Impaired AMPK Activity Drives Age-Associated Acute Lung Injury after Hemorrhage
Trauma is a significant cause of mortality worldwide. Hemorrhage is responsible for 30–40% of trauma mortality, and hemorrhagic shock is the second leading cause of death among the injured (1). Acute lung injury after hemorrhage is associated with an overwhelming systemic inflammatory response characterized by elevated systemic cytokine levels and activation of the innate immune system (2). In hemorrhagic shock, advanced age has been associated with more severe organ failure and an increased number of days spent on the ventilator and in the intensive care unit (3). It is also well known that older individuals (>65 yr old) have greater morbidity and mortality with all etiologies of acute lung injury (4). However, the mechanisms linking age to worse outcomes in acute lung injury are poorly understood.
创伤是全世界死亡率的一个重要原因。出血是30-40% 的创伤死亡原因，失血性休克是第二大死亡原因(1)。出血后的急性肺损伤与全身炎症反应有关，拥有属性因子水平升高，先天免疫系统激活。在失血性休克中，高龄患者常伴有更严重的器官衰竭，并且使用呼吸机和重症监护室的天数增加。众所周知，老年人(> 65岁)具有更高的发病率和死亡率与所有病因急性肺损伤(4)。然而，在急性肺损伤中，年龄与恶化结果之间的联系机制还知之甚少。
In this issue of the Journal, Klingbeil and colleagues (pp. 585–596) (5) explore the role of the 5′ adenosine monophosphate–activated protein kinase (AMPK) in age-related acute lung injury after hemorrhagic shock. AMPK is a sensor of cellular energy status that responds to decreases in energy (increased AMP/ATP or ADP/ATP ratios) by stimulating mitochondrial catabolic activity. Aging has been shown to decrease AMPK activity and signaling, which results in reduced mitochondrial biogenesis and function (6). These investigators conducted an in vivo/in vitro analysis of whether the predominant catalytic AMPK subunit in the lung (AMPKα1) was associated with age-related impaired mitochondrial biogenesis and autophagy in response to hemorrhage-induced lung injury.
在这一期的杂志上，Klingbeil 和他的同事们(第585-596页)(5)探讨了5′单磷酸腺苷激活蛋白激酶(AMPK)在年龄相关的失血性休克后急性肺损伤中的作用。AMPK 是一种细胞能量状态的传感器，通过刺激线粒体的分解代谢活性来反应能量的减少(增加 AMP/ATP 或 ADP/ATP 比率)。衰老会降低 AMPK 的活性和信号传导，从而导致线粒体的生物发生和功能减退。这些研究人员进行了一项体内/体外的分析，研究肺中主要的催化 AMPK 亚基(AMPKα1)是否与年龄相关的线粒体生物合成和自噬相关，以应对出血引起的肺损伤。
Arterial hemorrhage to a fixed blood pressure (from 60 to 30 mm Hg) was performed equally in young (3–5 mo; approximately 10–17-yr-old human equivalent) and older (9–12 mo; 30–40-yr-old approximate human equivalent) wild-type mice, followed by fluid/blood resuscitation, then treatment ± an AMPK activator. Hemorrhage induced convincing acute lung injury, as assessed by increases in bronchoalveolar lavage (BAL) total protein, BAL inflammatory cells, wet/dry lung weights, histopathologic lung injury scores, and myeloperoxidase activity in lung homogenates, along with increases in systemic cytokine levels. Overall, there was a more severe lung injury response in the older mice compared with that in younger mice. Furthermore, there was a greater percentage of structurally altered mitochondria in older mice.
