Chemical Hypoxia-induced Injury Research Articles

Propofol protects human cardiac cells against chemical hypoxiainduced injury by regulating the JNK signaling pathways.

Propofol is a widely used intravenous anesthetic shown to exert a cardioprotective role against oxidative stress and ischemia/reperfusion injury in rat cardiac H9c2 cells. However, the regulatory mechanisms and functions of propofol in human cardiomyocytes remain unknown. The present study chemically induced hypoxia with cobalt chloride (CoCl2) to mimic cardiomyocyte ischemic injury in human cardiac AC16 and HCM cells. To investigate its underlying mechanisms, propofol was added to the cells before the chemical hypoxia phase. The present results suggested that, in response to hypoxia, mitochondrial membrane potential was lost, and cardiomyocyte viability and superoxide dismutase levels decreased. However, the present results showed that reactive oxygen species and malondialdehyde levels increased. The present results suggested that these effects were significantly reversed following propofol treatment. Additionally, the present results suggested that the protective effect of propofol against CoCl2-induced injury may be inhibited by the activation of the JNK signaling pathways. The present results indicated that propofol pre-treatment inhibited CoCl2-induced myocardial injury by preventing mitochondrial dysfunction, which may be partially due to the activation of the JNK signaling pathways. Therefore, propofol may exert anti-oxidative effects in human cardiac cells. The present results suggested that propofol may be used as a treatment for oxidative stress-related cardiac disorders.

Propofol protects human cardiac AC16 cells from CoCl2-induced hypoxic injury

To explore the effect of propofol on human cardiac AC16 cells under CoCl2-induced hypoxic injury and the possible mechanisms. Methods: Human AC16 cardiomyocytes were treated with cobalt chloride (CoCl2) to mimic hypoxic condition in cultured cardiomyocytes. The AC16 cells were divided into 3 groups: a control group, a CoCl2 hypoxia group (CoCl2 group), and a propofol+CoCl2 group (propofol+ CoCl2 group). The cell viability was assessed by cell counting kit-8 (CCK-8). Cell apoptosis ratio (AR) and the mitochondrial membrane potential (Δψm) were detected by flow cytometry. The reactive oxygen species (ROS) production in AC16 cells were determined with the ROS-sensitive fluorescent probe. Meanwhile, total intracellular levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in AC16 cells were detected with commercially available kits. Western blot was used to evaluate the activation of c-Jun N-terminal kinase (JNK) and p38 signaling pathways. Results: 1) Compared with the control group, AC16 cell viability was decreased significantly in the CoCl2 group following the treatment with 500 μmol/L CoCl2 (P<0.01); 2) Compared with the control group, AR value in AC16 cells was increased significantly in the CoCl2 group, while Δψm was decreased significantly (all P<0.01). Compared with the CoCl2 group, AR value in AC16 cells was decreased significantly in the propofol+CoCl2 group, while Δψm was increased significantly (both P<0.05); 3) Compared with the control group, the levels of ROS and MDA were increased significantly, and the level of SOD was significantly decreased in the CoCl2 group (all P<0.01). Compared with the CoCl2 group, the ROS and MDA levels in the propofol+CoCl2 group were increased significantly and the SOD levels were decreased significantly (all P<0.05); 4) Compared with the control group, the phosphorylation levels of JNK and p38 were increased significantly (both P<0.05) in the CoCl2 group. Compared with the CoCl2 group, the phosphorylation levels of JNK and p38 were decreased significantly in the propofol+CoCl2 group (both P<0.05). Conclusion: The pretreatment with propofol may protect human cardiac AC16 cells from the chemical hypoxia-induced injury through regulation of JNK and p38 signaling pathways.

