第一部分

中、老年人群主要不良心血管事件血清代谢标志物的共同与差异：一项队列研究

背景：

心血管事件的发生是微循环障碍的严重后果之一。心血管疾病的病理生理涉及包括脂蛋白和氨基酸代谢在内的多种代谢异常。目前，在不同年龄人群中探讨主要不良心血管事件（major adverse cardiovascular events，MACE）风险相关代谢物的研究较为缺乏。本研究旨在评估血清代谢物与中、老年人群MACE发生的关联；探讨代谢组学在预测MACE中的价值；并比较中、老年组与MACE风险相关的代谢生物标志物的差异。

方法：

纳入具有基于核磁共振检测的血清代谢组学数据、且协变量与临床血脂参数完整的英国生物银行人群。依据基线时无缺血性心脏病、脑血管疾病、心力衰竭、心脏骤停病史，且未服用降脂药物的标准筛选研究人群。相关协变量包括社会人口学特征、生活方式、临床信息及空腹时间。通过Cox回归分析计算代谢物的校正风险比，包括各种脂蛋白颗粒的浓度、不同脂蛋白的组分、酮体、氨基酸、脂肪酸和其他低分子量代谢生物标志物。采用最小绝对收缩和选择算子（least absolute shrinkage and selection operator，LASSO）回归筛选特征代谢物加入到基础模型之中。将特征代谢物纳入MACE风险预测模型，评估模型的区分度（C指数）和重分类准确性（净重新分类改善指数 [net reclassification improvement，NRI]，综合判别改善指数[integrated differentiation improvement，IDI]）。

结果：

研究纳入共54,561位UK Biobank受试者（34,797名中年人和19,764名老年人），中位随访时间超过12年。其中，1799名中年人及2527名老年人发生MACE（缺血性心脏病、中风、心血管死亡）。调整相关协变量后，Cox回归得出与人群MACE发生相关的代谢生物标志物 （错误发生率校正P值小于0.05）。与中年组相比，老年组中与MACE风险增加相关的代谢物显著减少，老年组中强调中、小粒径HDL及其组分、二十二碳六烯酸和甘氨酸的保护作用。纳入特征代谢物的更全面的模型在中年个体显示出对MACE发生的区分能力和预测准确性增强，C统计量改善（从0.711 [0.699-0.722]到0.723[0.711-0.734]），连续NRI为0.247[0.207-0.315]，绝对IDI为0.005[0.004-0.008]。其评价价值优于老年人。

结论：

本研究探讨了循环代谢物与中、老年人MACE风险的关联，并进行了比较。代谢组学研究揭示了不同年龄人群中与新发MACE相关的生物标志物，强调了保护性代谢物在老年人群中的价值。这为可能实施精准医学以预防MACE提供了重要信息。

第二部分

高尿酸血症通过炎症途径诱导微循环功能障碍的作用与机制研究

背景：  

高尿酸血症（hyperuricemia，HUA）是一类较为常见的代谢紊乱，尿酸产生增加或排泄减少可导致HUA。HUA患病率呈现上升趋势，且与外周动脉疾病和慢性肾脏病进展相关。探索HUA与微循环障碍的关联具有一定的病理生理学价值，目前相关证据局限于临床获取的患者眼底成像。本研究旨在探讨HUA诱导的外周和肾脏微循环功能障碍，并揭示肾脏组织病理改变和血清代谢物差异。

方法：

接触式激光多普勒血流探测仪（moor vascular monitoring system-laser doppler perfusion and temperature monitor 2, MoorVMS-LDF2）和激光多普勒血氧气仪（moor vascular monitoring system-oxygenation monitor，moorVMS-OXY）采集HUA组（n=6）和对照组（n=6）小鼠的微循环参数。应用小波分析评估微血流动力学的不同生理来源贡献及振幅。免疫组织化学法对肾脏黏附因子进行半定量分析，蛋白免疫印迹法评估肾组织匀浆炎症因子的相对表达量。非靶向代谢组学检测血清代谢物，进行差异分析和通路分析。

