基于UPLC/Q-TOF-MS技术的大黄牡丹汤治疗IBD大鼠的血清代谢组学研究
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摘要
目的从代谢组学角度探讨大黄牡丹汤治疗炎症性肠病(IBD)的作用机制。方法采用灌肠给予2,4,6-三硝基苯磺酸(TNBS)-乙醇(90 mg·kg~(-1))诱导IBD模型;SD大鼠随机分正常组、模型组、美沙拉嗪组(430mg·kg~(-1))和大黄牡丹汤组(7.5 g·kg~(-1)),每组8只,连续灌胃给药7 d,每天1次。观察大鼠一般情况,测定体质量及疾病活动指数(DAI);全自动血液分析仪检测大鼠红细胞、白细胞、粒细胞和淋巴细胞数量;采用ELISA法检测血清中IL-6、TNF-α的含量;HE染色观察结肠组织病理变化。采用超高效液相色谱-串联四极杆飞行时间质谱(UPLC/Q-TOF-MS)技术建立大鼠血清代谢轮廓图,分析各组大鼠血清内源性代谢物的变化,鉴定潜在的生物标志物并进行生物学意义分析。结果与正常组比较,模型组大鼠的DAI评分明显升高(P <0.001),体质量明显降低(P <0.001),白细胞、红细胞、淋巴细胞及粒细胞数目均显著上升(P <0.05,P <0.01),血清中IL-6、TNF-α含量显著升高(P <0.01,P <0.001),结肠组织出现不同程度的水肿、糜烂,腺体破坏严重且紊乱,有隐窝炎及脓肿形成。与模型组比较,给药组大鼠的DAI评分明显降低(P <0.05,P <0.01,P <0.001),体质量明显升高(P <0.05,P <0.01),白细胞、红细胞、淋巴细胞和粒细胞数目均显著下降(P <0.05,P <0.01),血清中IL-6、TNF-α的含量显著降低(P <0.01),结肠黏膜上皮逐渐恢复,肠绒毛形态结构趋于完整,肠腺结构明显。结果鉴定出IBD大鼠血清中的8个生物标志物,大黄牡丹汤可以上调苯乙酰甘氨酸、鞘氨醇、植物鞘氨醇、黄嘌呤、色氨酸、吲哚的含量(P <0.05,P <0.01),下调甘氨酸、苯丙氨酸的含量(P <0.05,P <0.01)。结论大黄牡丹汤对TNBS-乙醇诱导的IBD大鼠具明显的治疗作用,可能与其改善血清中内源性代谢物水平,恢复体内正常代谢活动有关。
Objective To investigate the mechanism of Dahuang Mudan decoction(DMD)on the treatment of inflammatory bowel disease(IBD)by metabolomics approach. Methods The IBD model was induced by 2,4,6-trinitrobenzenesulfonic acid(TNBS)/ethanol(90 mg·kg~(-1)). SD rats were randomly divided into control group,model group,mesalazine group(430 mg·kg~(-1)) and DMD group(7.5 g·kg~(-1)),8 rats in each group,and were continuously given correspondent drugs by gavage once a day for 7 days. The general conditions of the rats were evaluated by measuring the body weight and disease activity index(DAI). The white blood cell counts(WBC),lymphocyte counts(Lymph),granulocyte counts(Gran)and red blood cell(RBC)counts were measured by automatic blood analyzer. The contents of inflammatory cytokines including IL-6 and TNF-α in serum were determined by ELISA. Pathological changes of colon tissues were observed by HE staining. Ultrahigh performance liquid chromatography-tandem quadrupole time-of flight mass spectrometry(UPLC/Q-TOF-MS)was used to establish the serum metabolic profile of rats,and analyze the changes of serum endogenous metabolites to identify the potential biomarkers. The biological significance of the treatments was then interpreted. Results Compared with the control group,DAI score of model group increased significantly(P < 0.001),and the body weight significantly decreased(P < 0.001). The counts of WBC,RBC,Lymph and Gran of model group rats were significantly increased(P <0.05,P < 0.01). Meanwhile,IL-6,TNF-α contents of model group rats increased significantly(P < 0.01,P <0.001). Different degree of edema, erosion, destroyed and disordered glands, fossae inflammation and abscess formation appeared in colon tissues of model group rats. Compared with model group, DAI scores of DMD and mesalazine groups rats significantly decreased(P < 0.05, P < 0.01, P < 0.001), and body weights increased significantly(P < 0.05,P < 0.01);WBC,RBC,Lymph and Gran counts were significantly decreased(P < 0.05,P < 0.01);and the contents of IL-6 and TNF-α significantly decreased(P < 0.01). Moreover,the colonic mucosa epithelial recovered gradually,and the morphological structure and intestinal structure tended to be normal. From the perspective of metabolomics,eight biomarkers in the serum of IBD rats were identified. DMD can up-regulate the contents of phenylacetylglycine, sphingomamine, phytosphingosine, xanthine, tryptophan and indole(P <0.05,P < 0.01),and down-regulate the contents of glycine and phenylalanine(P < 0.05,P < 0.01). Conclusion DMD has an obvious therapeutic effect on TNBS-ethanol-induced IBD rats,which may be related to improving the levels of endogenous metabolites in serum and then restoring the metabolism to be normal in vivo.
