缺氧诱导因子-1α与缺氧状态下胃癌细胞侵袭的相关性

目的探讨缺氧状态下及缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)RNA干扰后,HIF-1α的表达及其与胃癌细胞黏附、游走、侵袭能力的相关性。方法将HIF-1α shRNA真核表达质粒pGPU6/GFP/Neo-HIF-1α-2484转染胃癌细胞SGC-7901,构建靶向干扰HIF-1α表达的SGC-7901稳定转染细胞株;采用Western blot法检测缺氧及HIF-1αRNA干扰对SGC-7901细胞HIF-1α表达的影响;MTT法检测缺氧及HIF-1αRNA干扰对SGC-7901细胞黏附能力的影响;采用Boyden Chamber膜侵袭系统,观察缺氧及HIF-1αRNA干扰对SGC-7901细胞游走及侵袭能力的影响。结果 SGC-7901细胞在缺氧培养4、8和16h时,HIF-1α均呈阳性表达,并随时间的延长而显著增加;细胞粘贴率、游走和侵袭穿膜细胞数分别随时间延长而显著增加,与HIF-1α表达呈显著正相关。HIF-1αRNA干扰后,缺氧SGC-7901细胞HIF-1α表达显著下降,细胞粘贴率、游走和侵袭穿膜细胞数均显著下降。结论 HIF-1α的过度表达导致胃癌细胞黏附、游走及侵袭能力增强,可能是胃癌局部侵袭和远处转移的重要原因之一。     

Nox2 Upregulation and p38α MAPK Activation in Right Ventricular Hypertrophy of Rats Exposed to Long-Term Chronic Intermittent Hypobaric Hypoxia

One of the consequences of high altitude (hypobaric hypoxia) exposure is the development of right ventricular hypertrophy (RVH). One particular type of exposure is long-term chronic intermittent hypobaric hypoxia (CIH); the molecular alterations in RVH in this particular condition are less known. Studies show an important role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-induced oxidative stress and protein kinase activation in different models of cardiac hypertrophy. The aim was to determine the oxidative level, NADPH oxidase expression and MAPK activation in rats with RVH induced by CIH. Male Wistar rats were randomly subjected to CIH (2 days hypoxia/2 days normoxia; n = 10) and normoxia (NX; n = 10) for 30 days. Hypoxia was simulated with a hypobaric chamber. Measurements in the RV included the following: hypertrophy, Nox2, Nox4, p22phox, LOX-1 and HIF-1α expression, lipid peroxidation and H₂O₂ concentration, and p38α and Akt activation. All CIH rats developed RVH and showed an upregulation of LOX-1, Nox2 and p22phox and an increase in lipid peroxidation, HIF-1α stabilization and p38α activation. Rats with long-term CIH-induced RVH clearly showed Nox2, p22phox and LOX-1 upregulation and increased lipid peroxidation, HIF-1α stabilization and p38α activation. Therefore, these molecules may be considered new targets in CIH-induced RVH.     

Paricalcitol Improves Hypoxia-Induced and TGF-β1-Induced Injury in Kidney Pericytes

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-β1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-β1 (5 ng/mL) or hypoxia (1% O₂ and 5% CO₂). TGF-β1 increased α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-β1-induced cell migration of pericytes. Hypoxia increased TGF-β1, α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-β1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-β1, α-SMA upregulation, and PDGFRβ downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-β1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.     

