ObjectiveTo investigate the expression of C/EBP homologous protein (CHOP) in lung tissue of chronic intermittent hypoxia rats, and explore the intervention effect of edaravone and its possible mechanism.MethodsA total of 120 adult male Wistar rats were randomly divided into three groups: a normal control group (UC group), a chronic intermittent hypoxia group (CIH group), an edaravone intervention group (NE group), and a normal saline group (NS group). The above four groups were also randomly divided into five time subgroups of 3 days, 7 days, 14 days, 21 days and 28 days, respectively, with 6 rats in each time subgroup. The histopathological changes of lung tissue were observed by hematoxylin-eosin (HE) staining and the expression of CHOP in lung tissue was detected by immunohistochemical method.ResultsHE staining results showed that there was no obvious pathological change in UC group. The epithelial cells of lung tissue in CIH group showed edema, hyperemia, widening of alveolar septum and inflammatory cell infiltration. The pathological injury was more serious with the prolongation of intermittent hypoxia time. There were also pathological changes in NE group, but the degree of lung tissue injury was significantly lower than that in CIH group. The results of immunohistochemistry showed that the expression of CHOP in CIH group was significantly higher than that in UC group. The expression of CHOP in NE group was higher than that in UC group, but it was still significantly lower than that in CIH group.ConclusionsThe expression of CHOP protein in lung tissue of chronic intermittent hypoxic rats is enhanced and the high expression of CHOP protein plays a certain role in the lung injury of chronic intermittent hypoxia rats complicated with lung injury. Edaravone may protect lung tissue from chronic intermittent hypoxia by inhibiting the expression of CHOP.
Objective：To investigate the role of 17beta; estradiol on th e expressi on of vascular endothelial growth factor (VEGF) and on the releasing rate of lac tate dehydrogenase (LDH) in cultured anoxiainjured human retinal pigment epit h eliual (RPE) cells.
Methods:Established the anoxiainjuried m odel of human RPE c ells with Cobalt Chloride (CoCl2) after RPE cells were pretreated with 17beta;E 2 and tamoxife, 17beta;E2 antagonist. The expression of VEGF mRNA was detecte d by re v erse transcriptionpolymerase chain reaction technique (RTPCR). The cultured RP E cells were divided into four groups: normal control group, anoxiainjured gro u p, 17beta;E2 pretreatment group and 17beta;E2 with tamoxifen pretreatment grou p. The releasing rate of LDH was detected by chromatometry. The expression of VEGF pro tein were detected by cellular immunohistochemistry.
Results:T he expression of VEGF and LDH releasing rate were higher in anoxiainjured grou p than that in nor m al control group (P＜0.05), and were lower in 17beta;E2 pretreatment group than th at in anoxiainjured group （P＜0.05）. When the effect of 17beta;E2 was o bstructe d by tamoxifen, the expression of VEGF and LDH releasing rate increased but didn prime;t differ much from which in anoxiainjured group (P＞0.05).
Conc lusion:The ex pression of VEGF increases in anoxiainjured human RPE cells. 17beta;E2 can do wnr egulate the expression of VEGF and decrease the releasing rate of LDH, which can be blocked by tamoxifen.
Objective To develope a novel rabbit carotid body and carotid common artery model in vivo for the simulation of various intermittent hypoxia ( IH) intensities, IH durations, IH reoxygenation ( ROX) durations and continuous hypoxia ( CH) modes. Methods Forty-five adult New Zealand rabbits ( 2. 5-3. 0 kg) were anesthetized while spontaneous breathing kept intact. The tissue surrounding the right carotid common artery and carotid sinus nerve ( CSN) were cleared and " single" chemoreceptor bundle of the CSN was revealed. Then suction electrodes were placed and CSN afferent activity was monitored and recorded carefully. The right common carotid artery was exposed, cannulated to distal part and its proximal part was ligated. Preparations were challenged by changing the PO2 of the gas mixture equilibrating the perfusate. Alternatively perfusion ( 2 mL/min) of equilibrated perfusate bubbled with normoxia or hypoxia gas mixtures formed IH/ROX cycles in carotid common artery, simulating the pattern of hypoxic episodes seen in obstructive sleep apnea syndrome ( OSAS) , or with continuously perfusing hypoxia perfusate to form CH modes. All the perfusing procedures were regulated by a customized computer-controlled set and monitored using O2 gas analyzer. After the systematic exposures, carotid body, carotid common artery part distal to cannula, and carotid bifurcation were harvested as samples. Results The frequencies and average amplitudes of CSN chemoreceptor bundles afferent activities with normoxia perfusion were ( 0. 17 ±0. 03) impulse/ s and ( 46. 2 ±4. 4) μV, and with hypoxia perfusion were ( 0. 64 ±0. 09) impulse/ s and ( 87. 4 ±6. 6) μV, respectively. PO2 was ( 139 ±1. 5) mm Hg in normoxia perfusate and ( 35. 2 ±1. 3) mm Hg in hypoxia perfusate. Conclusion This new carotid body and carotid common arterymodel is a valuable tool to study neurological and biochemical changes in various IH and CH modes.
