背景

人类如何适应南极特殊环境是一项重要的研究课题。课题组从事南极考察队员医学研究20余年，并建立靶向低氧动物模型开展机制研究。南极昆仑站位于冰盖高原最高点（4087m，氧分压11.3kPa，氧含量相当于中低纬度5km），考察队员在低氧环境下出现一系列生理反应，包括心血管功能、神经内分泌激素、肠道微生物群组成等改变。

脂肪组织作为重要的代谢器官和内分泌器官，分泌的一些脂肪因子被认为是预测心血管结局的重要生物标志物。南极冰盖高原低氧环境下，与心血管功能变化密切相关的脂肪因子的变化和作用机制尚不清楚。

人类或啮齿类动物急性暴露于高原低氧环境，会出现下丘脑-垂体-肾上腺（HPA）轴和下丘脑-垂体-甲状腺（HPT）轴过度激活。肠道作为最大的代谢器官和潜在的内分泌器官，肠道微生物产生的代谢物质可能与宿主的HPA和HPT轴相互串扰。急性低氧环境下，肠道微生物-代谢物-神经内分泌相互作用机制仍不清楚。

目的

本论文旨在研究（1）高原低氧环境下，参与心血管功能调控的关键脂肪代谢因子和作用机制；（2）急性高原低氧环境下，介导肠道微生物与宿主神经内分泌激素相关性的关键代谢物。

方法

（1）在上海出发前和昆仑站考察20天两个时间点，检测中国南极冰盖高原考察队员的心肺功能、心电图、血氧饱和度、身体成分的变化，使用Milliplex蛋白多因子方法检测血浆心血管疾病风险因子和6种关键脂肪代谢因子的变化。为进一步研究脂肪代谢因子在低氧诱导心脏功能降低中的作用机制，构建靶向低氧大鼠模型。成年雄性Sprague-Dawley大鼠暴露于模拟低压低氧环境（5km），其氧分压与南极昆仑站相同，相应的对照组暴露于常氧环境，分为1和3天（急性期）、14和28天（慢性期）组。超声心动图详细检测大鼠心室结构和功能变化。ELISA检测心血管风险因子和脂肪代谢因子水平的变化。采用免疫组织化学、透射电子显微镜、qPCR和蛋白印迹等方法检测低氧大鼠心肌组织肥大、纤维化、脂质沉积、细胞凋亡、线粒体功能等病理学变化。最后，通过蛋白印迹深入研究关键脂肪代谢因子的下游信号通路在心肌组织中的变化，通过转录组测序技术进一步挖掘潜在的调控机制。

（2）雄性SD大鼠在模拟海拔5km的低压氧舱中暴露3天，对照组暴露在常氧环境下。检测血清HPA和HPT轴激素水平，分别采用16S rRNA测序和非靶向代谢组学，检测和分析粪便微生物组成和粪便/血清代谢物的变化。使用因果中介分析模型，挖掘介导肠道微生物与宿主HPA轴和HPT轴激素相互作用的潜在粪便和血清代谢物。

结果

对第31次南极冰盖高原昆仑站考察队研究发现，队员在昆仑站出现心肺功能降低、心血管风险因子升高，而脂肪代谢因子降低。肺功能检测发现，最大肺活量（VC_MAX）、用力肺活量（FVC）、一秒量（FEV₁）显著下降，而气体交换速率、用力呼吸流速显著增加。心功能检测发现，心率（HR）、血压和全身血管阻力（SVR）升高，心脏泵血和收缩功能降低。这些生理表型变化伴随血浆肌酸激酶同工酶MB（CK-MB）和血小板内皮细胞粘附分子-1（Pecam-1）增加，而血浆脂肪代谢因子瘦素（Leptin）、抵抗素（Resistin）和脂蛋白2（Lipocalin-2）显著减少。本研究第一次发现，低氧环境下血浆瘦素水平降低与心功能变化显著相关，深入研究脂肪代谢因子瘦素在心功能变化中的作用机制具有重要科学意义。

模拟海拔5km低压低氧环境下暴露14天和28天的大鼠出现明显的肺动脉高压、右心室肥大、左心室收缩和舒张功能减弱。血浆sPecam-1在低氧暴露1天、3天显著增加，CK-MB在低氧暴露14天显著增加，血浆瘦素在低氧暴露3天和14天显著降低。这与考察队员在昆仑站驻留20天后的表型变化基本一致。慢性低氧暴露导致大鼠出现心肌细胞肥大、纤维化、细胞凋亡和线粒体功能降低等病理学改变，同时降低心肌组织瘦素/瘦素受体、JAK2/STAT3、PI3K/AKT/GSK3β和ERK/JNK蛋白水平。转录组学分析揭示瘦素降低与生物钟、钠/钾离子运输和细胞骨架等相关基因的下调有关。

