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AIM:To investigate the effect of hypoxia-preconditioned human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSC-Exos)on pulmonary vascular endothelial-mesenchymal transition(EndMT)in hypoxic pulmonary hypertension(HPH).METHODS:(1)Primary hUCMSCs were isolated and cultured by tissue adhesion method,and hUCMSC-Exos were extracted by ultrafiltration and identified.(2)Twenty-four SPF male SD rats were ran-domly divided into normoxia(N)group,hypoxia(H)group,hypoxia+normoxic hUCMSC-Exos group and hypoxia+hypoxia-preconditioned hUCMSC-Exos group,with 6 rats in each group.The rats in H group and intervention groups were placed in a cabin that simulated the hypoxic environment at an altitude of 5 000 m,and normoxic hUCMSC-Exos,hypoxia-precon-ditioned hUCMSC-Exos or equivalent volume of PBS were injected through the tail vein on the 3rd,5th,7th,10th and 14th days in hypoxia environment.After 21 d of modeling,the right ventricular systolic pressure(RVSP)and right ven-tricular hypertrophy index(RVHI)of the rats were detected,and the pathological changes of lung tissues were observed by HE staining.(3)After starvation for 12 h,human pulmonary arteriole endothelial cells(HPAECs)were randomly di-vided into normoxic control(N-Con)group,hypoxic model(H-Con)group,hypoxia+normoxic hUCMSC-Exos group and hypoxia+hypoxia-preconditioned hUCMSC-Exos group.The migration ability and tube formation ability of HPAECs were detected by Transwell assay and tube formation experiment.The expression of CD31 and α-smooth muscle actin(α-SMA)in HPAECs was detected by immunofluorescence double-staining.The protein levels of CD31,VE-cadherin,α-SMA and vimentin in pulmonary vessels and HPAECs were assessed by Western blot.RESULTS:(1)The HPH rat model was suc-cessfully established after 21 d of hypoxia,and EndMT occurred in pulmonary vessels.Compared with N group,the levels of RVSP,RVHI,percentage of vascular wall area(WA%)and percentage of vascular wall thickness(WT%)in H group were significantly increased(P＜0.01),pulmonary vascular wall thickening and the protein levels of CD31 and VE-cad-herin were significantly decreased(P＜0.01),while the protein levels of α-SMA and vimentin were significantly increased in pulmonary vessels(P＜0.05 or P＜0.01).Compared with H group,the RVSP,RVHI,WA%and WT%(P＜0.01)were significantly decreased(P＜0.05 or P＜0.01),and pulmonary vascular remodeling was attenuated after normoxic or hypoxia-preconditioned hUCMSC-Exos intervention.After hypoxia-preconditioned hUCMSC-Exos intervention,HPH pul-monary vascular remodeling and EndMT formation were significantly inhibited.(2)After 48 h of hypoxic treatment,the migration,tubule formation and EndMT of HPAECs were induced.Compared with H-Con group,cell migration and tube formation were significantly decreased after hypoxia-preconditioned hUCMSC-Exos intervention(P＜0.01).The protein levels of CD31 and VE-cadherin were increased,while the protein levels of α-SMA and vimentin were decreased(P＜0.05 or P＜0.01).CONCLUSION:Hypoxia-preconditioned hUCMSC-Exos attenuate the formation of HPH pulmonary vascu-lar remodeling by inhibiting pulmonary vascular EndMT.
