IH-induced Apoptosis Research Articles

Suppression of CHOP Reduces Neuronal Apoptosis and Rescues Cognitive Impairment Induced by Intermittent Hypoxia by Inhibiting Bax and Bak Activation.

Our previous study showed that growth arrest- and DNA damage-inducible gene 153 (GAD153/CHOP) plays an important role in intermittent hypoxia- (IH-) induced apoptosis and impaired synaptic plasticity. This study is aimed at determining which signaling pathway is activated to induce CHOP and the role of this protein in mitochondria-dependent apoptosis induced by IH. In the in vivo study, mice were placed in IH chambers for 8 h daily over a period of 2 weeks; the IH chambers had oxygen (O2) concentrations that oscillated between 10% and 21%, cycling every 90 s. In the in vitro study, PC12 cells were exposed to 21% O2 (normoxia) or 8 IH cycles (25 min at 21% O2 and 35 min at 0.1% O2 for each cycle). After 2 weeks of IH treatment, we observed that the expression levels of phosphorylated protein kinase-like endoplasmic reticulum kinase (p-PERK), activating transcription factor 4 (ATF-4) and phosphorylated eukaryotic initiation factor 2 alpha (p-elf2α), were increased, but the levels of activating transcription factor 6 (ATF-6) and inositol-requiring enzyme 1 (IRE-1) were not increased. GSK2606414, a specific chemical inhibitor of the PERK pathway, reduced the expression of p-PERK, ATF-4, p-elf2α, and CHOP and rescued ER structure. In addition, Bax and Bak accumulated in the mitochondria after IH treatment, which induced cytochrome c release and initiated apoptosis. These effects were prevented by GSK2606414 and CHOP shRNA. Finally, the impaired long-term potentiation and long-term spatial memory in the IH group were rescued by GSK2606414. Together, the data from the in vitro and in vivo experiments indicate that IH-induced apoptosis and impaired synaptic plasticity were mediated by the PERK-ATF-4-CHOP pathway. Suppressing PERK-ATF-4-CHOP signaling pathway attenuated mitochondria-dependent apoptosis by reducing the expression of Bax and Bak in mitochondria, which may serve as novel adjunct therapeutic strategy for ameliorating obstructive sleep apnea- (OSA-) induced neurocognitive impairment.

Tempol alleviates chronic intermittent hypoxia-induced pancreatic injury through repressing inflammation and apoptosis.

Obstructive sleep apnea (OSA) has been demonstrated to be implicated in disorder of insulin secretion and diabetes mellitus. In this study, we aimed to evaluate the protective role of tempol, a powerful antioxidant, in chronic intermittent hypoxia (IH)-induced pancreatic injury. The rat model of OSA was established by IH exposure. The pathological changes, increased blood-glucose level, and raised proinsulin/insulin ratio in pancreatic tissues of rats received IH were effectively relieved by tempol delivery. In addition, the enhanced levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta, IL-6, and inflammatory mediators, PGE2, cyclooxygenase-2 (COX-2), NO, and inducible nitric oxide synthase (iNOS) in pancreatic tissue were suppressed by tempol. Moreover, tempol inhibited IH-induced apoptosis in pancreatic tissue as evidenced by upregulated Bcl-2 level, and downregulated Bax and cleaved caspase-3 levels. Finally, the abnormal activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) signaling pathways induced by IH was restrained by tempol administration. In summary, our study demonstrates that tempol relieves IH-induced pancreatic injury by inhibiting inflammatory response and apoptosis, which provides theoretical basis for tempol as an effective treatment for OSA-induced pancreatic injury.

Blocking C/EBP β protects vascular endothelial cells from injury induced by intermittent hypoxia.