结果：

HUA组相比对照组，肾脏血流灌注和血细胞聚集度无明显差异，血流速度（97.60±5.18 AU vs 80.27±5.18 AU，P<0.05）显著增加，血氧饱和度（75.58±1.05 % vs 71.88±0.73 %，P<0.05）显著增加。而HUA诱导的外周微循环障碍主要表现为血流灌注显著降低（肢体：145.68±7.93 PU vs 198.40±16.68 PU，P<0.05；足趾：272.52±12.52 PU vs 333.32±10.93 PU，P<0.05）。小波分析显示HUA组肾脏血细胞聚集度的NO依赖内皮源性贡献组分占比显著降低（P<0.05），而心源性、呼吸源性和肌源性贡献组分占比显著增加（P<0.05）。外周体表微血流动力学的不同生理来源贡献组分占比两组间无明显差异。肾脏组织血小板内皮细胞粘附分子-1（platelet endothelial cell adhesion molecule-1，PECAM-1）、细胞间黏附分子-1（intercellular cell adhesion molecule-1，ICAM-1）、血管细胞黏附分子-1（vascular cell adhesion molecule-1，VCAM-1）和白细胞介素-1β（interleukin-1 beta，IL-1β）在HUA组表达量显著高于对照组（P<0.05）。HUA组相比对照组，存在多种差异代谢物，具有统计学意义的通路改变为雌激素代谢通路和乳糖降解通路的下调。

结论：

HUA组小鼠存在微血流动力学改变，肾脏和外周体表的变化模式不同。肾脏表现为血流速度增加，生理来源贡献占比改变，IL-1β/VCAM-1上调和雌激素代谢通路下调。HUA诱导的外周微循环障碍主要表现为血流灌注降低。

Part Ⅰ

A Cohort Study Reveals Shared and Distinct Serum Metabolic Biomarkers for Major Adverse Cardiovascular Events in Middle-Aged and Older Adults

Background:

The occurrence of cardiovascular events represents a severe consequence of microcirculation disorders. The pathophysiology of cardiovascular diseases involves multiple metabolic abnormalities, including lipoprotein and amino acid metabolism. Current research that probes into metabolites associated with major adverse cardiovascular events (MACE) risk across different age groups remains scarce. This study aimed to assess the association of serum metabolites with the occurrence of MACE in middle-aged and elderly individuals; explore the value of metabolomics in predicting MACE; and compare the distinctions in MACE risk-related metabolic biomarkers between middle-aged and elderly groups.

Methods:

Among the participants of the UK Biobank who underwent baseline assessment through nuclear magnetic resonance (NMR)-based metabolomic profiling of 168 serum metabolites and had complete covariates and clinical lipid parameters, we included those without a previous diagnosis of ischemic heart disease, cerebrovascular disease, heart failure, or cardiac arrest and not on lipid-lowering medications. Relevant covariates included sociodemographic characteristics, lifestyle factors, clinical information, and fasting time. Cox regression gave adjusted hazard ratios for metabolites, including the concentrations of various lipoprotein particles, compositional profiles of different lipoproteins, ketone bodies, amino acids, fatty acids, and additional low-molecular-weight metabolic biomarkers. The least absolute shrinkage and selection operator (LASSO) regression was applied to these metabolites to screen characteristic metabolic variables in addition to the basic model. Selected feature metabolic biomarkers were added to the established model for predicting MACE risk; risk differentiation (C-statistic) and reclassification accuracy (net reclassification improvement improvement [NRI], integrated differentiation improvement [IDI]) were evaluated.

Results:

This study included 54,561 UK Biobank participants (34,797 middle-aged adults and 19,764 elderly adults) with a median follow-up period exceeding 12 years. Of these, 1,799 middle-aged individuals and 2,527 elderly individuals incident MACE (ischemic heart disease, stroke, and cardiovascular deaths). After adjusting for relevant covariates, Cox regression yielded metabolic biomarkers associated with the occurrence of MACE in the population (false discovery rate controlled P < 0.05). In the elderly, the metabolites associated with increased MACE risk were notably diminished compared to the middle-aged; and the elderly group underscored the protective function of medium and small HDL and their constituents, docosahexaenoic acid, and glycine. The more comprehensive model, which additionally includes the feature metabolic biomarkers, demonstrated enhanced discriminatory power and predictive accuracy for MACE occurrence among middle-aged individuals, evidenced by improved C-statistics (from 0.711 [0.699-0.722] to 0.723 [0.711-0.734]), a continuous NRI of 0.247 [0.207-0.315], and an absolute IDI of 0.005 [0.004-0.008]. Its evaluation value is superior to that in the elderly.