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Hypoxic preconditioning could improve the survival of mesenchymal stem cells (MSCs) in ischemic or hypoxic environments, but its exact mechanism remains to be further explored. This study aims to determine the role of lysine crotonylation (Kcr) in regulating the survival and proliferation of peripheral blood mesenchymal stem cells (PBMSCs) in the hypoxic culture. PBMSCs were isolated and cultured from rat peripheral blood mononuclear cells, and their surface markers were identified by flow cytometry. PBMSCs were first subjected to hypoxic/ normoxic preconditioning: hypoxic (1% O
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Objective To investigate the effect of hypoxic pretreatment on the angiogenesis of aged human bone marrow mesenchymal stem cells (hBMSCs), so to provide experimental support for more effective autologous stem cell transplantation therapy in aged patients with ischemic myocardial injury. Methods The aged hBMSCs were cultured in a hypoxic incubator, and then the cell morphology was observed under inverted phase-contrast microscope, the surface markers were detected by flow cytometry, and the differentiation potential was detected by osteogenic and adipogenic differentiation. Subsequently, the conditioned medium (CM) of young hBMSCs under normoxic culture (norCM), the conditioned media of aged hBMSCs under normoxic and hypoxic culture(hypoCM) were collected respectively. They were named as norCM-young, norCM-old and hypoCM-old. The equal volume of medium, which was not treated with stem cells, was set as control group. Human umbilical vein endothelial cells (HUVECs) were treated with 4 conditioned media, the cell survival rate was detected by CCK-8 assay, and the tube formation experiment was used to detect the angiogenesis ability in vitro. The BCA method was used to detect the total protein concentration of the conditioned medium of each group, and the Western blotting was used to detect the expression of hypoxia inducible factor-1α (HIF-1α) in the cells and the conditioned media. Results There was not significant effect of hypoxic pretreatment on the morphology, surface markers and differentiation ability of aged hBMSCs (P > 0. 05. n ≥ 3). Compared with the norCM-old group, hypoCM-old group significantly improved the survival of HUVECs under hypoxia-reoxygenation condition (P < 0. 05, n = 5), and the tube formation ability of it (P<0. 01, n = 5). The total protein concentration of hypoCM-old group was significantly higher than that of norCM-old group (P<0. 05, n = 3). The expression of HIF-1α in hBMSCs of hypo-old group was significantly higher than that of nor-old group (P<0. 05,n = 3), while the content of HIF-1α in conditioned medium of hypoCM-old group was significantly higher than that in norCM-old group (P<0. 01,n = 3). Conclusion The aged hBMSCs pretreated with hypoxia can promote the survival and tube formation of HUVECs through paracrine in vitro, which is HIF-1α-related.
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[Abstract] Objective To investigate the protective effects of hypoxic preconditioning (HPC) on cardiomyocytes after hypoxia/reoxygenation (H/R) by activating AMP-activated protein kinase (AMPK). Methods The primary cardiomyocytes were prepared from neonatal SD rats. The model of H/R of cardiomyocytes was established and randomly divided into control group, H/R group, HPC group, H/R+A-769662 (AMPK agonist) group and HPC+compound C (AMPK inhibitor) group. The survival rate of cardiomyocytes was detected by chromatometry with Cell Counting Kit-8 (CCK-8); the reactive oxygen species (ROS) and ATP contents in the cells were detected respectively by dihydroethidium (DHE) dyeing and ATP Detection Assay Kit measured by fluorescence microplate reader; the phosphorylation level of AMPK and activation level of caspase-3 were detected by Western blotting; the concentration of free Ca2+ in cardiomyocytes was detected by Fluo-3AM dyeing using laser scanning confocal microscopy; the apoptotic rate of cardiomyocytes was detected by TUNEL staining. Results CCK-8 assay showed that compared with H/R group, the survival rate of cardiomyocytes in HPC group and H/R+A-769662 group increased obviously (P0.05); compared with H/ R group, the AMPK phosphorylation levels elevated significantly in HPC group and H/R+A-769662 group respectively (P<0.05, P<0.01). Meanwhile, the activation levels of caspase-3 in H/R group and HPC+compound C group were increased obviously compared with that in control group (P<0.01), but in HPC group and H/R+A-769662 group were significantly lower than that in H/R group (P<0.01, P<0.05). The results of Fluo-3AM staining showed that compared with control group, the concentrations of intracellular free Ca2+ increased obviously in H/R group and HPC+compound C group (P<0.01); the concentrations of free Ca2+ in cardiomyocytes of HPC group and H/R+A-769662 group were less than that in H/R group (P<0.01), while the concentrations of free Ca2+ in HPC+compound C group much more increased than in HPC group (P<0.01). The results of TUNEL staining showed that compared with H/R group, the apoptotic rate of cardiomyocytes decreased obviously in HPC group and H/R+A-769662 group (P<0.01, P<0.05); while compared with HPC group, the apoptotic rate of cardiomyocytes increased significantly in HPC+compound C group (P<0.05). Conclusions H/R may lead to the increase of ROS production, the decrease of ATP contents, and increase the levels of free Ca2+ in cardiomyocytes, and thus activate caspase-3, lead to apoptosis eventually. While HPC may reduce the production of ROS by activating AMPK to ensure the energy supply of cardiomyocytes after H/R treatment, prevent apoptosis of cardiomyocytes, and then reduce the H/R injury to cardiomyocytes.