缺氧诱导因子-1α与Wnt/β-catenin信号通路在缺氧胃癌细胞中的相互作用

目的在缺氧环境下,探讨缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)与Wnt/β-catenin信号通路在胃癌细胞SGC-7901中的相互作用。方法将质粒β-catenin-micRNA转染SGC-7091细胞,筛选稳定转染细胞株SGC-7901-β-catenin-micRNA。将SGC-7901细胞分为对照组、脂质体组和阴性对照组,对照组:常氧培养48 h;脂质体组:常氧培养24 h后,加入10μl脂质体,培养24 h;阴性对照组:常氧培养24 h后,转染阴性对照质粒,培养24 h;设稳定转染细胞株为干扰组,常氧培养48 h。采用倍增时间试验、平板克隆形成试验及流式细胞术检测各组细胞倍增时间、平板克隆集落数及细胞周期的变化。另取SGC-7901细胞,分为对照组、缺氧组、双重缺氧组;对照组:常氧培养48 h;缺氧组:常氧培养32 h,物理缺氧培养16 h;双重缺氧组:常氧培养24 h,化学缺氧培养8 h后,再同时物理缺氧培养16 h。同时取稳定转染细胞株,分为对照干扰组、缺氧干扰组和双重缺氧干扰组。对照干扰组:常氧培养48 h;缺氧干扰组:常氧培养32 h,物理缺氧培养16 h;双重缺氧干扰组:常氧培养24 h,化学缺氧培养8 h后,再同时物理缺氧培养16 h。采用RT-PCR和Western blot法检测各组细胞中HIF-1α、β-catenin的mRNA及蛋白的表达水平。结果与对照组、脂质体组、阴性对照组比较,干扰组倍增时间延长,平板克隆集落数减少,细胞阻滞在G1期比例增加,S期减少,差异均有统计学意义(P<0.05)。缺氧组与对照组、双重缺氧组与缺氧组、缺氧干扰组与对照干扰组及双重缺氧干扰组与缺氧干扰组相比,HIF-1α、β-catenin的mRNA及蛋白表达水平均升高,差异有统计学意义(P均<0.05)。缺氧干扰组与缺氧组及双重缺氧干扰组与双重缺氧组比较,β-catenin、HIF-1α的mRNA及蛋白表达水平均降低,差异有统计学意义(P均<0.05)。结论 HIF-1α激活可调控Wnt/β-catenin通路,但也受控于Wnt/β-catenin通路,二者之间相互影响,相互调节。     

Differential Modulation of S1PR(1–5) and Specific Activities of SphK and nSMase in Pulmonary and Cerebral Tissues of Rats Exposed to Hypobaric Hypoxia

Recent preclinical and clinical studies have unfolded the potential of pharmacological modulation of activities of sphingosine‐1‐phosphate (S1P) receptors and S1P metabolizing enzymes for the development of therapeutic interventions against a variety of pathologies. An understanding of differential and temporal effects of hypoxia exposure on the key components of S1P signalling would certainly aid in designing improved drug development strategies in this direction. In view of this, the aim of the present study was to assess the effect of progressive hypobaric hypoxia exposure on expression of S1P receptors (S1PR_1–5) and specific activities of S1P synthesizing enzymes—neutral sphingomyelinase (nSMase) and sphingosine kinase (Sphk) in pulmonary and cerebral tissues of rats exposed to simulated altitude of 21,000 feet in an animal decompression chamber. Along with this, development of cerebral and pulmonary edema and markers of inflammation were studied at 12, 24, and 48 h to validate our study model of hypobaric hypoxia‐induced stress. The protein expression of S1PR_1–5 and activities of Sphk and nSMase enzymes were observed to be dramatically affected by simulated hypobaric hypoxia exposure, concurrent with deterioration of pathology, with 12 h of exposure appearing to be the most critical of the various time points studied.     

Melatonin Suppresses Hypoxia-Induced Migration of HUVECs via Inhibition of ERK/Rac1 Activation

Melatonin, a naturally-occurring hormone, possesses antioxidant properties and ameliorates vascular endothelial dysfunction. In this study, we evaluate the impact of melatonin on the migratory capability of human umbilical vein endothelial cells (HUVECs) to hypoxia and further investigate whether ERK/Rac1 signaling is involved in this process. Here, we found that melatonin inhibited hypoxia-stimulated hypoxia-inducible factor-1α (HIF-1α) expression and cell migration in a dose-dependent manner. Mechanistically, melatonin inhibited Rac1 activation and suppressed the co-localized Rac1 and F-actin on the membrane of HUVECs under hypoxic condition. In addition, the blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1-T17N suppressed HIF-1α expression and cell migration in response to hypoxia, as well, but constitutive activation of Rac1 mutant Rac1-V12 restored HIF-1α expression, preventing the inhibition of melatonin on cell migration. Furthermore, the anti-Rac1 effect of melatonin in HUVECs appeared to be associated with its inhibition of ERK phosphorylation, but not that of the PI3k/Akt signaling pathway. Taken together, our work indicates that melatonin exerts an anti-migratory effect on hypoxic HUVECs by blocking ERK/Rac1 activation and subsequent HIF-1α upregulation.     

