DI-3-n-butylphthalide exerts neuroprotective effects by modulating hypoxia-inducible factor 1-alpha ubiquitination to attenuate oxidative stress-induced apoptosis

DI-3-n-butylphthalide is used to treat mild and moderate acute ischemic stroke.However,the precise underlying mechanism requires further investigation.In this study,we investigated the molecular mechanism of DI-3-n-butylphthalide action by various means.We used hydrogen peroxide to induce injury to PC12cells and RAW264.7 cells to mimic neuronal oxidative stress injury in stroke in vitro and examined the effects of DI-3-n-butylphthalide.We found that DI-3-nbutylphthalide pretreatment markedly inhibited the reduction in viability and reactive oxygen species production in PC12 cells caused by hydrogen peroxide and inhibited cell apoptosis.Furthermore,DI-3-n-butylphthalide pretreatment inhibited the expression of the pro-apoptotic genes Bax and Bnip3.DI-3-nbutylphthalide also promoted ubiquitination and degradation of hypoxia inducible factor 1α,the key transcription factor that regulates Bax and Bnip3 genes.These findings suggest that DI-3-n-butylphthalide exhibits a neuroprotective effect on stroke by promoting hypoxia inducible factor-1α ubiquitination and degradation and inhibiting cell apoptosis.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

Effect of NADPH Oxidase Inhibitor Apocynin on the Expression of Hypoxia-induced Factor-1α and Endothelin-1 in Rat Carotid Body Exposed to Chronic Intermittent Hypoxia

The effects of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase inhibitor apocynin on the enhanced hypoxia induced factor-let （HIF-lct） and endothelin-1 （ET-1） expression, elevated systolic blood pressure under chronic intermittent hypoxia （CIH） condition and its action mechanism were investigated. Thirty healthy 8-week old Sprague-Dawley （SD） male rats were randomly divided into three groups （n=10 each）： sham group, CIH group, and apocynin-treated CIH group. Tail artery systolic blood pressure was measured by tail-cuff method. Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the mRNA expression of HIF-lu and ET-1 in the carotid body, and the HIF-1a protein expression was examined by using Western blotting. The levels of malondialdehyde （MDA） and superoxide dismutase （SOD） were determined by using colorimetric method. In addition, the plasma ET-1 and HIF-1a levels were measured by using enzyme-linked immunosorbent assay. It was found that CIH exposure was associated with increased MDA levels, and apo- cynin-treated CIH animals showed reduction in MDA levels. Apocynin treatment prevented CIH-induced hypertension as well as CIH-induced decrease in SOD. The increases of HIF-1a and ET-1 mRNA along with HIF-la protein expression in the carotid body, and elevated circulating HIF-1a and ET-1 levels were observed in CIH-exposed animals. Treatment with apocynin significantly decreased the ET-1 mRNA, HIF-lct protein expression and circulating HIF-la level in CIH-exposed animals, and there was no statistically significant difference in the HIF-lu mRNA expression between CIH group and apo- cynin-treated group. These results indicated that apocynin alleviated CIH-induced hypertension by inhibiting NADPH oxidase, further leading to the reduced vasoconstrictor ET-1 level and oxidative stress. HIF-1a/ET-1 system signal pathway may interact with CIH-induced NADPH oxidase-dependent oxidative stress. Inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for prevention and treatment of obstructive sleep apnea hypopnea syndrome-induced hypertension.

Is the hypoxia-inducible factor-1 alpha mRNA expression activated by ethanol-induced injury, the mechanism underlying alcoholic liver disease?

BACKGROUND: Excessive alcohol consumption can result in multiple organ injury, of which alcoholic liver disease (ALD) is the most common. With economic development and improvement of living standards, the incidence of diseases caused by alcohol abuse has been increasing in China, although its pathogenesis remains obscure. The aim of this study was to investigate the role of hypoxia in chronic ALD. METHODS: Twenty-eight male Sprague-Dawley rats were randomized into a control group (n=12) with a normal history and an experimental group (n=16) fed with 10 ml/ kg of 56% (vol/vol) ethanol once per day by gastric lavage for 24 weeks. At 24 weeks, blood samples were collected and then the rats were killed. Liver samples were frozen at -80 ℃ and used for RT-PCR; other liver samples were obtained for immunohistochemical staining. RESULTS: When the period of alcohol consumption increased, the positive rate of expression of hypoxia- inducible factor-1 alpha (HIF-1α) mRNA was more significantly elevated in the liver of the alcohol group than in the control group (P≤0.05). The HIF-1α protein located in the cytoplasm was seldom expressed in the control group, but significantly in the alcohol group (P≤0.01). CONCLUSION: HIF-1α mRNA expression was activated by ethanol-induced injury in this study, suggesting that hypoxia is involved in the underlying mechanism of ALD.

Quantitative analysis of hepatic hypoxia-inducible factor-1α and its abnormal gene expression during the formation of hepatocellular carcinoma

BACKGROUND:Hepatic hypoxia-inducible factor-1(HIF-1) is activated in the progression of hepatocellular carcinoma (HCC).This study aimed to investigate the dynamic alterations of HIF-1αand its gene expression so as to explore the relationship between HIF-1αexpression and hepatocarcinogenesis at the early stage of HCC. METHODS:A hepatoma model was made with 2-fluorenyl- acetamide(2-FAA)in male Sprague-Dawley rats.Morphological changes of rat hepatocytes were assessed pathologically (HE staining).The dynamic expression of hepatic and circulating HIF-1αwas quantitatively analyzed by ELISA. The gene fragments of hepatic HIF-1αmRNA were amplified by RT-PCR and confirmed by sequencing.The cellular distribution of hepatic HIF-1αexpression was confirmed by immunohistochemistry. RESULTS:Histological examination confirmed granulelike degeneration to atypical hyperplasia and HCC development in rat hepatocytes and progressive increases in the levels of hepatic and circulating HIF-1αand its gene expression during the course.The levels of HIF-1α expression in the liver and blood of rats with hepatoma were significantly higher than those in normal ratsand those with degeneration.Immunohistochemical analysis confirmed the positive expression and hepatocyte distribution of HIF-1αin the development of rat hepatoma. A positive relationship was found between HIF-1α expression in the liver and blood(P<0.01). CONCLUSIONS:The above observations support the hypothesis that the overexpression of HIF-1αand its gene are closely associated with the malignant transformation of hepatocytes and play an important role at the stage of hepatocarcinogenesis.

Hypoxia inducible factor-1αaccumulation in steatotic liver preservation:Role of nitric oxide

AIM:To examine the relevance of hypoxia inducible factor(HIF-1)and nitric oxide(NO)on the preservation of fatty liver against cold ischemia-reperfusion injury(IRI). METHODS:We used an isolated perfused rat liver model and we evaluated HIF-1αin steatotic and non-steatotic livers preserved for 24 h at 4℃in University of Wisconsin and IGL-1 solutions,and then subjected to 2 h of normothermic reperfusion.After normoxic reperfusion,liver enzymes,bile production,bromosulfophthalein clearance,as well as HIF-1αand NO[endothelial NO synthase(eNOS)activity and nitrites/nitrates]were also measured.Other factors associated with the higher susceptibility of steatotic livers to IRI,such as mitochondrial damage and vascular resistance were evaluated. RESULTS:A significant increase in HIF-1αwas found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage.Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters.These benefits were enhanced by the addition of trimetazidine(an antiischemic drug),which induces NO and eNOS activation, to IGL-1 solution.In normoxic reperfusion,the presence of NO favors HIF-1αaccumulation,promoting also the activation of other cytoprotective genes,such as hemeoxygenase-1. CONCLUSION:We found evidence for the role of the HIF-1α/NO system in fatty liver preservation,especially when IGL-1 solution is used.

Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury

Mild traumatic brain injury（TBI）, also called concussion, initiates sequelae leading to motor deficits, cognitive impairments and subtly compromised neurobehaviors. While the acute phase of TBI is associated with neuroinflammation and nitroxidative burst, the chronic phase shows a lack of stimulation of the neurorepair process and regeneration. The deficiency of nitric oxide（NO）, the consequent disturbed NO metabolome, and imbalanced mechanisms of S-nitrosylation are implicated in blocking the mechanisms of neurorepair processes and functional recovery in the both phases. Hypoxia inducible factor-1 alpha（HIF-1α）, a master regulator of hypoxia/ischemia, stimulates the process of neurorepair and thus aids in functional recovery after brain trauma. The activity of HIF-1α is regulated by NO via the mechanism of S-nitrosylation of HIF-1α. S-nitrosylation is dynamically regulated by NO metabolites such as S-nitrosoglutathione（GSNO） and peroxynitrite. GSNO stabilizes, and peroxynitrite destabilizes HIF-1α. Exogenously administered GSNO was found not only to stabilize HIF-1α and to induce HIF-1α-dependent genes but also to stimulate the regeneration process and to aid in functional recovery in TBI animals.

Expression of Nerve Growth Factor and Hypoxia Inducible Factor-1α and Its Correlation with Angiogenesis in Non-Small Cell Lung Cancer

Summary： In order to investigate the expression of nerve growth factor （NGF） and hypoxia inducible factor-1α （HIF-1α） and its correlation with angiogenesis in non-small cell lung cancer （NSCLC）, paraffin-embedded tissue blocks from 20 patients with NSCLC were examined. Twenty corresponding para-cancerous lung tissue specimens were obtained to serve as a control. The expression of NGF, HIF-1α, and vascular endothelial growth factor （VEGF） in the NSCLC tissues was detected by using immunohistochemistry. The microvascular density （MVD） was determined by CD31 staining. The resuits showed that the expression levels ofNGF, HIF-1α and VEGF in the NSCLC tissues were remarkably higher than those in the para-cancerous lung tissues （P〈0.05）. There was significant difference in the MVD between the NSCLC tissues （9.19±1.43） and para-cancerous lung tissues （2.23±1.19） （P〈0.05）. There were positive correlations between NGF and VEGF, between HIF-1α and VEGF, and between NGF and HIF-1α in NSCLC tissues, with the spearman correlation coefficient being 0.588, 0.519 and 0.588, respectively. In NSCLC tissues, the MVD had a positive correlation with the three factors （P〈0.05）. Theses results suggest that NGF and HIF-1α are synergically involved in the angiogenesis of NSCLC.

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.

Sirolimus increases the anti-cancer effect of Huai Er by regulating hypoxia inducible factor-1α-mediated glycolysis in hepatocellular carcinoma

BACKGROUND Glycolysis caused by hypoxia-induced abnormal activation of hypoxia inducible factor-1α(HIF-1α)in the immune microenvironment promotes the progression of hepatocellular carcinoma(HCC),leading to enhanced drug resistance in cancer cells.Therefore,altering the immunosuppressive microenvironment by improving the hypoxic state is a new goal in improving cancer treatment.AIM To analyse the role of HIF-1α,which is closely related to tumour proliferation,invasion,metastasis,and angiogenesis,in the proliferation and invasion of liver cancer,and to explore the HIF-1αpathway-mediated anti-cancer mechanism of sirolimus(SRL)combined with Huai Er.METHODS Previous studies on HCC tissues identified the importance of HIF-1α,glucose transporter 1(GLUT1),and lactate dehydrogenase A(LDHA)expression.In this study,HepG2 and Huh7 cell lines were treated,under hypoxic and normoxic conditions,with a combination of SRL and Huai Er.The effects on proliferation,invasion,cell cycle,and apoptosis were analysed.Proteomics and genomics techniques were used to analyze the HIF-1α-related signalling pathway during SRL combined with Huai Er treatment and its inhibition of the proliferation of HCC cells.RESULTS High levels of HIF-1α,LDHA,and GLUT-1 were found in poorly differentiated HCC,with lower patient survival rates.Hypoxia promoted the proliferation of HepG2 and Huh7 cells and weakened the apoptosis and cell cycle blocking effects of the SRL/Huai Er treatment.This was achieved by activation of HIF-1αand glycolysis in HCC,leading to the upregulation of LDHA,GLUT-1,Akt/mammalian target of rapamycin(mTOR),vascular endothelial growth factor(VEGF),and Forkhead box P3 and downregulation of phosphatase and tensin homolog deleted on chromosome ten(PTEN)and p27.The hypoxia-induced activation of HIF-1αshowed the greatest attenuation in the SRL/Huai Er(S50+H8)group compared to the drug treatments alone(P<0.001).The S50+H8 treatment significantly downregulated the expression of mTOR and HIF-1α,and significantly reduced the expression of VEGF mRNA.Meanwhile,the combined blocking of mTOR and HIF-1αenhanced the downregulation of Akt/mTOR,HIF-1α,LDHA,and GLUT-1 mRNA and resulted in the downregulation of PTEN,p27,and VEGF mRNA(P<0.001).CONCLUSION SRL increases the anti-cancer effect of Huai Er,which reduces the promotion of hypoxia-induced HIF-1αon the Warburg effect by inhibition of the PI3K/Akt/mTOR-HIF-1αand HIF-1α-PTEN signalling pathways in HCC.

Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells

AIM: To investigate the influence of phosphatidylinositol-3-kinase protein kinase B(PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. METHODS: Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, m RNA and activity levels of hypoxia inducible factor-1 alpha(HIF-1α), glucose transporter 1, hexokinase-Ⅱ, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting si RNA to assess impact of the high expression of HIF-1α on glycolysis.RESULTS: HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymesand the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions.CONCLUSION: The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.

Prognostic value of hypoxia-inducible factor-1 alpha and prolyl 4-hydroxylase beta polypeptide overexpression in gastric cancer

AIM To investigate the relationship between hypoxia-inducible factor-1α(HIF-1α), prolyl 4-hydroxylase beta(P4 HB) expression, and clinicopathologic parameters, as well as the prognostic value of these genes for patients with gastric cancer(Gc).METHODS Hypoxia is a critical factor that shapes the Gc microenvironment. In previous reports, we have demonstrated that P4 HB is a potential target of HIF-1α. In the present study, gene expression profiling interactive analysis(GEPIA) was used to analyze the relationship between P4 HB and hypoxia-associated genes. To this end, 428 Gc tissue samples were used to analyze the expression of HIF-1α and P4 HB via immunohistochemical staining. Patient samples were classified as having weak-expression or over-expression both in terms of HIF-1α and P4 HB. Correlations between biomarkers and clinicopathological factors were analyzed to predict survival. RESULTS P4 HB demonstrated a positive correlation with hypoxiaassociated genes(P < 0.05). HIF-1α and P4 HB overexpression have a significant correlation with TNM staging(χ2 = 23.32, P = 0.00; χ2 = 65.64, P = 0.00) and peritoneum cavity metastasis(χ2 = 12.67, P = 0.00; χ2 = 39.29, P = 0.00). In univariate analysis, patients with a high HIF-1α expression trend had a shorter disease-free survival(DFS: 44.80 mo vs 22.06 mo) and overall survival(OS: 49.58 mo vs 39.92 mo). P4 HB overexpression reflected similar results: patients with over-expression of P4 HB had a shorter survival time than those with weak-expression(DFS: 48.03 mo vs 29.64 mo, OS: 52.48 mo vs 36.87 mo). Furthermore, HIF-1α is also a clinicopathological predictor of dismal prognosis according to multivariate analysis(DFS, 95%c I: 0.52-0.88, P < 0.00; OS, 95%c I: 0.50-0.85, P < 0.00). However, P4 HB was meaningful in DFS(95%c I: 0.58-1.00, P < 0.05) but not in OS(95%c I: 0.72-1.23, P > 0.05).CONCLUSION Overexpression of HIF-1α and P4 HB is associated with poor prognosis in patients with Gc. Thus, these genes may be potential prognostic biomarker candidates in GC.

Metabolic shift in liver: Correlation between perfusion temperature and hypoxia inducible factor-1α

AIM: To study at what temperature the oxygen carried by the perfusate meets liver requirements in a model of organ perfusion. METHODS: in this study, we correlated hypoxia induciblefactor(Hi F)-1α expression to the perfusion temperature and the hepatic oxygen uptake in a model of isolated perfused rat liver. Livers from Wistar rats were perfused for 6 h with an oxygenated medium at 10, 20, 30 and 37 ℃. Oxygen uptake was measured by an oxygen probe; lactate dehydrogenase activity, lactate release and glycogen were measured spectrophotometrically; bile flow was gravitationally determined; p H of the perfusate was also evaluated; Hi F-1α m RNA and protein expression were analyzed by real time-polymerase chain reaction and ELi SA, respectively. RESULTS: Livers perfused at 10 and 20 ℃ showed no difference in lactate dehydrogenase release after 6 h of perfusion(0.96 ± 0.23 vs 0.93 ± 0.09 m U/min per g) and had lower hepatic damage as compared to 30 and 37 ℃(5.63 ± 0.76 vs 527.69 ± 45.27 m U/min per g, respectively, P s < 0.01). After 6 h, tissue ATP was significantly higher in livers perfused at 10 and 20 ℃than in livers perfused at 30 and 37 ℃(0.89 ± 0.06 and 1.16 ± 0.05 vs 0.57 ± 0.09 and 0.33 ± 0.08 nmol/mg, respectively, P s < 0.01). No sign of hypoxia was observed at 10 and 20 ℃, as highlighted by low lactate release respect to livers perfused at 30 and 37 ℃(121.4 ± 12.6 and 146.3 ± 7.3 vs 281.8 ± 45.3 and 1094.5 ± 71.7 nmol/m L, respectively, P s < 0.02), and low relative Hi F-1α m RNA(0.40 ± 0.08 and 0.20 ± 0.03 vs 0.60 ± 0.20 and 1.47 ± 0.30, respectively, P s < 0.05) and protein(3.72 ± 0.16 and 3.65 ± 0.06 vs 4.43 ± 0.41 and 6.44 ± 0.82, respectively, P s < 0.05) expression.CONCLUSION: Livers perfused at 10 and 20 ℃ show no sign of liver injury or anaerobiosis, in contrast to livers perfused at 30 and 37 ℃.

Meta-analysis of immunohistochemical expression of hypoxia inducible factor-1α as a prognostic role in gastric cancer

AIM: To conduct a meta-analysis to evaluate the prognostic role of hypoxia inducible factor-1&#x003b1; (HIF-1&#x003b1;) expression in gastric cancer.

Hypoxia-inducible factor-1 modulates upregulation of mutT homolog-1 in colorectal cancer

AIM: To investigate the roles and interactions of mut T homolog(MTH)-1 and hypoxia-inducible factor(HIF)-1α in human colorectal cancer(CRC).METHODS: The expression and distribution of HIF-1α and MTH-1 proteins were detected in human CRC tissues by immunohistochemistry and quantitative realtime polymerase chain reaction(q RT-PCR). SW480 and HT-29 cells were exposed to normoxia or hypoxia. Protein and m RNA levels of HIF-1α and MTH-1 were analyzed by western blotting and q RT-PCR, respectively. In order to determine the effect of HIF-1α on the expression of MTH-1 and the amount of 8-oxodeoxyguanosine triphosphate(d GTP) in SW480 and HT-29 cells, HIF-1α was silenced with small interfering RNA(si RNA). Growth studies were conducted on cells with HIF-1α inhibition using a xenograft tumor model. Finally, MTH-1 protein was detected by western blotting in vivo.RESULTS: High MTH-1 m RNA expression was detected in 64.2% of cases(54/84), and this was significantly correlated with tumor stage(P = 0.023) and size(P = 0.043). HIF-1α protein expression was correlated significantly with MTH-1 expression(R = 0.640; P < 0.01) in human CRC tissues. Hypoxic stress induced m RNA and protein expression of MTH-1 in SW480 and HT-29 cells. Inhibition of HIF-1α by si RNA decreased the expression of MTH-1 and led to the accumulation of 8-oxo-d GTP in SW480 and HT-29 cells. In the in vivo xenograft tumor model, expression of MTH-1 was decreased in the HIF-1α si RNA group, and the tumor volume was much smaller than that in the mock si RNA group.CONCLUSION: MTH-1 expression in CRC cells was upregulated via HIF-1α in response to hypoxic stress, emphasizing the crucial role of HIF-1α-induced MTH-1 in tumor growth.

Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND： Hypoxia inducible factor-1 alpha （HIF-1 （x） and erythropoietin（EPO）, possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance （IT）. OBJECTIVE：To observe the neuroprotective effect of cerebral ischemic preconditioning（IPC） of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN ： A randomized and controlled observation SETTING： Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University MATERIALS： Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd （Wuhan）; Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company （USA）. METHODS： This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot： IPC group （n=40）, sham-operation group （n=40） and control group （n=4）. In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1^st, 3^rd, 7^th, 14^th and 21^st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion （MCAO）. That was to say, after 10-minute preischemia, suture was exited to the extemal carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation（only common carotid artery and crotch were exposed, and MCAO by suture was omitted）, and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume （The total cerebral infarction area of each layerxinterspace ） was calculated. After brain tissue was stained by haematoxylin-esoin （HE）, the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α（and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES： ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS：Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group： Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively [（161.2±6.9） mm^3 vs （219.9±11.2） mm^3, （134.9±9.0） mm^3 vs （218.6±13.0） mm^3, （142.9±13.7） mm^3 vs （221.3±14.2） mm^3, t=-8.924, 10.587,7.947, P〈 0.01]. ② Observation of nerve cell form of brain tissue： HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus ： The expression of HIF-1αof IPC group was significantly higher than that of sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively （125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763,9.899,P 〈 0.05-0.01）. The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3^rd and 7^th days after perfusion respectively （141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18,t=5.499,3.970, P〈 0.05）. CONCLUSION ： ①IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.

Puerarin decreases hypoxia inducible factor-1 alpha in the hippocampus of vascular dementia rats

In this study, a rat vascular dementia model was established by permanent bilateral common carotid arterial occlusion. Rats were intraperitoneally injected with puerarin 3 days before modeling, for 45 successive days. Results demonstrated that in treated animals hippocampal structures were clear, nerve cells arranged neatly, and cytoplasm was rich in Nissl bodies. The number of cells positive for hypoxia inducible factor-1 alpha, erythropoietin and endothelial nitric oxide synthase was reduced; and the learning and memory abilities of rats were significantly improved. Our experimental findings indicate that puerarin can significantly improve learning and memory in a vascular dementia model, and that the underlying mechanism may be associated with the regulation of the expression of hypoxia inducible factor-1 alpha.

Expression of Hypoxia-inducible Factor-1α in Liver Tumors after Transcatheter Arterial Embolization in an Animal Model

To examine the effect of transcatheter arterial embolization （TAE） of liver tumors on hypoxia-inducible factor-1α （HIF-1α） expression in the residual viable tumor, a total of 30 New Zealand White rabbits implanted with VX2 liver tumor were divided into 2 groups. TAE-treated group animals （n=15） were subjected to TAE with 150–250 μm polyvinyl alcohol particles. Control group animals （n=15） underwent sham embolization with distilled water. Six hours, 3 days or 7 days after TAE, the animals were sacrificed, and samples of tumor and adjacent normal liver tissue were harvested. Expression of HIF-1α protein was examined immunohistochemically. Real-time PCR was performed to examine the HIF-1α mRNA levels. Our results showed that HIF-1α protein was expressed in the VX2 tumors but not in the adjacent normal liver tissue. The HIF-1α-positive tumor cells were located predominantly at the periphery of necrotic tumor regions. The mean levels of HIF-1α protein were significantly higher in TAE-treated tumors than those in control tumors （P=0.002）. Among the three sacrificing time points, the difference in increase in HIF-1α protein was significant between the two groups at the sacrificing time point of 6 h and 3 days after TAE （P=0.020, P=0.031, respectively）, whereas no significant increase was noted 7 days after TAE （P=0.502）. In contrast, although HIF-1α mRNA was expressed in TAE-treated and control VX2 tumors, there existed no significant difference in the HIF-1α mRNA level between the two groups （P=0.372）. It is concluded that TAE of liver tumors increases the expression of HIF-1α at protein level in the residual viable tumor, which could be attributed to hypoxia generated by the procedure.

