Investigation of the mechanisms of Genkwa Flos hepatotoxicity by a cell metabolomics strategy combined with serum pharmacology in HL-7702 liver cells

1. To investigate Genkwa Flos hepatotoxicity, a cell metabolomics strategy combined with serum pharmacology was performed on human HL-7702 liver cells in this study.

2. Firstly, cell viability and biochemical indicators were determined and the cell morphology was observed to confirm the cell injury and develop a cell hepatotoxicity model. Then, with the help of cell metabolomics based on UPLC-MS, the Genkwa Flos group samples were completely separated from the blank group samples in the score plots and seven upregulated as well as two down-regulated putative biomarkers in the loading plot were identified and confirmed. Besides, two signal molecules and four enzymes involved in biosynthesis pathway of lysophosphatidylcholine and the sphingosine kinase/sphingosine-1-phosphate pathway were determined to investigate the relationship between Genkwa Flos hepatotoxicity and these two classic pathways. Finally, the metabolic pathways related to specific biomarkers and two classic metabolic pathways were analyzed to explain the possible mechanism of Genkwa Flos hepatotoxicity.

3. Based on the results, lipid peroxidation and oxidative stress, phospholipase A₂/lysophosphatidylcholine pathway, the disturbance of sphingosine-1-phosphate metabolic profile centered on sphingosine kinase/sphingosine-1-phosphate pathway and fatty acid metabolism might be critical participators in the progression of liver injury induced by Genkwa Flos.     

创伤性脊髓损伤患者血清和尿液的代谢组学分析

背景:创伤性脊髓损伤在临床上主要依赖于量表评估与影像学检查,但对于损伤程度的预后评估具有一定局限性,利用代谢组学技术进行生物标志物筛选,对于估计病变范围、损伤与恢复程度以及开发新疗法具有重要意义。目的:使用代谢组学技术来表征创伤性脊髓损伤患者的代谢特征,探寻潜在的生物标志物及失调的代谢途径。方法:收集20例创伤性脊髓损伤患者(观察组)和10例健康受试者(对照组)的血清和尿液样本,进行代谢物分析,然后利用多元变量统计分析进行数据处理,筛选差异代谢物。通过MetaboAnalyst软件进行代谢通路富集,应用logistic回归构建生物标志物组合模型,并分析其与美国脊髓损伤协会(ASIA)分级的关系。结果与结论:两组受试者的血清和尿液中分别检测出160种和73种具有显著差异的代谢物。通路富集分析显示,创伤性脊髓损伤后脂质代谢出现明显的紊乱,包括鞘脂类、亚油酸、α-亚麻酸和花生四烯酸代谢以及糖基磷脂酰肌醇生物合成。识别出他索沙坦和葫芦素糖苷这组生物标志物,并且二者构成的代谢物组合在血清和尿液中的水平与ASIA分级存在相关性。由此可见,代谢组学技术为进一步理解创伤性脊髓损伤病理机制、筛选治疗靶点提供帮助。识别出的代谢生物标志物组合可能为评估创伤性脊髓损伤的严重程度提供参考。     

非靶向代谢组学揭示天葵不同部位差异代谢物及其主要代谢途径

目的:利用非靶向代谢组学挖掘天葵非药用部位潜在的药用价值。方法:以天葵茎、叶、花为材料,利用超高效液相色谱-质谱法对其进行非靶向代谢组学分析。结果:天葵茎、叶、花中共鉴定出16大类101个差异代谢物(DAMs),其中包含羧酸及其衍生物、有机含氧化合物、脂质、苯及其取代衍生物、生物碱、黄酮类、萜类、苯丙素类、酚酸类、核苷酸、有机盐、内酯、有机酸、内源性植物激素、氨基酸和13个其他物质。通过聚类和京都基因与基因组百科全书(KEGG)分析发现,天葵不同部位的DAMs代谢模式存在一定的差异;其主要代谢途径为苯丙氨酸代谢途径、ABC转运途径、氨酰-tRNA合成途径及苯丙氨酸、酪氨酸和色氨酸生物合成途径。结论:通过天葵不同部位间DAMs的鉴定及其生物合成途径的分析,确定天葵茎、叶、花富含多种具有药用功效的代谢活性物质,可为天葵茎、叶、花的资源开发利用提供参考。     

Neuroprotective effects of Longxue Tongluo Capsule on ischemic stroke rats revealed by LC-MS/MS-based metabolomics approach

Objective: The present study aimed to evaluate the therapeutic effect and explore the underlying mechanisms of Longxue Tongluo Capsule (LTC) on ischemic stroke rats. Methods: Twenty-six rats were randomly divided into four groups, including sham group, sham + LTC group, MCAO group, and MCAO + LTC group. Ischemic stroke rats were simulated by middle cerebral artery occlusion (MCAO), and LTC treatment group were orally administrated with 300 mg/kg of LTC once daily for seven consecutive days. LTC therapy was validated in terms of neurobehavioral abnormality evaluation, cerebral infarct area, and histological assessments. The plasma metabolome comparisons amongst different groups were conducted by UHPLC-Q Exactive MS in combination with subsequent multivariate statistical analysis, aiming to finding the molecules in respond to the surgery or LTC treatment. Results: Intragastric administration of LTC significantly decreased not only the neurobehavioral abnormality scores but also the cerebral infarct area of MCAO rats. The interstitial edema, atrophy, and pyknosis of glial and neuronal cells occurred in the infarcted area, core area, and marginal area of cerebral cortex were improved after LTC treatment. A total of 13 potential biomarkers were observed, and Youden index of 11 biomarkers such as LysoPC, SM, and PE were more than 0.7, which were involved in neuroprotective process. The correlation and pathway analysis showed that LTC was beneficial to ischemic stroke rats via regulating glycerophospholipid and sphingolipid metabolism, together with nicotinate and nicotinamide metabolism. Heatmap and ternary analysis indicated the synergistic effect of carbohydrates and lipids may be induced by flavonoid intake from LTC. Conclusion: The present study could provide evidence that metabolomics, as systematic approach, revealed its capacity to evaluate the holistic efficacy of TCM, and investigate the molecular mechanism underlying the clinical treatment of LTC on ischemic stroke.     