动脉出血导致固定血压(60-30毫米汞柱)在年轻(3-5个月; 大约10-17岁相当于人类年龄)和老年(9-12个月; 30-40岁相当于人类年龄)的野生型小鼠中进行，随后进行液体/血液复苏，然后治疗 ± AMPK 激活剂。通过增加支气管肺泡灌洗总蛋白、 BAL 炎症细胞、湿/干肺重量、组织病理学肺损伤评分、肺匀浆中髓过氧化物酶活性以及全身细胞因子水平来评估出血诱发的急性肺损伤。总的来说，与年轻老鼠相比，年长老鼠的肺损伤反应更为严重。此外，在老年鼠体内有更大比例的结构改变的线粒体。
Treatment with a pharmacologic AMPK activator (AICAR, an analog of AMP) after resuscitation had a restorative normotensive effect selectively in young mice. Whereas the specific AICAR treatment effects on hemorrhage-induced lung injury and cytokines were similarly beneficial in younger and older mice, the resultant injury phenotype remained more deranged in older mice. Concordant with the beneficial effects of AMPK activation, mice lacking AMPK 1α (AMPK KO) had a greater inflammatory response to hemorrhage (myeloperoxidase, lung pathologic changes, and plasma IL-1β and TNF-α levels) compared with wild-type mice, and AMPK KO mice had no response to AICAR. Taken together, these data demonstrate a beneficial effect of AMPK activation in younger mice, greater than that in older mice, and suggest that the AICAR’s beneficial effects were a result of AMPK activation, rather than off-target effects. This work extends the similar prior findings of the role of AMPK in LPS-induced lung injury (7, 8) to hemorrhage-induced lung injury.
复苏后使用药物学 AMPK 激活剂(AICAR，AMP 的类似物)治疗对年轻小鼠有选择性恢复正常血压的作用。AICAR 对出血性肺损伤和细胞因子的特异性治疗作用在年轻和老年小鼠中同样有效，而在老年小鼠中，由此产生的损伤表型仍然更加混乱。AMPK 1α (AMPK 1α，AMPK KO)缺失的小鼠对出血(髓过氧化物酶、肺组织病理改变、血浆 il-1β 和 tnf-α 水平)的炎症反应明显高于野生型小鼠，AMPK 缺失的小鼠对 AICAR 没有反应。综上所述，这些数据显示了 AMPK 激活在年轻小鼠中的有益作用，大于老年小鼠，并表明 AICAR 的有益作用是 AMPK 激活的结果，而不是靶外效应。这项工作扩展了 AMPK 在 lps 诱导的肺损伤(7,8)和出血诱导的肺损伤中的作用的相似的先前研究结果。
To begin to identify the mechanism of AMPK’s age-dependent effect on lung injury, the putative role of AMPK in both mitochondrial biogenesis and autophagy were measured. Prior work had demonstrated that energy depletion-induced activation of AMPK (by phosphorylation) is impaired with aging (6). This study extends this impaired response to alveolar epithelial cells in mice. AMPK activation is linked to mitochondrial biogenesis through its ability to activate peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1-alpha (PGC-1α) and the deactylase sirtuin-1 (SIRT1) (9). PGC-1α is deacetylated by SIRT1, thereby increasing its activity in the nucleus and inducing a mitochondrial biogenetic gene expression program. These investigators demonstrate that AMPK phosphorylation was impaired in primary alveolar epithelial cells of older mice. Further, PGC-1α and SIRT1 nuclear localization was decreased after hemorrhage and restored with AICAR, but to a lesser extent in older mice. This suggests that the observed impairment of AMPK phosphorylation drives the increase in structurally altered mitochondria in aged mice upon hemorrhage-induced lung injury. AICAR had a modest effect in restoring mitochondrial structure in both groups, with a greater effect in younger mice, suggesting the AMPK-initiated pathway is refractory to activation in older mice.
为了确定 AMPK 对肺损伤的年龄依赖性作用机制，我们测定了 AMPK 在线粒体生物发生和自噬中的推测作用。先前的工作已经证明，能量耗竭引起的活化 AMPK (通过磷酸化)是受损的老化(6)。这项研究扩展了小鼠肺泡上皮细胞的这种受损反应。AMPK 通过激活 PPAR-γ 辅激活过氧化物酶体增殖物活化受体1-alpha (pgc-1α)和去酰化酶 sirtuin-1(SIRT1)而与线粒体生物发生相关。Pgc-1α 被 SIRT1去乙酰化，从而增加了其在细胞核中的活性，并诱导了线粒体生物遗传基因表达程序。这些研究表明，AMPK 磷酸化受损的原发性肺泡上皮细胞的老年小鼠。此外，出血后 pgc-1α 和 SIRT1的核定位减少，AICAR 恢复，但在老年小鼠中程度较轻。这表明，AMPK 磷酸化损伤促进了老年小鼠肺损伤后结构改变的线粒体的增加。在两组小鼠中，AICAR 对恢复线粒体结构都有一定的作用，而在年轻小鼠中的作用更大，这表明 ampk 启动的途径在老年小鼠中是不易激活的。
Although lung injury increased the autophagy marker protein LC3-B in both young and older mice, lung injury, age, and AICAR treatment did not have an effect on the number of autophagic vesicles. These apparently contradictory results have yet to be resolved. Similarly, some of the beneficial effects of AICAR on lung injury could be a result of increased blood flow selectively in young mice on AICAR treatment. AMPK has pleotropic effects in multiple cell types that play a role in lung injury, so future studies could be directed toward identifying the specific cause, cell type, and pathway by which AMPK activation mediates lung injury protection.
