柞蚕蛹培养蛹虫草不同时间后的代谢组分析

为了解柞蚕蛹培养蛹虫草（简称柞蚕蛹虫草）不同时间后的代谢物变化规律,利用广泛靶向代谢组学技术,比较不同培养时期的柞蚕蛹虫草代谢产物成分,找出差异代谢物并进行代谢通路分析。在柞蚕蛹虫草的5个生长时期共检测到10类421种化合物,主要包括：氨基酸及其衍生物、核苷酸及其衍生物、萜类、酚酸、有机酸、糖及醇类、甾体、脂类、生物碱、醌类等多种化合物。主成分分析（PCA）结果显示,不同生长时期柞蚕蛹虫草的代谢组表现出不同的代谢特征,5个生长时期样本在得分图中可分为3个不同区域,对照（S1）与子座形成初期（S3）归为一区,菌核期（S2）独自归为一区,子座形成期（S4）和子囊壳形成期（S5）归为一区。S2-S5筛选到的特有差异代谢物有36种,包括虫草素、甘露醇等主要活性物质,其中香豆酰腐胺、五羟色胺、γ-生育三烯酚等物质首次被检测到。对不同生长阶段变化排在前20的差异显著代谢成分进行分析,结果表明上下调幅度较大的物质多为有机酸、酚酸、生物碱、核苷酸及其衍生物等次生代谢产物。5个生长时期筛选出的所有差异代谢物共富集在154条代谢通路上,差异代谢物数量富集最多的前5条通路,分别为代谢途径、次生代谢物的生物合成、ABC转运蛋白、嘌呤代谢和氨酰生物合成。整个生长周期中,具有显著影响的通路是嘌呤代谢与丙氨酸、天门冬氨酸和谷氨酸代谢途径。本研究结果可为柞蚕蛹虫草的深入研究与应用提供参考。     

基于代谢组学的功能性蛹虫草成分研究

对功能性蛹虫草、野生蛹虫草、柞蚕蛹虫草及冬虫夏草的代谢组学进行非靶向成分比较分析,并对功能性蛹虫草的常见生物活性成分进行定量分析。结果表明,功能性蛹虫草共检测出代谢产物成分2 213种,占成分总量的72.67%,其特有成分497种,与冬虫夏草、柞蚕蛹虫草和野生蛹虫草相比具有显著的成分优势。除目前已经报导的部分成分外,还含有丰富的特殊性代谢产物,如灵芝酸、茯苓新酸、人参皂苷、仙茅皂苷、苦玄参苷、黄芪皂苷、金铁锁环肽、王不留行环肽、甾醇、丹酚酸等。常规成分定量分析表明功能性蛹虫草子实体中虫草素和喷司他丁含量均较高,麦角甾醇、SOD酶、维生素E含量均显著高于对照组。大量药物活性物质在蛹虫草代谢产物成分体系中得到表达,对于揭示蛹虫草保健价值的物质基础具有重要意义。     

柞蚕蛹虫草继代培育其活性物质变化规律

柞蚕蛹虫草中含有虫草素、腺苷、多糖等多种活性成分,具有提高免疫力、抗疲劳、保护心脑血管、抗癌等方面的作用,是冬虫夏草的良好替代品。以柞蚕蛹虫草的继代培育为基础,分别检测蛹虫草菌在不同传代次数时柞蚕蛹虫草子实体生长状态、蛹虫草中腺苷、虫草素、虫草多糖含量及蛹虫草菌菌丝、分生孢子中活性氧的分布。第1代蛹虫草菌接种后柞蚕蛹出现腐烂现象;第2、3代培育的子实体生长量、腺苷及虫草素含量均较高;第4代子实体生长量、腺苷及虫草素含量均出现大幅下降的现象。柞蚕蛹虫草中虫草多糖含量在传代过程中也逐渐降低,但降低幅度较腺苷和虫草素缓慢。第2代培育的柞蚕蛹虫草子实体生长状态优于第3代,故第2代蛹虫草菌更适合应用于批量生产。蛹虫草菌退化后含有活性氧的分生孢子比例增大,这可能是发生退化的表象之一。     

