PERK signal pathway is involved in hypoxia-induced endoplasmic reti-culum stress and apoptosis in cultured cardiac myocytes

AIM:To investigate the role of endoplasmic reticulum(ER) stress in the process of hypoxia-induced neonatal rat myocardial injury through PERK signal pathway. METHODS:Neonatal rat cardiac myocytes were randomly divided into control group and hypoxia 1 h, 4 h, 8 h, 12 h and 24 h groups. Cell viability was evaluated by determining the intracellular content of ATP. Apoptosis was measured by high-content analysis(HCA) cell imaging system. The protein levels of GRP78, calreticulin, p-PERK, p-eIF2α, ATF4 and CHOP were detected by Western blotting at different time points. The primary cultured neonatal rat cardiac myocytes were treated with an agonist of PERK pathway salubrinal and the cell apoptosis was observed under hypoxia. RESULTS:In the early phase, hypoxia induced an increase in the expression of calreticulin and GPR78. In the middle phase of hypoxia, the levels of p-PERK, p-eIF2α and ATF4 were increased. In the later phase of hypoxia, increased CHOP level was also observed. Salubrinal effectively protected the cardiac myocytes from hypoxic injury. CONCLUSION:Hypoxia activates ER stress in cardiac myocytes and also activates PERK signal pathway. PERK signaling protects cardiac myocytes from hypoxic damage in the early stage and triggers apoptosis of the cells in the later phase.     

Effects of open- and closed-system temperature changes on blood O2-binding characteristics of Atlantic bluefin tuna (Thunnus thynnus)

We investigated the effects of open- and closed-system temperature changes on the O₂ affinity of Atlantic bluefin tuna (Thunnus thynnus) blood using in vitro methods essentially identical to those previously employed on tropical tuna species. Bluefin tuna blood has a general O₂ affinity (P ₅₀ = 2.6–3.1 kPa or 19–23 mm Hg at 0.5% CO₂) similar to that of skipjack tuna, yellowfin tuna, and kawakawa blood (P ₅₀ = 2.8–3.1 kPa at 0.5% CO₂) but significantly above that of bigeye tuna blood (P ₅₀ = 1.6–2.0 kPa at 0.5% CO₂). We therefore hypothesize that bluefin tuna are less tolerant of hypoxia than bigeye tuna. Further, we found the P ₅₀ of bluefin tuna blood to be slightly reduced by a 10°C open-system temperature increase (e.g., from 4.83 kPa at 15°C to 3.95 kPa at 25°C) and to be completely unaffected by a 10°C closed-system temperature change. Bluefin tuna blood, therefore, had a significantly reduced Bohr effect when subjected to the inevitable changes in P CO ₂ and plasma pH that accompany closed-system temperature shifts (0.04–0.09 Δlog P₅₀ΔpH⁻¹) compared with the effects of changes in plasma pH accomplished by changing P CO ₂ alone (0.81–0.94 Δlog P₅₀ Δ pH⁻¹). This response is similar to that of skipjack tuna blood, but different from yellowfin or bigeye tuna blood. During closed-system temperature changes at oxygen levels above P ₅₀, however, bluefin tuna blood showed a reversed temperature effect (i.e., P O ₂ decreased in response to an increase in temperature). Unlike in other tuna species, temperature effects on O₂ affinity of bluefin tuna whole blood were similar to those previously reported for hemoglobin solutions, suggesting that red cell-mediated ligand changes are not involved.     

黄精对小鼠耐缺氧的实验研究

目的研究黄精对小鼠耐缺氧能力的影响。方法选择常温常压耐缺氧实验和亚硝酸钠中毒耐缺氧存活实验两种方法。结果黄精高、中、低剂量组明显比对照组存活时间长,其中黄精高剂量组实验结果与氯丙嗪对照组最为接近.结论:黄精具有提高小鼠耐缺氧能力的作用。     

Effects of different β-adrenergic receptors on cardiac systolic and diastolic functions in hypoxic stress rats

AIM:To explore the effects of different β-adrenergic receptors (β-AR) on the left and right ventricular systolic and diastolic functions in rats under acute hypoxic stress. METHODS:The healthy male SD rats were randomly divided into 4 groups (n=7):control group, non-selected β-AR blocker propranolol group, selected β₁-AR blocker atenolol group and selected β₂-AR blocker ICI 118,551 group, and then the rats were exposed to normoxia (20.9% O₂, 79.1% N₂) and hypoxia (15.0% O₂, 85.0% N₂) condition respectively at the altitude of 2 260 m (Xining, China). The heart rate (HR), the left ventricular systolic pressure (LVSP), the right ventricular systolic pressure (RVSP), and the maximum raise/decline rate of left and right ventricular pressure (±dp/dt_max) were monitored, and the arterial blood gas in normoxia and hypoxia condition were compared to explore the effect of β-AR on the left and right ventricular systolic and diastolic functions in acute hypoxic stress rats. RESULTS:Under normoxia condition, the LVSP, ±dp/dt_max of left ventricular were decreased in propranolol group, atenolol group and ICI 118,551 group, the RVSP and ±dp/dt_max of right ventricle were decreased in propranolol group and atenolol group (P<0.05). Under hypoxia condition, the PaO₂, LVSP, ±dp/dt_max of left ventricle were decreased in all groups compared with the normoxia group, and the ±dp/dt_max of right ventricle was increased in all groups (P<0.05), also the degree of index change in control group was more obvious than that in propranolol group and atenolol group. CONCLUSION:The activation of β₁-AR is an important compensatory regulation for heart function during hypoxic stress. However, the compensatory enhancement of right heart function under acute hypoxia condition which through tonogenic dilation is more significant for maintaining the normal circulating blood flow.     