ObjectiveTo explore the effects of hypoxia inducible factor-1 alpha (HIF-1α) on the reverse differentiation of hepatocellular carcinoma cells into liver cancer stem cells, and the maintenance of malignant biological behavior in hypoxic environment.MethodsCD133-negative cells in HepG2 cells were separated by immunomagnetic beads and divided into two groups. The cells of siRNA group were transfected with siRNA-HIF-1α to silence the expression of HIF-1α gene, while cells of the blank control group did not transfect any siRNA fragments. The two groups of cells were cultured under normal and hypoxic conditions respectively. MTT, cloning and Transwell chamber experiments were used to detect the proliferation and invasion ability of cells. Western blot and real-time PCR (RT-PCR) were used to detect the expressions of HIF-1α, CD133, CD90, and CD44 protein and mRNA in cells.ResultsMTT results showed that the cell proliferation rate increased with the prolongation of hypoxia in four groups. Compared with the blank control group at 24, 32, 40, and 48 hours, the cell proliferation rate decreased significantly after siRNA-HIF-1a transfection, on both two kinds of cultured conditions (P<0.05). The results of plate cloning experiment showed that the number of cell-forming clones increased significantly after hypoxic culture (there were significant differences between the transfected normoxic group and transfected hypoxic group, blank control normoxic group and blank control hypoxic group, P<0.05); and the formation of transfected hypoxic condition group at the same time of hypoxia was also significant (P<0.05). The number of clones were significantly less than that of the blank control group at the hypoxic condition (P<0.05). Transwell lab experiment showed that after hypoxic culture, the number of cells migrated to the inferior chamber in the transfection group was significantly reduced compared with that of the blank control group (P<0.05). Western blot and RT-PCR results showed that the expression levels of HIF-1α protein and tumor stem cell markers (CD133, CD90, and CD44 protein) in the blank control hypoxic condition group were significantly higher than those in the other three groups (P<0.05); after siRNA-HIF-1a transfection, HIF-1α mRNA and tumor stem cell markers mRNA (CD133, CD90, and CD44 mRNA) in the transfected hypoxic condition group were significantly lower than those in the transfected normal condition group and the blank control normal condition group (P<0.05).ConclusionsIn hypoxia environment, HIF-1α can promote hepatocellular carcinoma cells to differentiate into liver cancer stem cells and enhance their malignant biological behavior.
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.
ObjectiveBy detecting the expression of the long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in myocardial tissue under different hypoxia patterns, to explore the possible mechanism of obstructive sleep apnea (OSA)-induced cardiovascular diseases.MethodsSD rats were randomly and equally divided into 4 groups namely a normal (N) group, a continuous hypoxia (CH) group, an intermittent hypoxia (IH) group and an intermittent hypoxia with hypercapnia (IHH) group, and were treated for 1, 2, and 3 weeks. The expression of MALAT1 and associated immune factors of the myocardial tissue were examined by qRT-PCR.ResultsAn elevation without significance was observed in those three hypoxia groups in contrast with N group after 1 week’s treatment. However, in 2 and 3 weeks’ groups, the mRNA expression of MALAT1 was significantly higher in IHH group than the other three groups (all P<0.01), while there was no significant difference among IH, CH or N groups despite an increasing tendency in IH and CH groups against N group were observed. Additionally, the expressions of hypoxia inducible factor-1α (P<0.05), Toll-like receptor 4 (P<0.01) and interleukin-6 (P<0.05) mRNA were also increased significantly in IHH group compared with IH, CH and IHH groups in 3 weeks’ treatment respectively, which were coordinated with the change of MALAT1 mRNA.ConclusionsThe expression of MALAT1 in myocardial tissue is elevated by intermittent hypoxia with hypercapnia, and the tendency is similar with hypoxia-induced inflammation factors. These findings indicate that MALAT1 is probably a regulatory factor of OSA induced myocardial immune injury.
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.
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.
ObjectiveTo study the influence of hemin on blood pressure of intermittent hypoxic rats and investigate the mechanism of hypertension caused by intermittent hypoxia.MethodsTwenty-four male SD rats were randomly divided into a hemin group, an intermittent hypoxia group (IH group) and a normal group. Thirty minutes after intraperitoneal injection of hemin, the rats in the hemin group were exposed to intermittent normobaric hypoxic environment (8 h/d). The rats in the IH group were intraperitoneal injected with normal sodium and then exposed to the same environment (8 h/d). The rats in the normal group were intraperitoneal injected with normal sodium and placed in the glass box. The three groups were bred in the same condition. Thirty-five days later, the mean carotid artery pressure (mCAP) of the rats was measured and their plasma carbon monoxide (CO) level was measured by Chalmer’s method. Reverse transcription polymerase chain reaction was performed to detect the levels of heme oxygenase-1 (HO-1) mRNA expression in lung, liver, spleen, kidney and other organs. The expression of HO-1 protein in the organs was detected by immunohistochemistry.ResultsThe mCAP in the IH group was significantly higher than the hemin group and the normal group (P<0.05), and was higher in the hemin group than the normal group (P<0.05). The concentration of plasma CO in the hemin group was higher than the IH group and the normal group (P<0.05). There was no significant difference in plasma CO between the IH group and the normal group (P>0.05). The expression of HO-1 mRNA of lung, liver, spleen and kidney in the hemin group and the IH group was higher than the normal group (P<0.05), and was higher in the hemin group than the IH group (P<0.05). The relationship between mCAP and HO-1 mRNA showed a curvilinear trend. The quadratic curve fitting equation was Y=39.715+446.640X-334.353X2.ConclusionsIntermittent hypoxia can cause hypertension in rats. The HO-1 expression is increased in hypoxic rats, but the plasma CO does not increase significantly. As an inducer of HO-1, hemin can increase the expression of HO-1 and CO in hypoxic rats, then lower their blood pressure to some extent.
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