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Objective To explore the effect of hypoxic preconditioning (HPC) on endoplasmic reticulum stress after traumatic brain injury in rats.Methods Forty-eight Sprague Dawley rats were randomly divided into HPC group (HPC modeling) and non-HPC group (without HPC modeling),with 24 rats in each group.And then,each of the group was further divided into four sub-groups (n=6):three sub-groups after traumatic brain injury (TBI) for one,3 and 7 days (TBI modeling,and drawing and observation after various TBI treatment times),and a control sub-group (without any treatment).HPC models were induced in the low-pressure oxygen chamber for 3 h daily continuing for 3 d.TBI models were established by modified Freeny's freely falling equipment.Modified neurological severity scale (mNSS) scores of the rats were recorded after brain injury.C/EBP homologous protein (CHOP) mRNA and protein expressions were detected by quantitative real-time PCR (qRT-PCR) and Western blotting.TUNEL was used to evaluate the apoptotic rate and the correlation between CHOP levels and apoptotic rate was analyzed.Results The rnNSS scores,relative CHOP mRNA and protein expressions,and apoptotic rate in the one,3 and 7 days subgroups after TBI were significantly higher than those in the control group (P＜0.05);and these levels peaked at 3 d;the mNSS scores,relative CHOP mRNA and protein expressions,and apoptotic rate in HPC group were significantly lower than those in the non-HCP group (P＜0.05);and the correlation analysis showed the CHOP expressions were positively correlated with apoptotic rate in the in HPC group and non-HCP group (r=0.957,P=0.000;r=0.966,P=0.000).Conclusion HPC can down-regulate the expression of pro-apoptotic protein CHOP which participates in endoplasmic reticulum stress pathway,reduce neuronal apoptosis and improve neurological function.
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Objective To investigate the effects of hypoxic preconditioning on learning and memory and the possible protective mechanism in mice with cerebral ischemia-reperfusion injury.Methods Healthy adult male Kunming mice were randomly divided into five groups by Random number table:normal group( N group),hypoxic preconditioning group (HPC group),sham operation group (C group),ischemia-reperfusion group(O group),hypoxic preconditioning and ischemia-reperfusion group(HPC+O group).HPC+O group were given hypoxic preconditioning before 24h of ischemia-reperfusion.The escape latency was detected by Morris water maze and the neuron apoptosis of CA 1 area of hippocampal was determined by immunofluores-cence techniqueR.e sults The escape latency in HPC+O group on the second,third and fourth day of MWM was (39.92±4.52)s,(30.98±2.44)s,(19.69±4.27)s,and significantly lower than that in O group((54.35± 3.66)s,(46.31±4.81)s,(36.81±3.86)s).Mice in HPC+O spent longer time in the target quadrant than that in O group((36.44±5.33)%and(24.5±2.59)%,respectively, P<0.05).Immunofluorescence showed that the apoptotic ration of nerve cells in hippocampal CA 1 was significantly lower than that in O group ( 11.7 ± 0.14 and 1.35±0.14, P<0.05).Conclusion Hypoxic preconditioning can increase hippocampal CA1 neurons hypoxia tolerance of ischemia reperfusion injury in mice,and reduce the incidence of neural cell apoptosis.
