Collagen is the major matrix protein in dermis and consists of proline and lysine, which are hydroxylated by prolyl hydroxylase (PH) and lysyl hydroxylase (LH) with cofactors such as vitamin C, oxygen, iron (Fe2+), ketoglutarate and silicon. The collagen degradation is regulated by matrix metalloproteinase-1 (MMP-1), of which is the major collagen-degrading proteinase whereas tissue inhibitors of metalloproteinase-1 (TIMP-1) bind to MMP-1 thereby inhibiting MMP-1 activity. In this study, we investigated the effects of vitamin C, silicon and iron on mRNA, protein expressions of PH, LH, MMP-1 and TIMP-1. The physiological concentrations of vitamin C (0-100 micrometer), silicon (0-50 micrometer) and iron (Fe2+:0-50 micrometer) were treated to human dermal fibroblast cells (HS27 cells) for 3 or 5days. The expression level of mRNA and protein was increased in not only PH but also LH when cells were incubated with vitamin C. A similar increase in LH mRNA or protein expression occurred when cells were incubated with silicon. Our results suggest that treatment of vitamin C and silicon increased mRNA and protein expression of PH and LH in human dermal fibroblast.
Ascorbic Acid ; Collagen ; Dermis ; Fibroblasts ; Humans ; Hydrogen-Ion Concentration ; Iron ; Lysine ; Matrix Metalloproteinase 1 ; Oxygen ; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase ; Procollagen-Proline Dioxygenase ; Proline ; RNA, Messenger ; Silicon ; Tissue Inhibitor of Metalloproteinase-1 ; Vitamins

Prolyl 4 hydroxylases (P4H) are iron- and 2-oxoglutamate-dependent dioxygenase enzymes and hypoxia-inducible transcription factor (HIF)-P4Hs play a critical role in the regulating oxygen homeostasis in the local tissues as well in the systemic circulation. Over a period of time, a number of prolyl hydroxylase inhibitors and activators have been developed. By employing the pharmacological tools and transgenic knock out animals, the critical role of these enzymes has been established in the pathophysiology of number of diseases including myocardial infarction, congestive heart failure, stroke, neurodegeneration, inflammatory disease, respiratory diseases, retinopathy and others. The present review discusses the different aspects of these enzymes including their pathophysiological role in disease development.
Animals ; Heart Failure ; Homeostasis ; Inflammation ; Ischemia ; Mixed Function Oxygenases ; Myocardial Infarction ; Oxygen ; Procollagen-Proline Dioxygenase ; Stroke ; Transcription Factors

OBJECTIVETo detect the expression of the plateau adaptablity gene(EPAS1, EGLN1 and PPARα) and proteins(HIF-2, PHD2 and PPARα) in rats blood, heart, liver, lung and kidney tissue after the rats exposed to high altitude.

METHODSThe Wistar rats were randomly divided into plain group(Shanghai, 55 m), acute exposure to high altitude 3400 m group, acute exposure to high altitude 4300 m group. Blood and organs of rats were collected in 1, 3, 5 days after arrival. Real time PCR and ELISA were used to compare the expression of plateau adaptablity gene and related protein between plain group and high altitude exposure groups.

RESULTSThe count of red blood cells, hemoglobin and HCT in high altitude 4300 m were higher than those in plain group. Compared with plain group, the expression of EPAS1 gene in blood, heart, liver and kidney tissue of rats at high altitude increased obviously(all P<0.05); the expression of EGLN1 in the heart, liver, brain and kidney increased, and PPARα gene in the heart, liver and kidney increased(all P<0.05). Compared with plain group, the expression of HIF-2 protein increased significantly at high altitudes in the liver, brain and kidney tissues. PHD2 and PPARα increased in the heart, liver and kidney.

CONCLUSIONPlateau adaptive genes(EPAS1, EGLN1 and PPARα) and protein(HIF-2, PHD2 and PPARα) differed in different altitude and different organizations. They might be used as target markers of plateau hypoxia.

