素特异性蛋白酶10在低氧性肺动脉高压中的表达及对USP10-AMPK信号通路的调控作用

目的 研究泛素特异性蛋白酶10（UPS10）在低氧性肺动脉高压（PAH）中表达的意义及对USP10-单磷酸腺苷活化蛋白激酶（AMPK）信号通路的调控作用。方法40只大鼠随机分为4组，空载组与沉默组分别经气管滴入USP10-lentivirus、NC-lentivirus，对照组与模型组滴入等量生理盐水。3 d后除对照组外均建立低氧PAH模型。以PowerLab压力记录分析系统检测各组平均肺动脉压（MPAP）、右心室收缩压（RVSP），计算肺血管管壁相对厚度指数（RTI）、右心肥大指数（RVHI），对比各组MPAP、RVSP、RTI、RVHI。实时荧光定量PCR技术检测USP10、AMPK、磷脂酰肌醇3激酶（PI3K）、蛋白激酶B（Akt）、内皮细胞型一氧化氮合成酶（eNOS）mRNA，比较各组USP10、AMPK、PI3K、Akt、eNOS mRNA相对表达量。蛋白质印迹法（Weston blot）检测USP10、AMPK、PI3K、Akt、eNOS蛋白及AMPK、PI3K、Akt、eNOS蛋白磷酸化表达情况，对比USP10蛋白相对表达量及p-AMPK/AMPK、p-PI3K/PI3K、p-Akt/Akt、p-eNOS/eNOS蛋白比值。结果沉默组、模型组与空载组大鼠均出现精神状态变差，活动量减少，毛发暗淡，口唇、眼眶发紫，沉默组更为严重。肺动脉血管组织HE染色观察，沉默组、模型组与空载组管壁增厚、管腔变窄，伴有平滑肌和弹力纤维层增厚，沉默组变化更为明显。与对照组比较，其余3组的MPAP、RVSP、RTI、RVHI均增加（P<0.05），沉默组均大于模型组与空载组（P<0.05）；与对照组比较，其余3组的USP10 mRNA、蛋白相对表达量及p-AMPK/AMPK、p-PI3K/PI3K、p-Akt/Akt、p-eNOS/eNOS蛋白比值均下降（P<0.05），沉默组均低于模型组与空载组（P<0.05）。结论USP10在低氧性PAH大鼠肺组织中表达低于正常肺组织，且其在肺组织中的表达下调刺激了PAH的发展，推测可能与抑制AMPK/PI3K/Akt/eNOS信号通路有关

Significance of ubiquitin-specific protease 10 expression in hypoxic pulmonary hypertension and regulation of USP10-AMPK signaling pathway

Objective To study the expression of ubiquitin-specific protease 10 (UPS10) in hypoxic pulmonary hypertension (PAH) and its regulation on the signal pathway of USP10-AMP-activated protein kinase (AMPK). Methods Forty rats were randomly divided into 4 groups. USP10-lentivirus and NC-lentivirus were intratracheally dripped into the no-load group and the silent group, respectively; while control group and model group were intratracheally dripped with the same amount of saline. After 3 days, the hypoxic PAH model was established except for the control group. The general condition of rats and the pathological changes of pulmonary arteries were observed. Mean pulmonary artery pressure (MPAP) and right ventricular systolic pressure (RVSP) were measured by PowerLab pressure recording analysis system, and the relative thickness index (RTI) and right ventricular hypertrophy index (RVHI) were calculated. MPAP, RVSP, RTI and RVHI were compared. Real-time fluorescence quantitative PCR was used to assess the relative expression of USP10, AMPK, phosphatidylinositol 3 kinase (P13K), protein kinase B (Akt) and endothelialnitric oxide synthase (eNOS). Western blot analysis of USP10, AMPK, PI3K, Akt, eNOS protein and AMPK, PI3K, Akt, eNOS protein phosphorylation were performed. The relative expression of USP10 and the ratio of p-AMPK/AMPK, p-PI3K/PI3K, p-Akt/Akt and p-eNOS/eNOS was compared. Results The silent group, model group and no-load group all showed mental state deterioration, decreased activity, dim hair, purple lips and orbitals, especially in the silent group. HE staining of pulmonary artery tissue showed that in silent group, model group and no-load group, the wall of pulmonary artery was thickened and lumen was narrowed, accompanied by smooth muscle and elastic fibrous layer thickening, and the change was more obvious in silent group. Compared with the control group, the MPAP, PVR, RTI and RVHI of the other three groups were significantly increased (P<0.05), while those in the silent group were significantly higher than those in the model group and the no-load group (P<0.05). Compared with the control group, the relative expression of USP10 and protein and the ratio of p-AMPK/AMPK, p-PI3K/PI3K, p-Akt/Akt and p-eNOS/eNOS protein in the other three groups were significantly reduced (P<0.05), while the silence group were significantly lower than those in the model group and the no-load group (P<0.05). Conclusion The expression of USP10 in lung tissues of hypoxic PAH rats is lower than that in normal lung tissues, and the down-regulation of USP10 in lung tissue stimulates the development of PAH, which may be correlated to the inhibition of AMPK/PI3K/Akt/eNOS signaling pathway.