虽然肺损伤增加了年轻和老年小鼠的自噬标记蛋白 LC3-B，但是肺损伤、年龄和 AICAR 治疗对自噬小泡的数量没有影响。这些显然相互矛盾的结果尚未得到解决。类似地，AICAR 对肺损伤的一些有益作用可能是在 AICAR 治疗中年轻小鼠选择性增加血流量的结果。AMPK 在多种细胞类型中具有多效性，这些细胞类型在肺损伤中起着重要作用，因此未来的研究可能会着眼于确定 AMPK 激活介导肺损伤保护的特定原因、细胞类型和途径。
Decreased AMPK activation capacity with aging has been attributed to reduced AMPK phosphorylation, but the exact mechanisms of the reduced phosphorylation have not been elucidated (6). Concordant with the current study, the downstream effects of impaired AMPK activation include impaired longevity/autophagy/mitochondrial biogenesis pathways (mTOR, FoxO, PCG-1α, SIRT1), impaired stress resistance pathways (SIRT1, HIF1), and increased inflammatory/immune responses (NF-κB) (10). The reduction of SIRT1 activity and elevation of NF-κB in older mice in this study extends these mechanistic pathways to lung injury after hemorrhage.
随着老化 AMPK 活化能力的降低被认为是 AMPK 磷酸化水平降低所致，但其确切机制尚未阐明(6)。与目前的研究一致的是，AMPK 激活受损的下游效应包括受损的寿命/自噬/线粒体生物发生途径(mTOR，FoxO，pcg-1α，SIRT1) ，受损的应激抵抗途径(SIRT1，HIF1) ，以及增加的炎症/免疫反应(NF-κB)。本研究中老年小鼠 SIRT1活性的降低和 NF-κB 的升高扩展了出血后肺损伤的机制。
As with most key regulatory proteins, there are consequences of both impaired and overexuberant AMPK activation. For example. AMPK is dysregulated in several cancers and can promote cancer cell survival under conditions of metabolic stress, such as hypoxia and glucose deprivation (11–13). Furthermore, AMPK is involved in a diverse panoply of metabolic and physiologic processes such as lipid and glucose metabolism, protein synthesis, and redox regulation. For example, it has been shown that hypercapnia plays a role in the AMPK-dependent atrophy of skeletal muscle in patients with COPD (14). As AMPK activation is cell type process- and context-dependent, one should be cautious in considering AMPK manipulations for therapeutic intent before thorough study.
与大多数关键调节蛋白一样，AMPK 激活受损和过度激活都会产生后果。例如。AMPK 在几种癌症中表现为失调，可以促进癌细胞在代谢应激条件下的存活，如缺氧和葡萄糖剥夺(11-13)。此外，AMPK 参与多种代谢和生理过程，如脂质和葡萄糖代谢、蛋白质合成和氧化还原调节。例如，有研究表明，高碳酸血症在 COPD 患者骨骼肌 ampk 依赖性萎缩中发挥作用。由于 AMPK 激活是细胞类型的过程和情境依赖性的，在彻底研究之前，在考虑 AMPK 操作的治疗意图时应该谨慎。
In addition to acute lung injury, AMPK has been shown to play diverse roles in other inflammatory lung diseases. Metformin has been shown to suppress eosinophilic airway inflammation and reduce peribronchial fibrosis via AMPK activation in asthmatic mice (15). AMPK-induced MMP8 secretion in neutrophils has been implicated in causing lung destruction in human tuberculosis (16). AMPKα1 KO mice exposed to cigarette smoke have increased production of IL-8, leading to increased lung inflammation and emphysema (17). Finally, in experimental pulmonary hypertension, AMPK activation has been shown to be both beneficial (18) and deleterious (19). The role of aging on the AMPK-mediated pathways in the aforementioned lung diseases has yet to be evaluated, and is an exciting area for future research.
