纯氧或空气环境复苏对新生大鼠缺氧性大脑皮质神经元超微结构的影响

目的应用缺氧动物模型比较纯氧环境(pure oxygen environment,POE,100%氧)与空气环境(roomair environment,RAE,21%氧)复苏对新生大鼠缺氧性大脑皮质神经元超微结构的影响。方法7日龄20只SD(Sprague Dawley)乳鼠建立缺氧2.5 h模型后,分为纯氧环境组(POE)和空气环境组(RAE),每组再根据复氧后时间点分为2个亚组,即24 h组和72 h组,每亚组5只。按时间点取各组乳鼠大脑半球右侧额顶皮质行电镜样品制备,透射电镜观察。结果复氧各组均可见神经元、神经毡和细胞间隙水肿。RAE 24 h组神经元核膜结构不清,细胞器减少,线粒体肿胀、空泡化、嵴断裂;粗面内质网扩张,常呈空泡状,核糖体减少;高尔基复合体囊泡扩张;溶酶体较多。RAE 72 h组细胞器改变同前,但类似凋亡的细胞核较多,坏死细胞亦较其他组多见。POE 24 h组病变较RAE 24 h组轻。POE 72 h组细胞内线粒体及粗面内质网较丰富,病变亦比RAE 72 h组轻。结论POE复苏较RAE复苏可更能缓解缺氧致神经元超微结构的损伤、减少细胞凋亡及减轻脑水肿。提示,纯氧环境对缺氧复苏后大脑皮质神经元有一定的保护作用,并表明本动物模型适合缺氧新生大鼠大脑皮质神经元研究。     

高原环境与肠道微环境间的关系研究

为了便于进一步认识和深入研究高原环境与机体肠道微环境间的关系,本文就高原环境的特点,肠道微环境的病理学改变,分类,治疗和预后等相关研究做一简要综述。     

Activation of Glucose-6-phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O₂ tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N₂, 5% CO₂) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD⁺ and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca²⁺ sensitivity to the myofilaments and diminished Ca²⁺-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca²⁺-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD^mut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP⁺ ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.     

Critical oxygen partial pressures and maximal tracheal conductances for Drosophila melanogaster reared for multiple generations in hypoxia or hyperoxia

In Drosophila melanogaster and other insects, increases in atmospheric oxygen partial pressure (aPO₂) tend to increase adult body size and decrease tracheal diameters and tracheolar proliferation. If changes in tracheal morphology allow for functional compensation for aPO₂, we would predict that higher aPO₂ would be associated with higher critical PO₂ values (CritPO₂) and lower maximal tracheal conductances (G_max). We measured CritPO₂ and G_max for adult and larval vinegar flies reared for 7-9 generations in 10, 21 or 40 kPa O₂. The CritPO₂, CO₂ emission rates and G_max values were generally independent of the rearing PO₂ these flies had experienced, suggesting that minimal functional changes in tracheal capacities occurred in response to rearing PO₂. Larvae were able to continue activity during 20 min of anoxia. The lack of multigenerational rearing PO₂ effects on tracheal function suggests that the functional compensation at the whole-body level due to tracheal morphological changes in response to aPO₂ may be minimal; alternatively the benefits of such compensation may occur in specific tissues or during processes not assessed by these methods. In larvae, the CritPO₂ and the capacity for movement in anoxia suggest adaptations for life in hypoxic organic matter.     

Zinc-induced Neurotoxicity Mediated by Transient Receptor Potential Melastatin 7 Channels

Transient receptor potential melastatin 7 (TRPM7) channels are novel Ca²⁺-permeable non-selective cation channels ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in cellular Mg²⁺ homeostasis, diseases caused by abnormal magnesium absorption, and in Ca²⁺-mediated neuronal injury under ischemic conditions. Here we show strong evidence suggesting that TRPM7 channels also play an important role in cellular Zn²⁺ homeostasis and in Zn²⁺-mediated neuronal injury. Using a combination of fluorescent Zn²⁺ imaging, small interfering RNA, pharmacological analysis, and cell injury assays, we show that activation of TRPM7 channels augmented Zn²⁺-induced injury of cultured mouse cortical neurons. The Zn²⁺-mediated neurotoxicity was inhibited by nonspecific TRPM7 blockers Gd³⁺ or 2-aminoethoxydiphenyl borate, and by knockdown of TRPM7 channels with small interfering RNA. In addition, Zn²⁺-mediated neuronal injury under oxygen-glucose deprivation conditions was also diminished by silencing TRPM7. Furthermore, we show that overexpression of TRPM7 channels in HEK293 cells increased intracellular Zn²⁺ accumulation and Zn²⁺-induced cell injury, while silencing TRPM7 by small interfering RNA attenuated the Zn²⁺-mediated cell toxicity. Thus, TRPM7 channels may represent a novel target for neurological disorders where Zn²⁺ toxicity plays an important role.     

Type II Collagen Expression Is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.     

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.     

Prevention of CCAAT/Enhancer-binding Protein �� DNA Binding by Hypoxia during Adipogenesis

Upon exposure to adipogenesis-inducing hormones, confluent 3T3-L1 preadipocytes express C/EBPβ (CCAAT/enhancer binding protein β). Early induced C/EBPβ is inactive but, after a lag period, acquires its DNA-binding capability by sequential phosphorylation. During this period, preadipocytes pass the G₁/S checkpoint synchronously. Thr¹⁸⁸ of C/EBPβ is phosphorylated initially to prime the factor for subsequent phosphorylation at Ser¹⁸⁴ or Thr¹⁷⁹ by GSK3β, which translocates into the nuclei during the G₁/S transition. Many events take place during the G₁/S transition, including reduction in p27^Kip1 protein levels, retinoblastoma (Rb) phosphorylation, GSK3β nuclear translocation, and C/EBPβ binding to target promoters. During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is stabilized, thus maintaining expression of p27^Kip1, which inhibits Rb phosphorylation. Even under normoxic conditions, constitutive expression of p27^Kip1 blocks the nuclear translocation of GSK3β and DNA binding capability of C/EBPβ. Hypoxia also blocks nuclear translocation of GSK3β and DNA binding capability of C/EBPβ in HIF-1α knockdown 3T3-L1 cells that fail to induce p27^Kip1. Nonetheless, under hypoxia, these cells can block Rb phosphorylation and the G₁/S transition. Altogether, these findings suggest that hypoxia prevents the nuclear translocation of GSK3β and the DNA binding capability of C/EBPβ by blocking the G₁/S transition through HIF-1α-dependent induction of p27^Kip1 and an HIF-1α/p27-independent mechanism.     

Hypoxia-induced Cell Death and Activation of Pro- and Anti-apoptotic Proteins in Developing Chick Optic Lobe

Exposure of the CNS to hypoxia is associated with cell death. Our aim was to establish a temporal correlation between cellular and molecular alterations induced by an acute hypoxia evaluated at different post-hypoxia (p-h) times and at two stages of chick optic lobe development: embryonic days (ED) 12 and 18. TUNEL assays at ED12 disclosed a significant increase (300%) in pyknotic cells at 6 h p-h, while at ED18 no morphological changes were observed in hypoxic versus controls. At ED12 there was a significant increase (48%) in Bcl-2 levels at the end of the hypoxic treatment, followed by a significant increase of active caspase-9 (49%) and active caspase-3 (58%) at 30 and 60 min p-h, respectively, while at ED18 no significant changes were observed. These findings indicate that prenatal hypoxia produces an equilibrated imbalance in both pro- and anti-apoptotic proteins that culminates in a process of cell death, present at earlier stages of development.