四味黄芪散降低低氧性肺动脉高压的机制研究

Mechanism of Siwei Huangqi powder on reducing hypoxic pulmonary hypertension

目的探讨低氧不同时相内皮型一氧化氮合酶(eNOS)信号通路参与藏药四味黄芪散降低低氧性肺动脉高压的机制。方法将110只SD大鼠随机分为常氧组、低氧对照组和低氧药物组,后两组根据暴露低氧时间又各分为5个组。常氧组大鼠暴露于西宁地区(海拔2260 m)大气环境,正常喂养;10个低氧组置于模拟海拔5000 m的低压氧舱,其中,5个低氧药物组灌胃给予四味黄芪散混悬液,5个低氧对照组灌胃给予生理盐水,每日1次,持续30 d。分别于暴露低氧第1、3、7、15、30天时测定肺动脉压(PAP)、左右心室比值;并采集肺组织标本,用WB法测定肺组织中腺苷酸活化蛋白激酶(AMPK)、eNOS、内皮素1(ET-1)蛋白的含量。结果与常氧组比较,低氧对照组大鼠的PAP和左右心室比值逐渐增高,于暴露低氧第7天起显著升高,并随暴露低氧时间的进一步延长而持续增高;低氧药物组大鼠的PAP也随暴露低氧时间的延长而增高,但其增高幅度明显低于低氧对照组。低氧药物组大鼠肺组织eNOS蛋白的水平在急性低氧15 d内均低于常氧组和低氧对照组;于低氧第15天后,低氧药物组eNOS蛋白的水平逐渐升高,超过低氧对照组,于低氧30 d时显著增高,甚至超过常氧组水平。低氧下,eNOS蛋白水平的表达趋势与肺组织中AMPK蛋白及Beclin-1的水平呈高度相关。低氧对照组大鼠肺组织中ET-1蛋白的水平随暴露低氧时间的延长而增高,于低氧第7天时达峰值;低氧药物组大鼠肺组织中ET-1的水平无论在急性低氧期或慢性低氧期均显著低于低氧对照组。结论eNOS蛋白在不同低氧时相呈现不同的水平,四味黄芪散降低低氧性肺动脉高压的机制也不同。急性低氧期主要通过下调ET-1的水平来降低PAP;慢性低氧期主要通过上调eNOS的水平及调控AMPK-eNOS信号通路,抑制平滑肌细胞增殖,降低PAP。

OBJECTIVE To investigate the mechanism of eNOS signaling pathway involved in the Tibetan medicine Siwei Huangqi powder on reducing hypoxic pulmonary hypertension at different hypoxic phases.METHODS A total of 110 SD rats were randomly divided into normoxic group,hypoxia control group and hypoxic drug group.The latter two groups were further divided into 5 groups according to the hypoxia exposure time.The rats in the normoxia group were exposed to atmosphere in Xining(altitude 2260 m)and fed normally without intervention.Ten hypoxia groups were placed in a hypobaric hypoxia chamber simulating an altitude of 5000 m,in which 5 hypoxic drug groups were given Siwei Huangqi powder suspension and other 5 hypoxia control groups were given normal saline by gavage once per day for 30 days.The Pulmonary artery pressure(PAP)and the ratio of left and right ventricle[RV/(LV+S)]were measured and lung tissue samples were collected at the 1st,3rd,7 th,15th and 30th day after exposure to hypoxia.Western Blotting method was used to determine the protein levels of AMPK,eNOS and ET-1 in lung tissues.RESULTS Compared with the normoxia groups,the PAP and RV/(LV+S)in hypoxia control groups increased gradually,and showed a significant increase from the 7th day after exposure to hypoxia,and continued to increase with further exposure.The PAP in hypoxia drug group also increased along with the hypoxia exposure time,but the increase amplitude was significantly lower than that of the hypoxia control group.The level of eNOS protein in lung tissue of hypoxic drug group was lower than that of normoxia group and hypoxic control group within 15 days of acute hypoxia.After 15 days of hypoxia exposure,eNOS protein level of hypoxic drug group gradually increased and exceeded hypoxia control group,and significantly increased at 30 days of hypoxia,even exceeding normoxia group.The expression trend of eNOS protein was highly correlated with AMPK and Beclin-1 in lung tissue of the rats during hypoxia.The level of ET-1 protein in the lung tissue of hypoxia control group increased with the prolongation of exposure to hypoxia,and reached a peak on the 7th day of hypoxia exposure.The level of ET-1 protein in lung tissue of hypoxic drug group was significantly lower than that of hypoxia control group in either acute hypoxia phase or chronic hypoxia phase.CONCLUSION The mechanism of Siwei Huangqi powder in reducing hypoxic pulmonary hypertension is different with different exposure time.In acute hypoxic stage,the PAP can be decreased mainly by down-regulating ET-1 level.In chronic hypoxic stage,the proliferation of smooth muscle cells is inhibited and PAP is reduced mainly through the up-regulation of eNOS level and the regulation of AMPK-eNOS signaling pathway.