxia. The whole brain was harvested 6, 18, or 36 h after the hypoxia to determine the histopathological outcome and cleaved caspase-3, Bax/Bcl, NF-κB, and COX-2 levels. Hypoxia treatment induced significant neurotoxicity, including destruction of the tissue structure and upregulation of the protein levels of caspase-3, the ratio of Bax/Bcl-2, NF-κB, and COX-2. Dexmedetomidine pretreatment effectively improved histological outcome and restored levels of caspase-3, the Bax/Bcl-2 ratio, NF-κB, and COX-2. Atipamezole reversed the neuroprotection induced by dexmedetomidine. Neuroprotection was achieved by PDTC and NS-398, inhibitors of NF-κB and COX-2, respectively. Dexmedetomidine use before hypoxia provides neuroprotection. Inhibition of NF-κB/COX-2 pathways activation may contribute to the neuroprotection of dexmedetomidine.

SummaryBackground Dexmedetomidine decreases cardiac complications in adults undergoing cardiovascular surgery. This systematic review assessed whether perioperative dexmedetomidine improves congenital heart disease (CHD) surgery outcomes in children. Methods The PubMed, Embase, and Cochrane Library databases were searched for randomized controlled trials (RCTs) or observational studies that were published until 16 April 2015 and compared dexmedetomidine with placebo or an alternative anesthetic agent during pediatric CHD surgery. The assessed outcomes included hemodynamics, ventilation length, intensive care unit (ICU) and hospital stays, blood glucose and serum cortisol levels, postoperative analgesia requirements, and postoperative delirium. Results Five RCTs and nine observational studies involving 2229 patients were included. In pooled analyses, dexmedetomidine was associated with shorter length of mechanical ventilation (mean difference: −93.36, 95% CI: −137.45, −49.27), lower postoperative fentanyl (mean difference: −24.11, 95% CI: −36.98, −11.24) and morphine (mean difference: −0.07, 95% CI: −0.14, 0.00) requirements, reduced stress response (i.e., lower blood glucose and serum cortisol levels), and lower risk of delirium (OR: 0.39, 95% CI: 0.21, 0.74). The hemodynamics of dexmedetomidine-treated patients appeared more stable, but there were no significant differences in the ICU or hospital stay durations. Dexmedetomidine may increase the bradycardia and hypotension risk (OR: 3.14, 95% CI: 1.47, 6.69). Conclusions Current evidence indicates that dexmedetomidine improves outcomes in children undergoing CHD surgery. However, this finding largely relies on data from observational studies; high-quality RCTs are warranted because of the potential for subject selection bias.

Subsequently to the publication of this article, the authors have realized that the address affiliation for the corresponding author, Chengheng Hu, and the authors Longyun Peng and Xinxue Liao appeared incorrectly. These authors' affiliation information should have appeared as follows (the corrected address affiliation is featured in bold): XIAO KE1,2*, JINGFU CHEN3*, LONGYUN PENG4, WEI ZHANG5, YIYING YANG5, XINXUE LIAO4, LIQIU MO6, RUIXIAN GUO7, JIANQIANG FENG6, CHENGHENG HU4 and RUQIONG NIE2 1Department of Cardiology, Shenzhen Sun Yat‑sen Cardiovascular Hospital, Shenzhen; 2Department of Cardiology, Sun Yat‑sen Memorial Hospital, Sun Yat‑sen University, Guangzhou, Guangdong; 3Department of Cardiovascular Medicine and Dongguan Cardiovascular Institute, The Third People's Hospital of Dongguan City, Dongguan; 4Department of Cardiology and Key Laboratory on Assisted Circulation, Ministry of Health, The First Affiliated Hospital, Sun Yat‑sen University; 5Department of Cardiovasology and Cardiac Care Unit (CCU), Huangpu Division of The First Affiliated Hospital, Sun Yat‑sen University; 6Department of Anesthesiology, Huangpu Division of The First Affiliated Hospital, Sun Yat‑sen University; 7Department of Physiology, Zhongshan School of Medicine, Sun Yat‑sen University, Guangzhou, Guangdong, P.R. China *Contributed equally In addition, the address for correspondence in the correspondence box should have appeared as follows: Correspondence to: Professor Chengheng Hu, Department of Cardiology and Key Laboratory on Assisted Circulation, Ministry of Health, The First Affiliated Hospital, Sun Yat‑sen University, Guangdong, 58 Zhongshan 2rd Road, Guangzhou 510080, P.R. China E‑mail: huchengheng138@163.com The authors regret this error in the affiliations, and apologize for any inconvenience caused. [the original article was published in the International Journal of Molecular Medicine 39: 1001‑1010, 2017; DOI: 10.3892/ijmm.2017.2891].