虫草素产量不同的蛹虫草菌株代谢组差异

为探究虫草素高产和低产的两株蛹虫草菌代谢差异及其与虫草素代谢的关联性,本研究采用UPLC-QTOF-MS广泛靶向代谢组学技术和多元统计分析方法,结合相同蛹虫草菌不同发酵时间的菌丝体差异代谢物相对含量的变化情况,比较了两株蛹虫草菌相同发酵时间的菌丝体代谢差异。结果表明：蛹虫草CICC 14014菌株发酵第20和10天的菌粉（H20和H10）及其发酵液的虫草素含量都显著高于蛹虫草CGMCC 3.4655菌株（其菌粉为L20和L10）（P<0.01）。主成分模型显示H10、L10、H20和L20间分散明显;通过正交偏最小二乘法分析,以t检验P<0.05并且VIP>1为标准,从H10 vs L10和H20 vs L20分别识别出190和158种差异代谢物,其中具有生物活性的亚精胺为首次在蛹虫草菌中被检出;以P<0.01为标准,从H10 vs L10、H20 vs L20中筛选出2条显著富集的通路,分别为烟酸-烟酰胺和精氨酸-脯氨酸代谢通路。对H10 vs L10和H20 vs L20进行代谢调控网络分析,获得7条显著互作的通路,相同的互作通路为碱基切除修复。代谢网络分析结果表明抑制三羧酸循环、核黄素代谢、赖氨酸降解、鞘脂类代谢通路以及促进碱基切除修复、β-丙氨酸代谢、精氨酸-脯氨酸代谢、脂质代谢、嘌呤代谢通路有利于虫草素的生成;虫草素高产蛹虫草菌甘油三酯代谢和合成腺苷的能力高于虫草素低产蛹虫草菌;在嘌呤代谢途径中,腺嘌呤通过黄嘌呤代谢反馈促进虫草素的生物合成。本研究为蛹虫草菌生物活性成分的挖掘提供理论基础,为深入解析蛹虫草菌虫草素的代谢机制提供参考。     

肠道菌群与高尿酸血症及痛风的相关性研究

高尿酸血症以及痛风的发病率持续升高,已经成为一个重大的公共卫生问题。肠道菌群的结构改变或失调可引起机体代谢紊乱,肠道微生态尤其与代谢性疾病的发生发展关系密切。目前研究发现高尿酸血症、痛风患者存在肠道菌群失调,降尿酸治疗后肠道菌群可发生相应改变,并且益生菌制剂具有降尿酸作用。本文概述高尿酸血症及痛风患者的肠道菌群特点,从高嘌呤及高果糖饮食对肠道菌群的影响、肠道参与嘌呤和尿酸的代谢、代谢性内毒素血症以及痛风相关炎症因子等方面探讨肠道菌群与高尿酸血症及痛风的关系,并展望肠道菌群可能成为未来诊治高尿酸血症以及痛风的一种新方法。     

降血尿酸益生菌株的筛选和降血尿酸机理的探索

【背景】高尿酸血症是人体内血尿酸含量显著高于正常水平的代谢性疾病,利用益生菌降解食物中外源性嘌呤类成分成为治疗高尿酸血症的新方法。【目的】筛选具有降低血尿酸作用的益生菌,并探索其作用机制。【方法】利用HPLC从多株实验菌株中筛选降解核苷酸(腺苷酸、鸟苷酸)、核苷(腺苷、鸟苷)、嘌呤(黄嘌呤、次黄嘌呤、鸟嘌呤)、尿酸能力最强的益生菌。首次利用质谱定性与定量检测菌株降解核苷与核苷酸过程中代谢物的变化,结合菌株对高尿酸血症模型大鼠血尿酸水平的影响,初步探索其降低血尿酸的机理。【结果】首次筛选出具有较强降解核苷酸与核苷能力的干酪乳杆菌ZM15(CGMCC No.13980),高尿酸血症模型大鼠验证其具有降低血尿酸的作用。结果显示菌株ZM15在胞内降解核苷酸、核苷后,胞内、外均测到鸟嘌呤、黄嘌呤、次黄嘌呤,且胞内3种嘌呤含量显著高于正常菌体内含量(P0.01),尿酸和尿囊素在胞内、外均未发现。【结论】干酪乳杆菌ZM15具有较强的降解核苷酸、核苷的能力,推测其主要通过与肠道上皮细胞竞争吸收核苷酸与核苷,从而对高尿酸血症模型大鼠具有降血尿酸作用。     

