ObjectiveTo study the changes of body weight, body length, tail length, femur length, bone mineral density, serum osteocalcin content and apoptosis of bone cells in rats under intermittent hypoxia condition, so as to explore the effects of intermittent hypoxia on bone growth.MethodsForty healthy male SD rats aged 3 to 4 weeks were selected and divided into 2 groups, 20 rats in each group. Group A: normoxic control group (normal diet and normoxic environment); group B: intermittent hypoxia group (normal feeding and was put into the hypoxic chamber to establish intermittent hypoxia environment), 8 hours a day (09:00 to 17:00), 4 weeks of modeling. The body weight, body length and tail length of the two groups were measured in every morning. At the end of 4 weeks after anesthesia, the body weight, body length, tail length and right femur length were measured. The body weight growth rate, body length growth rate and tail length growth rate were calculated. Blood samples were collected from the abdominal aortic, and the content of serum osteocalcin was measured by enzyme linked immunosorbent assay; the right femur bone mineral density was measured by automatic dual-energy X-ray bone densitometer; the apoptosis of bone cells was detected by immunofluorescence staining+TUNEL.ResultsThe body weight growth rate, body length growth rate, tail length growth rate and right femur length in group A were all higher than those in group B (P<0.05); serum osteocalcin content in group A was higher than that in group B (P<0.05); bone mineral density in group A was higher than that in group B (P<0.05); the apoptotic index of bone cells in group B was higher than that in group A (P<0.05). Pearson correlation analysis showed that the serum osteocalcin content was significantly positively correlated with the growth rate of body length, femoral length and bone mineral density (P< 0.01).ConclusionIntermittent hypoxia could reduce osteocalcin secretion, inhibit bone growth and sclerosis, and induce osteocyte apoptosis, thus delay the bone growth.
Objective To investigate the expression pattern of hypoxia-inducible factor 1α (HIF-1α) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI. Methods A total of 66 SD rats (female or male) with weight (250 ± 20) g were randomly divided into 3 groups: normal control group (group A, n=6), pseudo injury group (group B, n=6), and spinal cord injury (SCI) group (group C, n=54). In group A, no treatment was given as normal control. In groupB, only laminectomy was appl ied. In group C, laminectomy was appl ied and static compression model of SCI was built at T10 level. The expression of HIF-1α was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with l ight staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of gl ial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-1α was poorly expressed in group A and increased a l ittle in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of gangl ion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then decl ined. At 14 days, it appeared only in a small amount of gangl ion cells of white matter. There was no significant difference in the number of HIF-1α positive cells between groups A and B (t=1.325, P=0.137). The number of HIF-1α positive cells at each time point in group C was more than those in groups A and B (P lt; 0.05), and there were significant differences between all time points in group C (P lt; 0.05). Conclusion The expression of HIF-1α increases after SCI, it is related to the ischemia hypoxia after SSCI, and the expression pattern was correlated with the injury time.
ObjectiveTo investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells.MethodsSHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β (p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis.ResultsAfter identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly (P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher (P<0.05), the relative expression of p-GSK3β protein was significantly lower (P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased (P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group (P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower (P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups (P<0.05).ConclusionOverexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.
Objective To investigate the expression of miR-31a-5p in myocardial infarction (MI) mice and its potential mechanism. Methods A dataset was downloaded from the gene expression database, and miR-31a-5p and its predicted target gene hypoxia-inducible factor-1α (HIF-1α) were screened using bioinformatics methods. The MI model was established by ligating the left anterior descending branch of the coronary artery in C57BL/6J male mice which were randomly divided into sham and MI groups (n=6 in each group). The in vitro hypoxic cell model was induced by treatment of H9c2 cells with cobalt chloride (CoCl2) and divided into a control group, a model group, a NC group, a miR-31a-5p mimic group and a miR-31a-5p inhibitor group. The degree of myocardial tissue fibrosis was stained by Masson and analyzed. The expression levels of miR-31a-5p and HIF-1α mRNA in mouse myocardial tissues and H9c2 cells were detected by qRT-PCR. Western blotting was used to detect the expression levels of B-cell lymphoma 2 (Bcl-2), cleaved-caspase 3 apoptotic protein in mouse myocardial tissues and HIF-1α and apoptotic protein in H9c2 cells, respectively. The dual luciferase reporter gene assay was used to verify the targeting relationship between miR-31a-5p and HIF-1α. Results Masson staining showed significantly increased fibrosis in MI mice (P<0.000 1); miR-31a-5p, cleaved-caspase 3 were significantly elevated and Bcl-2 was decreased in MI mice and CoCl2 treated H9c2 (P<0.05). The results of dual luciferase reporter assay showed that the relative luciferase activity of miR-31a-5p mimic cotransfected with HIF-1α-3'-UTR WT plasmid was reduced (P<0.000 1); miR-31a-5p mimic decreased HIF-1α expression and increased apoptotic protein levels in CoCl2 induced H9c2 cells (both P<0.05), while miR-31a-5p exerted the opposite effect. Conclusion miR-31a-5p can aggravate apoptosis in myocardial ischemia by targeting HIF-1α.