基于肠道菌群和代谢组学研究化滞柔肝颗粒治疗非酒精性脂肪肝的作用机制

目的 探讨化滞柔肝颗粒治疗非酒精性脂肪肝（nonalcoholic fatty liver disease,NAFLD）的作用机制。方法 高脂饮食饲养8周构建大鼠NAFLD模型,化滞柔肝颗粒ig给药4周。半自动生化分析仪测定大鼠血脂相关指标;油红O染色观察肝脏组织病理变化;Illumina Miseq测序平台对V3～V4可变区进行扩增和测序,超高效液相色谱串联质谱（UPLCMS/MS）开展血浆代谢组学研究,并结合Spearman进行肠道菌群与代谢组学之间的关联分析。结果 与模型组相比,化滞柔肝颗粒中、高剂量组可显著降低大鼠体质量（P<0.05、0.01）和血清总胆固醇（totalcholesterol,TC）、三酰甘油（triacylglycerol,TG）水平（P<0.01）,升高高密度脂蛋白（high-density lipoprotein cholesterol,HDL-C）水平（P<0.05、0.01）。此外,化滞柔肝颗粒高剂量组还可显著降低大鼠低密度脂蛋白（low-density lipoprotein cholesterol,LDL-C）水平（P<0...     

Investigation of urinary components in rat model of ketamine-induced bladder fibrosis based on metabolomics

BackgroundKetamine abuse has been linked to the system''s damage, presenting with lower urinary tract symptoms (LUTS). While the pathogenesis of ketamine-induced urinary damage is not fully understood, fibrosis is believed to be a potential mechanism. A metabolomic investigation of the urinary metabolites in ketamine abuse was conducted to gain insights into its pathogenesis.MethodsA rat model of ketamine induced bladder fibrosis was established through tail vein injection of ketamine hydrochloride and control group was established through tail vein injection of the equivalent normal saline. Hematoxylin and eosin (H&E) staining and Masson trichrome staining were performed to evaluated bladder pathology. Urinary components were detected based on a metabolomic approach using ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS platform). Orthogonal projections analyzed the data to latent structures discriminant analysis (OPLS-DA) and bioinformatics analysis.ResultsThe rat model of ketamine induced bladder fibrosis was confirmed through H&E and Masson trichrome staining. There were marked differences in the urinary metabolites between the experimental group and the control group. Compared to the control group, 16 kinds of differential metabolites were up-regulated and 102 differential metabolites were down-regulated in the urine samples of the ketamine group. Bioinformatics analysis revealed the related metabolic pathways.ConclusionsUsing a ketamine-induced bladder fibrosis rat model, this study identified the differential urinary metabolites expressed following ketamine treatment. These results provide vital clues for exploring the pathogenesis of ketamine-induced LUTS and may further contribute to the disease''s diagnosis and treatment.     

辛弗林对CUMS大鼠抑郁样行为和血清代谢组学的影响

目的 通过观察分析大鼠慢性应激引起的行为学和血清代谢组变化以及辛弗林抗抑郁的有效治疗,发现潜在的抗抑郁新药先导化合物和生物标志物。方法 将24只大鼠分为正常组、模型组和辛弗林组。模型组为6周慢性温和不可预知应激(CUMS)模型大鼠,用体质量变化、糖水偏好、开场实验作为抑郁评估指标,后2周辛弗林(Syn 20 mg/kg)干预治疗后采集大鼠血清进行气相色谱-质谱联用(GC-MS/MS)测定,研究CUMS大鼠血清内源性代谢物变化,以及辛弗林的干预作用,寻找其抗抑郁潜在的先导化合物和生物标志物,并找到相关靶向代谢通路。结果 辛弗林干预后,在增加CUMS大鼠体质量、提高糖水偏好率、增加开场运动总距离和理毛次数的同时,还能逆转CUMS大鼠血清中磷酸乙醇胺、苯甲酸、胆固醇、谷氨酸等14种代谢物的变化使其趋于正常组,并发现谷氨酰胺和谷氨酸的代谢、缬氨酸和异亮氨酸的生物合成等9条主要牵涉辛弗林的靶向代谢通路。结论 辛弗林可能通过调节谷氨酰胺和谷氨酸的代谢、缬氨酸和异亮氨酸的生物合成等生物学途径发挥对CUMS大鼠抑郁样行为的调正作用。      

代谢组学研究在肿瘤药效学和耐药研究中的进展

随着核磁共振和质谱等检测手段,以及计算机分析技术的不断进步,代谢组学在肿瘤中的研究越来越广泛,特别是在肿瘤药物疗效判断、毒性作用以及耐药预测上有着重要作用,对临床个体化药物治疗起到指导作用。文章对此进行综述。     

Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer

Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene‐metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography–mass spectrometry (GC–MS)‐based metabolomics and RNA‐seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network‐based analyses to gain a comprehensive and in‐depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies.