Triiodothyronine prevents tissue hypoxia in experimental sepsis: potential therapeutic implications,Intensive Care Medicine Experimental

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Triiodothyronine prevents tissue hypoxia in experimental sepsis: potential therapeutic implications
Intensive Care Medicine Experimental ( IF 2.8 ) Pub Date : 2021-04-09 , DOI: 10.1186/s40635-021-00382-y
Iordanis S. Mourouzis , Athanasios I. Lourbopoulos , Athanasios G. Trikas , Ioulia K. Tseti , Constantinos I. Pantos

Tissue hypoxia occurs frequently in sepsis even after apparent restoration of stable systemic hemodynamics (macro- to micro-circulation uncoupling). Low cellular oxygen content results in neo-vessel formation with abnormal vasomotor response, increased vascular permeability and thrombosis (pathologic angiogenesis) [1] and ultimately in organ failure. Thyroid hormone (TH) which is critical regulator of organ maturation, physiologic angiogenesis and mitochondrial biogenesis can adapt heart and other organs to hypoxia during development and after tissue injury later in adult life via regulation of p38 MAPK and Akt signaling pathways [2]. Along this line, the present study explored the potential of triiodothyronine (T3) to prevent tissue hypoxia which occurs early in experimental sepsis despite cardiac hemodynamics being preserved.

The protocol was approved by the Institutional Animal Care and Use Committee of Medical School, National and Kapodistrian University of Athens. Sepsis was induced in adult male 10- to 12-week-old C57BL/6 N mice by ligation distal to the ileocecal valve (25% of total cecum length) and perforation by a single 21G puncture (CLP). Animals were treated with a single dose of either vehicle (n = 8, placebo group) or T3 (n = 8, 0.3 μg/g, T3 group) intraperitoneally immediately after surgery. Naive animals were used as control (n = 9, naive group). Animals were killed 18 h after the CLP procedure. Lactate was measured with L-lactate assay kit in serum (Sigma-Aldrich, MAK329). Cardiac and liver hypoxia at cellular level was detected using Hypoxyprobe™ Plus kit (pimonidazole hydrochloride, PMZ) on frozen, 4% paraformaldehyde fixed tissues. PMZ was administered intravenously 2 h before the killing at a dosage of 60 mg/kg. PMZ is reductively activated in hypoxic cells (pO2 < 10 mm Hg) and forms stable adducts (sulphydryl) groups in proteins and amino acids. A specific antibody (FITC-MAb1, 1:200, overnight, 4 °C) that binds to these adducts was used combined with a chromogenic anti-FITC secondary antibody (1:200, 1 h, RT) and allowed detection by immunoperoxidase staining. Captured microscopy images were analyzed with ImageJ by automated demarcation of the PMZ-positive compared to the PMZ-negative area. Cardiac performance was assessed by echocardiography. Cardiac output (ml/min) was 14.7 (SEM, 1.0), 12.1(0.7) and 14(1.0) and heart rate (beats/min) 444(23), 439(16) and 427(9) for naïve, placebo and T3, respectively (p = ns). CLP resulted in increased lactate levels and cardiac and liver hypoxia at cellular level (PMZ staining) in placebo, but not in T3-treated group (Fig. 1).

TH signaling seems to be crucial in the response to lung injury in experimental sepsis and ventilator-induced trauma [3]. In addition, the present study demonstrated that T3 treatment can prevent tissue hypoxia in cardiac and liver samples which occurs early in experimental sepsis (within 18 h) before cardiac output is impaired. PMZ staining was used to detect tissue pO2 < 10 mmHg. Oxygen below this level results in activation of HIF1α-dependent regulatory mechanisms which promote pathologic angiogenesis, changes in immune response and determine sepsis-induced injury progression [4]. T3 treatment was also shown to significantly lower circulating lactate levels probably due to the prevention of tissue hypoxia. However, favorable actions of T3 on cellular metabolism may also account for this effect. T3 can improve coupling of glycolysis to glucose oxidation and decrease H⁺ production via its action on pyruvate dehydrogenase activity (PDH) [5]. PDH is found to be suppressed during sepsis [6]. This experimental evidence may be of therapeutic relevance, particularly for COVID-19 therapy where tissue hypoxia prevails [7]. Triiodothyronine has previously been administered in dopamine-dependent shock to support hemodynamics [8]. More recently, the efficacy and safety of the use of triiodothyronine has been investigated in patients with anterior STEMI undergoing angioplasty (ThyRepair trial EudraCT: 2016-000631-40). This study has been successfully completed without major safety issues [9, Suppl.Material]. Accordingly, a phase II randomized double-blind placebo-controlled study is underway to demonstrate the safety and efficacy of T3 using the same dose in critically ill COVID-19 patients (Thy-Support study, NCT04348513, EudraCT: 2020-001623-13) [9].

Raw data and datasets used and/or analyzed during the current study are available from the corresponding author on request.

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We would like to thank Aggelos Pavlopoulos, Nefeli Zerva and Despoina Papassava for their technical support during experimentation.

This project was supported by a grant from Uni-Pharma Pharmaceutical Company. The study sponsor had no involvement in study design, collection, analysis, and interpretation of data.

Affiliations

Department of Pharmacology, National and Kapodistrian University of Athens, 75 MikrasAsias Ave., Goudi, 11527, Athens, Greece
Iordanis S. Mourouzis, Athanasios I. Lourbopoulos, Athanasios G. Trikas, Ioulia K. Tseti & Constantinos I. Pantos
Institute for Stroke and Dementia Research (ISD), University of Munich Medical Center, Munich, Germany
Athanasios I. Lourbopoulos
Department of Neurointensive Care, Schoen Klinik Bad Aibling, Kolbermoorerstrasse 72 Bad Aibling, Bayern, Germany
Athanasios I. Lourbopoulos

Authors

Iordanis S. MourouzisView author publications
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Athanasios I. LourbopoulosView author publications
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Constantinos I. PantosView author publications
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Contributions

I.M.: concept, experimentation, data gathering, interpretation and revision of the manuscript. A.L.: experimentation, data gathering, data analysis and revision of the manuscript. A.T.: data analysis, interpretation and revision of the manuscript. I.T.: concept and revision of the manuscript. C.P.: concept, study design, data gathering, interpretation and drafting of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Constantinos I. Pantos.

Ethics approval and consent to participate

The protocol was approved by the Institutional Animal Care and Use Committee of Medical School, National and Kapodistrian University of Athens. All animal experiments conformed to the revised Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council "Guide for the Care and Use of Laboratory Animals" National Academy Press, Washington, D.C. 1996.

Consent for publication

The work described has not been published before and is not under consideration for publication anywhere else. The submitted work is original and has been approved for publication by all co-authors.

Competing interests

The following pending patents are relevant to the work in this manuscript. PCT/EP2019/087056. L-triiodothyronine (T3) for use in limiting microvascular obstruction. Greek Patent Office, number of case: 22-0002577373. Composition comprising L-triiodothyronine (T3) for use in the treatment of critically ill patients with coronavirus infection. Greek Patent Office, number of case 22-0003823965. Composition comprising L-triiodothyronine (T3) for use in the treatment of tissue hypoxia. IT, managing director of Uni-Pharma, is the sponsor of these patents. CP and IM are the inventors and hold royalties in relation to these patents.

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Mourouzis, I.S., Lourbopoulos, A.I., Trikas, A.G. et al. Triiodothyronine prevents tissue hypoxia in experimental sepsis: potential therapeutic implications. ICMx 9, 17 (2021). https://doi.org/10.1186/s40635-021-00382-y

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Received: 18 January 2021
Accepted: 11 March 2021
Published: 09 April 2021
DOI: https://doi.org/10.1186/s40635-021-00382-y

中文翻译：

Triiodothyronine预防实验性败血症中的组织缺氧：潜在的治疗意义

即使在明显恢复稳定的系统血流动力学（宏循环至微循环解偶联）后，脓毒症中也经常发生组织缺氧。低的细胞氧含量导致新生血管形成，具有异常的血管舒缩反应，增加的血管通透性和血栓形成（病理性血管生成）[1]，并最终导致器官衰竭。甲状腺激素（TH）是器官成熟，生理性血管生成和线粒体生物发生的关键调节器，可以通过调节p38 MAPK和Akt信号通路使心脏和其他器官在发育过程中以及成年后的组织损伤后适应缺氧[2]。沿着这一思路，本研究探索了三碘甲腺嘌呤（T3）预防组织缺氧的潜力，尽管保留了心脏血液动力学，但组织缺氧发生在实验性脓毒症的早期。

该方案已由雅典国立和卡波迪斯安大学医学院的机构动物护理和使用委员会批准。通过结扎回盲肠瓣远端（盲肠总长度的25％）并通过单次21G穿刺穿孔，在成年雄性10至12周大的C57BL / 6 N小鼠中诱发败血症。手术后立即腹腔内用单剂量媒介物（n = 8，安慰剂组）或T3（n = 8，0.3μg/ g，T3组）治疗动物。幼稚的动物被用作对照（n = 9，天真小组）。CLP程序后18小时将动物处死。用血清中的L-乳酸盐测定试剂盒（Sigma-Aldrich，MAK329）测量乳酸盐。使用Hypoxyprobe™Plus试剂盒（盐酸吡莫尼唑，PMZ）在4％低聚甲醛固定的冷冻组织上检测到心脏和肝脏的细胞缺氧。在杀死前2小时静脉内给予PMZ，剂量为60mg / kg。PMZ在低氧细胞（pO2 <10 mm Hg）中被还原活化，并在蛋白质和氨基酸中形成稳定的加合物（巯基）。与这些加合物结合的特异性抗体（FITC-MAb1，1：200，过夜，4°C）与发色抗FITC二抗（1：200，1 h，RT）结合使用，并通过免疫过氧化物酶染色进行检测。与PMZ阴性区域相比，通过自动划定PMZ阳性区域，使用ImageJ分析捕获的显微镜图像。通过超声心动图评估心脏性能。天真的，安慰剂的心输出量（ml / min）是14.7（SEM，1.0），12.1（0.7）和14（1.0），心率（beats / min）444（23），439（16）和427（9）和T3分别（p = ns）。CLP导致安慰剂中细胞水平的乳酸水平升高以及心脏和肝脏缺氧（PMZ染色），但在T3治疗组中则没有（图1）。