蛹虫草饲料添加剂的研究现状及展望

蛹虫草饲料添加剂包括蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养残基提取物、蛹虫草菌固液发酵产物、 微生物发酵蛹虫草残基等产品。蛹虫草饲料添加剂含有粗蛋白、粗脂肪、氨基酸等营养成分,以及虫草素、腺苷、多糖等活性成分,在畜禽、反刍动物、水产品等动物养殖中的应用均获得较好的 效果。对蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养残基提取物、利用蛹虫草菌及培养残基制作发酵饲料等蛹虫草饲料添加剂在动物养殖中的研究应用进行了总结,对存在的问题及发展前景进行了探讨及展望。     

基于蛹虫草转录物组学的虫草素生物合成相关基因的表达和功能分析

蛹虫草Cordyceps militaris是我国著名的中药，而虫草素是蛹虫草重要的生物活性成分之一。通过转录物组分析野生型C.militaris、虫草素高产菌株C.militaris GYS60和低产菌株C.militaris GYS80虫草素生物合成相关基因的表达和功能变化。利用Illumina NovaSeq 6000测序获得3株菌株的转录物组数据，以鉴定差异表达基因。与C.militaris相比，在GYS60和GYS80中，分别有145和470个上调基因及96和594个下调基因；GYS60与GYS80相比，GYS60有306个上调基因和207个下调基因。这些基因经过GO和KEGG分析，与C.militaris相比，GYS60和GYS80分别有10和8个与嘌呤途径相关的基因差异表达；与GYS80相比，GYS60有12个基因差异表达。定量聚合酶链式反应验证8个关键酶的基因表达与转录物组分析一致。在GYS60中，参与虫草素生物合成的嘌呤核苷酸代谢中的氧化还原酶(CCM＿07507,cmORE)、3′,5′-环AMP磷酸二酯酶(CCM＿02777,cmAPE)、转移酶(CCM＿0472...     

蛹虫草组学研究进展

作为虫草属的模式种,蛹虫草是目前虫草类真菌中研究和应用最为广泛的物种之一。随着基因组序列的公布,蛹虫草组学水平的研究近年来取得了明显的进展。本文从基因组、线粒体基因组、甲基化组、转录组、蛋白质组、代谢网络等角度对蛹虫草组学水平的研究现状进行综述,期望对进一步推动蛹虫草的深入研究提供帮助。     

成人原发肾病综合征患者血尿酸与血脂代谢的关系

目的:研究成人原发肾病综合征(PNS)患者高尿酸血症的患病率及其与血脂代谢的关系。方法:将109例成人PNS患者根据其尿酸水平分为高尿酸血症组与血尿酸正常组,检测患者的血脂、血清脂蛋白、ALB及24小时尿蛋白定量,分析成人PNS患者高尿酸血症的患病率,比较高尿酸血症组与血尿酸正常组PNS患者的一般情况及血脂水平,并分析PNS患者血尿酸水平的相关因素。结果:成人PNS患者高尿酸血症的发病率为24.77%;高尿酸血症组患者TG及24小时尿蛋白定量水平明显高于血尿酸正常组(P0.01),两组患者TC、LDL-C、HDL-C、Apo AI及Apo B比较差异无统计学意义(P0.05);成人PNS患者血尿酸水平与TG及24小时尿蛋白定量水平呈正相关(r=0.350,P=0.001;r=0.533,P=0.014),与TC、LDL-C、HDL-C及ALB无明显相关性(P0.05)。结论:成人PNS患者高尿酸血症的发生率较高,高尿酸血症患者具有更高的TG及尿蛋白水平,且成人PNS患者血尿酸水平与TG及24小时尿蛋白定量具有相关性,应高度重视成人PNS患者的血尿酸水平。     

Metabonomic studies on potential plasma biomarkers in rats exposed to ionizing radiation and the protective effects of Hong Shan Capsule