Pathological Role of Phosphoglycerate Kinase 1 in Balloon Angioplasty-Induced Neointima Formation

Restenosis is a common vascular complication after balloon angioplasty. Catheter balloon inflation-induced transient ischemia (hypoxia) of local arterial tissues plays a pathological role in neointima formation. Phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate (ATP)-generating glycolytic enzyme, has been reported to associate with cell survival and can be triggered under hypoxia. The purposes of this study were to investigate the possible role and regulation of PGK1 in vascular smooth muscle cells (VSMCs) and balloon-injured arteries under hypoxia. Neointimal hyperplasia was induced by a rat carotid artery injury model. The cellular functions and regulatory mechanisms of PGK1 in VSMCs were investigated using small interfering RNAs (siRNAs), chemical inhibitors, or anaerobic cultivation. Our data indicated that protein expression of PGK1 can be rapidly induced at a very early stage after balloon angioplasty, and the silencing PGK1-induced low cellular energy circumstance resulted in the suppressions of VSMC proliferation and migration. Moreover, the experimental results demonstrated that blockage of PDGF receptor-β (PDGFRB) or its downstream pathway, the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis, effectively reduced hypoxia-induced factor-1 (HIF-1α) and PGK1 expressions in VSMCs. In vivo study evidenced that PGK1 knockdown significantly reduced neointima hyperplasia. PGK1 was expressed at the early stage of neointimal formation, and suppressing PGK1 has a potential beneficial effect for preventing restenosis.     

Interplay between Selenium,Selenoproteins and HIV-1 Replication in Human CD4 T-Lymphocytes

The infection of CD4 T-lymphocytes with human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), disrupts cellular homeostasis, increases oxidative stress and interferes with micronutrient metabolism. Viral replication simultaneously increases the demand for micronutrients and causes their loss, as for selenium (Se). In HIV-infected patients, selenium deficiency was associated with a lower CD4 T-cell count and a shorter life expectancy. Selenium has an important role in antioxidant defense, redox signaling and redox homeostasis, and most of these biological activities are mediated by its incorporation in an essential family of redox enzymes, namely the selenoproteins. Here, we have investigated how selenium and selenoproteins interplay with HIV infection in different cellular models of human CD4 T lymphocytes derived from established cell lines (Jurkat and SupT1) and isolated primary CD4 T cells. First, we characterized the expression of the selenoproteome in various human T-cell models and found it tightly regulated by the selenium level of the culture media, which was in agreement with reports from non-immune cells. Then, we showed that selenium had no significant effect on HIV-1 protein production nor on infectivity, but slightly reduced the percentage of infected cells in a Jurkat cell line and isolated primary CD4 T cells. Finally, in response to HIV-1 infection, the selenoproteome was slightly altered.     

Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells

Hypoxia is a major obstacle to gastric cancer (GC) therapy and leads to chemoresistance as GC cells are frequently exposed to the hypoxia environment. Apigenin, a flavonoid found in traditional medicine, fruits, and vegetables and an HDAC inhibitor, is a powerful anti-cancer agent against various cancer cell lines. However, detailed mechanisms involved in the treatment of GC using APG are not fully understood. In this study, we investigated the biological activity of and molecular mechanisms involved in APG-mediated treatment of GC under hypoxia. APG promoted autophagic cell death by increasing ATG5, LC3-II, and phosphorylation of AMPK and ULK1 and down-regulating p-mTOR and p62 in GC. Furthermore, our results show that APG induces autophagic cell death via the activation of the PERK signaling, indicating an endoplasmic reticulum (ER) stress response. The inhibition of ER stress suppressed APG-induced autophagy and conferred prolonged cell survival, indicating autophagic cell death. We further show that APG induces ER stress- and autophagy-related cell death through the inhibition of HIF-1α and Ezh2 under normoxia and hypoxia. Taken together, our findings indicate that APG activates autophagic cell death by inhibiting HIF-1α and Ezh2 under hypoxia conditions in GC cells.     

糖原合成酶激酶-3β介导低氧诱导因子-1α抗心肌缺血/再灌注损伤的作用

目的通过稳定低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的表达,探讨其是否通过糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)减轻大鼠心肌缺血/再灌注损伤。方法将雄性SD大鼠随机分为5组:假手术组(Sham组)、心肌缺血/再灌组(I/R组)、HIF-1α稳定剂二甲氧乙二酰甘氨酸(dimethyloxalyl glycine,DMOG)+心肌缺血/再灌注组(DMOG+I/R组)、HIF-1α抑制剂YC-1+I/R组(YC-1+I/R组)、DMOG+GSK-3β抑制剂SB216763+I/R组(DMOG+SB216763+I/R组)。各组进行相应处理后,处死大鼠并采集样本,Western blot法检测心肌组织中HIF-1α、总GSK-3β、p-GSK-3β(Ser9)、UCP3蛋白表达量;Real-time PCR法检测心肌组织中VEGF、HO-1、Bcl2基因mRNA转录水平;HE染色法检测心肌损伤情况;免疫组化法检测心肌细胞炎性浸润情况;TUNEL染色法检测心肌细胞凋亡情况。结果与I/R组相比,DMOG预处理组可上调I/R大鼠心肌组织中HIF-1α蛋白及其下游因子VEGF、HO-1、Bcl2 mRNA表达水平,并引起线粒体内膜UCP3蛋白及mRNA表达水平升高,同时明显上调p-GSK-3β(Ser9)蛋白表达,并减轻心肌损伤、炎性细胞浸润及降低心肌细胞凋亡率(P <0. 05);与DMOG预处理组相比,给予GSK-3β抑制剂SB216763后,HIF-1α的心肌保护效应明显下降(P <0. 05)。结论 HIF-1α可减轻心肌缺血/再灌注损伤,其心脏保护作用应与GSK-3β的介导有关。     

靶向人Gadd45α基因的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞生物学功能损伤的影响

目的研究靶向人生长阻滞和DNA损伤45α(Growth arrest and DNA damage 45 alpha,Gadd45α)基因和表达绿色荧光蛋白(Green fluorescert protein,GFP)的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞(Human umbilical vein endothelialcells,HUVECs)表达上调的Gadd45α的沉默作用,初步阐明Gadd45α在缺氧应激致HUVECs生物学功能损伤过程中的作用。方法慢病毒包装后感染HUVECs,筛选最适MOI及感染时间。感染72h后,采用Real-time PCR和Western blot检测细胞中Gadd45αmRNA和蛋白的表达;流式细胞术检测细胞的凋亡率;Transwell小室实验检测细胞的迁移率;ELISA检测细胞sFlt-1和sEng的分泌水平。结果包装后慢病毒载体的滴度为1×108TU/ml,最适MOI为20,最适感染时间为72h,对HUVECs的感染效率约为80%。Gadd45α shRNA慢病毒载体对Gadd45α基因的沉默效率可达80%,阴性对照慢病毒载体对Gadd45α的表达无抑制作用。缺氧可致HUVECs中Gadd45α的表达上调,靶向Gadd45α基因的shRNA慢病毒颗粒可有效抑制缺氧致HUVECs的凋亡,减少sFlt-1及sEng的释放,同时增强其体外迁移能力,与缺氧组相比,差异具有统计学意义(P<0.05)。Gadd45α蛋白的表达水平与sFlt-1及sEng的分泌水平呈正相关(r1=0.89,r2=0.77,P均<0.05)。结论沉默Gadd45α基因对缺氧应激条件下的HUVECs生物学功能具有保护作用;Gadd45α可能是一个关键上游位点,参与子痫前期时sFlt-1及sEng的释放。     

Oxidative Folding in the Mitochondrial Intermembrane Space in Human Health and Disease

Oxidative folding in the mitochondrial intermembrane space (IMS) is a key cellular event associated with the folding and import of a large and still undetermined number of proteins. This process is catalyzed by an oxidoreductase, Mia40 that is able to recognize substrates with apparently little or no homology. Following substrate oxidation, Mia40 is reduced and must be reoxidized by Erv1/Alr1 that consequently transfers the electrons to the mitochondrial respiratory chain. Although our understanding of the physiological relevance of this process is still limited, an increasing number of pathologies are being associated with the impairment of this pathway; especially because oxidative folding is fundamental for several of the proteins involved in defense against oxidative stress. Here we review these aspects and discuss recent findings suggesting that oxidative folding in the IMS is modulated by the redox state of the cell.