Hyperglycemia has been reported to activate the nuclear factor-κB (NF-κB) pathway. We have previously demonstrated that exogenous hydrogen sulfide (H2S) protects cardiomyocytes against high glucose (HG)-induced injury by inhibiting the activity of p38 mitogen-activated protein kinase (MAPK), which can activate the NF-κB pathway and induce interleukin (IL)-1β production. In the present study, we aimed to investigate the hypothesis that exogenous H2S protects cardiomyocytes against HG-induced injury and inflammation through the inhibition of the NF-κB/IL-1β pathway. H9c2 cardiac cells were treated with 35 mM glucose (HG) for 24 h to establish a model of HG-induced damage. Our results demonstrated that treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) or 100 µM pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) for 30 min prior to exposure to HG markedly attenuated the HG-induced increase in the expression levels of the phosphorylated (p)-NF-κB p65 subunit. Notably, pre-treatment of the H9c2 cardiac cells with NaHS or PDTC significantly suppressed the HG-induced injury, including cytotoxicity, apoptosis, oxidative stress and mitochondrial insults, as evidenced by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). In addition, pre-treatment of the cells with NaHS or PDTC ameliorated the HG-induced inflammatory response, leading to a decrease in the levels of IL-1β, IL-6 and tumor necrosis factor-α (TNF-α). Importantly, co-treatment of the H9c2 cells with 20 ng/ml IL-1 receptor antagonist (IL-1Ra) and HG markedly reduced the HG-induced increase in p-NF-κB p65 expression, cytotoxicity, the number of apoptotic cells, as well as the production of TNF-α. In conclusion, the present study presents novel mechanistic evidence that exogenous H2S protects H9c2 cardiac cells against HG-induced inflammation and injury, including cytotoxicity, apoptosis, overproduction of ROS and the dissipation of MMP, by inhibiting the NF-κB/IL-1β pathway. We also provide new data indicating that the positive interaction between the NF-κB pathway and IL-1β is critical in HG-induced injury and inflammation 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.

Hypoxia and/or ischemia are implicated in neurodegenerative disorders. In these diseases, hypoxia/ischemia may induce oxidative stress, including production of reactive oxygen species (ROS), which result in a decrease in glutamate transporter expression. Hydrogen sulfide (H2S), as the third gasotransmitter, has neuroprotective effects and potent antioxidant properties. In the present study, we investigated the role of glutamate transporter-1 (GLT-1) in the protection of H2S against chemical hypoxia-induced injury in PC12 cells. We found that cobalt chloride (CoCl2), a chemical hypoxia agent, reduced the expression of GLT-1 in a time-dependent manner. Pretreatment with NaHS (a donor of H2S) reversed the CoCl2-induced downregulation of GLT-1 expression. Pretreatment with DHK (a selective inhibitor of GLT-1) for 30 min prior to NaHS preconditioning significantly inhibited the cytoprotection of H2S against CoCl2-induced injuries, leading to an increase in cytotoxicity and apoptosis as well as to a loss of mitochondrial membrane potential (MMP). In addition, we found that similar to the effect of NaHS, pretreatment with NAC (a ROS scavenger) or U0126 (a MEK1/2 inhibitor) blocked the downregulation of GLT-1 expression induced by CoCl2. Collectively, we demonstrated for the first time that ROS and extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated reduction of GLT-1 expression may be involved in chemical hypoxia-induced neural injury and that H2S attenuates this injury partly by upregulating GLT-1 expression in PC12 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.

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.

Hyperglycemia, as well as diabetes mellitus, has been shown to impair ATP-sensitive K+ (KATP) channels in human vascular smooth muscle cells. Hydrogen sulfide (H2S) is also known to be an opener of KATP channels. We previously demonstrated the cardioprotective effects exerted by H2S against high-glucose (HG, 35 mM glucose)-induced injury in H9c2 cardiac cells. As such, we hypothesized that KATP channels play a role in the cardioprotective effects of H2S against HG-induced injury. In this study, to examine this hypothesis, H9c2 cardiac cells were treated with HG for 24 h to establish a model of HG-induced insults. Our findings revealed that treatment of the cells with HG markedly decreased the expression level of KATP channels. However, the decreased expression of KATP channels was reversed by the treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) for 30 min prior to exposure to HG. Additionally, the HG-induced cardiomyocyte injuries, including cytotoxicity, apoptosis, oxidative stress and mitochondrial damage, were ameliorated by treatment with NaHS or 100 µM diazoxide (a mitochondrial KATP channel opener) or 50 µM pinacidil (a non-selective KATP channel opener) for 30 min prior to exposure to HG, as indicated by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). Notably, treatment of the H9c2 cardiac cells with 100 µM 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP channel blocker) or 1 mM glibenclamide (Gli, a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and exposure to HG significantly attenuated the above-mentioned cardioprotective effects exerted by NaHS. Notably, treatment of the cells with 500 µM N-acetyl‑L‑cysteine (NAC, a scavenger of ROS) for 60 min prior to exposure to HG markedly reduced the HG-induced inhibitory effect on the expression of KATP channels. Taken together, our results suggest that KATP channels play an important role in the cardioprotective effects of exogenous H2S against HG-induced injury. This study also provides novel data demonstraring that there is an antagonistic interaction between ROS and KATP channels in HG-exposed H9c2 cardiac cells.