An ultra performance liquid chromatography coupled to mass spectrometry-based metabonomic approach, combined with pattern recognition methods including PCA, PLS-DA, RF and heatmap, has been developed to characterize the global serum metabolic profile associated with ionizing radiation (IR). As the VIP-value threshold cutoff of the metabolites was set to 2, metabolites above this threshold were filtered out as potential target biomarkers. Nineteen distinct potential biomarkers in rat plasma were identified. To further elucidate the pathophysiology of IR, related metabolic pathways have been studied. It was found that IR was closely related to disturbed fatty acid metabolism, taurine and hypotaurine metabolism, sphingolipid metabolism, purine metabolism, pyrimidine metabolism, phospholipid catabolism, tryptophan metabolism, phenylalanine metabolism, and bile acid metabolism. With the presented metabonomic method, we systematically analyzed the protective effects of Traditional Chinese Medicine Hong Shan Capsule (HSC). The results demonstrated that HSC administration could provide satisfactory effects on IR through partially regulating the perturbed metabolic pathways.     

A Pharmaco-Metabonomic Study on Chronic Kidney Disease and Therapeutic Effect of Ergone by UPLC-QTOF/HDMS

Chronic kidney disease (CKD) is an important public health problem. Ergone has been proved to prevent the progression of CKD. UPLC-QTOF/HDMS was employed for metabolic profiling of adenine-induced CKD and to investigate the nephroprotective effects of ergone. Pharmacology parameters including blood biochemistry, histopathological evaluation and Western blot analysis were performed concurrently. The UPLC-MS data were analyzed by partial least squares-discriminate analysis, correlation analysis, heatmap analysis and mapped to KEGG pathways. Blood and serum biochemistry were observed to be significantly different in the CKD group than in the control group. In conjunction with biochemistry, histopathology and protein expression results, identified metabolites indicated perturbations in fatty acid metabolism, purine metabolism and amino acid metabolism as changes associated with adenine-induced CKD and the interventions of ergone. Upregulated expression of TGF-β1, ED-1, CTGF, bFGF and collagen I was observed in the CKD group. However, downregulated expression of these proteins was observed after oral administration of ergone. These results suggest that expression changes in these proteins had implications for fatty acid metabolism, purine metabolism and amino acid metabolism in the development of CKD and that ergone treatment could delay the development of CKD by normalizing or blocking abnormal changes in biomarker metabolites and protein expression in the CKD group.     

Effects of dopamine D2 receptor antagonists on retinal pigment epithelial/choroid complex metabolism in form-deprived myopic guinea pigs

The retinal pigment epithelial (RPE)/choroid complex regulates myopia development, but the precise pathogenesis of myopia remains unclear. We aimed to investigate the changes in RPE/choroid complex metabolism in a form deprivation myopia model after dopamine D2 receptor (D2R) modulation. Guinea pigs were randomly divided into normal (NC), form deprivation myopia (FDM), and FDM treated with dopamine D2R antagonist groups. Differential metabolites were screened using SIMCA-P software and MetaboAnalyst metabolomics analysis tool. Functions of differential metabolites were analyzed using KEGG enrichment pathways. Relative to the NC group, 38 differential metabolites were identified, comprising 29 increased metabolites (including nicotinic acid, cytosine, and glutamate) and 9 decreased metabolites, of which proline exhibited the largest decrease. Pathway analysis revealed regulation of arginine/proline and aspartate/glutamate metabolism. Intravitreal D2R antagonist injection increased proline concentrations and activated arginine/proline and purine metabolism pathways. In sum, D2R antagonists alleviated the myopia trend of refractive biological parameters in form deprivation myopic guinea pigs, suggesting the involvement of dopamine D2R signaling in myopia pathogenesis. The RPE/choroid may provide glutamate to the retina by activating proline metabolism via metabolic coupling with the retina. Dopamine D2R antagonism may modulate proline/arginine metabolic pathways in the RPE/choroid and regulate metabolism, information presentation, and myopia.     

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki' values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 muM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.     

Metabolomics Analysis Reveals that AICAR Affects Glycerolipid,Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells

AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using ¹³C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.