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| 基于非靶代谢组学研究痰热腑实证型出血性中风后发生二次出血的生物标志物 |
| Research on biomarkers of secondary bleeding after hemorrhagic stroke with phlegm heat and fu syndrome based on non target metabolomics |
| 投稿时间：2024-03-08 录用日期：2024-04-11 |
| DOI： |
| 中文关键词: 出血性中风 痰热腑实 二次脑出血 代谢组学 |
| 英文关键词: Hemorrhagic stroke Phlegm heat and visceral excess Secondary cerebral hemorrhage Metabonomics |
| 基金项目:国家自然科学基金项目（82074099）：黄芪-葛根药对配伍广陈皮激活SIRT6-PPARα信号通路改善NAFLD的机制研究 |
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| 中文摘要: |
| 摘要 目的: 通过非靶代谢组学研究痰热腑实证型出血性中风后发生二次出血的生物标志物，通过代谢谱与脑出血表型的关联研究揭示发生二次出血的中西医病理本质，并筛选出具有预测二次出血价值的生物标志物，为今后临床预测疾病发生提供新的参考指标。方法：收集广州中医药大学第一附属医院神经外科临床西医诊断为高血压脑出血，中医诊断为痰热腑实证型出血性中风患者共60例，其中仅发生一次出血、发生二次出血患者各30例；同时招募30位健康患者作为空白对照。应用QExactive 台式四极杆 - 轨道阱高分辨质谱仪（UHPLC-Q-Orbitrap HRMS）技术对一次脑出血（NCX）、二次出血（NCX+）及健康组（CON）之间血浆差异代谢物进行鉴定。采用主成分分析（PCA）、正交偏最小二乘法判别分析（OPLS-DA）、差异物的热图制作、ROC曲线绘制，对代谢谱数据进行多维统计分析，筛选出具有预测二次出血潜力的生物标志物。结果：健康组/一次出血组差异代谢物9种，其中N-苄基甲酰胺、DL-色氨酸、4-吲哚甲醛、5-羟基吲哚-3-乙酸、L-犬尿氨酸与健康组相比表达上调，虾青素、荆芥内酯、大豆苷元、维生素K表达下调。健康组/二次出血组差异代谢物共22种，与健康组相比2,2-双(羟甲基)丙酸、DL-甲羟戊酸、2-叔丁基-4，6-二硝基苯酚、2,2,6,6-四甲基-4-哌啶醇、N-苄基甲酰胺、4-庚基苯酚、8-甲基-2(1H)-喹啉酮、DL-色氨酸、4-吲哚甲醛、5-羟基吲哚-3-乙酸、L-犬尿氨酸表达上调，二氢松柏醇、3-羟基十四烷酸、2-乙酰基环己酮、白花丹素、劳丹素、虾青素、(Z)-肉桂醛、荆芥内酯、大豆苷元、维生素K、芥酸酰胺表达下调。一次出血/二次出血组差异代谢物共11种，表达上调的代谢物为DL-甲羟戊酸、2-叔丁基-4，6-二硝基苯酚、4-庚基苯酚、8-甲基-2(1H)-喹啉酮、5-羟基吲哚-3-乙酸、L-犬尿氨酸；表达下调的代谢物为3-羟基十四烷酸、2-乙酰基环己酮、劳丹素、大豆苷元、苏氨酸。DL-甲羟戊酸、2-叔丁基-4，6-二硝基苯酚、4-庚基苯酚、8-甲基-2(1H)-喹啉酮、5-羟基吲哚-3-乙酸、L-犬尿氨酸、苏氨酸ROC分析曲线下面积分别为0.95、0.97、0.98、0.96、0.97、0.97、0.85。色氨酸代谢通路P值为0.02，Impact值为0.12：缬氨酸、亮氨酸和异亮氨酸降解通路P值为0.07，Impact值为0.03。结论：DL-甲羟戊酸、2-叔丁基-4，6-二硝基苯酚、4-庚基苯酚、8-甲基-2(1H)-喹啉酮、5-羟基吲哚-3-乙酸、L-犬尿氨酸、苏氨酸7种差异代谢物均具有预测痰热腑实证型出血性中风后发生二次出血的潜力。色氨酸代谢为显著差异代谢通路。二次出血的发生与疾病发生后机体炎症程度、能量供应、氧化应激程度、自由基含量、神经系统损害程度、血管情况、凝血功能等密切相关。从祖国医学角度而言，痰热腑实证型出血性中风发生后是否会进展为二次出血与机体痰热症候程度联系紧密。 |
| 英文摘要: |
| Abstract Objective：By using non target metabolomics to study the plasma differential metabolites that cause secondary bleeding after hemorrhagic stroke with phlegm heat and fu syndrome, and by studying the correlation between metabolic spectrum and cerebral hemorrhage phenotype, the pathological essence of secondary bleeding is revealed, and biomarkers with predictive value for secondary bleeding are screened, providing new reference indicators for clinical prediction of disease occurrence in the future.Methods:Collect a total of 60 patients with hemorrhagic stroke diagnosed by Western medicine as hypertensive intracerebral hemorrhage and traditional Chinese medicine as phlegm heat and viscera syndrome. Among them, there were 30 patients with only one episode of bleeding and 30 patients with two episodes of bleeding each; Recruit 30 healthy patients as blank controls simultaneously. Identification of plasma differential metabolites between primary cerebral hemorrhage (NCX), secondary hemorrhage (NCX+), and healthy group (CON) using QExactive desktop quadrupole orbital