Changes of hypoxia-inducible factor-1 signaling and the effect of cilostazol in chronic cerebral ischemia

Hypoxiainducible factor1 and its specific target gene heme oxygenase1, are involved in acute cerebral ischemia. However, very few studies have examined in detail the changes in the hy poxiainducible factor1/heme oxygenase1 signaling pathway in chronic cerebral ischemia. In this study, a rat model of chronic cerebral ischemia was established by permanent bilateral common carotid artery occlusion, and these rats were treated with intragastric cilostazol （30 mg/kg） for 9 weeks. Morris water maze results showed that cognitive impairment gradually worsened as the cerebral ischemia proceeded. Immunohistochemistry, semiquantitative PCR and western blot analysis showed that hypoxiainducible factorla and heme oxygenase1 expression levels in creased after chronic cerebral ischemia, with hypoxiainducible factorla expression peaking at 3 weeks and heme oxygenase1 expression peaking at 6 weeks. These results suggest that the elevated levels of hypoxiainducible factorla may upregulate heine oxygenase1 expression fol lowing chronic cerebral ischemia and that the hypoxiainducible factor1/heme oxygenase1 sig naling pathway is involved in the development of cognitive impairment induced by chronic cerebral ischemia. Cilostazol treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, decreased hypoxiainducible factorla and heme oxygenase1 expression levels, and reduced apoptosis in the frontal cortex. These findings demonstrate that cilostazol can protect against cognitive impairment induced by chronic cerebral ischemic injury through an antiapoptotic mechanism.

Inhibition of Expression of Hypoxia-inducible Factor-1α mRNA by Nitric Oxide in Hypoxic Pulmonary Hypertension Rats

In order to study the effect of nitric oxide (NO) on the expression of hypoxia inducible factor 1 alpha (HIF 1α) mRNA in hypoxic pulmonary hypertension (HPH) rats, 30 healthy male Wistar rats were randomly divided into normoxic control group, chronic hypoxic group and hypoxia plus L argine (L Arg) group. The animal model of HPH was developed. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The HIF 1α mRNA expression levels were detected by in situ hybridization (ISH) and semiquantitative RT PCR. It was found that after 14 days hypoxia, the mPAP in normoxic control group (17.6±2 7 mmHg,1 mmHg=0 133 kPa) was significantly lower than that in chronic hypoxic group(35.8±6.1 mmHg, t =0.2918, P <0.05) and mPAP in chronic hypoxic group was higher than that in hypoxia plus L argine group(24.4±3.8 mmHg, t =0.2563, P <0.05). ISH showed that the expression of HIF 1α mRNA in the intraacinar pulmonary arteriolae (IAPA) in normoxic control group (0.1076±0.0205) was markedly weaker than that in chronic hypoxic group (0.3317±0.0683, t =3.125, P <0.05) and that in chronic hypoxic group was stronger than that in hypoxia plus L argine group (0.1928±0.0381, t =2.844, P <0.05). RT PCR showed that the content of HIF 1α mRNA in chronic hypoxic group (2.5395±0.6449) was 2.16 times and 1.75 times higher than that in normoxic control group (1.1781±0.3628) and hypoxia plus L argine group (1.4511±0.3981), respectively. It is concluded that NO can reduce the mPAP by the inhibition of the expression of HIF 1α mRNA, which may be one of the mechanisms through which NO affects the pathogenesis of HPH.