与常氧组相比，模拟海拔5km低压低氧环境急性暴露3天组大鼠出现HPA轴的激活和HPT轴的抑制、肠道微生物群组成改变和粪便/血清代谢物的显著变化。血清促肾上腺皮质激素释放激素（CRH）、促肾上腺皮质激素（ACTH）和皮质酮（CORT）升高，促甲状腺激素释放激素刺激激素（TRH）和甲状腺素（tT₄）降低。低氧组显著富集拟杆菌属（Bacteroides）、乳杆菌属（Lactobacillus）、副拟杆菌属（Parabacteroides）、丁酸单胞菌属（Butyricimonas）、SMB53、阿克曼氏菌属（Akkermansia）、管状杆菌属（Phascolarctobacterium）和气球菌属（Aerococcus）。常氧组显著富集普雷沃氏菌属（[Prevotella]）、普雷沃氏菌属（Prevotella）、凯斯托杆菌属（Kaistobacter）、盐杆菌属（Salinibacterium）和沃格氏菌属（Vogesella）。代谢组学分析表明，急性低氧显著影响粪便和血清脂质代谢。因果中介分析发现，5种粪便代谢物可能介导TRH、tT₄和CORT与[Prevotella]、Kaistobacter、Parabacteroides和Aerococcus的相互作用，6种血清代谢物可能介导TRH和tT₄对[Prevotella]和Kaistobacter的影响。

结论

本研究第一次提出慢性高原低氧下，脂肪代谢因子降低与心肺功能的适应性调节有关。其中，脂肪代谢因子瘦素在调节高原低氧下心脏功能和心肌病理学变化中起着重要的作用。瘦素可能通过瘦素经典信号通路（JAK2/STAT3、PI3K/AKT/GSK3β和ERK/JNK）和与生物钟、钠/钾离子运输以及细胞骨架相关的基因，调节心脏功能。

急性高原低氧环境下，神经内分泌反应与肠道微生物群组成的紊乱密切相关。因果中介分析发现，5种粪便代谢物和6种血清代谢物介导TRH、tT₄和CORT与肠道菌属的相互交流。

Background

How humans adapt to the extreme environment of Antarctica is an important research question. The research team has been studying the medical research of Antarctic expeditioners for over 20 years and has established targeted hypoxic animal models to conduct mechanistic studies. Kunlun Station in Antarctica is located at the highest point of the ice plateau (4087m, oxygen partial pressure is 11.3kPa, equivalent to 5km at mid-low latitudes), where expeditioners exhibited a series of physiological responses under hypoxic conditions, including changes in cardiovascular function, neuroendocrine hormones, and gut microbiota composition.

Adipose tissues are considered as important metabolic and endocrine organs. Several adipokines secreted by adipose are considered important biomarkers for predicting cardiovascular outcomes. The changes and mechanisms of adipokines on cardiovascular function alterations under hypoxic environments at Antarctic ice plateau remain unclear.

Previous studies have found hyperactivation of hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-thyroid (HPT) axis in humans and animal models acute exposed to high altitudes, but the mechanism of activation is still unknown. The gut microbiota, as the largest metabolic organ and potential endocrine organ, may produce important metabolites that could interact with the host's HPA and HPT axis. However, the mechanisms of interaction among gut microbiota, metabolites, and neuroendocrine factors under acute hypoxic stress are not well understood.

Aims

The study aims to investigate (1) the alterations of key adipokines and underlying mechanisms in regulating cardiovascular function under prolonged high-altitude exposure, and (2) key metabolites mediating the correlation between gut microbiota and host neuroendocrine hormones under acute exposure to high-altitude hypoxic environments.

Methods

(1) We investigated alteration of cardiopulmonary function, electrocardiogram, blood oxygen saturation, and body composition changes of the Chinese Antarctic expeditioners at departure (Shanghai, 5 m) and staying at Kunlun Station for 20 days. The changes in plasma cardiovascular disease risk factors and six key adipose metabolism factors were detected using the Milliplex protein assay. To further investigate the role of key adipokines in hypoxia-induced cardiac alteration, we established a targeted rat model exposed to simulated hypobaric hypoxia (5 km), mimicking the oxygen partial pressure as Kunlun Station, for durations of 1 and 3 days (acute phase), 14 and 28 days (chronic phase). Echocardiography was conducted to analyze the alteration of ventricular structure, diastolic and contract function. Plasma cardiovascular risk factors and adipose metabolic factor levels were measured by ELISA. Myocardial pathological changes in hypoxic rats, including hypertrophy, fibrosis, lipid deposition, cell apoptosis, and mitochondrial dysfunction, were analyzed by immunohistochemistry, transmission electron microscopy, qPCR, and western blot. Furthermore, we studied the changes in signaling pathways of key adipose metabolic factors in cardiac tissues using western blot and further explored potential regulatory mechanisms through transcriptome sequencing analysis.

