基于血清代谢组学探究色胺酮抗小鼠溃疡性结肠炎的作用机制

Mechanism of tryptanthrin in treatment of ulcerative colitis in mice based on serum metabolomics

采用液相色谱-质谱(liquid chromatograph-mass spectrometer,LC-MS)联用的方法探究色胺酮对葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的溃疡性结肠炎(ulcerative colitis,UC)小鼠血清中潜在代谢生物标志物的影响,并分析其相关的代谢通路。随机将C57BL/6小鼠分为色胺酮组、柳氮磺胺吡啶组、对照组、模型组,采用自由饮用3%DSS 11 d的方式造模,边造模边给药,从第1天起观察小鼠体征,并进行疾病活动指数(disease activity index,DAI)评分;实验结束后取结肠组织,采用苏木素-伊红(hematoxylin-eosin,HE)染色观察结肠组织的病理变化;酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)试剂盒检测血清中白细胞介素-4(interleukin-4,IL-4)、白细胞介素-10(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-8(interleukin-8,IL-8)的水平;每组各收集6份小鼠血清进行广泛靶向代谢组学分析,并借助MetaboAnalyst 5.0数据库富集代谢通路。结果表明,与模型组相比,色胺酮组小鼠的DAI评分显著降低(P<0.05),结肠组织损伤减轻,炎性细胞浸润减少,血清中促炎因子水平下降,抑炎因子水平升高。代谢组学分析出28个差异代谢物,涉及3条代谢通路,包括嘌呤代谢、花生四烯酸代谢和色氨酸代谢。色胺酮干预后使DSS诱导的UC小鼠的代谢物向对照组方向回调,其治疗机制可能与嘌呤代谢、花生四烯酸代谢、色氨酸代谢有关。该研究利用代谢组学技术分析色胺酮治疗UC的作用机制,为色胺酮的利用与开发提供实验依据。

This study aims to explore the effect of tryptanthrin on potential metabolic biomarkers in the serum of mice with ulcerative colitis(UC)induced by dextran sulfate sodium(DSS)based on liquid chromatography-mass spectrometry(LC-MS)and predict the related metabolic pathways.C57BL/6 mice were randomly assigned into a tryptanthrin group,a sulfasalazine group,a control group,and a model group.The mouse model of UC was established by free drinking of 3%DSS solution for 11 days,and corresponding drugs were adminsitrated at the same time.The signs of mice were observed and the disease activity index(DAI)score was recorded from the first day.Colon tissue samples were collected after the experiment and observed by hematoxylin-eosin(HE)staining.The levels of interleukin-4(IL-4),interleukin-10(IL-10),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-8(IL-8)in the serum were measured by enzyme linked immunosorbent assay(ELISA).The serum samples were collected from 6 mice in each group for widely targeted metabolomics.The metabolic pathways were enriched by MetaboAnalyst 5.0.The results showed that compared with the model group,tryptanthrin treatment decreased the DAI score(P<0.05),alleviated the injury of the colon tissue and the infiltration of inflammatory cells,lowered the levels of proinflammatory cytokines,and elevated the levels of anti-inflammatory cytokines in the serum.The metabolomic analysis revealed 28 differential metabolites which were involved in 3 metabolic pathways including purine metabolism,arachidonic acid metabolism,and tryptophan metabolism.Tryptanthrin may restore the metabolism of the mice with UC induced by DSS to the normal level by regulating the purine metabolism,arachidonic acid metabolism,and tryptophan metabolism.This study employed metabolomics to analyze the mechanism of tryptanthrin in the treatment of UC,providing an experimental basis for the utilization and development of tryptanthrin.