Genistein Protects H9c2 Cardiomyocytes against Chemical Hypoxia-Induced Injury via Inhibition of Apoptosis

Background/Aims: Hypoxia can induce cell injury in cardiomyocytes and further lead to cardiovascular diseases. Genistein (Gen), the predominant isoflavone found in soy products, has shown protective effects on cardiovascular system. The aim of the present study was to investigate the cardioprotective effect of Gen against chemical hypoxia-induced injury. Methods: Cobalt chloride (CoCl₂) was administrated to trigger chemical hypoxia in H9c2 cardiomyocytes. Cell proliferation was detected by using MTT assay. The expression level of hypoxia-related proteins (hypoxia-inducible factor [HIF]-1α and Notch-1) and apoptosis-related proteins (B cell lymphoma [Bcl]-2, Bax, and caspase-3) were evaluated by Western blot analysis. Results: In response to hypoxia, cell viability was reduced dramatically, whereas the expression of HIF-1α was upregulated. Hypoxia also induced cardiomyocytes apoptosis by reducing the ratio of Bcl-2/Bax and increasing expression of caspase-3. Interestingly, Gen attenuated CoCl₂-induced cell death and suppressed HIF-1α expression, as well as upregulated the expression of Notch-1. Furthermore, Gen could antagonize CoCl₂-induced apoptosis through upregulating Bcl-2/Bax ratio and inhibiting caspase-3 expression. Conclusions: Gen prevents chemical hypoxia-induced cell apoptosis through inhibition of the mitochondrial apoptotic pathway, exerting protective effects on H9c2 cardiomyocytes.

Hydrogen Sulfide Protects against Chemical Hypoxia-Induced Injury via Attenuation of ROS-Mediated Ca2+ Overload and Mitochondrial Dysfunction in Human Bronchial Epithelial Cells.

Oxidative stress induced by hypoxia/ischemia resulted in the excessive reactive oxygen species (ROS) and the relative inadequate antioxidants. As the initial barrier to environmental pollutants and allergic stimuli, airway epithelial cell is vulnerable to oxidative stress. In recent years, the antioxidant effect of hydrogen sulfide (H2S) has attracted much attention. Therefore, in this study, we explored the impact of H2S on CoCl2-induced cell injury in 16HBE14o- cells. The effect of CoCl2 on the cell viability was detected by Cell Counting Kit (CCK-8) and the level of ROS in 16HBE14o- cells in response to varying doses (100–1000 μmol/L) of CoCl2 (a common chemical mimic of hypoxia) was measured by using fluorescent probe DCFH-DA. It was shown that, in 16HBE14o- cells, CoCl2 acutely increased the ROS content in a dose-dependent manner, and the increased ROS was inhibited by the NaHS (as a donor of H2S). Moreover, the calcium ion fluorescence probe Fura-2/AM and fluorescence dye Rh123 were used to investigate the intracellular calcium concentration ([Ca2+]i) and mitochondria membrane potential (MMP) in 16HBE14o- cells, respectively. In addition, we examined apoptosis of 16HBE14o- cells with Hoechst 33342. The results showed that the CoCl2 effectively elevated the Ca2+ influx, declined the MMP, and aggravated apoptosis, which were abrogated by NaHS. These results demonstrate that H2S could attenuate CoCl2-induced hypoxia injury via reducing ROS to perform an agonistic role for the Ca2+ influx and MMP dissipation.

Involvement of miR-455 in the protective effect of H2S against chemical hypoxia-induced injury in BEAS-2B cells

The protective effect of hydrogen sulfide (H2S) against hypoxia-induced injury via anti-apoptosis is well established, but the underlying mechanism remains unclear. The present study aimed to investigate whether miR-455 participated in the H2S protection of lung epithelial cells against CoCl2-induced apoptosis by regulating endoplasmic reticulum stress (ERS)-related genes. Human lung epithelial cells BEAS-2B were subjected to hypoxia injury with or without H2S preconditioning. It was found that hypoxia injury increased apoptosis of BEAS-2B cells, down-regulated the expression of miR-455, and upregulated the expression of calreticulin (Calr). H2S preconditioning attenuated lung epithelial cells apoptosis, enhanced cell viability, up-regulated the expression of miR-455, as well as down-regulated the expression of Calr following hypoxia injury. In addition, Calr, GRP78, C/EBP homologous protein (CHOP) and Caspase-12 protein was down-regulated by the miR-455 mimic and up-regulated by the miR-455 inhibitor. These results implicate miR-455 regulated H2S protection of lung epithelial cells against hypoxia-induced apoptosis by stimulating Calr.

