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.

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.

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.

Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB

AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.

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 ℃.

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.

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.

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.

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.

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.

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.

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.

Expression and Implication of Hypoxia Inducible Factor-1α in Prostate Neoplasm

Summary: To study the expression of hypoxia inducible factor-1α (HIF-1α) protein in prostate cancer (Pca) and its biological significance, the expression of HIF-1α was assayed by means of immunohistochemical technique in 42 prostate cancer, 12 prostatic intraepithelial neoplasm (PIN) and 9 normal prostate tissue (NP) specimens. Western blot was used to examine the expression of HIF-1α in prostate cancer cell line (PC-3M) induced by different oxygen tension. HIF-1α expression was positive in 33 Pca and 9 PIN specimens, and the positive rate of HIF-1α was higher in distant metastasis patients than in patients without metastasis of prostate cancer (P<0.05), while there was no expression of HIF-1α in NP. The level of HIF-1α in PC-3M significantly increased with the decrease of oxygen tension (P<0.01). Overexpression of HIF-1α is the preliminary event of the formation of Pca, which may induce carcinoma into malignant phenotype. Thus it may serve as an early diagnosis marker and the novel target for Pca treatment.

Abnormal expression of hypoxia inducible factor-1α and clinical values of molecular-targeted interference in hepatocellular carcinoma

Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection,the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results:Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%,χ2=22.35,P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection,the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level,with VEGF and ANG-2 decreased to 54% and 36%,respectively.After RNA interference combined with anti-cancer drug,the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%,with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion:Abnormal expression of HIF-1α is associated with development of HCC,and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.

EXPRESSION OF HYPOXIA INDUCIBLE FACTOR-1α AND ITS REGULATORY ROLE IN DEVELOPING VERTEBRAE

Objective To explore the expression pattern and possible role of hypoxia inducible factor-1α （ HIF-1α ） in fetal vertebrae development of mouse. Methods The developmental stages of mice fetal vertebrae were observed from embryonic days 13. 5 to 18. 5 （ E13. 5 to E18. 5 ） by stereoscopic and light microscopes respectively, and the expressions of HIF-1α at various times were also detected at levels of mRNA and protein by using methods of RT-PCR and Western blotting. Distribution of HIF-1α in the vertebrae was examined by immunohistochemical assay. Vascular endothelia growth factor （VEGF） mRNA and other chondro-osteoblast marker genes as type II collagen al （ Coll2al ） and osteocalcin （OCN） were detected by RT-PCR too. Results The cartilaginous spine column began to form at E13. 5, followed by the arising of the primary ossification center in vertebrae at E15. 5, then the osteogenesis expanded and extended to both sides of the vertebrae. HIF-1α mRNA began to express at E13. 5, and showed significantly higher level at E14. 5 （ P 〈 O. 05 ）, then declined to a low level. VEGF mRNA expressed coincidently with HIF-1α. While HIF-1α protein expression was observed at E14. 5 and lasted at low level till to birth. The expression pattern of Coll2al and OCN elucidated the cell evolution from chondrocyte to osteoblast. Conclusion The developmental pattern of vertebrae appears to be an endochondral osteogenesis process. Existed hypoxia microenviroment in the vertebrae may increase HIF-1α mRNA and protein contents thus activate VEGF expression, as may be related to the activation of other downstream genes of hypoxia inducible factor-1α and initiate the cascade of endochondral osteogenesis.

瑞马唑仑调节HIF-1α/BNIP3信号通路对OGD/R诱导神经细胞自噬和凋亡的影响

目的探讨瑞马唑仑对OGD/R诱导的神经细胞自噬和凋亡的影响及作用机制。方法体外培养小鼠海马神经元细胞(HT22)并进行神经细胞氧糖剥夺/再复氧(OGD/R),筛选实验用瑞马唑仑浓度;将HT22细胞分为对照组、OGD/R组、瑞马唑仑组、2-ME2组、瑞马唑仑+2-ME2组;CCK8法检测5组HT22细胞活力;流式细胞术检测5组HT22细胞凋亡率;透射电子显微镜观察5组HT22细胞自噬小体的形成;Western blot检测5组HT22细胞HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ的表达。结果确定实验用瑞马唑仑浓度为50μg/mL;与对照组比较,OGD/R组HT22细胞OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平下调,凋亡率上调(P<0.05);与OGD/R组比较,瑞马唑仑组HT22细胞自噬小体增加,OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平上调,凋亡率下调(P<0.05);2-ME2组HT22细胞OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平下调,凋亡率上调(P<0.05)。与瑞马唑仑组比较,瑞马唑仑+2-ME2组HT22细胞自噬小体数量减少,OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平下调,凋亡率上调(P<0.05);与2-ME2组比较,瑞马唑仑+2-ME2组HT22细胞OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平上调,凋亡率下调(P<0.05)。结论瑞马唑仑可通过激活HIF-1α/BNIP3信号通路促进OGD/R诱导的神经细胞自噬,抑制细胞凋亡,从而减轻OGD/R诱导的神经细胞损伤。

