2. 海军军医大学(第二军医大学)海军特色医学中心高压氧神经内科,上海 200052
2. Department of Hyperbaric Oxygen and Neurology, Naval Medical Center, Naval Medical University (Second Military Medical University), Shanghai 200052, China
氧稳态对机体功能的维持至关重要,氧稳态破坏可导致基因表达、能量供应、血管再生及干细胞增殖分化等生物学功能的异常[1]。缺氧是机体生命活动中常见的损伤因素之一,主要指体内游离氧浓度而非绝对氧水平的降低。当机体处于病理状态时,单纯靠呼吸系统增加氧气输送往往不能满足生命活动需要,还需依靠细胞内的缺氧调节机制[2]。其中,缺氧诱导因子(hypoxia-inducible factor,HIF)氧感知信号通路在细胞感知与适应氧气浓度变化中发挥关键作用[3-4]。
氧浓度的波动包括低压低氧环境中间断常压吸氧、常压常氧环境中间歇性高压氧暴露等,可使细胞反复处于相对缺氧状态,进而触发通常由缺氧引发的细胞内级联反应,增强细胞对抗缺氧的能力,减轻缺氧损伤,这种现象被称为“高氧-低氧悖论”(hyperoxic-hypoxic paradox,HHP)[5]。HHP已在动物实验和临床实践中得到验证。动物实验表明,间歇性低氧刺激比持续低氧更易促进体内促红细胞生成素的生成,显著缩短提高网织红细胞计数和血细胞比容所需的时间,使机体快速启动缺氧调节机制[6]。临床实践同样发现,在高原低氧环境中往往需要数天的时间才可诱导红细胞增多,而间歇性高原环境锻炼仅需36~48 h即可达到相似效果,因此往往采用“高住低练”方案来促进机体快速适应高原低氧环境[7]。目前认为HHP与HIF氧感知信号通路密切相关[8],本文就HIF氧感知信号通路与HHP的研究进展进行综述。
1 HIF氧感知信号通路HIF作为低氧反应的主要转录因子,是由1个α亚基(HIF-α)和1个β亚基(HIF-β)构成的二聚体[4],依据α亚基的不同分为HIF-lα、HIF-2α和HIF-3α亚型,其中HIF-1α存在于人和哺乳动物细胞内,HIF-2α仅在脊椎动物谱系中的部分组织中表达(主要包括髓系细胞、肝实质、血管内皮细胞、Ⅱ型肺泡细胞和肾间质细胞等),而HIF-3α的生物学功能目前仍未完全阐明[9-10]。低氧可增加HIF-1α和HIF-2α的表达,其中HIF-1α对缺氧更灵敏,而HIF-β为组成性表达,不受氧浓度的影响[11]。由此可见,HIF-1α是HIF氧感知信号通路的核心转录因子,是低氧条件下诱导基因表达的重要调控因子。低氧可通过腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、PI3K/Akt、MAPK信号通路调控HIF通路,但不同时期、不同组织的细胞在低氧环境中可受到不同信号通路或多种信号通路调控。
研究发现,脯氨酰羟化酶(prolyl hydroxylase,PHD)可诱导HIF-1α的氧依赖性降解结构域中的脯氨酸/天冬氨酸羟基化,羟基化的HIF-1α极易与E3泛素连接酶冯希佩尔-林道(von Hippel-Lindau,VHL)蛋白结合,最终通过泛素-蛋白酶体途径快速降解[12-13]。作为转录因子,HIF-1α可调控下游多达200种目的基因的表达,在机体糖代谢、细胞增殖分化、细胞凋亡、血管生长和血管重塑等生物学过程的调节方面发挥重要甚至核心的作用[9]。在缺氧条件下,HIF-1α可诱导内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)表达上调,促进一氧化氮合成,改善血管舒张功能,维持组织灌注[14];HIF-1α可诱导血管内皮生长因子(vascular endothelial growth factor,VEGF)表达上调,促进微血管新生[15-16];HIF-1α可通过上调促红细胞生成素,促进红细胞分化发育,最终增加血液携氧能力[16];在免疫调控和细胞增殖等方面,HIF-1α可通过调节免疫细胞活性及炎症通路中关键信号分子(如NF-κB、基质金属蛋白酶9等)的表达,增强细胞的抗炎症损伤能力[17];HIF-1α还可通过活化AMPK及沉默信息调节因子2(silencing information regulator 2,SIRT2)激酶,促进线粒体的生物发生,增强细胞的抗缺氧损伤能力[18]。