ObjectiveTo identify genes associated with resistance to programmed cell death protein 1 (PD-1) inhibitors in colorectal cancer and elucidate their underlying mechanisms using bioinformatics approaches. MethodsGenes expression datasets were downloaded from the Gene Expression Omnibus (GEO) database to screen hypoxia-related genes (HRGs) and differentially expressed genes (DEGs). The intersection of HRGs and DEGs was defined as hypoxia-related differentially expressed genes (HDGs). The gene expression data of patients with colorectal cancer from The Cancer Genome Atlas (TCGA) were analyzed using Pearson correlation to identify the PD-1-related genes, further the STRING analysis (minimum interaction score was greater than 0.7) and Cytoscape were subsequently employed to screen the key PD-1-related genes. The relation between the screened key PD-1-related genes and the prognosis of colorectal cancer patients was analyzed to screen out the target genes. The real-time fluorescence reverse transcription quantitative polymerase chain reaction was used to analyze the expression of the target genes in the cancer tissues and their corresponding adjacent tissues of 20 patients with colorectal cancer. The Kaplan-Meier Plotter database and the ROC Plotter database were used to analyze the relation between the high and low expression of the target genes and the prognosis in different patients. The significance level was set as α=0.05. ResultsA total of 651 HRGs and 329 DEGs were screened out. By taking the intersection of these two sets, 37 HDGs were obtained for subsequent analysis. Through Pearson correlation analysis, 25 key PD-1-related genes were screened out and 10 and 14 key PD-1-related genes were screened out by the MCC algorithm and the MCODE algorithm respectively. By taking the intersection of these three sets, 3 key PD-1-related genes were obtained, then survival analysis, the Aurora kinase A (AURKA) gene was finally screened out as the target gene. The expression level of the AURKA gene in the pan-cancer patients who responded to PD-1 inhibitor treatment was significantly higher than that in non-responders (P＜0.001), and was significantly lower in the six colorectal cancer cells treated with hypoxia than in six colorectal cancer cells treated with normoxia (P＜0.001). The AURKA expression in the colorectal cancer tissues was significantly higher than that in the corresponding adjacent colorectal tissues (P=0.008). The overall survival of pan-cancer patients with high AURKA expression was better than that of those with low AURKA expression [HR (95%CI)=0.67 (0.49, 0.93), P=0.015]. Among the colorectal cancer patients with MMR deficiency, the patients with low AURKA gene expression had worse overall survival [HR (95%CI)=2.596 (1.028, 6.332), P=0.043] and recurrence-free survival [HR (95%CI)=4.201 (1.092, 16.150), P=0.037] as compared with those with high AURKA gene expression. The low AURKA expression was associated with significantly worse overall survivals in the colorectal cancer patients harboring wild-type or mutant TP53, BRAF, and KRAS as compared with high AURKA expression (P＜0.05), while no statistically significant difference was found in the overall survival of the normal MMR patients between with high AURKA expression and low AURKA expression (P=0.307). ConclusionThe results of this bioinformatics analysis suggest that hypoxia down-regulated AURKA expression, and low AURKA expression is associated with worse prognosis in colorectal cancer patients, and worse reactivity and prognosis in patients treated with PD-1 inhibitors.
