• Biomedical Science Letters
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• v.11 no.4
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• pp.441-446
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• 2005

The molecular mechanism of ischemia/reperfusion injury remains unclear. Reactive oxygen species (ROS) are implicated in cell death caused by ischemia/reperfusion in vivo or hypoxia in vitro. Poly (ADP-ribose) polymerase (PARP) activation has been reported to be involved in hydrogen peroxide-induced cell death in renal epithelial cells. This study was therefore undertaken to evaluate the role of P ARP activation in chemical hypoxia in opossum kidney (OK) cells. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport. Exposure of OK cells to chemical hypoxia resulted in a time-dependent cell death. In OK cells subjected to chemical hypoxia, the generation of ROS was increased, and this increase was prevented by the $H_2O_2$ scavenger catalase. Chemical hypoxia increased P ARP activity and chemical hypoxia-induced cell death was prevented by the inhibitor of PARP activation 3-aminobenzamide. Catalase prevented OK cell death induced by chemical hypoxia. $H_2O_2$ caused PARP activation and $H_2O_2-induced$ cell death was prevented by 3-aminobenzamide. Taken together, these results indicate that chemical hypoxia-induced cell injury is mediated by PARP activation through H202 generation in renal epithelial cells.

• Biomedical Science Letters
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• v.10 no.4
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• pp.341-346
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• 2004

Tumor necrosis factor-α (TNF-α) or its mRNA expression are increased in acute nephrosis of various types including ischemia/reperfusion injury. This study was undertaken to determine whether pentoxifylline (PTX), an inhibitor of TNF-α production, provides a protective effect against hypoxia-induced cell injury in rabbit renal cortical slices. To induce hypoxia-induced cell injury, renal cortical slices were exposed to 100% N₂ atmosphere. Control slices were exposed to 100% O₂ atmosphere. The cell injury was estimated by measuring lactate dehydrogenase (LDH) release and p-aminohippurate (PAH) uptake. Exposure of slices to hypoxia increased the LDH release in a time-dependent manner. However, when slices were exposed to hypoxia in the presence of PTX, the LDH release was decreased. The protective effect of PTX was dose-dependent over the concentrations of 0.05∼1 mM. Hypoxia did not increase lipid peroxidation, whereas an organic hydroperoxide t-butylhydroperoxide (tBHP) resulted in a significant increase in lipid peroxidation. PTX did not affect tBHP-induced lipid peroxidation. Hypoxia decreased PAH uptake, which was significantly attenuated by PTX and glycine. tBHP-induced inhibition of PAH uptake was not altered by PTX, although it was prevented by antioxidant deferoxarnine. The PAH uptake by slices in rabbits with ischemic acute renal failure was prevented by PTX pretreatment. These results suggest that PTX may exert a protective effect against hypoxia-induced cell injury and its effect may due to inhibition of the TNF-α production, but not by its antioxidant action.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.1
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• pp.93-100
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• 1999

The present study was designed to assess the roles of $PLA_2$ activation and arachidonic acid (AA) metabolites in hypoxia-induced renal cell injury. Hypoxia increased LDH release in a dose-dependent manner in rabbit renal cortical slices, and this increase was significant after 20-min hypoxia. The hypoxia-induced LDH release was prevented by amino acids, glycine and alanine, and extracellular acidosis (pH 6.0). Buffering intracellular $Ca^{2+}$ by a chelator, but not omission of $Ca^{2+}$ in the medium produced a significant reduction in hypoxia-induced LDH release. The effect of hypoxia was blocked by $PLA_2$ inhibitors, mepacrine, butacaine, and dibucaine. A similar effect was observed by a 85-kD $cPLA_2$ inhibitor $AACOCF_3.$ AA increased hypoxia-induced LDH release, and albumin, a fatty acid absorbent, prevented the LDH release, suggesting that free fatty acids are involved in hypoxia-induced cell injury. These results suggest that $PLA_2$ activation and its metabolic products play important roles in pathogenesis of hypoxia-induced cell injury in rabbit renal cortical slices.

• Archives of Pharmacal Research
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• v.20 no.2
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• pp.103-109
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• 1997

The effects of ginseng total saponins (GTS) on hypoxic damage of primary cultures of astrocytes were studied. Hypoxia was created by placing cultures in an air tight chamber that was flushed with 95% $N_2/5%CO_2$ for 15 min before being sealed. Cultures showed evidence of significant cell injury after 24 h of hypoxia (increased lactate dehydrogenase (LDH) content in the culture medium, cell swelling and decreased glutamate uptake and protein content). Addition of GTS (0.1, 0.3 mg/ml) to the cultures during the exposure to hypoxic conditions produced dose-dependent inhibition of the LDH efflux. GTS (0.1, 0.3 mg/ml) also produced significant inhibition of the increased cell volume of astrocytes measured by $[^3H]$ O-methyl-D-glucose uptake under the hypoxic conditions. Decreased glutamate uptake and protein content was inhibited by GTS. These data suggest that GTS prevents astrocytic cell injury induced by severe hypoxia in vitro.