2 氧稳态与HIF氧感知信号通路在正常生理条件下,进入细胞内的氧气有80%被线粒体利用,与线粒体内膜上的电子呼吸链末端复合体Ⅳ的电子相结合,促进ATP形成,为细胞的生命活动提供能量来源。在常氧条件下,HIF中的脯氨酸残基在细胞内PHD作用下发生羟基化,进而与VHL特异性结合,导致HIF降解;而在缺氧条件下,进入细胞内的氧气显著不足,使得PHD活性下降,HIF-1α降解减少,更多的HIF-1α进入细胞核内与HIF-β结合形成二聚体,进而执行转录因子的功能。此外,缺氧还可通过以下途径促进HIF-1α转录:缺氧可抑制缺氧诱导因子抑制因子(factor-inhibiting hypoxia-inducible factor,FIH)羟基化,继而抑制HIF-1α的转录激活结构域与共激活蛋白cAMP反应元件结合蛋白的结合蛋白(cAMP-response element binding protein binding protein,CBP)、p300等结合,使HIF-1α的转录活性提高;缺氧可上调线粒体内还原型烟酰胺腺嘌呤二核苷酸(reduced nicotinamide adenine dinucleotide,NADH)浓度,抑制组蛋白去乙酰化酶,从而诱导HIF-1α的乙酰化,增强其转录活性;缺氧通过激活MAPK通路分子,如ERK、p38 MAPK等,间接提高HIF-1α的转录活性[19-20]。
研究表明不同氧环境下,线粒体内活性氧(reactive oxygen species,ROS)也可调控PHD/HIF-1α氧感知信号通路[21-22]。静息状态下约有0.2%的氧气在线粒体内膜呼吸链反应中转变为ROS。在正常生理条件下细胞内维持着一定浓度的ROS,ROS作为一种重要的信号分子参与细胞内多种信号通路的调节,对细胞功能稳态具有重要作用[23-24]。但在某些病理因素刺激下,ROS可暴发式地大量合成,使细胞发生氧化应激性损伤。在常氧条件下,进入线粒体内的氧气含量充分,ROS的合成大于清除,细胞质中游离状态的ROS可促进PHD对HIF-1α的羟基化,增强HIF-1α的快速降解,最终使HIF-1α的生物学效应下降[22-24]。而在缺氧状态下,由于进入线粒体内氧气的减少及线粒体功能的抑制,经呼吸链电子还原合成的ROS减少,而ROS的清除受到的影响较小,导致ROS生成与清除的比值降低,从而使PHD对HIF-1α的羟基化及降解减少,更多的HIF-1α进入细胞核内与HIF-β结合形成二聚体,增强细胞的抗缺氧能力[25-26]。
3 HHP与HIF氧感知信号通路目前研究认为,HHP现象的产生可能与细胞线粒体内ROS的产生和清除有关。一次高氧暴露后恢复至常氧(以下简称“单次HHP”),进入线粒体内的氧气大大增多,ROS的合成速度大于其清除速度,过多游离状态的ROS促进HIF-1α羟基化,HIF-1α与VHL蛋白结合后经泛素化-蛋白酶体途径被迅速降解,减弱HIF氧感知信号通路介导的生物学效应;反复多次高氧暴露后恢复至常氧(以下简称“反复多次HHP”),ROS还原酶(包括超氧化物歧化酶、谷胱甘肽合成酶等)的含量逐渐增多,且其半衰期明显长于ROS合成酶,从而大大增强ROS的清除能力,降低ROS生成与清除的比值,减少游离ROS含量,最终减缓HIF-1α的降解,使更多的HIF-1α进入细胞核内发挥生物学效应[5]。