Histone Deacetylase 1 Inhibition Protects Against Hypoxia-Induced Swelling in H9c2 Cardiomyocytes Through Regulating Cell Stiffness.

The process of cardiomyocyte swelling involves changes of biomechanical properties and profiles of cellular genes. Although many genes have been proved to regulate cell edema of cardiomyocyte, the mechanisms involved in this event, as well as the biomechanical properties of swelling cell, remain unknown.Methods and Results:Whether histone deacetylase 1 (HDAC1) inhibition protects against hypoxia-induced H9c2 cardiomyocyte swelling is examined in this study. Hypoxia-induced changes in the biomechanical properties and cytoskeletal structure that are relevant to cell swelling were also determined. H9c2 cells were treated under a chemical hypoxia situation (cobalt chloride) with HDAC1 inhibition (chemical inhibitor or siRNA) for 5 h, followed by in vitro biological and mechanical characterization. The results showed that expression of HDAC1 instead of HDAC4 was upregulated by chemical hypoxia. HDAC1 inhibition protects H9c2 cells against chemical hypoxia-induced hypoxic injury and cell swelling. HDAC1 inhibition improved cell viability, decreased lactate dehydrogenase leakage, cell apoptosis, malondialdehyde concentration, cell volume, and particles on the cell surface, and increased superoxide dismutase activity. Moreover, chemical hypoxia induced a decrease of Young's modulus, accompanied by alterations in the integrity of acetylated histone and organization of the cytoskeletal network. HDAC1 inhibition significantly reversed these processes. Based on the ideal physical model, HDAC1 inhibition protects against hypoxia-induced swelling in H9c2 cardiomyocytes through enhancing cell stiffness. Overall, HDAC1 is a potential therapeutic target for myocardial edema.

Cyclosporin A Protects H9c2 Cells Against Chemical Hypoxia-Induced Injury via Inhibition of MAPK Signaling Pathway

This study aimed to investigate the effects and molecular mechanism of cyclosporin A (CsA) on cobalt chloride (CoCl2)-induced injury in H9c2 embryonic rat cardiac cells. The results showed that CsA could protect H9c2 cells against CoCl2-induced hypoxic injury. CsA effectively improved cell viability, and decreased LDH leakage, cell apoptosis, MDA concentration, and ROS generation, and increased SOD activity, GSH production, and CAT activity in a dosedependent manner. In addition, CsA treatment blocked the CoCl2-induced increases in ROS production and mitochondrial dysfunction, including a decrease in membrane potential, cytochrome c (cyto-c) release, Bax/Bcl-2 imbalance, as well as the ratios of cl-casp-9/casp-9 and cl-casp-3/casp-3 ratios, via the inhibition of p38 and ERK MAPK signaling pathways. The results also suggested that CsA protected H9c2 cells against CoCl2-induced hypoxic injury, possibly by suppressing the MAPK signaling pathway. Thus, CsA is a potential therapeutic agent for cardiac hypoxic injury.

Anti-apoptotic effect of fermentedCitrus sunkipeel extract on chemical hypoxia-induced neuronal injury

Purpose: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimers disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride (CoCl₂)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. Methods: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2,7- dichlorofluorescin diacetate (DCF-DA) and 4,6-diamidino-2-phenylindole (DAPI) staining. Results: Exposure to CoCl₂, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the CoCl₂-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. Conclusion: Based on these data, fermented C. sunki peel extract might have a protective effect against CoCl₂-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.

Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes

Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2-induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.

