目的 探究肺心汤通过调控腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路对低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)大鼠的治疗作用及机制.方法 36 只SD雄性大鼠随机分为正常组、模型组、西地那非组以及肺心汤低、中、高剂量组.除正常组外,将其余 5 组置于氧气浓度为 10.0%±0.5%的低氧舱内造模 28 d,每天 8h,造模同时灌胃给药.4周后检测大鼠心肺血流动力学指标[右心室收缩压(right ventricular systolic pressure,RVSP),右心室肥厚指数(right ventricular hypertrophy index,RVHI),肺动脉加速时间/肺动脉射血时间(pulmonary acceleration time/pulmonary ejection time,PAT/PET)、三尖瓣环收缩期位移(tricuspid annulus plane systolic excursion,TAPSE)、右心室前壁厚度(right ventricular anterior wall thickness,RVAWT)];HE染色法检测肺动脉重塑;免疫荧光法检测α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白共定位表达;透射电镜观察自噬溶酶体的数量;免疫组织化学法检测肺组织中p-AMPK、p-mTOR、微管相关蛋白1轻链3B(microtubule-as-sociated protein light chain 3B,LC3B)、Beclin1 和p62蛋白阳性表达.结果 与正常组相比,模型组大鼠RVSP、RVHI及RVAWT显著升高(P<0.01),PAT/PET和TAPSE显著降低(P＜0.01);肺小动脉壁明显增厚,管腔狭窄,肺小动脉管壁厚度占血管直径的百分比(the percentage of wall thickness of pulmonary arterioles to vascular diameter,WT%)显著升高(P＜0.01);α-SMA和PCNA共定位表达显著增加;自噬溶酶体数量增加;肺组织中p-AMPK、LC3B和Beclin1蛋白阳性表达上调(P＜0.01),p-mTOR及p62蛋白阳性表达下调(P＜0.01).与模型组相比,各给药组大鼠RVSP、RVHI及RVAWT 显著下降(P＜0.01),PAT/PET、TAPSE显著升高(P＜0.05,P＜0.01);肺小动脉壁增厚程度减轻,WT%显著降低(P＜0.01);α-SMA和PCNA共定位表达减少;自噬溶酶体数量减少;肺组织中p-AMPK、LC3B和Beclin1蛋白阳性表达显著下降(P＜0.05,P＜0.01),p-mTOR及p62蛋白阳性表达显著上调(P＜0.01).与西地那非组相比,肺心汤低、中剂量组TAPSE降低(P＜0.01);肺心汤低、中剂量组肺动脉中膜增厚程度增加,WT%增加(P＜0.01);肺心汤低、中剂量组p-AMPK蛋白阳性表达升高(P＜0.01),肺心汤高剂量组p-AMPK蛋白阳性表达降低(P＜0.05);肺心汤低剂量组p-mTOR蛋白阳性表达降低(P＜0.01).结论 肺心汤可能通过下调AMPK/mTOR信号通路抑制自噬,发挥改善HPH的作用.
Protective effects of Feixin Decoction on rats with hypoxic pulmonary hypertension by inhibiting autophagy through regulating AMPK/mTOR signaling pathway
Objective To investigate the therapeutic efficacy and mechanism of Feixin Decoction(FXD)on hypoxic pulmonary hypertension(HPH)rats through regulating adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Thirty-six SD rats were randomized into control group,model group,sildenafil group and low-,medium-,high-dose FXD groups.Except control group,the remaining five groups were placed in a hypoxic chamber with an oxygen concentration of 10%±0.5%for modeling,8 hours per day for 28 d,with simultaneous intragastric administration.After 4 weeks,the rat cardiopulmonary hemodynamic indexes[right ventricular systolic pressure(RVSP),right ventricular hypertrophy index(RVHI),the ratio of pulmonary acceleration time to pulmonary ejection time(PAT/PET),tricuspid annulus plane systolic excursion(TAPSE),right ventricular anterior wall thickness(RVAWT)]were determined.Pulmonary artery remodeling was checked by HE staining;the co-localization of α-smooth muscle actin(α-SMA)and proliferating cell nuclear antigen(PCNA)was examined by immunofluorescence assay;the number of autolysosomes was observed by transmission electron microscope(TEM);the positive expressions of p-AMPK,p-mTOR,microtubule-associated protein 1 light chain 3B(LC3B),Beclin1,and p62 in the lung tissue were measured by immunohistochemical assay.Results Compared with the control group,in the model group,RVSP,RVHI,and RVAWT were significantly increased(P<0.01),while PAT/PET and TAPSE were significantly decreased(P<0.01);the wall of the pulmonary arterioles was markedly thickened and the lumina narrowed,with a significant increase in the per-centage of wall thickness of pulmonary arterioles to vascular diameter,WT%(P<0.01);the co-localization of α-SMA and PCNA was significantly enhanced(P<0.01);the number of autolysosomes was increased;the protein positive expressions of p-AMPK,LC3B,and Beclin1 in the lung tissue were up-regulated(P<0.01),while those of p-mTOR and p62 were down-regulated(P<0.01).Compared with the model group,in all medication groups,RVSP,RVHI,and RVAWT were significantly decreased(P<0.01),while PAT/PET and TAPSE were significantly increased(P<0.05,P<0.01);the thickness of the pulmonary arterioles was reduced with a significant reduction in WT%(P<0.01);the co-localization of α-SMA and PCNA was significantly reduced;the number of autolysosomes decreased;the protein positive expressions of p-AMPK,LC3B,and Beclin1 in the lung tissue were significantly down-regulated(P<0.05,P<0.01),while those of p-mTOR and p62 were significantly up-regulated(P<0.01).Compared with the sildenafil group,TAPSE decreased in the low-and medium-dose FXD groups(P<0.01);the degree of pulmonary arterial intima-media thickening was increased and the WT%was higher in the low-and medium-dose FXD groups(P<0.01);the protein positive expression of p-AMPK was elevated in the low-and medium-dose FXD groups(P<0.01),while that was reduced in the high-dose FXD group(P<0.05);the protein positive expression of p-mTOR was decreased in the low-dose FXD group(P<0.01).Conclusion FXD may play a role in ameliorating hypoxic pulmonary hypertension by inhibiting autophagy through down-regulation of the AMPK/mTOR signaling pathway.
Feixin Decoctionhypoxic pulmonary hypertensionadenosine monophosphate-activated protein kinasemamm-alian target of rapamycinautophagy
万方数据
维普