Adaptation, Physiological ; Altitude ; Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Brain ; China ; Heart ; Hypoxia ; Hypoxia-Inducible Factor-Proline Dioxygenases ; metabolism ; Kidney ; Liver ; Lung ; PPAR alpha ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Rats ; Rats, Wistar

Animals ; Basic Helix-Loop-Helix Transcription Factors ; physiology ; Chronic Disease ; Humans ; Hypoxia ; complications ; Hypoxia-Inducible Factor 1 ; physiology ; Kidney ; metabolism ; Kidney Diseases ; etiology ; Oxidative Stress ; Procollagen-Proline Dioxygenase ; physiology

AIMTo investigate the dynamic expression of hypoxia-inducible factor 1alpha, PHDs and OS-9 in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

METHODSSD rats were randomly divided into 5 groups (n = 8) and exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively. RT-PCR and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of protein.

RESULTSHIF-1alpha protein was poorly positive in control, markedly up-regulated after 3 d and 7 d of hypoxia (P < 0.05, vs group C), and then declined slightly after 14 d and 21 d of hypoxia. HIF-1alpha mRNA increased dramatically after 14 d of hypoxia (P < 0.05, vs group C). PHD1, PHD2 mRNA and protein was positive in group C. PHD2 mRNA and protein were up-regulated after 3 d of hypoxia (P < 0.05, vs group C), reaching its peak after 14 d of hypoxia while PHD1 protein declined after 14 d of hypoxia (P < 0.05, vs group C) without statistic mRNA changing. PHD3 mRNA and protein were detected at low level in control, markedly up-regulated after 3 d of hypoxia (P < 0.05, vs group C), and then PHD3 mRNA kept at high level while PHD3 protein declined after 14 d of hypoxia (P < 0.05, vs 7 d). OS-9 mRNA was positively in control, markedly decreased after 3 d of hypoxia (P < 0.05, vs group C), reaching its lowest lever after 14 d of hypoxia. Linear correlation analysis showed that OS-9 protein was positively correlated with OS-9 mRNA (r = 0.82, P < 0.01) and HIF-1alpha protein (r = 0.57, P < 0.01).

CONCLUSIONHIF-1alpha, PHDs and OS-9 are all involved in the pathogenesis of hypoxic pulmonary hypertension in rats. OS-9 may interact with both HIF-1alpha and PHDs to promote PHD-mediated hydroxylation of HIF-1alpha.

Animals ; Female ; Hypertension, Pulmonary ; metabolism ; physiopathology ; Hypoxia ; complications ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Lectins ; genetics ; metabolism ; Male ; Procollagen-Proline Dioxygenase ; genetics ; metabolism ; Pulmonary Artery ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo study the predicting effect of proly 4-hydroxylase beta polypeptide (P4HB) in treating non-small cell lung cancer (NSCLC) patients by Yiqi Chutan Recipe (YCR).

METHODSTotally 46 stage III and IV NSCLC patients were treated by YCR for 4 therapeutic courses. Effect was assessed by RECIST of solid tumor. P4HB expression was detected in the lung cancer tissue by immunohistochemical assay. Factors affecting disease control rates (DCR) of YCR were analyzed by Logistic regression analysis. The correlation between P4HB expression and the effect of YCR was analyzed.

RESULTSThe DCR of advanced NSCLC treated by YCR was 36.96% (17/46 cases). P4HB was high expressed in advanced lung cancer tissue (6/15 cases). Gender, initial treatment, and retreatment are independent factors for affecting DCR of treating lung cancer by YCR.

CONCLUSIONP4HB might be taken as a factor for predicting the effect of YCR in treating NSCLC.

Carcinoma, Non-Small-Cell Lung ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Lung ; Lung Neoplasms ; drug therapy ; metabolism ; Male ; Procollagen-Proline Dioxygenase ; metabolism ; Protein Disulfide-Isomerases ; metabolism