(2) Adult male SD rats were exposed to a simulated altitude of 5 km for 3 days in a hypobaric-hypoxic chamber. Serum levels of HPA and HPT axis hormones were measured, fecal microbiota was analyzed by using 16S rRNA sequencing and fecal and serum metabolites were analyzed by untargeted-metabolomics. Finally, we applied a causal mediation analysis model to explore potential fecal and serum metabolites mediating the interaction between gut microbiota and host HPT and HPA axis hormones.

Results

Antarctic expeditioners at 4087m exhibited decreased cardiopulmonary function, increased cardiovascular risk factors, and decreased adipose metabolic factors. Specifically, vital capacity (VC_MAX), forced vital capacity (FVC), and forced expiratory volume in one second (FEV₁) significantly decreased, while gas exchange rate and forced expiratory flow rate significantly increased. Heart rate (HR), blood pressure, and systemic vascular resistance (SVR) increased, whereas cardiac pumping and systolic function decreased. Additionally, plasma creatine phosphokinase-MB (CK-MB) and platelet-endothelial cell adhesion molecule-1 (Pecam-1) significantly increased and plasma leptin, resistin, and lipocalin-2 significantly decreased. This study is the first to find that decreased plasma leptin is significantly correlated with hypoxic-induced changes in cardiac function indicators, and further research on the mechanism of adipokines leptin in cardiac function under hypoxia has important scientific significance.

Chronic hypoxia-exposed rats exhibited pulmonary arterial hypertension, right ventricular hypertrophy, and impaired left ventricular systolic and diastolic function in 14 days and 28 days. Plasma CK-MB elevated in 14 days, and sPecam-1 increased in 1 day and 3 days. Plasma leptin decreased in 3 days and 14 days. The phenotype of hypoxic rats is similar to Chinese Antarctic expeditioners. Chronic hypoxia exposure led to significant pathological changes, including increases in myocyte hypertrophy, fibrosis, apoptosis, and mitochondrial dysfunction, coupled with reduced protein levels of leptin/leptin receptor, JAK2/STAT3, PI3K/AKT/GSK3β, and ERK/JNK in myocardial tissues. Transcriptome analysis revealed leptin was associated with downregulated genes involved in circadian rhythm, sodium/potassium ion transport, and cell skeleton.

Compared with the normoxic group, rats exposed to acute hypobaric hypoxia for 3 days exhibited increased serum corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), corticosterone (CORT), and decreased thyroxine (tT₄) and thyrotropin-releasing hormone (TRH). Bacteroides, Lactobacillus, Parabacteroides, Butyricimonas, SMB53, Akkermansia, Phascolarctobacterium, and Aerococcus were enriched in hypoxic group, whereas [Prevotella], Prevotella, Kaistobacter, Salinibacterium, and Vogesella were enriched in normoxic group. Metabolomic analysis indicated that acute hypoxia significantly affected fecal and serum lipid metabolites. In addition, we found 5 fecal metabolites may mediate the cross-talk between TRH, tT₄, and CORT with [Prevotella], Kaistobacter, Parabacteroides, and Aerococcus, and 6 serum metabolites may mediate the effect of TRH and tT₄ on [Prevotella] and Kaistobacter by causal mediation analysis.

Conclusions

For the first time, we proposed that the decreased levels of adipose metabolic factors are related to adaptive regulation of cardiopulmonary function under chronic hypoxia. Among these adipokines, leptin plays a critical role in regulating cardiac alterations at chronic exposure to high altitudes, potentially through leptin’s classical signaling pathway (JAK2/STAT3, PI3K/AKT/GSK3β, ERK/JNK) and genes related to circadian rhythm, sodium/potassium ion transport, and the cell skeleton.

Under acute high-altitude hypoxia exposure, neuroendocrine responses are closely related to disturbances in gut microbiota composition. Causal mediation analysis identified multiple fecal/serum metabolites involved in mediating the cross-talk between gut microbiota and the HPA and HPT axes.