Effect of minocycline on glutamate uptake in periventricular zone of neonatal rats after hypoxia

AIM: To investigate the role of minocycline on glutamate uptake in the periventricular zone and its putative mechanism after hypoxic exposure in neonatal rats. METHODS: A model of hypoxic-ischemic brain damage (HIBD) was developed by putting postnatal 1 d rat pups in 5% O₂ for 3.5 h. The glutamate level in periventricular zone was measured by liquid chromatography coupled with tandem mass spectrometry assay (LC-MS/MS) after hypoxic exposure for 4 h and 1 d. The dynamic changes of glutamate transporters EAAT1, and EAAT2 during developmental period in periventricular zone were determined by Western blot. Moreover, the expression of EAAT1, EAAT2, Iba-1, IL-1β, TNF-α and TGF-β1 was also detected by Western blot after hypoxic exposure for 4 h and 1 d in that region. The effects of minocycline on all parameters mentioned above were tested after minocycline treatment at the same time points and in the same region. RESULTS: After hypoxic exposure, glutamate level was increased, but it was decreased after minocycline treatment. EAAT1 and EAAT2 kept a low expression level at the first postnatal week, but a predominant elevation was found at the end of the second postnatal week. The expression of EAAT1, EAAT2, Iba-1, IL-1β and TGF-β1 was increased at 1 d after hypoxic exposure. EAAT1 and TNF-α expression was significantly up-regulated, while EAAT2 was down-regulated after minocycline treatment. CONCLUSION: Minocycline inhibits the increase in the glutamate level after hypoxia in periventricular region of the neonatal rats. The mechanism may relate to the selective regulation of glutamate transporters, rather than the inhibition of neuroinflammation in periventricular zone.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Cloning and sequencing of heme oxygenase-1 gene from Ochotona curzo-niae

[ABSTRACT] AIM: To clone and analyze the heme oxygenase-1 (HO-1) gene from Ochotona curzoniae (plateau pika). METHODS: The cDNA of HO-1 was cloned by RT-PCR and rapid amplication of cDNA ends (RACE) from the liver of Ochotona curzoniae. The bioinformatic analysis of HO-1 gene was performed. RESULTS: The cDNA of HO-1 gene in Ochotona curzoniae was obtained. The data of the sequence was deposited into GenBank and the accession number is JX035934. The full length of cDNA was 1 466 bp, including 873 bp encoding sequence (290 amino acids). Homology comparison showed that the DNA sequence of the HO-1 gene was highly homologous with Oryctolagus cuniculus (89%), Homo sapiens (87%), Bos taurus (85%), Mus musculus (79%), Rattus norvegicus (84%), Sus scrofa (85%) and Equus caballus (85%), and the amino acid sequence of HO-1 was identified with the homology of 89%, 85%, 84%, 80%, 79%, 82% and 67%, respectively. Phylogenetic analysis suggested that the structure of HO-1 was highly similar to that in Oryctolagus cuniculus. CONCLUSION: The HO-1 gene of Ochotona curzoniae was successfully cloned and provides essential information for elucidating the possible roles of HO-1 in adapting of Ochotona curzoniae to extremely high altitude.     

Hypoxic preconditioning increases tolerant ability of old human bone marrow mesenchymal stem cells to oxidative stress injury through AKT pathway

AIM: To investigate the protective effect of hypoxic preconditioning on human bone marrow mesenchymal stem cells (hBM-MSCs), and to provide basic experimental support for more effective autologous stem cell transplantation in aged patients. METHODS: The old hBM-MSCs were subjected to hypoxic preconditioning using a hypoxia incubator chamber for 24 h. The cells were divided into young group, old group and old+hypoxia group (with 24 h hypoxic preconditioning). Hydrogen peroxide (H₂O₂, 300 μmol/L) was applied to simulate the oxidative stress. The cells were treated with 50 μmol/L LY294002 for 2 h to inhibit PI3K/AKT pathway. BrdU incorporation and CCK-8 assay were used for analyzing the cell proliferation and viability. The protein levels of Bax, Bcl-2 and p-AKT were measured by Western blot. RESULTS: BrdU-positive cells, which represented the cell proliferation, and the cell viability were significantly increased in old+hypoxia group compared with old group (P<0.05). The protein level of Bax decreased (P<0.05) and Bcl-2 increased (P<0.05) in old+hypoxia group compared with old group after using 300 μmol/L H₂O₂ simulate. the oxidative stress. The phosphorylation of AKT was enhanced by hypoxic preconditioning in old group (P<0.05). The protective effect of hypoxic preconditioning on the cell survival was decreased after treated with LY294002 (inhibitor of the PI3K/AKT pathway) (P<0.05). CONCLUSION: Hypoxic preconditioning increases the survival and proliferation of old hBM-MSCs by activation of AKT pathway.