OBJECTIVE: To observe the effect of Biantie (bian stone plaste) pretreatment on serum level of prolyl hydroxylase domain 2 (PHD2) and hypoxia-inducible factor-1α (HIF-1α) in rats with acute hypobaric hypoxia induced-brain injury, and to explore the possible mechanism of Biantie on preventing brain injury at high altitude. METHODS: Forty-five male SD rats were randomly divided into a blank group, a model group, a Biantie group, a medication group and a Biantie+inhibitor group, 9 rats in each group. The rats in the Biantie group the and the Biantie+inhibitor group were pretreated with Biantie at "Taiyuan" (LU 9), "Neiguan" (PC 6) and "Renying" (ST 9), 2 h each time, once a day; the rats in the medication group were treated with intragastric administration of rhodiola capsule solution (280 mg/kg) for 14 d; the rats in the Biantie+inhibitor group were intraperitoneally injected with the PHD inhibitor dimethyloxalyl glycine (DMOG) at a dose of 40 mg/kg 24 h before the establishment of the model. After the intervention, except for the blank group, the rats in the remaining 4 groups were placed in the oxygen chamber to simulate a high-altitude environment to establish the acute hypobaric hypoxia brain injury model. The arterial blood-gas analysis indexes [blood oxygen saturation (SaO₂), lactic acid (Lac), blood sodium (Na⁺), blood potassium (K⁺)] and brain water content were detected in each group; the histomorphology of cerebral cortex was observed by HE staining; the serum levels of PHD2 and HIF-1α as well as vascular endothelial growth factor (VEGF) were detected by ELISA; the VEGF protein expression in brain tissue was detected by Western blot; the VEGF mRNA expression in brain tissue was detected by real-time fluorescent quantitative PCR. RESULTS: Compared with the blank group, the levels of SaO₂ and Na⁺ in the model group were decreased (P<0.05), while the levels of Lac and K⁺ as well as the water content of brain tissue were increased (P<0.05). Compared with the model group, the level of SaO₂ in the Biantie group and the medication group was increased (P<0.05), while the levels of Lac, K⁺ and the water content of brain tissue were decreased (P<0.05); the level of Na⁺ in the Biantie group was increased (P<0.05). Compared with the Biantie group, the level of SaO₂ in the Biantie+inhibitor group was decreased (P<0.05), and the level of Lac and the water content of brain tissue were increased (P<0.05). In the model group, the cortical tissue cells were loose and disordered, the cortical blood vessels were dilated, and the cells were obviously swollen; the anoxic injury in the Biantie group and the medication group was lighter, and the anoxic injury in the Biantie+inhibitor group was more obvious than that in the Biantie group. Compared with the blank group, the serum PHD2 content in the model group was decreased and the HIF-1α content was increased (P<0.05), and the content of VEGF in serum and VEGF protein and mRNA expressions in brain were increased (P<0.05). Compared with the model group, the content of PHD2 in serum in the Biantie group and the medication group was increased (P<0.05), and the level of HIF-1α was decreased (P<0.05), and the content of VEGF in serum as well as VEGF protein and mRNA expressions in brain were decreased (P<0.05). Compared with the Biantie group, the serum PHD2 content in the Biantie+inhibitor group was decreased and HIF-1α level were increased (P<0.05), and the content of VEGF in serum as well as VEGF mRNA expression in brain were increased (P<0.05). CONCLUSION Biantie at "Taiyuan" (LU 9), "Neiguan" (PC 6) and "Renying" (ST 9) could regulate serum PHD2/HIF-1α to down-regulate VEGF expression, reduce brain edema and enhance anti-hypoxia ability, so as to achieve the purpose of preventing brain injury at high altitude.
Animals ; Rats ; Male ; Prolyl Hydroxylases/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Rats, Sprague-Dawley ; Procollagen-Proline Dioxygenase/metabolism* ; Brain Injuries ; Brain/metabolism* ; RNA, Messenger ; Water

OBJECTIVETo investigate the protective effect of Crocin against hypoxia damage of cardiac myocytes of neonatal rats and the regulation of HIF-1 and prolyhydroxylase (PHDs).

METHODSA model of CoCl2 simulated hypoxia damage was established in primary cultural myocardial cell. Expression levels of HIF-1alpha, VEGF, iNOS, as well as PHD1, 2, 3 protein in myocardial cells were detected by Western blot.

RESULTSCompared with CoCl2 group, the viability of myocardial cell was significantly increased after treated 24 h at 10(-5)mol/L Crocin (P < 0.01), HIF-1alpha, VEGF and iNOS were expressed higher than those in Crocin + CoCl2 group (P < 0.01), the expression of PHD2 was significantly increased (P < 0.01), while the expression of PHD3 was remarkably reduced in Crocin + CoCl2 Group (P < 0.01).

