Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Angiogenic effect of HIF-1α on extranodal nasal-type NK/T-cell lymphoma

AIM: To study the angiogenic effect of hypoxia inducible factor 1α(HIF-1α) and its significance on human extranodal nasal-type NK/T-cell lymphoma. METHODS: The protein levels of HIF-1α, vascular endothelial growth factor(VEGF) and VEGF receptor 2(VEGFR2) in human extranodal nasal-type NK/T-cell lymphoma were detected by immunohistochemistry. Microvessel density (MVD) of the tumor tissues was determined by labeling of microvessel endothelium with CD34 antibody. The correlation between the expression of HIF-1α, VEGF and VEGFR2 and MVD was analyzed with SPSS 13.0 statistical software. RESULTS: The positive expression of HIF-1α was observed in 39 cases (39/50, 78%) and the positive expression of VEGFR2 was 27 cases (27/50, 54%) of human extranodal nasal-type NK/T-cell lymphoma. A statistical difference of HIF-1α and VEGFR2 expression between tumor tissues and normal lymphocytes in lymph node was observed (P<0.05). In the tumor tissues, the co-expression of VEGF or VEGFR2 with HIF-1α was 72% and 64%, respectively, significantly higher than that without HIF-1α co-expression (P<0.05). The expression of HIF-1α, VEGFR and VEGFR2 was positively correlated with MVD of the tumor tissues (P<0.01). HIF-1α was expressed in all 15 cases of extranodal nasal-type NK/T-cell lymphoma with angiocentric infiltration.CONCLUSION: HIF-1α may promote angiogenesis of extranodal nasal-type NK/T-cell lymphoma through VEGF/VEGFR2 signaling pathway.     

Effect of hyperbaric oxygen on HIF-1α expression in rat experimental periodontitis with psychological stress

AIM: To investigate the effect of hyperbaric oxygen (HBO) on hypoxia-inducible factor-1α (HIF-1α) expression in rat experimental periodontitis with psychological stress. METHODS: Male special pathogen-free Wistar rats (n=120) were randomly divided into 4 groups: normal control group; psychological stress stimulation group; experimental periodontitis group: the periodontitis model was induced by wrapping 3/0 silk ligature inoculated with Porphyromonas gingivalis around the left maxillary second molar of the rats; periodontitis model with stress stimulation group. Psychological stress was removed at the 9th weeks after ligature, 6 rats from each experiment group were randomly chosen to HBO treatment. The rats were sacrificed at the 2nd, 4th, 8th and 10th weeks after ligature. Gingival index (GI) and attachment loss (AL) were measured before sacrifice. The histological changes of periodontal tissues were observed under microscope with HE staining. The expression of HIF-1α was observed by the method of immunohistochemistry. RESULTS: The sites of gingival attachment were normal in control group and psychological stress stimulation group. Periodontal pocket, and periodontal attachment loss were observed in experimental periodontitis group. The tissue damage was much serious in periodontitis model with stress stimulation group. No significant difference of GI and AL among psychological stress stimulation group and normal control group during the experiment was observed. GI and AL in periodonitis model with stress stimulating group were significantly higher than those in experimental periodontitis group at the 4th and 8th weeks (P < 0.01). The levels of GI and AL were significantly lower at the 10th weeks after HBO treatmnt than those in untreated groups (P < 0.05). No significant difference of HIF-1α expression scores among psychological stress stimulation group and normal control group was found. HIF-1α expression scores in periodonitis model with stress stimulating group was significantly higher than that in experimental periodontitis group at the 4th and 8th weeks (P < 0.01). At the 10th weeks after HBO treatment the levels of HIF-1α were significantly lower than that in untreated groups (P < 0.01). CONCLUSION: Stress stimulation may aggravate periodontitis by decreasing tissue oxygenation in rats. HBO may represent a useful way in psychological stress periodontitis therapy.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Effect of psychological stress on development of periodontitis and expression of periodontal hypoxia-inducible factor 1α in rats

AIM: To investigate the effect of psychological stress on the development of periodontitis and the expression of periodontal hypoxia-inducible factor 1α(HIF-1α) in rats.METHODS: Forty-eight male Wistar rats(SPF grade) were randomly divided into 4 groups:(1) normal control group, i.e. naive rats;(2) experimental periodontitis group: the periodontitis model was induced by wrapping 3-0 silk ligature inoculated with putative periodontopathic bacteria around the left maxillary second molar of the rats;(3) stress group: the rats were treated with stress alone;(4) periodontits with stress group, the periodontitis model was induced as above,and the rats were treated with stress. The rats were sacrificed at week 1, 4, 6 and 8 after the ligature. The attachment losses(AL) were measured by home-made probe. The histological changes of periodontal tissues stained with hematoxylin and eosin(HE) were observed under microscope. The HIF-1α expression level in the periodontal epithelium was determined by immunohistochemistry that was used to evaluate the severity of hypoxia by measuring the average rate of HIF-1α-positive cells.RESULTS: No significant difference of AL between stress group and normal control group was observed(P>0.05).The AL and the average rate of HIF-1α-positive cells in periodontitis with stress group were significantly higher than those in experimental periodontitis group at time points of week 4,6 and 8 after ligature(both P<0.01).CONCLUSION: Psychological stress is one of the periodontitis inducing factors in the animal model. Psychological stress may aggravate periodontitis by decreasing tissue oxygenation in rats.     