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AIM: To examined the effects of hypoxic preconditioning ( HPC) on oxygen-glucose deprivation ( OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy .METHODS: Cultured PC12 cells were randomly divided into control group , HPC group, 3-methyladenine (3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group .CCK-8 assay was used to detect the cell viability .The caspase-3 activity was also tested . TUNEL staining and flow cytometry were used to detect the cell apoptosis .The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot .RESULTS:Compared with control group, the viability of PC12 cells was significantly reduced , and the activity of caspase-3 was significantly increased in OGD group.Compared with 3-MA+HPC+OGD group and OGD group , the viability of PC12 cells was significantly in-creased, and the activity of caspase-3 was significantly reduced in HPC +OGD group (P<0.05).The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05).Compared with OGD group, the apoptotic rate significantly decreased in HPC +OGD group ( P<0.05 ) .Compared with control group , the protein level of cleaved caspase-3 was significantly increased in OGD group ( P<0.05) .Compared with OGD group , the protein level of cleaved caspase-3 was significantly decreased , and the levels of LC3-2 and beclin-1 were significantly increased in HPC +OGD group (P<0.05).CONCLUSION:OGD decreases cell survival and induces apoptosis .Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC 12 cells from OGD induced injury .
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AIM:To elucidate whether ZFP580 is involved in the cardioprotective effects of hypoxic precondi-tioning (HPC) against hypoxia-reoxygenation (H/R) injury in H9c2 myocardial cells.METHODS: Rat heart-derived H9c2 cells were cultured in DMEM .H/R was induced by incubation under ischemic hypoxia for 3 h and reoxygenation for 2 h.HPC was induced by exposing the H 9c2 cells to 10 min of hypoxia and 20 min of reoxygenation for 3 cycles before H/R treatment.MTT staining and LDH leakage detection were used to evaluate the effects of HPC .Western blotting was used to detect the protein levels of ZFP580, phosphorylated ERK1/2 and cleaved caspased-3.The effects of ZFP580 overexpre-ssion or knockdown on H/R induced apoptosis were determined .RESULTS:The results of MTT staining and LDH leakage detection showed evidence of HPC cytoprotection against H /R-induced cell death in H9c2 cells.ZFP580 protein level and ERK1/2 phosphorylation were significantly increased in the HPC group compared with control group and H /R group. PD98059, an inhibitor of ERK1/2 phosphorylation , significantly suppressed the HPC-induced up-regulation of ZFP580 pro-tein expression.ZFP580 overexpression significantly inhibited apoptosis and caspase-3 activation in H9c2 cells.CON-CLUSION:HPC exhibits cytoprotection against H/R and leads to high level of ZFP 580 protein in H9c2 cells.ZFP580 is regulated by ERK1/2 activation and mediates the anti-apoptotic effect of the ERK1/2 signaling pathway in HPC cytoprotec-tion.
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Objective To explore the impact and significance of hypoxia preconditioning on the expression of cytochrome C and caspase 3 protein in rats after hepatic resection.Methods A hepatectomy model was used to study the ischemia reperfusion injury in hepatic resection.Sprague-Dawley rats were randomly divided into the following three groups:normal control (NC) group,hepatic resection(HR) group,and hypoxia preconditioning (HP) group,there were twenty four rats in each group.HP Group was given an 10％ oxygen-mixed gas for 90 minutes before the operation.At 1,6,12 and 24 hours after the operation,the rats were killed and the following tests were conducted:(1) Liver tissue was sampled to observe the expression of cytochrome C and caspase 3 protein; (2) blood was drawn to conduct a chemical examination; (3) Liver tissue and morphology was observed by transmission electron microscopy.Results The serum levels of ALT and AST in HP group were significantly lower than that of HR group (P＜0.05) at 1,6,12 and 24 hours after the hepatic resection.In each time,liver function of the HP group was significantly better than the HR group; The expression of cytochrome C and caspase 3 protein was decreased significantly in HP group at each measurement point.Hepatic cells in HR group showed typical apoptosis signs under transmission electron microscopy (TEM),but no apoptosis was found in HP group.Conclusion HP has marked inhibition to apoptosis by down-regulating the expression of Cyt C and Caspase-3protein and protection to chondrosomes after a hepatic resection.