Objective To investigate the mechanism of Dahuang Mudan decoction(DMD)on the treatment of inflammatory bowel disease(IBD)by metabolomics approach. Methods The IBD model was induced by 2,4,6-trinitrobenzenesulfonic acid(TNBS)/ethanol(90 mg·kg~(-1)). SD rats were randomly divided into control group,model group,mesalazine group(430 mg·kg~(-1)) and DMD group(7.5 g·kg~(-1)),8 rats in each group,and were continuously given correspondent drugs by gavage once a day for 7 days. The general conditions of the rats were evaluated by measuring the body weight and disease activity index(DAI). The white blood cell counts(WBC),lymphocyte counts(Lymph),granulocyte counts(Gran)and red blood cell(RBC)counts were measured by automatic blood analyzer. The contents of inflammatory cytokines including IL-6 and TNF-α in serum were determined by ELISA. Pathological changes of colon tissues were observed by HE staining. Ultrahigh performance liquid chromatography-tandem quadrupole time-of flight mass spectrometry(UPLC/Q-TOF-MS)was used to establish the serum metabolic profile of rats,and analyze the changes of serum endogenous metabolites to identify the potential biomarkers. The biological significance of the treatments was then interpreted. Results Compared with the control group,DAI score of model group increased significantly(P < 0.001),and the body weight significantly decreased(P < 0.001). The counts of WBC,RBC,Lymph and Gran of model group rats were significantly increased(P <0.05,P < 0.01). Meanwhile,IL-6,TNF-α contents of model group rats increased significantly(P < 0.01,P <0.001). Different degree of edema, erosion, destroyed and disordered glands, fossae inflammation and abscess formation appeared in colon tissues of model group rats. Compared with model group, DAI scores of DMD and mesalazine groups rats significantly decreased(P < 0.05, P < 0.01, P < 0.001), and body weights increased significantly(P < 0.05,P < 0.01);WBC,RBC,Lymph and Gran counts were significantly decreased(P < 0.05,P < 0.01);and the contents of IL-6 and TNF-α significantly decreased(P < 0.01). Moreover,the colonic mucosa epithelial recovered gradually,and the morphological structure and intestinal structure tended to be normal. From the perspective of metabolomics,eight biomarkers in the serum of IBD rats were identified. DMD can up-regulate the contents of phenylacetylglycine, sphingomamine, phytosphingosine, xanthine, tryptophan and indole(P <0.05,P < 0.01),and down-regulate the contents of glycine and phenylalanine(P < 0.05,P < 0.01). Conclusion DMD has an obvious therapeutic effect on TNBS-ethanol-induced IBD rats,which may be related to improving the levels of endogenous metabolites in serum and then restoring the metabolism to be normal in vivo.
引文
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[14]SCHOLLBüRGI S,SCHROECKSNADEL S,JENNY M,et al.Chronic immune stimulation may cause moderate impairment of phenylalanine 4-hydroxylase[J].Pteridines,2011,22(1):120-125.
[15]SHON W J,LEE Y K,SHIN J H,et al.Severity of DSS-induced colitis is reduced in ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks[J].Sci Rep,2015,5:17305.
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[19]CLERC F,NOVOKMET M,DOTZ V,et al.Plasma N-Glycan signatures associate with features of inflammatory bowel diseases[J].Gastroenterology,2018,155(3):829-843.
[20]YU X,YANG G,JIANG H,et al.Patchouli oil ameliorates acute colitis:A targeted metabolite analysis of 2,4,6-trinitrobenzenesulfonic acid-induced rats[J].Exp Ther Med,2017,14(2):1184-1192.
[21]MAINES L W,FITZPATRICK L R,FRENCH K J,et al.Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase[J].Dig Dis Sci,2008,53(4):997-1012.
[22]DON-DONCOW N,ZHANG Y,MATUSKOVA H,et al.The emerging alliance of sphingosine-1-phosphate signaling and immune cells:From basic mechanisms to implications in hypertension[J].Br J Pharmacol,2018.DOI:10.1111/bph.14381.
[23]BALDISSERA M D,SOUZA C F,DOLESKI P H,et al.Xanthine oxidase activity exerts pro-oxidative and pro-inflammatory effects in serum of silver catfish fed with a diet contaminated with aflatoxin B1[J].J Fish Dis,2018,41(7):1153-1158.