Regulatory effect of astragaloside IV on SDF-1α/CXCR4 in EPCs

AIM: To investigate the effects of astragaloside IV (AS-IV) on chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1α (SDF-1α) in endothelial progenitor cells (EPCs) and its mechanism. METHODS: Rat bone marrow-derived EPCs were cultured in vitro. The proliferation, adhesion, migration, apoptosis and tube formation capacity of EPCs treated with AS-IV and AMD3100, a specific blocker of CXCR4, were observed. The effects of AS-IV on the expression of SDF-1α/CXCR4 at mRNA and protein levels and the protein level of p-CXCR4 in the EPCs were determined. RESULTS: AS-IV significantly enhanced the proliferation, adhesion, migration and tube formation abilities of EPCs, reduced the apoptosis of EPCs, and up-regulated the mRNA and protein expression of SDF-1α and CXCR4 and the p-CXCR4 protein level in the EPCs. On the other hand, AMD3100 blocked the up-regulating effect of AS-IV on the mRNA and protein expression of CXCR4 and the p-CXCR4 protein level in the EPCs, but did not affect the effect of AS-IV on the expression of SDF-1α. CONCLUSION: AS-IV might enhance the biological function of EPCs by regulating the expression of SDF-1α/CXCR in EPCs.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

Effect of bFGF on promoting angiogenesis in infarct area and improving myocardial fibrosis in mouse myocardial infarction model

AIM: To investigate the protective effect of basic fibroblast growth factor (bFGF) on the heart of mice with myocardial infarction and its mechanism. METHODS: The model of myocardial infarction was established by the ligation of left anterior descending artery of C57/B6 mice (8~12 weeks old) after lateral thoracotomy. The mice were divided into sham operation group, myocardial infarction group and bFGF administration group. bFGF at 0.5 μg was intraperitoneally injected on alternate days after myocardial infarction for 7 d. Cardiac Doppler ultrasonography was used to detect cardiac function after myocardial infarction for 28 d, and left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction and left ventricular shortening fraction (LVFS) were used to evaluate cardiac function. After myocardial infarction for 28 d, the mice were sacrificed and the hearts were collected for preparing pathological sections. The degrees of myocardial fibrosis and angiogenesis in the myocardial infarction area were observed. Western blot was used to detect the indicators of angiogenesis. RESULTS: The results of Masson staining showed that bFGF administration significantly reduced myocardial fibrosis at 28 d after myocardial infarction. Cardiac ultrasound data showed that cardiac functions in myocardial infarction group were poorer than those in sham group, and bFGF administration significantly improved cardiac functions. Immunofluorescence staining showed that neovascularization in myocardial infarction area of bFGF administration group was more than that in myocardial infarction group. The results of Western blot showed that bFGF activated AKT/HIF-1α/VEGF signaling pathway. CONCLUSION: Intraperitoneal injection of bFGF reduces myocardial fibrosis and improves cardiac function in myocardial infarction mice. bFGF may promote angiogenesis by activating AKT/HIF-1α/VEGF signaling pathway.     

Oxidized α1-antitrypsin induces IL-8 and MCP-1 production in HBE cells

AIM:To investigate the effect of oxidized α1-antitrypsin (Ox-AT) on interleukin 8 (IL-8) and monocyte chemotactic protein 1(MCP-1) production in cultured human bronchial epithelial (HBE) cells. METHODS:Plasma native α1-antitrypsin (N-AT) was purified from human plasma by 50% and 75% ammonium sulfate fractionation followed by glutathione and anion exchange chromatography. Ox-AT was prepared by incubating N-AT (0.5 g/L) with N-chlorosuccinimide in a 25-fold molar excess to N-AT in PBS at room temperature for 30 min. HBE cells were cultured in the presence of Ox-AT (0.5 g/L) for 4 h, 10 h and 24 h, and the levels of IL-8 and MCP-1 in the supernatant were assayed using respective DuoSet kits. The effect of NF-κB inhibitor Bay11-7082 on the inflammatory cytokine release induced by Ox-AT was also evaluated. RESULTS:Ox-AT concentration-dependently and time-dependently increased the production of IL-8 and MCP-1 in HBE cells. The concentrations of IL-8 and MCP-1 in HBE cells induced by 0.5 g/L Ox-AT at 4 h, 10 h and 24 h were significantly higher than those in blank control and N-AT groups. Ox-AT increased the activity of NF-κB in a dose-dependent manner. The proinflammatory effect of by Ox-AT was inhibited by NF-κB inhibitor Bay11-7082. CONCLUSION: Ox-AT is a strong proinflammatory factor for HBE cells. The mechanism is related to NF-κB signaling pathway activation.     

