Changes Of HIF-1α、ET-1、PGI2and The Effect Of Traditional Chinese Drug In HPAECs In Hypoxic Hypercapnia Condition

Background:Chronic obstructive pulmonary disease (COPD) is one of the most common and progressive disease in the respiratory system, and its’ Lung function often has diminished, Ventilation and ventilatory dysfunction of lung can lead to hypoxia and carbon dioxide retention. To clarify the formation mechanism of PH and look for the ideal pharmacy have been the key point of slowing down its occurrence and development. Vascular endothelial cell exert very significant effect in the pathogenesis and it is the target when treating PH. Recent research shows that hypoxia will lead to shrinkage of endothelial cell, wider distance between cells and increase of vascular permeability. Hypoxia will damage the protective function of endothelial cell parclose,which deteriorate the degree of hypoxia of tissue and cell, meanwhile, the inflammatory cells locate more in the hypoxia area, which will lead to the injury of tissues and cells directly. Besides, hypoxia will induce the unbalance of secretion of vascular active substances which produced by pulmonary vascular endothelial cells,damage of endothelial cell structure and disorder of hemostasis, etc. PH start with endothelial cell injury, during this period, pulmonary vasoconstriction enhancement and pulmonary vascular structure reconstruction are considered as the main vascular characters right after the injury of endothelial cell. All the factors exert function separated or interact with each other, deteriorate the situation of pulmonary arterial hypertension. The pulmonary heart disease will follow right after that. Recent years, the exploration of ischemia pathway have been becoming wider and deeper. HIF-1α, the key of mediated and local organization anaerobic reaction and specific intermediary factor, widely participates in mammalian cells induced by ischemia specific response. It plays an important role in hypoxia lung injury with high expression transcription activity of nucleoprotein. HIF-1α is a different source dimer, composed of the oxygen sensitive a and stable beta subunit, The expression of it in cells depends on the oxygen partial pressure directly and degradate rapidly under the oxygen. HIF-1α protein accumulate rapidly in low oxygen environment,as index increased form, and launched a variety of ischemia adaptive gene transcription, such as ET1,EPO, etc. There is a specific sequence containd in these gene promoter enhancer or other regulatory regions of HIF-1α. And in lung metabolism, endothelin is the most effective shrinkage vasoactive peptides. It can expresss hrinkage vascular promote growth factor, adhesion molecules and stimulate the production of extracellular matrix protein and fiber connection protein. It not only participate in vascular tension adjustment, but also participate in low oxygen under the condition of pulmonary artery reconstruction. Animals research at home and abroad found when exposed to hypoxic hypercapnia environment, rat plasma ET-1concentration increased significantly than normal control group. They also found that expression of big rat pulmonary ET-1mRNA enhanced in situ hybridization in chronic low oxygen high carbon dioxide group than normal control group significantly. Therefore, they speculated the increased ET-1synthesis secretion may be the reason of chronic hypoxic hypercapnia pulmonary artery pressure and pulmonary vascular structure reconstruction.Towards the regulation of endothelial cell function, we have gained such progresses as follow:(1) ET receptor antagonist, the drug we used in the treatment of pulmonary hypertension has made satisfied clinical efficacy. It changes the treatment of PH from simple expansion of pulmonary vascular to control pulmonary vascular remodeling.(2) NO and its precursor, which can reduce the endothelial cell injury and expand vascular smooth muscle cells.(3) the prostacycline, which increase the concentration of cAMP, enlarge the role of blood vessels directly, inhibiti proliferation of vascular smooth muscle cell;(4) angiotensin converting enzyme inhibitor and angiotensin II receptor antagonist, which may improve the NO mediated endothelial cell function;(5) calcium antagonists, which can increase the role of NO diastolicing smooth muscle, inhibit shrinkage vascular function of ET-1, a certain of antioxidant effect that can protect endothelial cell function;(6) traditional Chinese medicine;(7) endothelial progenitor cells grow and gene therapy. In a word, along with the increasing role position of the pulmonary vascular endothelial cells in pulmonary arterial hypertension, to protect pulmonary vascular endothelial cell function becomes a important goal in the therapy for pulmonary arterial hypertension.Yifei Huoxue Compound is the treatment for CP by Dong Jianhua, China’s famous doctor of traditional Chinese medicine, which has been confirmed effectively for many years of clinical practice. Previous studies show, the animal experiment level, YFHXC can suppression thickening in membrane at animal experiment level, reduce vascular stenosis and reduce pulmonary arterial hypertension. At cell level, YFHXC could inhibit the rat smooth muscle proliferation of low oxygen in vitro and promote the formation of NO. COPD patients often in alveolar low oxygen at the same time with carbon dioxide retention, and carbon dioxide retention plays a certain role in the formation of pulmonary hypertension. In our experiment, we put HPAECs into the low oxygen with high carbon dioxide environment to observe the change of the endocrine function of HPAECs and the function of YFHXC.Objective:The research the change of HIF-1α, ET-1and PGI2of human pulmonary artery endothelial cells in hypoxic hypercapnia