화학적 허혈에 의해 손상된 마우스 간세포에 대한 hydrogen sulfide의 간세포 보호 효과

본 연구는 화학적 허혈에 의해 손상된 마우스 간세포에서 hydrogen sulfide (<TEX>$H_2S$</TEX>)의 효과를 규명하기 위해 수행되었다. 본 연구에서 허혈 모방 화합물로 알려져 있는 cobalt chloride (<TEX>$CoCl_2$</TEX>)는 간세포 손상을 시간 및 농도 의존적으로 유의성 있게 증가 시켰다. <TEX>$CoCl_2$</TEX>에 의한 간세포 손상은 Sodium sulfide (NaHS, <TEX>$H_2S$</TEX> 공여제)의 전처리에 의해 유의적으로 감소 되었다. <TEX>$CoCl_2$</TEX>는 세포 내 활성산소(reactive oxygen species, ROS)의 농도를 증가시켰으며, 이는 NaHS 및 N-acetyl-cysteine (NAC, a ROS 제거제)에 의해 감소하였다. 또한, <TEX>$CoCl_2$</TEX>에 의해 증가된 p38 MAPK 인산화가 NaHS 및 NAC에 의해 억제되었다. <TEX>$CoCl_2$</TEX>에 의해 증가된 Bax/Bcl-2 비율은 NaHS, NAC 및 SB 203580 (p38 MAPK 저해제)에 의해 차단되었으며, <TEX>$CoCl_2$</TEX>에 의해 유발된 간세포의 손상 또한 NaHS, NAC 및 SB 203580의 전처리에 의해 억제되었다. NaHS는 <TEX>$CoCl_2$</TEX>에 의해 증가된 COX-2의 발현을 억제하였다. 또한, NaHS의 효과와 유사하게 <TEX>$CoCl_2$</TEX>에 의해 증가된 COX-2의 발현이 NAC에 의해 억제되었다. 더욱이, NS-398 (COX-2 선택적 억제제)는 <TEX>$CoCl_2$</TEX>에 의한 Bax/Bcl-2 비율의 증가를 억제하였을 뿐 아니라, 간세포의 세포 손상 또한 억제하였다. 결론적으로, <TEX>$H_2S$</TEX>는 초대배양 된 마우스 간세포에서 <TEX>$CoCl_2$</TEX>에 의해 유발된 간세포의 손상을 ROS에 의해 유발된 p38 MAPK 및 COX-2 경로의 활성화를 억제함으로써 세포보호효과를 수행하는 것을 알 수 있었다. We examined the effect of hydrogen sulfide (<TEX>$H_2S$</TEX>) in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased by cobalt chloride (<TEX>$CoCl_2$</TEX>), a well-known hypoxia mimetic agent in a time- and dose- dependent manner. Sodium hydrosulfide (NaHS, a donor of <TEX>$H_2S$</TEX>) pretreatment before exposure to <TEX>$CoCl_2$</TEX> significantly attenuated the <TEX>$CoCl_2$</TEX>-induced decrease of cell viability. <TEX>$CoCl_2$</TEX> treatment resulted in an increase of intracellular ROS generation, which is inhibited by NaHS or N-acetyl-cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by NaHS or NAC. The <TEX>$CoCl_2$</TEX>-induced increase of the Bax/Bcl-2 ratio was attenuated by NaHS, NAC, and SB 203580 (p38 MAPK inhibitor). The <TEX>$CoCl_2$</TEX>-induced decrease of cell viability was also attenuated by NaHS, NAC, and SB 203580 pretreatment. Additionally, NaHS inhibited the <TEX>$CoCl_2$</TEX>-induced COX-2. Similar to the effect of NaHS, NAC blocked <TEX>$CoCl_2$</TEX>-induced COX-2 expression. Furthermore, NS-398 (a selective COX-2 inhibitor) attenuated not only the <TEX>$CoCl_2$</TEX>-induced increase of the Bax/Bcl-2 ratio, it also decreased cell viability. Taken together, <TEX>$H_2S$</TEX> protects primary cultured mouse hepatocytes against <TEX>$CoCl_2$</TEX>-induced cell injury through inhibition of the ROS-activated p38 MAPK cascade and the COX-2 pathway.