除了急性肺损伤外，AMPK 在其他炎症性肺疾病中也表现出不同的作用。二甲双胍已被证明可以抑制嗜酸性气道炎症和减少哮喘小鼠支气管周围的纤维化(15)。Ampk 诱导的 MMP8在中性粒细胞中的分泌有可能导致人类肺结核(16)的肺部破坏。AMPKα1 KO 小鼠暴露于香烟烟雾中可增加 IL-8的产生，导致肺部炎症和肺气肿(17)。最后，在实验性肺部高压中，AMPK 激活已被证明是有益的(18)和有害的(19)。老化在 ampk 介导的通路在上述肺部疾病中的作用尚未被评估，这是未来研究的一个令人兴奋的领域。
The results of this study agree with numerous other studies that demonstrate that AMPK basal activity or phosphorylation is impaired with aging in diverse tissues, including myocardium, skeletal muscle, and endothelium. For example, the capacity for AMPK activation in skeletal muscle in response to exercise was decreased in older rats. As in this study, AICAR administration only increased the activity of AMPK in younger rats (20). In contrast to studies involving LPS-induced lung injury (21), AICAR administration in this study did not affect NF-κB or TNF-α levels after hemorrhage-induced lung injury.
这项研究的结果与许多其他的研究一致，这些研究表明，随着年龄的增长，包括心肌、骨骼肌和内皮细胞在内的各种组织中 AMPK 基础活性或磷酸化受损。例如，老年大鼠运动后骨骼肌中 AMPK 激活的能力下降。在这项研究中，AICAR 只增加了年轻大鼠(20) AMPK 的活性。与 lps 诱导的肺损伤(21例)相比，本研究中 AICAR 对出血性肺损伤后 NF-κB 和 tnf-α 水平没有影响。
In summary, for the first time, the authors show that hemorrhage-induced lung injury is more severe in older mice than younger mice. Further, the level of AMPK activation in response to AICAR and its beneficial effects are impaired in older mice. The age-dependent effect of AMPK activation on selected measures of lung injury suggests that AMPK is an important determinant of the age-dependent effect of hemorrhage-induced lung injury (Figure 1). It remains to be seen whether AMPK activation is regulatable in older humans. Studies directed into the mechanism by which AMPK activation is impaired in aging would be potentially therapeutically transforming. This work adds to the literature showing that AICAR treatment improves LPS-induced lung injury, and cardiovascular and gastrointestinal injury and survival after hemorrhagic shock in young rats/pigs (22–24). As aspirin, metformin, and statins have all been shown to activate AMPK, the potential for effective therapeutic interventions in hemorrhage-induced lung injury may be on the horizon.
总之，作者首次表明，出血引起的肺损伤在老年小鼠中比年轻小鼠更为严重。此外，对 AICAR 反应的 AMPK 激活水平及其有益作用在老年小鼠中受损。AMPK 激活对肺损伤的选择性指标的年龄依赖性效应表明，AMPK 是出血性肺损伤年龄依赖性效应的一个重要决定因素(图1)。AMPK 激活是否在老年人中是可调节的还有待观察。针对 AMPK 激活在衰老过程中受损的机制的研究可能是潜在的治疗转化。这项工作增加了文献表明 AICAR 治疗可以改善 lps 诱导的肺损伤、心血管和胃肠道损伤以及幼年大鼠/猪失血性休克后的存活(22-24)。由于阿司匹林、二甲双胍和他汀类药物都能激活 AMPK，因此对出血性肺损伤进行有效治疗的可能性即将出现。
Figure 1.Working model for the role of 5′ adenosine monophosphate–activated protein kinase (AMPK) in aging and hemorrhage-induced lung injury. Hemorrhage and aging conspire to reduce AMPK phosphorylation, leading to the increased inflammation and impaired mitochondrial regulation that drives lung injury. NF-κB, nuclear factor-κB; PCG-1α, peroxisome proliferator-activated receptor-γ coactivator 1-α; SIRT1, sirtuin 1.
图1。5′单磷酸腺苷激活蛋白激酶(AMPK)在衰老和出血性肺损伤中作用的工作模型。出血和衰老合谋减少 AMPK 磷酸化，导致增加炎症和损害线粒体调节，驱动肺损伤。NF-κB、核因子 -κb、 pcg-1α、过氧化物酶体增殖物激活受体 γ 辅激活因子1-α、 SIRT1、 sirtuin 1。