Influence of Tangshenfang on diabetic liver injury and the expression of SIRT1 and PGC-1α in the liver tissue

AIM: To observe the effect of Tangshenfang (TS) on the liver protection and the levels of silent information regulator 1 (SIRT1) and peroxisom proliferator-activated receptor γ coactivator-1α (PGC-1α) in the liver tissue. METHODS: The rat model of diabetes mellitus (DM) was established by intravenous injection of streptozotocin (STZ;30 mg/kg) after having the high fat/high glucose diets for 1 month. The diabetic rats were randomly divided into DM group, DM with high-dose TS (TS^Hi) group, medium-dose TS (TS^Med) group and low-dose TS (TS^Low)group. The normal rats were served as control group. There were 8 rats in each group. After treatment with TS for 12 weeks, the serum biochemical indices including fasting blood glucose (FBG), triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested. Fasting insulin (FINS) was also detected by radioimmunoassay, and homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) were measured by ELISA. The activity of SOD and content of MDA in the liver tissues were measured by the methods of hydroxylamine and thiobarbituric acid. The liver pathological changes were observed under light microscope with HE and Masson staining. The protein expression of SIRT1and PGC-1α in the liver tissues was determined by Western blot. RESULTS: In DM group, serum FBG, TG, ALT, AST, FINS, HOMA-IR, TNF-α and IL-1 were obviously increased compared with the control group (P<0.01). The fatty changes, local necrosis, inflammation and fibrosis in the liver tissues were observed. The content of MDA in liver increased, while the activity of SOD decreased markedly. The protein expression of SIRT1 and PGC-1α was decreased (P<0.05). In TS treatment groups, all these changes in DM rats were markedly reversed by TS, and the protein expression of SIRT1 and PGC-1α in the liver tissues was markedly increased. CONCLUSION: TS may protect the rats from diabetic liver injury by increasing the expression of SIRT1 and PGC-1α, and thereby improving insulin resistance and oxidative stress.     

Effects of fucoidan on angiogenesis of human multiple myeloma RPMI 8226 cells

AIM: To investigate the effects of fucoidan on the angiogenesis of multiple myeloma cells in vitro, and its related mechanisms. METHODS: The human multiple myeloma RPMI 8226 cells and human endothelial cells were cultured in vitro. The growth inhibition rate of RPMI 8226 cells was examined by MTT assay. The cell cycle and apoptosis rate were measured by flow cytometry. RPMI 8226 cells were treated with fucoidan for 72 h, and the cell culture supernatant was collected. The VEGF concentration was examined by ELISA, and the tube formation assay was applied to assess the angiogenic activity. After treatment with fucoidan for 72 h at different concentrations, the protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 were detected by Western blot. RESULTS: Fucoidan inhibited the growth of RPMI 8226 cells in a dose- and time-dependent manner. After treatment with fucoidan for 72 h, the cell cycle was arrested at G₁ phase, and the apoptotic rate of RPMI 8226 cells was increased with the increasing concentration of fucoidan, which was much higher than that in control group (P<0.05). The VEGF concentration was significantly decreased with the increa-sing concentration of fucoidan. The numbers and areas of the capillary-like structures decreased while the concentration of fucoidan increased, and those at 100 mg/L were less than those in the control (P<0.05). The protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 in fucoidan group were significantly lower than those in control group (P<0.05). CONCLUSION: Fucoidan inhibits the secretion of VEGF in multiple myeloma cells, and reduces angiogenesis induced by multiple myeloma cells. It inhibits the protein expression of HIF-1α and VEGF, which may be related to inhibiting the phosphorylation of AKT and ERK1/2.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

αvβ6-ERK pathway participates in norcantharidin-induced apoptosis of HT-29 colon cancer cells