Rosmarinic acid inhibits chemical hypoxia-induced cytotoxicity in primary cultured rat hepatocytes

We examine the effect of rosmarinic acid (RA) in chemical hypoxia-induced injury in rat hepatocytes. Cell viability was significantly decreased by cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose- dependent manner. RA pretreatment before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with RA potentiated the decrease of Bcl-2 expression and attenuated the increase of Caspase-3 expression by CoCl2. CoCl2 treatment resulted in an increase of intracellular ROS generation, which is inhibited by RA or N-acetyl-cysteine (NAC, a ROS scavenger), and p38MAPK phosphorylation, which is also blocked by RA or NAC. CoCl2-induced increase of Bax/Bcl-2 ratio and Caspase-3 expression was attenuated by RA, NAC and SB203580 (p38MAPK inhibitor). CoCl2-induced decrease of cell viability was also attenuated by RA, NAC and SB203580 pretreatment. Additionally, RA inhibited CoCl2-induced COX-2 expression and prostaglandin E2 (PGE2) secretion. Similar to the effect of RA, both NAC and NS-398 (COX-2 inhibitor) blocked CoCl2-induced COX-2 expression and PGE2 secretion. NS-398 attenuated not only CoCl2-induced increase of Bax/Bcl-2 ratio and Caspase-3 expression, but decrease of cell viability. Taken together, RA protects primary cultured rat hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38MAPK and COX-2/PGE2 pathway.

Inhibition of ROS-Activated p38MAPK Pathway is Involved in the Protective Effect of H2S Against Chemical Hypoxia-Induced Inflammation in PC12 Cells

We have demonstrated the neuroprotection of hydrogen sulfide (H2S) against chemical hypoxia-induced injury by inhibiting p38MAPK pathway. The present study attempts to evaluate the effect of H2S on chemical hypoxia-induced inflammation responses and its mechanisms in PC12 cells. We found that treatment of PC12 cells with cobalt chloride (CoCl2, a hypoxia mimetic agent) enhanced IL-6 secretion, nitric oxide (NO) generation and expression levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). L-canavanine, a selective iNOS inhibitor, partly blocked CoCl2-induced cytotoxicity, apoptosis and mitochondrial insult. In addition, 7-Nitroindazole (7-NI), an inhibitor of nNOS, also partly attenuated the CoCl2-induced cytotoxicity. The inhibition of p38MAPK by SB203580 (a selective p38MAPK inhibitor) or genetic silencing of p38MAPK by RNAi (Si-p38) depressed not only CoCl2-induced iNOS expression, NO production, but also IL-6 secretion. In addition, N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, conferred a similar protective effect of SB203580 or Si-p38 against CoCl2-induced inflammatory responses. Importantly, pretreatment of PC12 cells with exogenous application of sodium hydrosulfide (a H2S donor, 400 μmol/l) for 30 min before exposure to CoCl2 markedly attenuated chemical hypoxia-stimulated iNOS and nNOS expression, NO generation and IL-6 secretion as well as p38MAPK phosphorylation in PC12 cells. Taken together, we demonstrated that p38MAPK-iNOS pathway contributes to chemical hypoxia-induced inflammation and that H2S produces an anti-inflammatory effect in chemical hypoxia-stimulated PC12 cells, which may be partly due to inhibition of ROS-activated p38MAPK-iNOS pathway.