• The Journal of Internal Korean Medicine
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• v.40 no.3
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• pp.425-442
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• 2019

Objectives: This study aimed to reveal the pharmacological properties of the newly prescribed herbal mixture, Chenmadansamgamibokhap-tang(CDBT), against hypoxia-induced neuronal cell injury (especially mouse hippocampal neuronal cell line, HT-22 cells) and their corresponding mechanisms. Methods: A cell-based in vitro experiment, in which a hypoxia condition induced neuronal cell death, was performed. Various concentrations of the CDBT were pre-treated to the HT-22 cells for 4 h before 18 h in the hypoxia chamber. The glial cell BV-2 cells were stimulated with $IFN{\gamma}$ and LSP to produce inflammatory cytokines and reactive oxygen species. When the neuronal HT-22 cells were treated with this culture solution, the drug efficacy against neuronal cell death was examined. Results: CDBT showed cytotoxicity in the normal condition of HT-22 cells at a dose of $125{\mu}g/mL$ and showed a protective effect against hypoxia-induced neuronal cell death at a dose of $31.3{\mu}g/mL$. CDBT prevented hypoxia-induced neuronal cell death in a dose-dependent manner in the HT-22 cells by regulating $HIF1{\alpha}$ and cell death signaling. CDBT prevented neuronal cell death signals and DNA fragmentation due to the hypoxia condition. CDBT significantly reduced cellular oxidation, cell death signals, and caspase-3 activities due to microglial cell activations. Moreover, CDBT significantly ameliorated LPS-induced BV-2 cell activation and evoked cellular oxidation through the recovery of redox homeostasis. Conclusions: CDBT cam be considered as a vital therapeutic agent against neuronal cell deaths. Further studies are required to reveal the other functions of CDBT in vivo or in the clinical field.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.2
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• pp.173-183
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• 2020

An in vitro model for ischemia/reperfusion injury has not been well-established. We hypothesized that this failure may be caused by serum deprivation, the use of glutamine-containing media, and absence of acidosis. Cell viability of H9c2 cells was significantly decreased by serum deprivation. In this condition, reperfusion damage was not observed even after simulating severe ischemia. However, when cells were cultured under 10% dialyzed FBS, cell viability was less affected compared to cells cultured under serum deprivation and reperfusion damage was observed after hypoxia for 24 h. Reperfusion damage after glucose or glutamine deprivation under hypoxia was not significantly different from that after hypoxia only. However, with both glucose and glutamine deprivation, reperfusion damage was significantly increased. After hypoxia with lactic acidosis, reperfusion damage was comparable with that after hypoxia with glucose and glutamine deprivation. Although high-passage H9c2 cells were more resistant to reperfusion damage than low-passage cells, reperfusion damage was observed especially after hypoxia and acidosis with glucose and glutamine deprivation. Cell death induced by reperfusion after hypoxia with acidosis was not prevented by apoptosis, autophagy, or necroptosis inhibitors, but significantly decreased by ferrostatin-1, a ferroptosis inhibitor, and deferoxamine, an iron chelator. These data suggested that in our SIR model, cell death due to reperfusion injury is likely to occur via ferroptosis, which is related with ischemia/reperfusion-induced cell death in vivo. In conclusion, we established an optimal reperfusion injury model, in which ferroptotic cell death occurred by hypoxia and acidosis with or without glucose/glutamine deprivation under 10% dialyzed FBS.

• BMB Reports
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• v.46 no.1
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• pp.47-52
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• 2013

Intestinal epithelial cell (IEC) apoptosis induced by hypoxia compromise intestinal epithelium barrier function. Both Akt and Hsp90 have cytoprotective function. However, the specific role of Akt and $Hsp90{\beta}$ in IEC apoptosis induced by hypoxia has not been explored. We confirmed that hypoxia-induced apoptosis was reduced by $Hsp90{\beta}$ overexpression but enhanced by decreasing $Hsp90{\beta}$ expression. $Hsp90{\beta}$ overexpression enhanced BAD phosphorylation and thus reduced mitochondrial release of cytochrome C. Reducing $Hsp90{\beta}$ expression had opposite effects. The protective effect of $Hsp90{\beta}$ against apoptosis was negated by LY294002, an Akt inhibitor. Further study showed that Akt phosphorylation was enhanced by $Hsp90{\beta}$, which was not due to the activation of upstream PI3K and PDK1 but because of stabilization of pAkt via direct interaction between $Hsp90{\beta}$ and pAkt. These results demonstrate that $Hsp90{\beta}$ may play a significant role in protecting IECs from hypoxia-induced apoptosis via stabilizing pAkt to phosphorylate BAD and reduce cytochrome C release.