CONCLUSIONCrocin has better protective effect on hypoxic damage of myocardial cell. The mechanisms of protective effect of Crocin may be related to the activation of HIF-1-mediated pathway of the hypoxia response. PHDs may be involved in the pathophysiology regulated process of myocardial cells.

Animals ; Carotenoids ; pharmacology ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Homeodomain Proteins ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Hypoxia-Inducible Factor-Proline Dioxygenases ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

This study was aimed to investigate the effect of low dose radiation (LDR) on human bone marrow mesenchymal stem cells (MSCs) by using proteomic analysis. The bidirectional gel electrophoresis was used to establish the two-dimensional gel electrophoresis patterns of proteome in group of MSCs exposed to LDR and in group of sham irradiated MSCs, the matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was used to identify the differentially expressed proteins in two groups. The results showed that among the differentially expressed proteins in the two groups, the expressions of 12 proteins were up-regulated, the expressions of 12 protein were down-regulated, 3 proteins disappeared after LDR, 12 proteins had been identified by MALDI-TOF-MS. In conclusion, the identified 12 proteins, such as prolyl 4-hydroxylase, dihydropyrimidinase-like 2 variant, ARP3 (actin-related protein 3, yeast) homolog, guanine nucleotide binding protein (G protein), phosphoglycerate mutase 1 may be related to mechanism of LDR effect. The study provides some new explanation for the mechanism of low dose radiation injury.
Actin-Related Protein 3 ; analysis ; Bone Marrow Cells ; radiation effects ; Dose-Response Relationship, Radiation ; GTP-Binding Proteins ; analysis ; Humans ; Mesenchymal Stromal Cells ; metabolism ; radiation effects ; Procollagen-Proline Dioxygenase ; analysis ; Proteins ; analysis ; Proteomics ; methods ; Radiation Dosage ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo observe the expression of hypoxia-inducible factor-lalpha subunit (HIF-1alpha), HIF prolyl hydroxylase domain-containing protein(PHDs) and factor inhibiting HIF-1(FIH) in pulmonary arteries of patient with chronic obstructive pulmonary disease (COPD).

METHODSPulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, 12 had concurrent COPD (COPD group) and 14 without COPD (control group). The ratio of vascular wall area to total vascular area (WA%) and pulmonary artery media thickness (PAMT) was observed, and HIF-1alpha and its hydroxylases(PHD1, PHD2, PHD3, FIH) mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively.

RESULTSWA% and PAMT of COPD patients(50 microm +/- 9 microm, 40% +/- 5%, were statistically different from those of the control subjects (39 microm +/- 6 microm, 31% +/- 4%, P < 0.01). Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-lalpha mRNA and protein levels in COPD group (0.230 +/- 0.036,0.275 +/- 0.039) were statistically higher than those of the control subjects (0.174 +/- 0.029, 0.102 +/- 0.015, P < 0.01 ), and that the protein level increased more markedly. PHD1 mRNA in COPD subjects (0.180 +/- 0.030) was comparable to that in control group (0.191 +/- 0.029, P > 0.05); PHD2 and PHD3 mRNA levels in COPD (0.245 +/- 0.044, 0.252 +/- 0.023) were significantly higher than those in control group(0.182 +/- 0.028, 0.127 +/- 0.017, P < 0.01). On the other hand, in COPD subjects PHD1 protein (0.104 +/- 0.015) was significantly lower(P < 0.01), whereas PHD2 protein (0.274 +/- 0.044) was significantly higher(P < 0.01) than those in control group(0.209 +/- 0.023, 0.219+/- 0.043). As for PHD3 protein, no significant changes were observed between the two groups (0.161+/- 0.023 in COPD, 0.146 +/- 0.021 in control, P > 0.05). FIH mRNA and protein both showed no differences between the two groups. Linear correlation analysis showed that HIF1alpha protein was positively correlated with WA%, PAMT, PHD2 mRNA and protein, PHD3 mRNA, and that HIF1alpha protein was negatively correlated with PHD1 protein.

CONCLUSIONPHDs may be involved in the process of hypoxic pulmonary vascular remodeling in COPD via regulation of HIF-1alpha gene expression

Aged ; Case-Control Studies ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Lung ; blood supply ; metabolism ; Male ; Middle Aged ; Mixed Function Oxygenases ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Pulmonary Artery ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; RNA, Messenger ; genetics ; Repressor Proteins ; metabolism