Neuroprotective effects of quercetin in chemical hypoxia: in silico evaluation of the hypothesis exploring PKC inhibition-mediated pharmacotherapy

The effect of chemical hypoxia, i.e., KCN-induced hypoxia on protein kinase C (PKC) translocation and cell injury was well reported. It has been well documented that activation of PKC is regulated by calcium and unsaturated free fatty acids which are actively involved in the phosphorylation of various cellular proteins, thereby initiating a variety of physiologic as well as cellular processes. The involvement of PKC has been reported in the regulation of neuronal excitability, neurotransmitter release, cellular proliferation, and gene expression. PKC can affect Ca2+ homeostasis by increasing the activity of Na+/Ca2+ exchanger and Ca2+-ATPase. These effects are most probably neuroprotective since they promote Ca2+ sequestration, thereby decreasing free cytosolic Ca2+ level. On the other side, PKC-mediated phosphorylation of NMDA receptor increases the probability of the channel opening and reduces the Mg2+ block increasing the channel’s conductivity. It has been proposed that sustained elevation of free cytosolic Ca2+ levels could result in prolonged activation of PKC which, in turn, may decrease neuronal energy due to excessive utilization of ATP by the activated enzyme. However, all known protein kinase inhibitors target the ATP-binding site. Owing to this reason, search for strong natural inhibitors that selectively target the protein kinase domains continues to be a daunting challenge. Past studies reiterated that quercetin being a dietary flavonoid of plant origin which has antioxidant, anti-inflammatory properties, and capability to cross the blood brain barrier. The binding energy of quercetin with PKCθ was −9.28 kcal/mol. Thus, we hypothesized that Quercetin, a plant-originated natural polyphenolic compound that can act as a neuroprotective agent in chemical hypoxia-induced injury due to its inhibitory effects on PKC. The aim of our proposed hypothesis is to find out a successful modality of effective natural and dietary compound for the treatment of chemical hypoxia available till date. Hence, we have tried to put forward a hypothesis on the basis of our in silico studies which involves the clue for regulation of PKC by quercetin.

Hydrogen sulfide protects PC12 cells against reactive oxygen species and extracellular signal-regulated kinase 1/2-mediated downregulation of glutamate transporter-1 expression induced by chemical hypoxia

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.

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.

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.

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.

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.

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.

Oxidative stress mediates chemical hypoxia-induced injury and inflammation by activating NF-κb-COX-2 pathway in HaCaT cells.

Hypoxia of skin is an important physiopathological process in many diseases, such as pressure ulcer, diabetic ulcer, and varicose ulcer. Although cellular injury and inflammation have been involved in hypoxia-induced dermatic injury, the underlying mechanisms remain largely unknown. This study was conducted to investigate the effects of cobalt chloride (CoCl(2)), a hypoxia-mimicking agent, on human skin keratinocytes (HaCaT cells) and to explore the possible molecular mechanisms. Exposure of HaCaT cells to CoCl(2) reduced cell viability and caused overproduction of reactive oxygen species (ROS) and oversecretion of interleukin-6 (IL-6) and interleukin-8 (IL-8). Importantly, CoCl(2) exposure elicited overexpression of cyclooxygenase-2 (COX-2) and phosphorylation of nuclear factor-kappa B (NF-κB) p65 subunit. Inhibition of COX-2 by NS-398, a selective inhibitor of COX-2, significantly repressed the cytotoxicity, as well as secretion of IL-6 and IL-8 induced by CoCl(2). Inhibition of NF-κB by PDTC (a selective inhibitor of NF-κB) or genetic silencing of p65 by RNAi (Si-p65), attenuated not only the cytotoxicity and secretion of IL-6 and IL-8, but also overexpression of COX-2 in CoCl(2)-treated HaCaT cells. Neutralizing anti-IL-6 or anti-IL-8 antibody statistically alleviated CoCl(2)-induced cytotoxicity in HaCaT cells. N-acetyl-L-cysteine (NAC), a well characterized ROS scavenger, obviously suppressed CoCl(2)-induced cytotoxicity in HaCaT cells, as well as secretion of IL-6 and IL-8. Additionally, NAC also repressed overexpression of COX-2 and phosphorylation of NF- B κ p65 subunit induced by CoCl(2) in HaCaT cells. In conclusion, our results demonstrated that oxidative stress mediates chemical hypoxia-induced injury and inflammatory response through activation of NF-κB-COX-2 pathway in HaCaT cells.