Conclusions:

This study explored the association of circulating metabolites with MACE risk in middle-aged and elderly adults and made comparisons. Metabolomic insights have revealed biomarkers associated with new-onset MACE in different age populations, highlighting the value of protective metabolites in the elderly. This provides instrumental information to possibly implement precision medicine for preventing MACE.

Part Ⅱ

Study on the Role and Mechanism of Hyperuricemia in Inducing Microcirculatory Dysfunction via Inflammatory Pathways

Background:

Hyperuricemia (HUA) is a prevalent metabolic disorder resulting from either increased production or reduced excretion of uric acid. The prevalence of HUA is increasing, and it has been associated with both peripheral arterial disease and the progression of chronic kidney disease. Understanding the association between HUA and microcirculatory dysfunction is of significant pathophysiological interest. Prior evidence has primarily been derived from patient fundus imaging; therefore, this study aims to investigate the impact of HUA on both peripheral and renal microcirculation, as well as to elucidate corresponding renal histopathological changes and alterations in serum metabolites.

Methods: 

Microcirculatory parameters were obtained from mice in both the HUA (n = 6) and control (n = 6) groups using a contact laser Doppler flowmeter (moor vascular monitoring system-laser doppler perfusion and temperature monitor 2, MoorVMS-LDF2) and a laser Doppler oxygenation monitor (moor vascular monitoring system-oxygenation monitor，moorVMS-OXY). Wavelet analysis was conducted to assess the contributions and amplitudes of different physiological sources to microvascular hemodynamics. Renal adhesion molecules were semi-quantitatively analyzed by immunohistochemistry, and the relative expression of inflammatory factors in kidney homogenates was evaluated via Western blotting. Additionally, untargeted metabolomics was performed on serum samples to identify differential metabolites and to conduct pathway enrichment analysis.

Results:

Compared with the control group, the HUA group showed no significant differences in renal perfusion or blood cell aggregation; however, renal blood flow velocity (97.60±5.18 AU vs 80.27±5.18 AU, P<0.05) and oxygen saturation (75.58±1.05% vs 71.88±0.73%, P<0.05) were significantly elevated. In contrast, HUA-induced peripheral microcirculatory dysfunction was primarily characterized by a significant decrease in perfusion, with both limb perfusion (145.68±7.93 PU vs 198.40±16.68 PU, P<0.05) and toe perfusion (272.52±12.52 PU vs 333.32±10.93 PU, P<0.05) significantly lower than in the control group. Wavelet analysis revealed that, in the HUA group, the proportion of the NO-dependent endothelial component contributing to renal blood cell aggregation was significantly reduced (P<0.05), whereas the contributions from cardiac, respiratory, and myogenic sources were significantly increased (P<0.05). There was no significant difference between the two groups in the proportional contributions of the various physiological sources to peripheral superficial microvascular hemodynamics. Immunohistochemical and Western blot analyses further revealed markedly elevated renal levels of platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and interleukin-1β (IL-1β) in the HUA group compared to controls (P<0.05). In addition, metabolomic profiling identified distinct metabolites, with pathway analyses demonstrating significant downregulation of both estrogen metabolism and lactose degradation in the HUA group relative to controls.

Conclusion:

In the mice of the HUA group, changes in microhemodynamics were observed, with differences between renal and peripheral microcirculation alterations. Renal changes were characterized by increased in blood flow velocity, shifts in the relative contributions of different physiological sources, upregulation of IL-1β and VCAM-1 expression, and downregulation of estrogen metabolism pathways. In contrast, peripheral microcirculatory dysfunction induced by HUA was mainly marked by reduced blood perfusion.