益气升清方调节HIF-1α/NLRP3信号通路对缺血性脑卒中大鼠神经元焦亡的影响

目的 探究益气升清方调节缺氧诱导因子-1α(HIF-1α)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路对缺血性脑卒中大鼠神经元焦亡的影响。方法 将SD大鼠随机分为假手术组(S组),模型组(M组),益气升清方低、中、高剂量组(3.465、6.930、13.860 g/kg)及益气升清方高剂量+HIF-1α激活剂DMOG组(13.860 g/kg益气升清方+40 mg/kg DMOG),每组15只。采用线栓法构建脑卒中模型。造模成功后进行神经功能缺陷评估;TTC染色评估脑梗死体积;ELISA法检测血清白细胞介素(IL)-1β、IL-18水平;HE染色检测缺血皮质区病理变化;TUNEL染色检测神经元凋亡;Western blot检测焦亡及HIF-1α/NLRP3通路相关蛋白表达。结果 与S组比较,M组神经功能缺陷评分、梗死体积、IL-1β含量、IL-18含量、神经细胞凋亡率、胞膜穿孔蛋白D-N端(GSDMD-N)、胱天蛋白酶1(Caspase-1)、HIF-1α、NLRP3蛋白水平均上升(P<0.05);与M组比较,低、中、高剂量益气升清方组神经功能缺陷评分、梗死体积、IL-1β含量、IL-18含量、神经细胞凋亡率、GSDMD-N、Caspase-1、HIF-1α、NLRP3蛋白水平均下调(P<0.05);DMOG减弱了高剂量益气升清方对缺血性脑卒中大鼠神经元焦亡的改善作用。结论 益气升清方可能通过下调HIF-1α/NLRP3信号通路减轻缺血性脑卒中大鼠神经元焦亡。

Mechanism of piR-1245/PIWI-like protein-2 regulating Janus kinase-2/signal transducer and activator of transcription-3/vascular endothelial growth factor signaling pathway in retinal neovascularization

Inhibiting retinal neovascularization is the optimal strategy for the treatment of retina-related diseases, but there is currently no effective treatment for retinal neovascularization. P-element-induced wimpy testis(PIWI)-interacting RNA(piRNA) is a type of small non-coding RNA implicated in a variety of diseases. In this study, we found that the expression of piR-1245 and the interacting protein PIWIL2 were remarkably increased in human retinal endothelial cells cultured in a hypoxic environment, and cell apoptosis, migration, tube formation and proliferation were remarkably enhanced in these cells. Knocking down piR-1245 inhibited the above phenomena. After intervention by a p-JAK2 activator, piR-1245 decreased the expression of hypoxia inducible factor-1α and vascular endothelial growth factor through the JAK2/STAT3 pathway. For in vivo analysis, 7-day-old newborn mice were raised in 75 ± 2% hyperoxia for 5 days and then piR-1245 in the retina was knocked down. In these mice, the number of newly formed vessels in the retina was decreased, the expressions of inflammationrelated proteins were reduced, the number of apoptotic cells in the retina was decreased, the JAK2/STAT3 pathway was inhibited, and the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor were decreased. Injection of the JAK2 inhibitor JAK2/TYK2-IN-1 into the vitreous cavity inhibited retinal neovascularization in mice and reduced expression of hypoxia inducible factor-1α and vascular endothelial growth factor. These findings suggest that piR-1245 activates the JAK2/STAT3 pathway, regulates the expression of hypoxia inducible factor-1α and vascular endothelial growth factor, and promotes retinal neovascularization. Therefore, piR-1245 may be a new therapeutic target for retinal neovascularization.