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Objective To research the influence of hypoxic preconditioning in anti-oxidative ability of brain tissues and neurological functions in rats after traumatic brain injury.Methods Forty eight adult male Sprague Dawley rats were randomly divided into sham-operated group,hypoxic preconditioning group (HPC),traumatic brain injury group (TBI) and hypoxic preconditioning+traumatic brain injury group (HPCT,n=12).The HPC rat models were made by hypobaric chamber for 3 d (50 kPa,3 h/d) and TBI were induced by Feeney's improved equipment.All rats were killed 24 h after injury,and the neurological functions of each group were evaluated by modified neurologieal severity scale (mNSS);the neuronal survival around contusion area was detected by NeuN immunohistochemical staining,and the protein and mRNA expressions of nuclear transcription factor-related factor 2 (Nrf2) and thioredoxin reductases 2 (TrxR2) in the brain tissues were detected by Western blotting and real-time quantitative PCR.Results The mNSS scores in the TBI and HPCT groups were significantly higher as compared with those in the sham-operated and HPC groups (P＜0.05),but those in the HPCT group was significantly lower than those in the TBI group (P＜0.05).The neuronal survival in the TBI and HPCT groups was decreased as compared with that in the sham-operated and HPC groups (NeuN expressions:0.274±0.033,0.281±0.042 vs 0.124±0.014,0.150±0.019),with significant difference (P＜0.05),while that in the HPCT group was statistically increased as compared with that in the TBI group (P＜0.05).The expressions of Nrf2 and TrxR2 in the brain tissues of TBI and HPCT groups were significantly increased as compared with those in the sham-operated and HPC groups (P＜0.05),and those in the HPCT group was significantly up-regulated as compared with those in the TBI group (P＜0.05).Conclusion HPC can increase the anti-oxidative ability and relieve the neurological functions missing after TBI.
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Objective To explore the effect ofhypoxic preconditioning (HPC) on the expression of sphingosine kinase-1 (SphK-1) and blood-brain barrier (BBB) permeability in rats after traumatic brain injury (TBI).Methods Two hundred and four SD rats were randomly assigned into TBI group (n=96),HPC group (giving HPC and TBI,n=96) and blank control group (n=12).The rats in the TBI group were subjected to TBI with freefall impact method,while the rats of HPC group were treated with the same methods after HPC (50.47 kpa,3 d,3 h/d).And then,they were sacrificed at each time point (1,4,8 and 12 h,and 1,3,7 and 14 d after injury).RT-PCR and Western blotting were employed to detect the mRNA and protein expression changes of SphK-1 in brain contusion area at each time points after injury.Immumohistochemical staining (IgG method) was used to detect the BBB permeability changes of the rats.Results IgG scores and Sphk-1 mRNA and protein expressions in rats of TBI group and HPC group began to increase at 1 h after injury and reached the highest level 1 d after injury; they were still higher than the normal levels,with significant differences (P＜0.05); SphK-1 mRNA and protein expressions in all the three groups had the same increased trend at all the time points excepted on the 14th d of injury,with significant differences (P＜0.05).IgG scores showed that the BBB permeability in the TBI group at each time point was significantly higher than that in the HPC group (P＜0.05).Conclusion Hypoxic preconditioning can increase SphK-1 expressions after TBI to promote sphingosine 1-phosphate sphingosine transformation so as to protect of the integrity of BBB.
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Objective To investigate the effects of intermittent hypoxic preconditioning on residual liver regeneration after parital hepatectomy in rats.Methods Fifty-four Sprague-Dawley rats were randomly divided into 3 groups (each group contained eighteen animals):sham operation group (SO group),parital hepatectomy group (PH group)and intermittent hypoxic preconditioning group (IHP group).The rats in PH group underwent the left and middle lobectomy of liver(70％ hepatectomy).The rats in IHP group were exposed to hypoxic environment of 10％ oxygen for 1 h/d.And after a week,the rats underwent parital hepatectomy.Six rats in each group were sacrificed respectively on postoperative day 1,3 and 5 (POD 1,3 and 5).The resected liver and the regenerated liver were weighed to calculate liver regeneration degree and regeneration index.The values of alaninea minotransferase (ALT) and aspartate aminotransferase (AST) in the inferior vena venous blood were examined by automatic biochemical analyzer.The positive ratio of hepatocellular proliferating cell nuclear antigen (PCNA) in the residual liver was investigated immunohistochemically.Results The degree and index of liver regeneration in IHP group were respectively higher than those in PH group on POD 1 and 3(P ＜0.05),but there were no statistical differences between the two groups on POD 5.The levels of ALT and AST in PH and IHP group began to decline after surgery,but they remined higher than those in SO group.Moreover,the ALT and AST levels in IHP group were significantly lower than those in PH group on POD 1 (P ＜0.05).The positive ratios of hepatocellular PCNA were respectively higher than those in SO and PH group on POD 1,3 and 5 (P ＜ 0.05).Conclusions To some extent,preoperative intermittent hypoxic preconditioning could prevent hepatocellular damage after parital hepatectomy,what is more,it also could promote the remnant liver regeneration.But the mechanism still needs to be studied furter.