• Journal of Korean Neurosurgical Society
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• v.40 no.2
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• pp.103-109
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• 2006

Objective : Activated endothelial cells mediate the cascade of reactions in response to hypoxia for adaptation to the stress. It has been suggested that hypoxia, by itself, without reperfusion, can activate the endothelial cells and initiate complex responses. In this study, we investigated whether hypoxia-induced endothelial products alter the endothelial permeability and have a direct cytotoxic effect on nerve cells. Methods : Hypoxic condition of primary human umbilical vein endothelial cells[HUVEC] was induced by $CoCl_2$ treatment in culture medium. Cell growth was evaluated by 3,4,5-dimethyl thiazole-3,5-diphenyl tetrazolium bromide [MTT] assay Hypoxia-induced products [$IL-1{\beta},\;TGF-{\beta}1,\;IFN-{\gamma},\;TNF-{\alpha}$, IL-10, IL-6, IL-8, MCP-l and VEGF] were assessed by enzyme-linked immunosorbent assay. Endothelial permeability was evaluated by Western blotting. Results : Prolonged hypoxia caused endothelial cells to secrete IL -6, IL -8, MCP-1 and VEGF. However, the levels of IL -1, IL -10, $TNF-{\alpha},\;TGF-{\beta},\;IFN-{\gamma}$ and nitric oxide remained unchanged over 48 h hypoxia. Hypoxic exposure to endothelial cells induced the time-dependent down regulation of the expression of cadherin and catenin protein. The conditioned medium taken from hypoxic HUVECs had the cytotoxic effect selectively on neuroblastoma cells, but not on astroglioma cells. Conclusion : These results suggest the possibility that endothelial cell derived cytokines or other secreted products with the increased endothelial permeability might directly contribute to nerve cell injury followed by hypoxia.

• Journal of Microbiology and Biotechnology
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• v.31 no.8
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• pp.1069-1078
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• 2021

Sevoflurane postconditioning (SPostC) has been proved effective in cardioprotection against myocardial ischemia/reperfusion injury. It was also reported that heat shock protein 70 (HSP70) could be induced by sevoflurane, which played a crucial role in hypoxic/reoxygenation (HR) injury of cardiomyocytes. However, the mechanism by which sevoflurane protects cardiomyocytes via HSP70 is still not understood. Here, we aimed to investigate the related mechanisms of SPostC inducing HSP70 expression to reduce the HR injury of cardiomyocytes. After the HR cardiomyocytes model was established, the cells transfected with siRNA for HSP70 (siHSP70) or not were treated with sevoflurane during reoxygenation. The lactate dehydrogenase (LDH) level was detected by colorimetry while cell viability and apoptosis were detected by MTT and flow cytometry. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect HSP70, apoptosis-, cell cycle-associated factors, iNOS, and Cox-2 expressions. Enzyme-linked immuno sorbent assay (ELISA) was used to measure malondialdehyde (MDA) and superoxide dismutase (SOD). SPostC decreased apoptosis, cell injury, oxidative stress and inflammation and increased viability of HR-induced cardiomyocytes. In addition, SPostC downregulated Bax and cleaved caspase-3 levels, while SPostC upregulated Bcl-2, CDK-4, Cyclin D1, and HSP70 levels. SiHSP70 had the opposite effect that SPostC had on HR-induced cardiomyocytes. Moreover, siHSP70 further reversed the effect of SPostC on apoptosis, cell injury, oxidative stress, inflammation, viability and the expressions of HSP70, apoptosis-, and cell cycle-associated factors in HR-induced cardiomyocytes. In conclusion, this study demonstrates that SPostC can reduce the HR injury of cardiomyocytes by inducing HSP70 expression.

• Journal of Life Science
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• v.23 no.11
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• pp.1342-1350
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• 2013

We examined the effect of hydrogen sulfide ($H_2S$) in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased by cobalt chloride ($CoCl_2$), a well-known hypoxia mimetic agent in a time- and dose- dependent manner. Sodium hydrosulfide (NaHS, a donor of $H_2S$) pretreatment before exposure to $CoCl_2$ significantly attenuated the $CoCl_2$-induced decrease of cell viability. $CoCl_2$ 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 $CoCl_2$-induced increase of the Bax/Bcl-2 ratio was attenuated by NaHS, NAC, and SB 203580 (p38 MAPK inhibitor). The $CoCl_2$-induced decrease of cell viability was also attenuated by NaHS, NAC, and SB 203580 pretreatment. Additionally, NaHS inhibited the $CoCl_2$-induced COX-2. Similar to the effect of NaHS, NAC blocked $CoCl_2$-induced COX-2 expression. Furthermore, NS-398 (a selective COX-2 inhibitor) attenuated not only the $CoCl_2$-induced increase of the Bax/Bcl-2 ratio, it also decreased cell viability. Taken together, $H_2S$ protects primary cultured mouse hepatocytes against $CoCl_2$-induced cell injury through inhibition of the ROS-activated p38 MAPK cascade and the COX-2 pathway.