由此可见,反复多次HHP可在没有缺氧的情况下产生一种相对缺氧状态,通过上调HIF氧感知信号通路增强细胞的抗缺氧损伤能力[27]。但过度增加HHP频次,细胞将始终处于氧稳态不断变换的环境中,反而不利于细胞维持正常功能,将会加剧细胞损伤。HHP的暴露频次、供氧水平及时间之间是否存在最优组合,仍需进一步研究。
4 HHP在疾病治疗中的应用目前HHP最常采用反复间歇性常压高氧或高压氧暴露来实现[8]。高压氧即在超过1个绝对大气压(absolute atmosphere,ATA)条件下吸入100%的氧气,通过增加组织内物理溶解的氧气量,增大毛细血管向线粒体扩散的氧浓度梯度。随着研究经验的积累,高压氧治疗方案逐渐完善,即采用较低高压氧氧分压(通常低于2.4 ATA)、每天暴露60~90 min(每20~30 min间歇换为空气1~2 min)、持续1~6周,研究结果证实该方案可显著改善疾病进程,同时减少不良反应的发生[28-29]。
反复间歇性高压氧治疗可诱导多个受损器官(如大脑、胃肠道和肝脏)及干细胞内HIF-1α表达,有利于维持能量代谢、血管生成和细胞存活等生命活动[8]。研究表明高压氧可通过促进大脑皮质及海马组织内HIF-1α的表达对大鼠急性脑梗死起到治疗作用,其中每天给予大鼠2.5 ATA的高压氧暴露60 min、隔日1次、反复暴露3次的方案HIF-1α表达升高最显著,对脑组织的保护作用最强[30]。高压氧还可作为一种预处理保护措施,通过预先调节HIF-1α表达及其相关靶基因发挥抑制炎症损伤、增强抗氧化能力、抑制凋亡等有益作用[31]。另有研究发现高压氧治疗还可减少脑出血灶周围组织中HIF-1α的过量表达,逆转脑组织的缺氧损伤,且其干预时间以脑出血后6 h最佳[32]。上述研究表明,高压氧暴露治疗的效果与暴露方案(包括压力、时间、频率和干预时机等)有关,需要进一步研究HIF-1α蛋白表达量与不同高压氧暴露方案的剂量-反应曲线及相关性,以最大限度地发挥高压氧的保护作用。
高压氧治疗对HIF-1α的作用机制包括:(1)类似于反复多次HHP调节机制,反复高压氧暴露可促进超氧化物歧化酶、线粒体转录因子、解偶联蛋白2等抗氧化蛋白的表达,且ROS清除半衰期远短于其生成半衰期,导致游离的ROS减少,从而抑制PHD对HIF-1α的羟基化及降解,使得更多的HIF-1α进入细胞核内发挥生物学效应[5, 33];(2)高压氧通过促进硫氧还蛋白1(thioredoxin 1,Trx1)的合成提高HIF-1α的转录活性[29];(3)高压氧还可诱导PI3K/Akt、Ras/Raf-1/MAPK/ERK等的磷酸化进一步上调HIF-1α的转录与表达[34]。另有证据表明,单次高压氧疗配合外源抗氧化剂可在减少ROS含量的同时增加HIF-1α的蛋白表达量,从而改善组织损伤[35]。此外,也有研究发现单次高压氧治疗可改善局灶性脑梗死大鼠的神经功能,其机制可能与稳定线粒体膜电位、抑制凋亡诱导因子由线粒体向细胞核转移有关,提示高压氧可通过非HIF氧感知信号通路机制发挥保护作用[36]。
5 小结HIF氧感知信号通路是缺氧环境下细胞内的重要调节机制。相对缺氧是HIF基因表达的有效诱导因素,细胞氧浓度的相对变化(如常氧至缺氧、高氧至常氧的转变)可不引起细胞缺氧性损伤,即HHP。反复多次HHP可降低ROS生成与清除的比值,减少HIF-1α降解,同时提高HIF-1α的转录活性,增强细胞的抗缺氧损伤能力。基于此,反复间歇性高压氧暴露已被用于缺血缺氧性疾病的治疗并被证实可适度上调HIF-1α的表达,采用反复间歇性高压氧暴露预处理可增强组织的缺氧耐受性。然而,HIF-1α的表达与HHP暴露方案(包括压力、时间、频率和干预时机等)的剂量-反应曲线及相关性仍未完全明确,需要进一步研究是否存在最佳的HHP暴露方案。
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