低氧通过抑制HectH9介导的肺动脉平滑肌细胞己糖激酶2泛素化和降解诱导肺血管重塑

Hypoxia induces pulmonary vascular remodeling by inhibiting HectH9-mediated ubiquitination and degradation of hexokinase 2 in pulmonary artery smooth muscle cells

    摘要
  • 摘要:
    目的  探讨己糖激酶（HK）2及其泛素化修饰在低氧诱导的大鼠肺动脉高压（PH）和肺动脉平滑肌细胞（PASMCs）过度增殖中的作用及潜在机制。
    方法  纳入 24 只雄性SD大鼠（4周龄，180～200 g），随机分为常氧对照组、低氧2 周组（常氧14 d，10% O2低氧14 d）与低氧4 周组（10% O2低氧28 d），每组 8只。右心导管法测量大鼠右心室收缩压（RVSP），计算右心室肥厚指数（RVHI）、肺小动脉血管壁面积百分比（WA%）和厚度百分比（WT%），Western-blot法检测肺动脉HK2、E3连接酶HectH9蛋白表达，比色法检测HK活性。体外以1% O2低氧培养PASMCs，Western-blot法检测HK2、HectH9、线粒体动力相关蛋白1（DRP1）、线粒体分裂因子（MFF）和线粒体分裂蛋白1（FIS1）的蛋白表达，比色法检测HK活性、葡萄糖-6-磷酸（G6P）含量，细胞计数试剂盒-8（CCK-8）法检测细胞增殖水平；免疫荧光法观察线粒体形态，免疫共沉淀分析HK2与HectH9相互作用及HK2 泛素化水平；通过小干扰RNA抑制HectH9观察HK2泛素化、HK2表达及活性、G6P含量及细胞增殖的变化。
    结果  低氧暴露2周和4周的大鼠RVSP2周（33.72±3.84），4周（50.54±5.30）比常氧（27.34±3.77）mmHg，t=2.96，10.41，均P＜0.01、RVHI2周（38.49±2.13）%，4周（42.38±2.34）% 比常氧（23.65±1.54）%，t=14.61，18.65，均P＜0.01、WA%2周（74.14±4.53）%，4周（88.63±3.81）% 比常氧（55.47±5.60）%，t=7.75，13.76，均P＜0.01及WT%2周（43.61±4.13）%，4周（58.66±3.24）% 比常氧（27.54±4.22）%，t=8.02，15.52，均P＜0.01比常氧组高，肺动脉组织中HK2蛋白表达（均P＜0.05）及HK活性比常氧组强（均P＜0.05），HectH9蛋白表达比常氧组低（均P＜0.05）。低氧48 h的PASMCs中HK2蛋白表达、HK活性、G6P含量、细胞增殖水平和线粒体碎片化程度均高于常氧对照（均P＜0.05），而HectH9表达（P＜0.05）及HK2的泛素化水平低于常氧对照。进一步在低氧条件下转染HectH9质粒后HK2的泛素化水平高于低氧PASMCs；而敲低HectH9的PASMCs中HK2蛋白表达、HK活性、G6P含量及细胞增殖水平均高于常氧对照（均P＜0.05）。
    结论  低氧通过下调HectH9表达，抑制HK2的泛素化降解，导致HK2蓄积并扰乱DRP1/MFF依赖的线粒体分裂稳态，促进PASMCs增殖和肺血管重构。

     

    Abstract:
    Objective To investigate the role and potential mechanism of hexokinase (HK) 2 and its ubiquitination in hypoxia-induced pulmonary hypertension (PH) in rat and proliferation of pulmonary artery smooth muscle cells (PASMCs).
    Methods  Twenty-four male SD rats (4 weeks old, 180–200 g) were included and randomly divided into a normoxic control group, a 2-week hypoxia group (14 days of normoxia followed by 14 days of 10% O2 hypoxia), and a 4-week hypoxia group (28 days of 10% O2 hypoxia), with 8 rats in each group. Right ventricular systolic pressure (RVSP) of rats was measured by right heart catheterization. Right ventricular hypertrophy index (RVHI), pulmonary arteriole wall area percentage (WA%) and wall thickness percentage (WT%) were calculated. Protein levels of HK2 and the E3 ubiquitin ligase HectH9 in pulmonary artery tissues were assessed by Western-blot, and the activity of HK was assessed by colorimetric method. In vitro, PASMCs were cultured under 1% O2 hypoxic conditions. Protein expression of HK2, HectH9, mitochondrial dynamin-related protein 1 (DRP1), mitochondrial fission factor (MFF) and mitochondrial fission 1 protein (FIS1) were detected by Western-blot. Colorimetric assays were employed to measure HK activity and glucose-6-phosphate (G6P) content. Cell proliferation levels were assessed using the cell counting kit 8 (CCK-8) assay. Mitochondrial morphology was observed via immunofluorescence. Co-immunoprecipitation was used to examine the interaction between HK2 and HectH9 and the level of HK2 ubiquitination. The effects of HectH9 silencing on HK2 ubiquitination, protein expression, activity, G6P content, and cell proliferation were further evaluated.
    Results Compared with the rats in the normoxic group, rats exposed to hypoxia for 2 and 4 weeks showed increased in RVSP 2-week: (33.72±3.84) mmHg, 4-week: (50.54±5.30) mmHg vs normoxia: (27.34±3.77) mmHg; t=2.96, 10.41, both P＜0.05, RVHI 2-week: (38.49±2.13)%, 4-week: (42.38±2.34)% vs normoxia: (23.65±1.54)%, t=14.61, 18.65, both P＜0.05, WA% 2-week: (74.14±4.53)%, 4-week: (88.63±3.81)% vs normoxia: (55.47±5.60)%, t=7.75, 13.76, both P＜0.05, and WT% 2-week: (43.61±4.13)%, 4-week: (58.66±3.24)% vs normoxia: (27.54±4.22)%, t2w=8.02, 15.52, both P＜0.05, accompanied by upregulated HK2 protein expression and HK activity, and downregulated HectH9 expression in pulmonary arteries (all P＜0.05). The expression of HK2 protein, HK activity, G6P content, cell proliferation level and mitochondrial fragmentation degree in PASMCs treated with hypoxia for 48 hours were all higher than those in the normoxia controls (all P＜0.05), whereas the HectH9 expression (P＜0.05) and HK2 ubiquitination levels were lower. Overexpression of HectH9 via plasmid transfection under hypoxia resulted in higher HK2 ubiquitination levels than hypoxic PASMCs; whereas PASMCs with HectH9 knockdown showed higher HK2 protein expression, enzymatic activity , G6P content, and proliferation levels compared to normoxic controls (all P＜0.05).
    Conclusion Hypoxia promotes the accumulation of HK2 by downregulating HectH9 expression and inhibiting HK2 ubiquitination and degradation, thereby disturbing DRP1/MFF-mediated mitochondrial fission homeostasis and promoting PASMC proliferation and pulmonary vascular remodeling.

     

    • HTML全文
    • 参考文献(36)
    • 相关文章
    • 施引文献
  • 资源附件(0)

/

返回文章
返回