[2]赵忠正.大黄牡丹汤对实验性结肠炎Treg细胞的影响[D].广州:广州中医药大学,2011.
[3]温如燕,罗霞,郑彦懿,等.大黄牡丹汤对葡聚糖硫酸钠诱导的溃疡性结肠炎小鼠的治疗作用[J].中药新药与临床药理,2016,27(5):649-654.
[4]WANG X,CUI D N,DAI X M,et al.HuangQin Decoction attenuates CPT-11-induced gastrointestinal toxicity by regulating bile acids metabolism homeostasis[J].Front Pharmacol,2017,8:156.
[5]YIN S,GUO P,HAI D,et al.Optimization of GC/TOF MSanalysis conditions for assessing host-gut microbiota metabolic interactions:Chinese rhubarb alters fecal aromatic amino acids and phenol metabolism[J].Anal Chim Acta,2017,995:21-33.
[6]朱维娜,隆红艳.代谢组学在炎症性肠病研究中的应用[J].世界华人消化杂志,2015,23(13):2084-2090.
[7]KANG J,ZHU L,LU J,et al.Application of metabolomics in autoimmune diseases:insight into biomarkers and pathology[J].Journal of Neuroimmunology,2015,279:25-32.
[8]LIAN J S,LIU W,HAO S R,et al.A serum metabolomic analysis for diagnosis and biomarker discovery of primary biliary cirrhosis and autoimmune hepatitis[J].Hepatobiliary Pancreat Dis Int,2015,14(4):413-421.
[9]侯婉儿.基于LC-MS/MS技术的克罗恩病模型大鼠的靶标定量代谢组学研究[D].广州:广州中医药大学,2016.
[10]FENG Q,LIU Z,ZHONG S,et al.Integrated metabolomics and metagenomics analysis of plasma and urine identified microbial metabolites associated with coronary heart disease[J].Sci Rep,2016,6:22525.
[11]SCOVILLE E A,ALLAMAN M M,BROWN C T,et al.Alterations in lipid,amino acid,and energy metabolism distinguish crohn's disease from ulcerative colitis and control subjects by serum metabolomic profiling[J].Metabolomics,2018,14(1):S557.
[12]彭琳秀,陈良慧,狄留庆,等.基于UPLC/LTQ-Orbitrap-MS技术的复方祖师麻片抗类风湿关节炎的血浆代谢组学研究[J].中草药,2017,48(10):1964-1970.
[13]MADSEN R K,LUNDSTEDT T,GABRIELSSON J,et al.Diagnostic properties of metabolic perturbations in rheumatoid arthritis[J].Arthritis Res Ther,2011,13(1):R19.
[14]SCHOLLBüRGI S,SCHROECKSNADEL S,JENNY M,et al.Chronic immune stimulation may cause moderate impairment of phenylalanine 4-hydroxylase[J].Pteridines,2011,22(1):120-125.
[15]SHON W J,LEE Y K,SHIN J H,et al.Severity of DSS-induced colitis is reduced in ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks[J].Sci Rep,2015,5:17305.
[16]AGUS A,PLANCHAIS J,SOKOL H.Gut microbiota regulation of tryptophan metabolism in health and disease[J].Cell Host Microbe,2018,23(6):716-724.
[17]LEE J H,WOOD T K,LEE J.Roles of indole as an interspecies and interkingdom signaling molecule[J].Trends Microbiol,2015,23(11):707-718.
[18]GIACOMIN P,AGHA Z,LOUKAS A.Helminths and intestinal flora team up to improve gut health[J].Trends Parasitol,2016,32(9):664-666.
[19]CLERC F,NOVOKMET M,DOTZ V,et al.Plasma N-Glycan signatures associate with features of inflammatory bowel diseases[J].Gastroenterology,2018,155(3):829-843.
[20]YU X,YANG G,JIANG H,et al.Patchouli oil ameliorates acute colitis:A targeted metabolite analysis of 2,4,6-trinitrobenzenesulfonic acid-induced rats[J].Exp Ther Med,2017,14(2):1184-1192.
[21]MAINES L W,FITZPATRICK L R,FRENCH K J,et al.Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase[J].Dig Dis Sci,2008,53(4):997-1012.
[22]DON-DONCOW N,ZHANG Y,MATUSKOVA H,et al.The emerging alliance of sphingosine-1-phosphate signaling and immune cells:From basic mechanisms to implications in hypertension[J].Br J Pharmacol,2018.DOI:10.1111/bph.14381.
[23]BALDISSERA M D,SOUZA C F,DOLESKI P H,et al.Xanthine oxidase activity exerts pro-oxidative and pro-inflammatory effects in serum of silver catfish fed with a diet contaminated with aflatoxin B1[J].J Fish Dis,2018,41(7):1153-1158.