Siah1对大鼠低氧性肺动脉高压发病的调控机制

Regulatory mechanism of Siah1 in the pathogenesis of rat hypoxic pulmonary hypertension

目的 探讨Siah1对大鼠低氧性肺动脉高压（HPH）发病的调控机制.方法 40只成年雄性Wistar大鼠按随机数字表法随机分为对照组和低氧3、7、14、21 d组,每组8只,常压间断低氧复制HPH大鼠模型,对照组大鼠常氧下饲养.测各组大鼠平均肺动脉压（mPAP）、右室肥厚指数（RVHI）、管壁面积与血管总面积比值（WA％）、管腔面积与血管总面积比值（LA％）;原位杂交检测肺内低氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF） mRNA相对表达量,免疫组化检测其蛋白质相对表达量;原位杂交、逆转录-PCR检测肺内低氧诱导因子抑制因子（FIH）、Siah1 mRNA相对表达量,免疫组化、Western印迹法检测其蛋白质相对表达量.结果 低氧7d组mPAP、WA％、LA％显著高于对照组[（21.3±1.6） mmHg比（15.9 ±1.3）mmHg（1 mmHg =0.133 kPa）、（41.4±2.8）％比（35.0±2.2）％、（58.6±2.8）％比（65.0±2.2）％,均P＜0.05],低氧14 d组达高水平并维持.低氧14d组RVHI显著高于对照组[（27.0±1.8）％比（23.2±2.1）％,P＜0.05].低氧14 d组HIF-1αmRNA相对表达量显著高于对照组[（0.188 ±0.014）比（0.150 ±0.014）,P＜0.05];低氧3d组HIF-1α蛋白相对表达量显著高于对照组（0.186±0.014比0.067±0.008,P＜0.05）.低氧7d组VEGFmRNA、蛋白相对表达量显著高于对照组（0.152±0.019比0.057±0.007、0.176±0.017比0.083±0.010,均P＜0.05）.FIH mRNA表达在低氧后无显著变化,低氧7d组FIH蛋白相对表达量显著低于对照组（0.166 ±0.015比0.200 ±0.017,P＜0.05）.低氧7d组Siah1 mRNA、蛋白表达均显著高于对照组（0.144 ±0.014比0.067 ±0.010、0.136±0.017比0.084±0.019,均P＜0.05）.HIF-1α蛋白与VEGF mRNA、VEGF蛋白表达均呈正相关（r=0.545、0.523,均P＜0.01）;FIH蛋白与VEGFmRNA、VEGF蛋白表达均呈负相关（r=-0.785、-0.788,均P＜0.01）;Siah1 mRNA与Siah1蛋白表达呈正相关（r =0.823,P＜0.01）;Siah1蛋白与FIH蛋白表达呈负相关（r=-0.671,P＜0.01）.结论 慢性低氧诱导肺小动脉壁Siah1表达增加,进而可能降低FIH蛋白水平,减弱FIH对HIF-1α转录活性的抑制,导致VEGF等HIF-1 α靶基因转录激活,从而参与HPH发生发展.

Objective To explore the regulatory mechanism of Siah1 in the pathogenesis of hypoxic pulmonary hypertension （HPH） in rats.Methods According to the random number table,40 adult male Wistar rats were randomly divided into 5 groups （n =8 each）.And the animals were exposed to normoxia or hypoxia for 3,7,14 or 21 days respectively.The HPH model was established by normobaric intermittent hypoxia.Mean pulmonary arterial pressure （mPAP）,ratio of vascular wall area to total vascular area （WA%）,ratio of vascular lumen area to total vascular area （LA%） and right ventricle hypertrophy index （RVHI） were measured.The mRNA and protein relative levels of hypoxia-inducible factor-1 α （HIF-1α） and vascular endothelial growth factor （VEGF） were detected by in situ hybridization and immunohistochemistry respectively.Reverse transcriptase-polymerase chain reaction （RT-PCR） and in situ hybridization were used to determine the relative expressions of mRNA of hypoxia-inducible factor-1 （FIH） and Siah1.Immunohistochemistry and Western blot were employed to determine the relative expressions of proteins of FIH and Siah1.Results The levels of mPAP,WA% and LA% were significantly higher after 7-day hypoxia than those in normoxic control （（21.3 ± 1.6） vs （15.9 ± 1.3） mmHg （1 mmHg =0.133 kPa）,（41.4±2.8）% vs （35.0±2.2）%,（58.6±2.8）% vs （65.0±2.2）%,all P〈0.05）.The level of RVHI was significantly higher after 14-day hypoxia than that in normoxic control （（27.0 ± 1.8） % vs （3.2 ±2.1） %,P 〈0.05）.The relative expression of HIF-1α mRNA was significantly higher after 14-day hypoxia than that in normoxic control （0.188 ± 0.014 vs 0.150 ± 0.014,P 〈 0.05）.The relative expression of HIF-1o protein was significantly higher after 3-day hypoxia than that in normoxic control （0.186±0.014 vs 0.067 ± 0.008,P 〈 0.05）.The relative levels of VEGF mRNA and protein were significantly higher after 7-day hypoxia than those in normoxic control （0.152 ± 0.019 vs 0.057 ± 0.007,0.176 ±0.017 vs 0.083 ±0.010,both P 〈0.05）.The relative expression of FIH mRNA had little changes after exposure to hypoxia compared with normoxia.However the related expression of FIH protein was markedly lower after 7-day hypoxia than that in normoxic control （0.166 ± 0.015 vs 0.200 ± 0.017,P 〈 0.05）.The relative levels of Siah1 mRNA and protein were markedly higher after 7-day hypoxia than those in normoxic control （0.144 ±0.014 vs 0.067 ±0.010,0.136 ±0.017 vs 0.084 ±0.019,both P 〈0.05）.Linear correlation analysis showed that HIF-1 α protein was positively correlated with the relative levels of VEGF mRNA and VEGF protein （r =0.545,0.523,both P 〈 0.01） while FIH protein was negatively correlated with the relative levels of VEGF mRNA and VEGF protein （r =-0.785,-0.788,both P 〈 0.01）.There was a positive correlation between the relative levels of Siahl mnRNA and Siah1 protein （r =0.823,P 〈0.01） while a negative correlation existed between the relative levels of Siah1 protein and FIH protein （r =-0.671,P 〈 0.01）.Conclusions Under chronic hypoxia,Siah1 is transcriptionally induced in pulmonary arterioles and it facilitates the degradation and decline of FIH in rats.And deceased FIH protein in pulmonary arterioles under hypoxia may attenuate its inhibitory effect on the transactivational activity of HIF-1 c and promote the transactivation of such HIF-1c target gene as VEGF.Thus it is probably implicated in the pathogenesis of HPH.