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INTRODUCCION: El estrés oxidativo (EO) es importante en la génesis de diversas patologías. Su rol en patología cardiovascular es reconocido, particularmente en isquemia-reperfusión, fenómeno asociado al uso de circulación extracorpórea (CEC) en cardiocirugía. Complicación frecuente es la fibrilación auricular post-operatoria(FAPO), que ha demostrado participación del EO. Estrategias que lo atenúen podrían reducir incidencia de FAPO. Este trabajo busca determinar efectos de un esquema de suplementación para prevenir el EO y FAPO. METODOLOGIA: Ensayo clínico, doble ciego, aleatorizado. A 80 pacientes programados para cardiocirugía con CEC se administró placebo (n=40) o suplementación(n=40), consistiendo desde 7 días antes de la cirugía ácidos grasos poli-insaturados omega-3 (n-3) (2 g/día), y 2 días pre-cirugía se agrega vitamina C (1 g/día) y E (400 UI/día), todo hasta el alta. Se obtuvieron muestras sanguíneas (al ingreso, en suplementación, en cirugía, en postoperatorio y al alta) y auriculares durante cirugía. El estado antioxidante fue medido por la habilidad plasmática para reducir hierro férrico (FRAP) y el índice GSH/GSSG. Se midió actividad de enzimas catalasa, superóxido-dismutasa y glutatión-peroxidasa. Lipoperoxidación fue medida por niveles de malondialdehído. Para variables paramétricas se usó t de student, entre grupos se usó ANOVA-Bonferroni. Significancia fue p<0.05. RESULTADOS: Suplementación con n-3 disminuyó índice GSH/GSSG en 25 por ciento. En postoperatorio hubo 21 por ciento menos de lipoperoxidación y niveles de FRAP 30 por ciento mayores. Actividad de enzimas mostró incremento significativo. Además disminuyó FAPO desde 25 por ciento a 7,5 por ciento. CONCLUSION: Suplementar con n-3 y vitaminas antioxidantes disminuye ocurrencia de FAPO evitando daño miocárdico bioquímico y funcional por EO.
INTRODUCTION: Oxidative stress is important in the genesis of several diseases. Their role in cardiovascular disease is recognized, particularly in ischemia-reperfusion, a phenomenon associated with the use of cardiopulmonary bypass (CPB) in cardiac surgery. Common complication is postoperative atrial fibrillation (FOAP), which has demonstrated participation of oxidative stress, strategies to mitigate what could reduce the occurrence of FOAP. This paper tries to determine the effect of a supplementation scheme to prevent oxidative stress and its consequences. MATERIALS AND METHODS: Randomized, double-blind, controlled trial. Eighty patients scheduled for CCEC received placebo (n = 40) or supplementation (n = 40). Inclusion criteria: Age 30-80 years, sinus rhythm. Exclusion criteria: previous cardiosurgery, paroxysmal atrial fibrillation, congenital heart disease, chronic diseases. The supplementation consisting of n-3 (2 g / day), vitamins C (1 g /day) and E (400 IU / day) from 7, 2 and 2 days before surgery, respectively, until discharge. In atrial tissue and blood samples the plasma ferric reducing ability (FRAP), index GSH/GSSG, activity of catalase, superoxide dismutase and glutathione-peroxidase, and malondialdehyde levels were measured. Protein carbonylationwas measured in atrial tissue. Parametric variables expressed as mean and standard error were analyzed with students t-test, groups were compared using ANOVA-Bonferroni. Significance was p <0.05. RESULTS: The supplementation reduced the incidence of FOAP, lipid peroxidation and protein carbonylation in 73, 21 and 19 percent (p <0.05), respectively, and increased the FRAP (30 percent) and activity of antioxidant enzymes (p <0.05). CONCLUSIONS: Antioxidant supplementation decreases FOAP probably avoiding damage by oxidative stress.