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 μg, 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.

Summary 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.

Leptin, an adipokine synthesized mainly by non‑neuronal tissues, has been reported to contribute to the pathogenesis of neuropathic pain. It has been hypothesized that morphine tolerance and neuropathic pain share some common pathological mechanisms. The present study was designed to examine whether spinal leptin is implicated in the development of morphine antinociceptive tolerance, and whether spinal leptin induces the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathway and the NR1 subunit of N‑methyl‑D‑aspartate (NMDA) receptor, in morphine antinociceptive tolerance in rats. The results demonstrated that intrathecal (i.t.) administration of a leptin antagonist (LA) prevented the development of morphine antinociceptive tolerance in rats. Further studies revealed that the levels of the spinal leptin and the leptin receptor (Ob‑R) were time‑dependently increased following chronic morphine treatment. Mechanistic examination indicated that chronic morphine triggered activation of the STAT3 pathway and an increase in the expression of the NR1 subunit of the NMDA receptor, which was ameliorated by i.t. administration of AG490 [a Janus kinase (JAK)‑STAT inhibitor]. The increased activation of STAT3 and the NR1 subunit was markedly attenuated by i.t. treatment with LA. In addition, the spinal administration of AG490 or MK‑801 (a non‑competitive NMDA receptor inhibitor) blocked the development of morphine antinociceptive tolerance. Taken together, these results have demonstrated, for the first time, to the best of our knowledge, that spinal leptin contributes to the development of morphine antinociceptive tolerance by activating the spinal STAT3‑NMDA receptor pathway.

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 μg 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.

We previously demonstrated the protective effect of hydrogen sulfide (H2S) against doxorubicin (DOX)-induced cardiotoxicity through inhibition of endoplasmic reticulum stress. The aim of the present study was to explore the role of p38 mitogen-activated protein kinase (MAPK) in DOX-induced cardiotoxicity and ascertain whether exogenous H2S protects DOX-induced injury by inhibiting p38 MAPK in cardiomyoblasts (H9c2). We observed that exposure of H9c2 cells to 5 µM DOX not only markedly induced injuries, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and dissipation of mitochondrial membrane potential (MMP), but also enhanced the expression level of phosphorylated (p)-p38 MAPK. The DOX-induced increase in expression of p-p38 MAPK was significantly attenuated by pretreatment of H9c2 cells with either 400 µM sodium hydrogen sulfide (NaHS) (a donor of H2S) or 1,000 µM N-acetyl-L-cysteine (NAC, an ROS scavenger) prior to exposure to DOX. Pretreatment with either 400 µM NaHS or 3 µM SB203580, a selective inhibitor of p38 MAPK, ameliorated DOX-induced cardiomyocyte injuries, as evidenced by an increase in cell viability, and decreases in the number of apoptotic cells, ROS generation as well as dissipation of MMP. In conclusion, the findings of the present study demonstrated that the activation of p38 MAPK contributes to DOX-induced injuries, including cytotoxicity, apoptosis, mitochondrial damage and oxidative stress in H9c2 cells. We also provide novel evidence that exogenous H2S protects H9c2 cells against DOX-induced cardiotoxicity by inhibition of the p38 MAPK pathway.

Both the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and heme oxygenase-1 (HO-1) create a survival signal against oxidative stress-induced injuries. Although we have demonstrated that hydrogen peroxide (H2O2) preconditioning confers adaptive cytoprotection against oxidative stress-induced injury in PC12 cells, it remains unknown whether these defense systems are involved in the protective effect of H2O2 preconditioning. In the current study, PC12 cells were preconditioned with 100 µM H2O2 for 90 min, followed by 24 h recovery and subsequent exposure to 300 µM H2O2 for further 12 h. The findings showed that preconditioning with 100 µM H2O2 upregulated HO-1 expression. Zinc protoporphyrin IX (ZnPP), a selective inhibitor of HO-1, at a concentration of 15 µM, significantly attenuated H2O2 preconditioning-elicited cytotoxicity, apoptosis, oxidative stress and mitochondrial membrane potential (ΔΨm) loss in PC12 cells. In addition, H2O2 preconditioning enhanced phosphorylation of Akt. Treatment with 25 µM LY294002, a selective inhibitor of PI3K, for 20 min before H2O2 preconditioning blocked not only H2O2 preconditioning-induced HO-1 induction, but also the protective effect of H2O2 preconditioning against cytotoxicity. The present study provides novel evidence for the effect of preconditioning with H2O2 on the induction of HO-1, which contributes to the adaptive cytoprotection of H2O2 preconditioning against oxidative stress-induced cellular injury via a PI3K/Akt-dependent mechanism in PC12 cells.

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 (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-β 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.