trap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) technology. Using Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), differential heatmap production, and ROC curve plotting, multidimensional statistical analysis was performed on the metabolic spectrum data to select biomarkers with potential for predicting secondary bleeding.Results:There are 9 different metabolites in the healthy group/single bleeding group, among which N-benzylformamide, DL tryptophan, 4-indole-3-aldehyde, 5-hydroxyindole-3-acetic acid, and L-caninurine are upregulated compared to the healthy group, while astaxanthin, resveratrol, daidzein, and vitamin K are downregulated. There are a total of 22 different metabolites in the healthy group/secondary bleeding group. Compared with the healthy group, the expression of 2,2-bis (hydroxymethyl) propionic acid, DL methylhydroxyvaleric acid, 2-tert butyl-4,6-dinitrophenol, 2,2,6,6-tetramethyl-4-piperidinol, N-benzylformamide, 4-heptanylphenol, 8-methyl-2 (1H) quinolone, DL tryptophan, 4-indole-3-aldehyde, 5-hydroxyindole-3-acetic acid, and L-caninurenine is upregulated. Dihydropine, 3-hydroxytetradecanoic acid, and 2-acetylcyclohexanone are also upregulated The expression of baihuadansu, laudansu, astaxanthin, (Z) - cinnamaldehyde, resveratrol, daidzein, vitamin K, and erucinamide was downregulated. There are a total of 11 differentially expressed metabolites in the first/second bleeding group, with the upregulated metabolites being DL methylhydroxyvaleric acid, 2-tert butyl-4,6-dinitrophenol, 4-heptanylphenol, 8-methyl-2 (1H) quinolone, 5-hydroxyindole-3-acetic acid, and L-caninurenine; The down regulated metabolites are 3-hydroxytetradecanoic acid, 2-acetylcyclohexanone, laudantin, daidzein, and threonine. The areas under the ROC analysis curves for DL hydroxyvaleric acid, 2-tert butyl-4,6-dinitrophenol, 4-heptylphenol, 8-methyl-2 (1H) quinolone, 5-hydroxyindole-3-acetic acid, L-caninurine, and threonine were 0.95, 0.97, 0.98, 0.96, 0.97, 0.97, and 0.85, respectively. The P value of tryptophan metabolism pathway is 0.02, with an Impact value of 0.12; the P value of valine, leucine, and isoleucine degradation pathway is 0.07, with an Impact value of 0.03.Conclusion:DL mevalonate, 2-tert-butyl-4,6-dinitrophenol, 4-heptylphenol, 8-methyl-2 (1H) - quinolinone, 5-hydroxyindole-3-acetic acid, L-kynurenine and threonine have the potential to predict secondary bleeding after hemorrhagic stroke with phlegm heat Fu syndrome. Tryptophan metabolism is a significantly different metabolic pathway. The occurrence of secondary bleeding is closely related to the degree of inflammation, energy supply, oxidative stress, free radical content, nervous system damage, vascular condition, coagulation function, etc. From the perspective of traditional Chinese medicine, whether the hemorrhagic stroke of phlegm heat Fu excess type will progress to secondary bleeding is closely related to the degree of phlegm heat syndrome. |
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