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Humans , Male , Female , Adult , Middle Aged , Aged, 80 and over , /administration & dosage , Antioxidants/administration & dosage , Extracorporeal Circulation , Atrial Fibrillation/prevention & control , Ischemic Preconditioning, Myocardial/methods , Cardiac Surgical Procedures/adverse effects , Analysis of Variance , Ascorbic Acid/administration & dosage , Double-Blind Method , Atrial Fibrillation/etiology , Lipid Peroxidation , Oxidative Stress , Protein Carbonylation , Time Factors , Vitamin E/administration & dosageABSTRACT
Objective To investigate whether conventional protein kinase C (cPKC ) βⅡ-interacting collapsin response mediator protein-2 (CRMP-2) provides neuroprotection against cerebral ischemic (I) injuries. Methods Male BALB/c mice were randomly divided into normoxic control (Nor) , HPC, Nor + Sham, HPC + Sham, Nor + I and HPC + I groups (n = 6 per group). Using our HPC and MCAO mouse models, we applied immunoprecipita-tion, two-dimensional electrophoresis and mass spectrometry to characterize cPKCβⅡ-interacting proteins and combined with SDS-PAGE and Western blot to quantitatively analyze CRMP-2 phosphorylation and degradation levels in the brain of mice after HPC and MCAO. Results The expression level of 10 cPKCβⅡ-interacting proteins changed obviously in cerebral cortex of HPC mice when compared with Nor group. One of these proteins, CRMP-2 protein level increased in particulate fraction and decreased in cytosolic fraction of cerebral cortex of HPC mice. CRMP-2 phosphorylation level in ischemic core (Ic) of cerebral cortex decreased significantly ( P < 0. 05 , n = 6) as compared with that of Nor + sham group, but CRMP-2 phosphorylation level in HPC +I group increased significantly as compared with that of Nor +I group ( P < 0. 05, n = 6). In ischemic cortex, CRMP-2 degradation (proteolysis) was observed as the appearance of 55 ku breakdown products (BDP). However, the CRMP-2 degradation level, BDPs products decreased significantly in penumbra ( P) of ischemic cortex from HPC +I group when we compared with that of Nor +I group (P < 0. 05, n = 6 ). Conclusion CRMP-2 is involved in attenuating the decrease of CRMP-2 phosphorylation in ischemic core and in inhibiting its degradation in penumbra of cerebral cortex of mice thereby to lessen the ischemic injuries.
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Objective To explore the effect of hypoxic preconditioning on the activity and gene expressions of glu-cose transporters in the cultured rat hippocampal neurons and astrocytes under anoxic condition. Methods The cultured rat hippocampal neurons and astrocytes were treated for 6 days by intermittently exposing to hypoxic gas mixture (1% O_2, 10% CO_2, 89% N_2) for20 min each day. 24 h after the last hypoxic exposure, the cells were exposed to anoxic gas mixture (10% CO_2, 90% N_2) for 6 h, and the uptake rate of [~3H]-2-deoxyglucose (2-DG), the levels of glucose transporter GLUT1 and GLUT3 mRNAs and the cell survival rate were examined im-mediately after anoxic exposure. Results Neurons and astrocytes preconditioned with hypoxia showed higher 2-DG uptake rates and increased expressions of GLUT 1 mRNA in the astrocytes and GLUT 1 and GLUT 3 mRNA in the neurons. The preconditioned neurons also showed an increased tolerance to anoxia. Conclusion Hypoxic precon-ditioning up-regulates the activity and gene expressions of glucose transporters of hippocampal neurons and astro-cytes under anoxic condition.