Intermittent hypoxia (IH) can damage endothelial cells and lead to apoptosis in obstructive sleep apnea-hypopnea syndrome (OSAHS). Hypoxia induces apoptosis in endothelial cells via upregulation of endothelin-1 (ET-1) and hypoxia inducible factor-1 alpha (HIF-1α) plays a key role in the hypoxic stress response. We investigated an approach to diminish the negative effect of HIF-1α while maintaining its protective effect. Human umbilical vein endothelial cells (HUVECs) were subjected to sustained hypoxia (SH) or IH for 24h, and the responses of HIF-1α, CCAAT/enhancer binding protein beta (C/EBP β), and endothelin-1 (ET-1) were assessed by western blotting. A luciferase reporter system was employed to verify the potential binding site (transcription factor binding site, TFBS) for C/EBP β in the ET-1 promoter. The specificity of regulation of ET-1 by HIF-1α via C/EBP β was evaluated by a lentiviral system. The effects of silencing of C/EBP β on IH-induced apoptosis, vascular endothelial growth factor (VEGF) protein levels, proliferation, and in vitro tube formation were studied. We found that IH significantly increased HIF-1α, C/EBP β, and ET-1 in HUVECs. Knockdown of HIF-1α or C/EBP β inhibited the upregulation of ET-1 induced by IH. Blocking C/EBP β impaired IH-induced apoptosis but did not affect VEGF expression, proliferation, or in vitro tube formation. C/EBP β was shown to mediate increased ET-1 transcription by HIF-1α through the TFBS, 5'-GTTGCCTGTTG-3', in ET-1 promoter. Silencing of C/EBP β can suppress apoptosis but does not affect the protective role of HIF-1α in the hypoxic stress response.

4-phenylbutyric acid attenuates endoplasmic reticulum stress-mediated apoptosis and protects the hepatocytes from intermittent hypoxia-induced injury.

To investigate the effect of 4-phenylbutyric acid (4-PBA) on intermittent hypoxia (IH)-induced liver cell injury and to clarify the underlying mechanisms. L02 cells (normal human liver cells) were cultured in normoxic condition or subjected to intermittent hypoxia for 4, 8, and 12h. A part of hypoxia-treated L02 cells was applied with 4-PBA 1h before exposure to hypoxia. The effect of 4-PBA on liver injury, hepatocyte apoptosis, endoplasmic reticulum stress (ERS), and PERK-eIFa2-ATF4-CHOP apoptotic pathway was investigated. (1) IH caused apoptosis in hepatocyte; (2) IH caused ERS in hepatocyte; (3) IH caused hepatic injury; (4) 4-PBA attenuated IH-induced liver cell injury; (5) 4-PBA protected liver cell from IH-induced apoptosis; (6) 4-PBA suppressed ERS-related apoptotic pathway (PERK-eIFa2-ATF4-CHOP), but did not suppress IH-induced unfold protein reaction (UPR). 4-PBA could protect liver cells by suppressing IH-induced apoptosis mediated by ERS, but not by reducing the UPR.

Inhibition of microRNA-218 reduces HIF-1α by targeting on Robo1 in mice aortic endothelial cells under intermittent hypoxia.

ObjectiveTo investigate the effects of miR-218 on expression of hypoxia-inducible factors 1α (HIF-1α), vascular endothelial growth factor (VEGF) and cell apoptosis in normal mice aortic endothelial cells under intermittent hypoxia (IH) condition.MethodsAnti-miR-218 inhibitor, miR-negative control and miR-218 mimic were used to tranfect the cells in different groups under IH condition. Both RT-PCR and Western blot were used to determine the expressions of HIF-1α and VEGF. Akt, p-Akt and cell apoptosis related proteins bcl-2, bax and caspase-3 and roundabout 1 (Robo1) were measured using Western blot. Cell apoptosis was evaluated by flow cytometry. Statistical analysis was performed using SPSS 18.0.ResultsExpression of miR-218 was significantly up-regulated in the IH group and was significantly inhibited when cells were transfected with miR-218 inhibitor. Down regulation of miR-218 could reduce the expression of HIF-1α and VEGF under intermittent hypoxia condition. In cells transfected with miR-218 mimic, expression of HIF-1α and VEGF significantly increased compared with the control. However, when treated with LY294002, the expression of HIF-1α and VEGF both decreased. Apoptosis assay showed that down regulation of miR-218 could inhibit intermittent hypoxia induced cell apoptosis, decrease expression of caspase-3 and bax and increase expression of bcl-2 under intermittent hypoxia condition. At last, silencing Robo1 could significantly enhance the expression of HIF-1α under IH condition.ConclusionInhibition of miR-218 could reduce the expression of HIF-1α and protect against IH-induced apoptosis in mice aortic endothelial cells. The effects were associated with PI3K/AKT pathway and might through targeting of Robo1.