Objective To compare the effects of oxygen therapy and local pressurization in alleviating plateau hypoxia at high altitude. Methods Forty-five healthy male soldiers were investigated at an altitude of 3992 meters. The subjects were randomly divided into three groups, ie. an oxygen inhalation group, a single-soldier oxygen increasing respirator ( SOIR) group and a BiPAP group. The oxygen inhalation group was treated with oxygen inhalation via nasal catheter at 2 L/ min. SOIR was used to assist breath in the SOIR group. The BiPAP group were treated with bi-level positive airway pressure ventilation, with IPAP of 10 cm H2O and EPAP of 4 cmH2 O. PaO2, PaCO2, SpO2 and heart rate were measured before and 30 minutes after the treatment. Results There were continuous increase of PaO2 from ( 53. 30 ±4. 88) mm Hg to( 58. 58 ±5. 05) mm Hg and ( 54. 43 ±3. 01) mm Hg to ( 91. 36 ±10. 99) mm Hg after BiPAP ventilation and oxygen inhalation, respectively ( both P lt; 0. 01) . However, the PaO2 of the SOIR group was decreased from( 56. 00 ±5. 75) mm Hg to ( 50. 82 ±5. 40) mm Hg( P lt; 0. 05 ) . In the other hand, the PaCO2 was increased from ( 30. 41 ±1. 51) mmHg to ( 32. 56 ±2. 98) mm Hg in the oxygen inhalation group ( P lt; 0. 05) , declined from( 28. 74 ±2. 91) mm Hg to ( 25. 82 ±4. 35) mm Hg in the BiPAP group( P lt;0. 05) ,and didn’t change significantly from( 28. 65 ±2. 78) mm Hg to ( 29. 75 ±3. 89) mmHg in the SOIR group ( P gt;0. 05) . Conclusions Both BiPAP ventilation and oxygen inhalation can alleviate plateau hypoxia by improving PaO2 at 3992 meter altitude while SOIR has no significant effect.
Objective To compare the effects of hypoxia-inducible drugs using deferoxamine (DFO) and accordion technique (AT) on activating the hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway to promote bone regeneration and remodelling during consolidation phase of distraction osteogenesis (DO). Methods Forty-five specific-pathogen-free adult male Sprague-Dawley (SD) rats were randomly divided into the control group, DFO group, and AT group, with 15 rats in each group. All rats underwent osteotomy to establish a right femur DO model. Then, continuous distraction was started for 10 days after 5 days of latency in each group. During the consolidation phase after distraction, no intervention was performed in the control group; DFO was locally perfused into the distraction area in the DFO group starting at the 3rd week of consolidation phase; cyclic stress stimulation was given in the AT group starting at the 3rd week of consolidation phase. The general condition of rats in each group was observed. X-ray films were conducted at the end of the distraction phase and at the 2nd, 4th, and 6th weeks of the consolidation phase to observe the calcification in the distraction area. At the 4th and 6th weeks of the consolidation phase, peripheral blood was taken for ELISA detection (HIF-1α, VEGF, CD31, and Osterix), femoral specimens were harvested for gross observation, histological staining (HE staining), and immunohistochemical staining [HIF-1α, VEGF, osteopontin (OPN), osteocalcin (OCN)]. At the 6th week of the consolidation phase, Micro-CT was used to observe the new bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the distraction area, and biomechanical test (ultimate load, elastic modulus, energy to failure, and stiffness) to detect bone regeneration in the distraction area. Results The rats in all groups survived until the termination of the experiment. ELISA showed that the contents of HIF-1α, VEGF, CD31, and Osterix in the serum of the AT group were significantly higher than those of the DFO group and control group at the 4th and 6th weeks of the consolidation phase (P<0.05). General observation, X-ray films, Micro-CT, and biomechanical test showed that bone formation in the femoral distraction area was significantly better in the DFO group and AT group than in the control group, and complete recanalization of the medullary cavity was achieved in the AT group, and BMD, BV/TV, Tb.Sp, Tb.N, and Tb.Th, as well as ultimate load, elastic modulus, energy to failure, and stiffness in the distraction area, were better in the AT group than in the DFO group and control group, and the differences were significant (P<0.05). HE staining showed that trabecular bone formation and maturation in the distraction area were better in the AT group than in the DFO group and control group. Immunohistochemical staining showed that at the 4th week of consolidation phase, the expression levels of HIF-1α, VEGF, OCN, and OPN in the distraction area of the AT group were significantly higher than those of the DFO group and control group (P<0.05); however, at 6th week of consolidation phase, the above indicators were lower in the AT group than in the DFO group and control group, but there was no significant difference between groups (P>0.05). Conclusion Both continuous local perfusion of DFO in the distraction area and AT during the consolidation phase can activate the HIF-1α/VEGF signaling pathway. However, AT is more effective than local perfusion of DFO in promoting the process of angiogenesis, osteogenesis, and bone remodelling.