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Objective To observe the protective effect of hypoxic preconditioning against cerebral ischemic injury induced by acute cerebral infarction in mice. Methods Blb/c mice were randomized into normal control, sham-operated, acute cerebral infarction (CI), and hypoxic preconditioning with CI (HP+CI) groups. In the latter two groups, acute cerebral cortical infarction was induced photochemically by cold light exposure of the brain tissue following intravenous Rose Bengal injection. The mice in the sham-operated group received only cold light exposure without Rose Bengal injection. Behavioral test, immunofluorescence assay and confocal laser scanning microscopy were performed to evaluate the neurological deficits of the mice and assess the cell apoptosis, and the cerebral infarction volume was measured. Results No infarct was found in the normal control and sham-operated groups, whereas obvious isehemic infarction foci were found in CI and HP+CI groups, but the infarction volume was significantly smaller in HP+CI group (P<0.05). The mice in the normal control and sham-operated groups presented with no neurological impairment, but in CI and HP+CI groups, obvious symptoms of neurological impairment were observed with lowered neurological scores. The neurological scores were significantly higher in HP+CI group than in CI group (P<0.05). Occasional apoptotic cells were found in the normal control and sham-operated groups, while in the other two groups, the TUNEL-positive cell number increased significantly. The apoptotic cell number was significantly smaller in HP+CI group than in CI group (P<0.05). Conclusion Hypoxic preconditioning may protect against cerebral ischemic injury induced by acute cerebral infarction in mice possibly by reducing the cerebral infarction volume and cell apoptosis.
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Objective To explore the role of conventional protein kinase C(cPKCs) in delayed hypoxic preconditioning.Methods The biochemistry techniques of SDS-PAGE,Western bolt and Gel Doc imagine were applied to analyze the effect of repetitive hypoxic exposure(H5,H6) on the level of cPKC?,? membrane translocation and protein expression in murine brain.Results We found that cPKC? protein expression was significantly decreased(P
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Objective To explore the role of P38 mitogen-activated protein kinase (P38 MAPK) in the development of cerebral hypoxic preconditioning. Methods Healthy male BALB/C mice weighted as18～20 g were randomly divided into 7 groups as follows: normoxic control (H0), early (H1～H4) and delayed (H5 and H6) hypoxic preconditioned mice groups. SDS-PAGE, Western blot and Gel Doc imagine systems were applied to quantitatively analyze the level of P38 MAPK phosphorylation and protein expression in the brain of mice. Results The phosphorylation levels of P38 MAPK increased in cortex, hippocampus and hypothalamus of mice in both early (H1～H4) and delayed (H5 and H6) hypoxic preconditioned groups, and the statistic significance (P
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Objective To determine the changes in phosphorylation of conventional protein kinase C?(cPKC?)-interacting extracellular signal regulated kinase 1/2(ERK1/2) in the hippocampus of hypoxic preconditioned(HPC) mice.Methods Healthy male BalB/c mice were used to develop "auto-hypoxia"-induced HPC mice and randomly divided into 3 groups as follows: normoxic control(H0),early(H3) and delayed hypoxic preconditioned groups(H6).Co-immunoprecipitation and Western blot were applied to quantitatively analyze the phosphorylation level of cPKC?-interacting ERK1/2 in cytosolic and particulate fractions of hippocampus of HPC mice.ResultsERK1/2 was co-immunoprecipitated by cPKC? in hippocampus of mice.The phosphorylation level of cPKC? interacting ERK1/2 at tyrosine 204 decreased significantly in the hippocampus of H3 and H6 groups as compared with that of the H0 group(P
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Objective To observe the protective effects of hypoxic preconditioning(HPC) on the improvement of the cognitive dysfunction(learning and memory) and the damage in hippocampus induced by global cerebral ischemia/reperfusion(I/R) in CA1 and CA3 for 5 days in rats,and on the regulation of expression of erythropoietin(EPO) protein to approach the mechanism of the protection.Methods One hundred and twenty adult male Sprague-Dawley(SD) rats were divided into four groups randomly: sham group,I/R group,HPC24 group(hypoxia for 24 hours before I/R) and HPC48 group(hypoxia for 48 hours before I/R).Hang(motor function),passive avoidance and Morris water maze tests were carried out on the 5th day after I/R to measure the motor and cognition functions;hematoxylin-eosin(HE) staining was used to detected histopathological changes in hippocampus tissues;and the contents of EPO were tested by immunohistochemistry at 1 hour and 4 hours after I/R from hippocampus CA1 and CA3 regions.Results Hang,passive avoidance and Morris water maze tests showed that I/R can injure rat cognition;the improvement of cognition was marked in HPC groups, and it was shown that the effects were more significant in HPC48 group than those in the HPC24 group(P