MuRF1在低氧性肺动脉高压中的作用及机制

目的 探讨肌肉环指蛋白1(MuRF1)在低氧性肺动脉高压(HPH)中的作用及可能机制。方法 将MuRF1转基因敲除小鼠(MuRF1 KO)及其同窝野生型(WT)小鼠随机分到对照(nWT)组、对照+HPH(hWT)组、MuRF1 KO(nMuRF1-KO)组、MuRF1 KO+HPH(hMuRF1-KO)组。其中,hWT组和hMuRF1-KO组置于低压低氧人工实验舱内,nWT组和nMuRF1-KO组置于常压常氧SPF环境中,维持28 d。检测小鼠右心功能及右心室重塑水平、远端肺小动脉血管重塑水平、肺泡灌洗液炎症因子表达、MuRF1和大电导钙激活钾通道β1亚基(BK-β1)蛋白表达。结果 与nWT组相比,hWT组右室内径(RVID)显著降低(P<0.01),右室前壁厚度(RVAW)、右心室收缩压(RVSP)、右心室肥厚指数(RVHI)、胶原容积分数(CVF)显著增加(P<0.01),远端肺动脉壁厚度比(WT%)、肺动脉壁面积比(WA%)、肺动脉肌化水平、相对肺重量显著增加(P<0.01),肺泡灌洗液中炎症因子IL-1β、IL-6、TNF-α显著增加(P<0.01),MuRF1表达显著增加(P<0.05),BK-β1表达降低(P<0.05)。与hWT组相比,hMuRF1-KO组RVID显著增加(P<0.05),RVAW、RVSP、RVHI、CVF显著降低(P<0.05),WT%、WA%、肺动脉肌化水平、相对肺重量显著降低(P<0.05,P<0.01),肺泡灌洗液中炎症因子IL-1β、IL-6、TNF-α也显著减少(P<0.05,P<0.01),BK-β1表达显著增加(P<0.05)。结论 敲除MuRF1可改善HPH小鼠右心功能障碍和右室重塑,减轻肺血管重塑和肺血管炎性环境,其机制可能与敲除MuRF1抑制BK-β1降解有关。

丁苯酞软胶囊联合瑞舒伐他汀治疗急性脑梗死患者的临床观察及对血清HIF-1α、Lp-PLA、Sestrin2水平的影响

目的探究丁苯酞软胶囊联合瑞舒伐他汀治疗急性脑梗死患者的疗效及对血清缺氧诱导因子-1α(HIF-1α)、脂蛋白相关磷脂酶A2(Lp-PLA)、应激诱导蛋白2(Sestrin2)水平的影响。方法前瞻性选取2021年1月至2022年12月在秦皇岛市第一医院治疗的急性脑梗死患者101例作为研究对象,按照信封法将患者分为对照组(n=50)和研究组(n=51)。对照组行常规治疗并瑞舒伐他汀治疗,研究组在对照组基础上联合丁苯酞软胶囊治疗。统计分析两组患者治疗前及治疗14 d后的美国国立卫生研究院脑卒中量表(NIHSS)评分、FuglMeyer评测法(FMA)评分、改良Rankin量表(mRS)评分、血流动力学指标(血浆黏度、全血高切黏度、低切黏度、红细胞压积、红细胞变形指数)、血清HIF-1α、Lp-PLA、Sestrin2水平并比较临床疗效的组间差异。结果治疗14 d后,两组患者的NIHSS评分和mRS评分均较治疗前降低,FMA较治疗前升高,且研究组患者的NIHSS评分和mRS评分分别为(4.03±0.38)、(3.01±0.45)分,均低于对照组,FMA为(80.64±9.16)分,高于对照组,差异均有统计学意义(P<0.05)。治疗14 d后,两组患者的血浆黏度、全血高切黏度、低切黏度、红细胞压积均较治疗前降低,红细胞变形指数均较治疗前升高,且研究组的血浆黏度、全血高切黏度、低切黏度、红细胞压积分别为(1.56±0.33)mPa·s、(4.78±0.31)mPa·s、(9.81±0.64)mPa·s、(0.37±0.05)%,均低于对照组,红细胞变形指数为0.78±0.11,高于对照组,差异均有统计学意义(P<0.05)。治疗14 d后,两组患者的血清HIF-1α、Lp-PLA、Sestrin2水平均降低,且研究组患者血清HIF-1α、Lp-PLA、Sestrin2水平分别为(690.56±65.12)ng/mL、(13.65±2.13)μg/L、(11.33±1.45)ng/mL,均显著低于对照组,差异均有统计学意义(P<0.05)。两组近期疗效比较,差异有统计学意义(P<0.05);研究组总有效率为90.20%,高于对照组(64.00%),差异有统计学意义(P<0.05)。结论采用丁苯酞软胶囊联合瑞舒伐他汀治疗急性脑梗死患者可显著提高患者临床治疗效果,提高肢体活动能力,缓解神经功能损伤,降低血清HIF-1α、Lp-PLA、Sestrin2水平,具有较高的临床应用潜力。