TH信号似乎在实验性败血症和呼吸机诱发的创伤对肺部损伤的反应中至关重要[3]。此外，本研究表明，T3处理可预防心脏和肝脏样本中的组织缺氧，这种缺氧发生在实验性败血症的早期（18小时内），而心输出量受损。PMZ染色用于检测组织pO2 <10 mmHg。低于此水平的氧气会激活HIF1α依赖性调节机制，从而促进病理性血管生成，改变免疫反应并确定败血症诱导的损伤进展[4]。还表明，T3处理可显着降低循环乳酸盐水平，这可能是由于预防了组织缺氧。但是，T3对细胞代谢的有利作用也可以解释这种作用。⁺通过其对丙酮酸脱氢酶活性（PDH）的作用来生产[5]。发现败血症期间PDH被抑制[6]。该实验证据可能具有治疗意义，特别是对于组织缺氧普遍存在的COVID-19治疗[7]。先前已在多巴胺依赖性休克中给予三碘甲状腺素以支持血流动力学[8]。最近，已对三碘甲甲状腺氨酸在行血管成形术的前STEMI患者中使用的有效性和安全性进行了研究（ThyRepair试验EudraCT：2016-000631-40）。这项研究已成功完成，没有重大安全问题[9，补充材料]。因此，正在进行一项II期随机双盲安慰剂对照研究，以证明在重症COVID-19患者中使用相同剂量的T3的安全性和有效性（Thy-Support研究，NCT04348513，

当前研究中使用和/或分析的原始数据和数据集可应要求从相应的作者处获得。

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下载参考

我们要感谢Aggelos Pavlopoulos，Nefeli Zerva和Despoina Papassava在实验过程中提供的技术支持。

该项目得到了Uni-Pharma Pharmaceutical Company的资助。研究发起人没有参与研究设计，数据收集，分析和解释。

隶属关系

雅典国立和卡波迪斯式大学药理学系，希腊古迪MikrasAsias Ave. 75，11527，希腊雅典
Iordanis S. Mourouzis，Athanasios I. Lourbopoulos，Athanasios G. Trikas，Ioulia K. Tseti和Constantinos I. Pantos
慕尼黑大学医学中心卒中和痴呆症研究所（ISD），德国慕尼黑
Athanasios I. Lourbopoulos
Schoen Klinik Bad Aibling神经重症监护系，Kolbermoorerstrasse 72 Bad Aibling，德国拜仁
Athanasios I. Lourbopoulos

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会费

IM：手稿的概念，实验，数据收集，解释和修订。AL：实验，数据收集，数据分析和手稿修订。AT：数据分析，手稿的解释和修订。IT：手稿的概念和修订。CP：手稿的概念，研究设计，数据收集，解释和起草。所有作者阅读并认可的终稿。

通讯作者

对应于君士坦丁堡一世·潘托斯。

道德规范的批准和参与同意

该方案已由雅典国立和卡波迪斯安大学医学院的机构动物护理和使用委员会批准。所有动物实验均符合经修订的实验动物资源研究所，生命科学委员会，国家研究委员会的“实验动物的护理和使用指南”，国家科学院出版社，华盛顿特区，1996年。

同意发表

所描述的作品之前从未发表过，也没有考虑在其他任何地方发表。提交的作品是原始作品，并且已被所有共同作者批准发表。

利益争夺

以下正在申请的专利与本手稿的工作有关。PCT / EP2019 / 087056。L-triiodothyronine（T3）用于限制微血管阻塞。希腊专利局，案件编号：22-0002577373。包含L-三碘甲状腺素（T3）的组合物，用于治疗患有冠状病毒感染的重症患者。希腊专利局，案号22-0003823965。用于治疗组织缺氧的包含L-三碘甲状腺素（T3）的组合物。Uni-Pharma的常务董事IT是这些专利的赞助商。CP和IM是发明人，并拥有与这些专利有关的特许权使用费。

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