ObjectiveTo investigate the correlations among the cadual homeobox gene 2 (CDX2), hypoxia inducible factor-1α (HIF-1α) protein expressions, and tumor budding in the colorectal cancer (CRC). MethodsIn this study, 63 CRC specimens surgically removed in the First Affiliated Hospital of Xi’an Jiaotong University from January 2012 to September 2015 were collected. The CDX2 and HIF-1α protein expressions were detected by immunohistochemical staining streptavidin-biotin peroxidase two-step method. The staining and the grade of tumor budding were observed under an optical microscope, and the correlation was analyzed using Spearman test. ResultsThe positive expressions of CDX2 and HIF-1α proteins in the CRC tissues were 35 (55.6%) and 47 (74.6%) cases, respectively, which was a negative correlation in the CRC (rs=–0.302, P=0.017). The positive expressions of CDX2 and HIF-1α proteins in the tumor budding of colorectal cancer were 21 (51.2%) and 26 (63.4%) cases, respectively, which was also a negative correlation in the tumor budding of CRC (rs=–0.336, P=0.031), but there was no statistic correlation between the grade of tumor budding and CDX2 or HIF-1α positive protein expression in the CRC (rs=0.113, P=0.370; rs=–0.026, P=0.838). ConclusionsThe positive expression between CDX2 and HIF-1α has a negative correlation in the same CRC specimen and which has a negative correlation in tumor budding. There is no statistic correlation between grade of tumor budding and CDX2 or HIF-1α protein expression in the CRC. Hypoxia environment may be involved in the downregulation of CDX2 level during the malignant progression of CRC.
ObjectiveTo investigate the mechanism of the early kidney injury in rats caused by intermittent hypoxia, and investigate the intervention effect of edaravone.MethodsEighty male Wistar rats were randomly divided into a control group (NC), an intermittent hypoxia group (IH), an intermittent hypoxia edaravone treatment group (IH+NE), and an intermittent hypoxia normal saline matched group (IH+NS). After 4 weeks of model establishment, serum urea nitrogen and creatinine concentration were determined. Pathological changes of kidney were observed under light microscope, and ultrastructural changes of glomeruli and renal tubules were observed under electron microscope. The kidney injury molecule 1 (KIM-1) protein was detected by immunohistochemistry. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), hydroxyl radical and Bcl-2 mRNA, Caspase-3 mRNA, Bax mRNA in homogenate of kidney tissue were measured.ResultsSerum urea nitrogen in each group showed no significant difference. Serum creatinine increased significantly in IH group and significantly decreased after edaravone treatment. There were no significant pathological damages in NC group under light and electron microscopy. IH group showed varying degrees of renal tubule damages compared with NC group. Compared with NC group, the mean optical density of KIM-1 protein in IH group and IH + NS group significantly increased (P<0.01), and the mean optical density of KIM-1 protein in IH+NE group significantly decreased (P<0.01). Compared with NC group, the activity of SOD in IH group and IH+NS group significantly decreased, the content of MDA and hydroxyl radical increased, the expression of Bcl-2 mRNA decreased, the expression of Caspase-3 mRNA and Bax mRNA increased, Bcl-2/Bax?decreased.?After edaravone intervention, the activity?of SOD in?kidney tissue?of rats significantly?increased, the content of MDA and hydroxyl radical?significantly?decreased, the expression?of Bcl-2 mRNA?increased, the expression of Caspase-3 mRNA?and Bax mRNA decreased, Bcl-2/Bax increased.ConclusionsIntermittent hypoxia can cause kidney injury through oxidative stress and regulation of Bcl-2, Bax and Caspase-3. KIM-1 may be used as a sensitive indicator for monitoring early kidney injury. Edaravone can prevent kidney injury induced by intermittent hypoxia though scavenging oxygen free radical, improving antioxidant capacity, regulating cell apoptosis mediated by regulating Bcl-2/Bax and Caspase-3.
Hypoxia inducible factor-1 (HIF-1) is the main transcription factor and the core regulator for cells to adapt to hypoxia, and oxygen homeostasis is achieved by controlling and utilizing oxygen delivery. Autophagy and apoptosis play an important role in determining cell fate and maintaining cell homeostasis. In recent years, it has been found that the dynamic change of HIF-1 expression plays a key role in the hypoxic adaptive response of cardiomyocytes. The regulation of HIF-1 on autophagy and apoptosis of hypoxic cardiomyocytes determines the survival of cardiomyocytes, which is of great significance for the prognosis of ischemic heart disease.
