| Objective: Pulmonary arterial hypertension(PH) is a clinical syndrome that characterized by increased pulmonary vascular resistance, and it is caused by different etiology and pathogenesis. It is considered that the formation of hypoxic pulmonary hypertension is about hypoxic pulmonary vasoconstriction earlier and pulmonary vascular remodeling later. Hypoxia-inducible factor-1(HIF-1), as an important oxygen dependence transcription activating factor in vivo, plays an important role in the occurrence and development of PH, via activation of downstream target genes to regulate gene expression and protein synthesis of a series cytokines and growth medium.It produces three kinds of natural estrogen in our body, namely 17beta-estradiol(E2),estrone and estriol,17beta-estradiol biological activity in the strongest among the three.and is considered the main physiological role of estrogen.Through the establishment of long-term chronic hypoxic pulmonary hypertension rats model, the study aims to explore the effects of 17 beta- estrogen( E2) and 2-methoxyestradiol(2ME) on HIF-1α and Alk B in ovariectomy and hypoxic pulmonary hypertension rats.Method:1 Model building: Sixty Sprague-Dawdley rats were divided randomly into six groups after Castrated surgery. Castration operation steps are as follows:to open the abdominal cavity,to find bilateral ovaries fallopian tube, to ligature ovaries fallopian tubes and nutrient vessels,to cut off ovaries, to close the abdominal cavity, to use gentamicin to prevent infection,to suture of the skin by “U”.Then rats were randomly divided into 6 groups:oxygen group, oxygen+E2 group,oxygen+2ME group,hypoxia group,hypoxia+2ME group, hypoxia+E2 group.Application of homemade organic glass anaerobic tank, the anaerobic tank 24 h continuous supply air and nitrogen gas atmospheric mixed gas, the oxygen concentration of oxygen box was controlled by oxygen monitoring control system(10±0.5)%, CO2 concentration less than 0.5%;Then the hypoxic rats were placed in the hypoxic chamber, control group rats were kept in the room air, and both group rats were put in the same room with the same diet feeding. oxygen+2ME group and hypoxia+2ME group since the date of building, were daily subcutaneous injected of 2-methoxyestradiol 240 ug/kg. oxygen+E2 group and hypoxia+E2 group were daily subcutaneous injected of estrogen 20 mu g/kg. oxygen group and hypoxia group were daily subcutaneous injected of the same amount of saline solution.2 Long-term changes in hypoxic pulmonary hypertension rats: Mean pulmonary artery pressure(m PAP), was measured by right-heart catheterizetion, right ventricular hypertrophy index(RVHI) was calculated by the ratio of right ventricle to the left ventricle plus septum, and hypoxic pulmonary vascular remodeling(HPSR) was observed by microscope.3 The level of alk B and HIF-1 m RNA in lungs was measured by reverse transcriptase polymerase chain reaction(RT-PCR).4 The level of alk B and HIF-1 protein in lungs was measured by Western blot.Results:1 Established rat model of PAH successfully: The m PAP of hypoxia group rats increased obviously after eight weeks, and compared with controls has significant difference.2 Right ventricular morphology changes of long-term hypoxia pulmonary hypertension rats: Blood vessel walls in lungs are thicken obviously, smooth muscle cells(SMC) of pulmonary small artery increased, the membrane(PAMT) thickening and luminal narrowing; and right ventricular hypertrophy, right ventricule hypertrophy became to develop significantly after eight weeks in hypoxia group the difference is statistically significant.3 Application of RT-PCR analysis Alk B and HIF-1 m RNA expression in the lung tissue: Compared with oxygen group,the expression levels of HIF-1αm RNA in hypoxia group rose markedly(P<0.05).The expression levels of HIF-1αm RNA in hypoxia+E2 and oxygen+2ME group rose markedly( both P<0.05),and amount of increase in hypoxia+E2 and oxygen+2ME group lower than that in hypoxia group. The expression levels of HIF-1αm RNA in oxygen +E2 group,oxygen+2ME group had no significant difference( both P>0.05);Compared with oxygen group,the expression levels of Alk Bm RNA in hypoxia group fell markedly(P<0.05).The expression levels of Alk Bm RNA in hypoxia+E2 and oxygen+2ME group fell markedly( both P<0.05),and amount of decrease in hypoxia+E2 and oxygen+2ME group lower than that in hypoxia group. The expression levels of Alk Bm RNA in oxygen +E2 group,oxygen+2ME group had no significant difference( both P>0.05).4 Application of Western blot analysis Alk B and HIF-1 expression in the lung tissue: Compared with oxygen group,the expression levels of HIF-1α in hypoxia group rose markedly(P<0.05).The expression levels of HIF-1α in hypoxia+E2 and oxygen+2ME group rose markedly(both P<0.05),and amount of increase in hypoxia+E2 and oxygen+2ME group lower than that in hypoxia group. The expression levels of HIF-1α in oxygen +E2 group, oxygen+2ME group had no significant difference(both P>0.05); Compared with oxygen group,the expression levels of Alk B in hypoxia group fell markedly( P<0.05).The expression levels of Alk B in hypoxia+E2 and oxygen+2ME group fell markedly( both P<0.05),and amount of decrease in hypoxia+E2 and oxygen+2ME group lower than that in hypoxia group. The expression levels of Alk B in oxygen +E2 group,oxygen+2ME group had no significant difference( both P>0.05).Conclusisons:1 The differential expression of HIF-1 and Alk B in pulmonary tissue may play an important role in hypoxic pulmonary hypertension development.2 In the condition of low oxygen, ALKB may by epigenetics, in turn, affects the activity of HIF- 1.3 The intervention effect of E2 and 2ME for hypoxic pulmonary hypertension may be partly mediated by the up-regulated expressions of Alk B in lung tissue resulting in the reduced activity of HIF-1α. |
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