SENP1 inhibits the IH-induced apoptosis and nitric oxide production in BV2 microglial cells

To reveal SUMOylation and the roles of Sentrin-specific proteases (SENP)s in microglial cells under Intermittent hypoxia (IH) condition would provide more intensive view of understanding the mechanisms of IH-induced central nervous system (CNS) damage. Hence, in the present study, we detected the expression levels of SENPs in microglial cells under IH and normoxia conditions via RT-PCR assay. We found that SENP1 was significantly down-regulated in cells exposure to IH. Subsequently, the effect of IH for the activation of microglia and the potential roles of SENP1 in the SENP1-overexpressing cell lines were investigated via Western blotting, RT-PCR and Griess assay. The present study demonstrated the apoptosis-inducing and activating role of IH on microglia. In addition, we revealed that the effect of IH on BV-2 including apoptosis, nitric oxide synthase (iNOS) expression and nitric oxide (NO) induction can be attenuated by SENP1 overexpression. The results of the present study are of both theoretical and therapeutic significance to explore the potential roles of SENP1 under IH condition and elucidated the mechanisms underlying microglial survival and activation.

2,3-Oxidosqualene cyclase protects liver cells from the injury of intermittent hypoxia by regulating lipid metabolism.

Purpose2,3-Oxidosqualene cyclase (OSC), an important enzyme of cholesterol biosynthesis, catalyzes the highly selective cyclization of 2,3-monoepoxysqualene to lanosterol. Intermittent hypoxia (IH) is a hallmark feature in obstructive sleep apnea (OSA) which is increasingly recognized as an independent risk factor for liver injury. The aim of this study was to determine the effect of IH on OSC expression and evaluate the role of OSC in the IH-induced apoptosis in hepatic cell line human liver cell (HL-02).MethodsHL-02 cells were exposed to normoxia or IH. Cell Counting Kit-8 (CCK-8) assay was used to value cell proliferation, and flow cytometry was used to determine cell apoptosis. The expression of OSC messenger RNA (mRNA) was evaluated by quantitative real-time PCR, and the expression of OSC protein was determined by Western blot. To further investigate the function of OSC in IH-induced apoptosis, oxidosqualene cyclase-enhanced green fluorescence protein (OSC-EGFP) plasmid was constructed to over-express OSC protein. Triglyceride content in HL-02 cells was analyzed by oil red staining or Triglyceride Quantification Kit.ResultsWe found that IH inhibited HL-02 cell proliferation and accelerated cell apoptosis. IH decreased OSC expression, and over-expression of OSC could protect HL-02 cells against the IH-induced hepatic cell injury. Moreover, over-expression of OSC could attenuate IH-induced cellular triglyceride accumulation.ConclusionsThese findings suggest that OSC are involved in IH-induced hepatic cell injury. These results may contribute to the further understanding of the mechanism underlying the liver injury in OSA patients.

Cyclooxygenase 2 and intermittent hypoxia-induced spatial deficits in the rat.

Intermittent hypoxia (IH) during sleep, a critical feature of sleep apnea, induces significant neurobehavioral deficits in the rat. Cyclooxygenase (COX)-2 is induced during stressful conditions such as cerebral ischemia and could play an important role in IH-induced learning deficits. We therefore examined COX-1 and COX-2 genes and COX-2 protein expression and activity (prostaglandin E2 [PGE2] tissue concentration) in cortical regions of rat brain after exposure to either IH (10% O2 alternating with 21% O2 every 90 seconds) or sustained hypoxia (10% O2). In addition, the effect of selective COX-2 inhibition with NS-398 on IH-induced neurobehavioral deficits was assessed. IH was associated with increased COX-2 protein and gene expression from Day 1 to Day 14 of exposure. No changes were found in COX-1 gene expression after exposure to hypoxia. IH-induced COX-2 upregulation was associated with increased PGE2 tissue levels, neuronal apoptosis, and neurobehavioral deficits. Administration of NS-398 abolished IH-induced apoptosis and PGE2 increases without modifying COX-2 mRNA expression. Furthermore, NS-398 treatment attenuated IH-induced deficits in the acquisition and retention of a spatial task in the water maze. We conclude that IH induces upregulation and activation of COX-2 in rat cortex and that COX-2 may play a role in IH-mediated neurobehavioral deficits.