condition in vitro and the protective role of YFHXC.Methods:1. The YFHXG crude drugs were manufactured and checked by Clinical Pharmacy Research Institute of Beijing Municipal Health Bureau. The sliced crude drug materials were extracted by decocting in decuple boiling water for3h each time, then the clear supernatant liquid was removed and condensed. Physic liquor flowed at300ml/min and was centrifuged at4000r/min, to yield YFHXG extracts. YFHXG was dissolved in a small amount of neutral saline and then diluted with the culture medium to the following final concentrations:the dose:1mg/ml. 2. Tweenty New Zealand male rabbits were equally divided into four groups in random:the normal control group and three Yifei Huoxue Granule Containing Drug-serum groups (crude drug10/20/40g.kg-1),5in each group. They were gastric perfused daily with normal saline and Yifei Huoxue Granule Containing Drug-serum in low and moderate and high concentration (crude drug10/20/40g.kg-1) respectively. Blood drawn from rabbits’ abdominal aorta2h after ending perfusion on the7th day, and the serum separated (that is the drug-serum) was taken for testing.Then mixed the same group of Yifei Huoxue Granule Containing Drug-serum and filtered them with microporous membrane.56inactivated30minutes, with0.22u m microfiltration membrane filtration, packaging,-80save backup.3. The attached method training HPAECs:HPAECs cell line recovery, every other day a replacement nutrient solution. After reaching80%confluence, cells were passaged using trypsin/EDTA. Passages3through6were used for the study. The harvested cells were randomly divided into Blank control group; model group; hypoxic hypercapnia+low, medium, high concentration group of Yifei Huoxue Granule Containing Drug-serum.4. HIF-1a protein expression in HPAECs was measured by SABC method. Briefly, after stimulated with hypoxia and YFHXG for12h, the cells were washed by PBS for three times and fixed with4%paraformaldehyde for30min at room temperature before the incubation in0.3%H2O2and5%normal goat serum for20min at room temperature. Finally, the samples were washed by PBS for another three times and incubated with other three agentia and detected with HRP-conjugated anti-rabbit antibody with diaminobenzidine(DAB).5. Radiation immunoassay measurement cell of supernatant fluid ET-1:the cells to4×104/well into a6-well plate, the cells in test group processing, after12hours for each cell nutrient solution supernatant, and refrigerator storage for measurement at20℃below0℃.6. Radiation immunoassay measurement cell supernatant fluid of PGI2:according to the above methods inoculation cells, the cells in test group processing, after12hours for each cell nutrient solution supernatant, and refrigerator storage for measurement at20℃below0℃.7. RT-PCR method was used for determination of ET-1mRNA expression: according to the above methods to cells, the cells in test group processing, after12hours, collect the cells,according to the total RNA extraction kit to extract total RNA, use ultraviolet spectrophotometer to detect RNA concentration, A260/A280=1.8～2.0, reverse transcription; use agarose gel electrophoresis to observe integrity of RNA, then take1ugrna RNA, according to reverse transcription-polymerase chain reaction kit to synthesis cDNA synthesis, polymerase chain reaction; PCR products were observed by Agarose Gel Electrophoresis.8. The results were expressed as mean±standard deviation(SD), all data were processed with SPSS13.0software. Analysis of variance (One-way ANOVA) followed by Dunnett’s multiple comparison test was used in order to compare more than two groups. Statistical significance was accepted at P<0.05.Results:1. There is almost no expression of HIF-1α protein in the Blank control group. The express of HIF-1α protein in model group is higher than that in the Blank control group(n=6,P<0.01). There is no difference between the model group and the YFHXC Containing group of low concentration (n=6, P>0.05). The express of HIF-1α protein in the YFHXC Containing group of moderate, high concentration is lower than that in the model group(n=6,P<0.05).2. There is basal expression of ET-1in the Blank control group. The express of ET-1in model group is higher than that in the Blank control group(n=6,P<0.01). There is no difference between the model group and the YFHXC Containing group of low concentration (n=6, P<0.05). The express of ET-1in the YFHXC Containing group of moderate, high concentration is lower than that in the model group(n=6,P<0.01).3. The expression of PGI2in model group is lower than that in the Blank control group(n=6,P<0.01). There is no difference between the model group and the YFHXC Containing group of low concentration (n=6, P>0.05). The express of PGI2in the YFHXC Containing group of moderate, high concentration is higher than that in the model group(n=6,P<0.01). 4. There is low expression of ET-1mRNA in the Blank control group. The express of ET-1mRNA in model group is higher than that in the Blank control group(n=6,P <0.05). There is no difference between the model group and the YFHXC Containing group of low concentration (n=6, P>0.05). The express of ET-1mRNA in the YFHXC Containing group of moderate, high concentration is lower than that in the model group(n=6,P<0.05).Conclusions:It inceased ET-1levels,the expression of HIF-la and ET-1mRNA to increase, and PGI2levels to decrease of HPAECs on hypoxic hypercapnia condition. YFHXC Containing (moderate, high concentration) Drug-serum can directly decrease ET-1levels, the expression of HIF-1α, ET-1mRNA and increase PGI2levels of HPAECs on hypoxic hypercapnia conditions, thus relieving the degree of endothelial dysfunction. It may play a crucial role in the protecting of hypoxic hypercapnia polmonary arterial hypertension.