基于UPLC-Q-TOF/MS的脾虚湿困型溃疡性结肠炎大鼠造模及参苓白术散给药不同阶段的血浆代谢组学分析

Metabolomics Analysis of Plasma at Different Stages Based on UPLC-Q-TOF/MS:Modeling of Spleen Deficiency with Dampness Retention-ulcerative Colitis Rats and Administration of Shenling Baizhusan

目的:探讨脾虚湿困型溃疡性结肠炎(SDDR-UC)造模和给药不同阶段大鼠血浆中差异代谢物的变化,以及参苓白术散治疗UC的作用机制。方法:造模阶段将大鼠随机分为正常组、SDDR-UC组和单纯UC(P-UC)组。在给药阶段,对上述2个不同模型给予参苓白术散治疗。采用超高效液相色谱-四级杆-飞行时间质谱法(UPLC-Q-TOF/MS)对造模及给药结束后大鼠血浆进行检测,流动相为0.1%甲酸水溶液(A)-乙腈(B)梯度洗脱(正离子模式:0~2 min,99%A;2~9 min,99%~73%A;9~10 min,73%~44%A;10~13 min,44%~38%A;13~19 min,38%~28%A;19~21 min,28%~2%A;21~23 min,2%A;23~25 min,2%~10%A;25~27 min,10%~99%A。负离子模式:0~2 min,85%A;2~3 min,85%~65%A;3~5.5 min,65%~44%A;5.5~8 min,44%~25%A;8~10 min,25%~2%A;10~16 min,2%~85%A);电喷雾离子源,正、负离子模式检测,采集范围m/z 50~1000。通过偏最小二乘法-判别分析探索2个UC模型组大鼠在不同阶段潜在生物标志物的变化,对已鉴定的代谢物通过MetaboAnalyst 5.0进行代谢通路分析。结果:造模阶段鉴定出了16个潜在生物标志物,其中2个模型组大鼠共有11个潜在生物标志物,主要影响了初级胆汁酸生物合成通路。给药阶段筛选鉴定出23个潜在生物标志物,其中2个模型组共有3个潜在生物标志物,此外,SDDR-UC和P-UC模型大鼠中发生显著变化的潜在生物标志物还分别有11、9个,主要影响了嘌呤代谢、磷酸戊糖途径、嘧啶代谢、视黄醇代谢、初级胆汁酸生物合成、类固醇激素合成6条通路。结论:大鼠造模和给药阶段均出现了初级胆汁酸生物合成通路,呈现出动态变化过程,且参苓白术散对SDDR-UC大鼠的治疗作用可能与抑制核转录因子-κB(NF-κB)信号通路的表达、激活法尼醇X受体(FXR)、增强细胞色素P450的表达有关。

Objective:Based on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS),the changes of endogenous markers in rat plasma at the different stage,namely modeling and administration of Shenling Baizhusan(SLBZS),and the mechanism of SLBZS in the treatment of ulcerative colitis(UC)were studied.Method:In the modeling stage,rats were randomly divided into normal group,spleen deficiency with dampness retention-UC(SDDR-UC)and pure-UC(P-UC)model group.In the administration stage,SLBZS was given to the above two different model groups.After modeling and administration,rat plasma was collected and determined by UPLC-Q-TOF/MS.The mobile phase was 0.1%formic acid aqueous solution(A)-acetonitrile(B)for gradient elution(in positive ion mode:0-2 min,99%A;2-9 min,99%-73%A;9-10 min,73%-44%A;10-13 min,44%-38%A;13-19 min,38%-28%A;19-21 min,28%-2%A;21-23 min,2%A;23-25 min,2%-10%A;25-27 min,10%-99%A;in negative ion mode:0-2 min,85%A;2-3 min,85%-65%A;3-5.5 min,65%-44%A;5.5-8 min,44%-25%A;8-10 min,25%-2%A;10-16 min,2%-85%A).The electrospray ionization(ESI)temperature was 120 ℃ under the positive and negative ion modes,and the acquisition range was 50-1 000.Partial least squares-discriminant analysis(PLS-DA)was used to analyze the changes of endogenous metabolites in the above two different model rats from the different stage.MetaboAnalyst 5.0 was used to analyze the metabolic pathways of these identified metabolites.Result:Sixteen potential biomarkers were screened and identified in the modeling stage,among which 11 potential biomarkers were common in the two model rats,which mainly affected the primary bile acid biosynthesis pathway.Twenty-three potential biomarkers were screened and identified during the administration stage,among which 3 potential biomarkers were shared by the two model rats,and SDDR-UC and P-UC model rats had 11and 9 potential biomarkers,respectively.It mainly affected 6 pathways such as purine metabolism,pentose phosphate pathway,pyrimidine metabolism,retinol metabolism,primary bile acid biosynthesis and steroid hormone synthesis.Conclusion:The primary bile acid biosynthesis pathway appears in the different stage of modeling and administration of UC,showing a dynamic change process.The therapeutic effect of SLBZS on SDDR-UC rats may be related to inhibiting the expression of nuclear transcription factor-κB(NF-κB)signaling pathway,activating farnesoid X receptor(FXR)and enhancing the expression of cytochrome P450.