AIM: To investigate the pharmacological mechanism of norcantharidin (NCTD)-induced apoptosis of HT-29 colon cancer cells. METHODS: Hoechst 33258 staining was used to analyze the apoptosis of HT-29 cells treated with NCTD. The effects of NCTD on the expression of integrin in HT-29 cells were determined by flow cytometry. The effects of several functional blocking antibodies on HT-29 cells were detected by MTT method. The expression and the phosphorylation of mitogen activated protein kinases (MAPKs) in HT-29 cells were measured by Western blotting. Co-immunoprecipitation assay was used to detect the activity of αvβ6-extracellular signal-regulated kinase (ERK) direct linkage in HT-29 cells.RESULTS: NCTD induced the apoptosis of HT-29 colon cancer cells. The expression of integrin αvβ6 in HT-29 cells treated with NCTD was reduced, but the expression of αvβ3 and αvβ5 was not changed. A function-blocking antibody to αvβ6,10D5,strengthened the growth inhibitory effect of NCTD on HT-29 cells ,but LM609 (a function-blocking antibody to αvβ3) and P1F6 (a function-blocking antibody to αvβ5) did not. The level of phosphorylated ERK (p-ERK) was decreased substantially after treated with NCTD in a dose-and time-dependent manner. NCTD also affected the association of αvβ6 and ERK. CONCLUSION: NCTD decreases the expression of integrin αvβ6 and interferes with the phosphorylation of ERK. As a result, the formation of αvβ6-ERK direct linkage is affected and the signal transduction mediated by αvβ6 is disturbed. The mechanism of NCTD-induced HT-29 cell apoptosis is involved in the αvβ6-ERK signaling pathway.     

Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

Activation of NF-κB in rat vascular smooth muscle cells by autoantibodies against α1-adrenergic receptor from hypertensive patients

AIM：To study the effects of autoantibodies against α1-adrenergic receptor (α1-AAs) isolated from the hypertensive patients, which showed the agonist-like activity similar to norepinephrine, on the signal mechanism of vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta. METHODS：Rat VSMCs were cultured and identified. The serum of hypertensive patients was purified by immunoaffinity chromatography. The autoantibodies were detected by ELISA and used to activate the cells with the titer of 1∶80. The total protein was extracted and the expression of NF-κB in different treatment groups was detected by Western blotting. Meanwhile, the activation of NF-κB in the nucleus was analyzed by immunofluorescence method. RESULTS：The expression of NF-κB in VSMCs was obviously higher in α1-AAs group than that in control group. Meanwhile, the expression of NF-κB was inhibited by prasozin and PDTC. The autoantibodies caused a significant increase in NF-κB expression in the nucleus. The fluorescence intensity in α1-AAs group was high than that in control group and α1-AAs+prasozin group (P<0.01). CONCLUSION:The α1-AAs from hypertensive patients increase NF-κB expression in rat VSMCs.     

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

Improving effects of ginsenoside Rb1 on glucose metabolism in cardiomyocytes under hypoxia by hypoxia-inducible factor 1α

AIM: To elucidate the effect of ginsenoside Rb1 (Gs-Rb1) on the glucose metabolism to improve the viability of the cardiomyocytes under hypoxia, and whether hypoxia-inducible factor 1α (HIF-1α) and/or AMPKα are involved in the process.METHODS: The neonatal rat cardiomyocytes were cultured, and randomly divided into control group, hypoxia (1% O₂, 94% N₂ and 5% CO₂) group, Gs-Rb1 (200 μmol/L) group, Ara-A (500 μmol/L) group, Gs-Rb1+Ara-A group, YC-1 (5 μmol/L) group, Gs-Rb1+YC-1 group, Ara-A+YC-1 group and Gs-Rb1+YC-1+Ara-A group. After the intervention for 8 h, the cell viability was analyzed by MTT assay. The protein levels of AMPK, HIF-1α and glucose transporter-4 (GLUT-4) were determined by Western blot. The activities of heterophosphatase (HK), phosphofructokinase (PFK) and lactic dehydrogenase (LDH) were measured by ELISA.RESULTS: Gs-Rb1 significantly improved the viability of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 and Ara-A. In addition, YC-1 and Ara-A had a synergistic effect. Gs-Rb1 increased the protein levels of AMPK and HIF-1α in the hypoxic cardiomyocytes, which was significantly inhibited by Ara-A and YC-1. Gs-Rb1 significantly increased the expression of GLUT-4 on the cytomembrane of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 or Ara-A, especially Ara-A+YC-1. Gs-Rb1 significantly increased the activities of HK, PFK and LDH, all those were significantly inhibited by YC-1 or Ara-A. Besides, YC-1 and Ara-A had a synergistic effect.CONCLUSION: Gs-Rb1 improves the viability of hypoxic cardiomyocytes, which may be related to the regulation of glucose uptake and enhancement of glycolysis by synergy of both HIF-1α and AMPK.