利用广泛靶标代谢组学表征熊胆粉对大鼠胆汁酸组的影响

Effects of bear bile powder on bile acids pool in rats by widely targeted metabolomics

目的:构建全面的胆汁酸组定量表征方法,探究熊胆粉对大鼠胆汁酸组的影响。方法:通过混合不同时间点的大鼠血浆样品构建模式样品,利用预测多反应离子监测模式结合高分辨质谱分析模式样品中的胆酸类成分,构建定制胆汁酸组检测方法。然后用该方法分析熊胆粉给药后72h内大鼠血浆中胆汁酸组含量变化,结合主成分分析找出显著变化的胆酸类成分,构建胆汁酸组"迁移路径",分析熊胆粉对大鼠胆汁酸组的影响。结果:通过构建裂解曲线获得胆汁酸磺酸化和葡萄糖醛酸化产物特征离子对的最佳碰撞能,从模式样品中初步鉴定出78种胆酸类成分。并发现熊胆粉给药后会引起胆汁酸组显著扰动,12h时变化最大,然后逐渐恢复至生理状态。结论:本研究构建了78种胆酸类成分的定量表征方法,发现熊胆粉可能通过纠正病理状态下受扰动的胆汁酸组而发挥疗效,为阐明熊胆粉利胆保肝的药效物质和作用机制奠定基础。

Objective:To propose a strategy enabling bile acids(BAs)-focused quantitative metabolomics and subsequently to lucubrate BA pool fluctuation trajectory in rats after dosing Bear bile powder(BBP).Methods:A so-called universal metabolome standard(UMS)sample containing numerous natural BAs was built by mixing rat plasma samples at different time points,in-depth chemical characterization was conducted for UMS to capture as many BAs as possible.Predictive multiple-reaction-monitoring(pMRM)with enhanced product ion(EPI)experiment was performed on Qtrap-MS for mining BAs,and IT-TOF-MS was deployed to provide high-resolution m/z values for precursor and fragment ion species.The validated quantitative program was then applied to pursue BA pool shift trajectories in 72 h in BBP-and vehicle-treated rats,the significantly changed BAs were found out combined with principal component analysis.Results:Optimum collision energy for certain BAs were obtained and a total of 78 bile acids were characterized in UMS.Mild variations were observed for the quantitative pattern of BA pool from vehicle group,whereas BBP could significantly enlarged the entire BA pool,and the pool showed recovery tendency until 12 h after gavage.Conclusion:In current study,a strategy allowing simultaneously quantitative analysis of as many as 78 BAs was proposed by successively conducting in-depth qualitative characterization and comprehensively quantitative analysis.And the therapeutic benefits of BBP should rely on their potentials of holistically rectifying the perturbed BA pool at pathological status.More importantly,the strategy offers a high-quality analytical choice for in-depth quantitative profiling of BA pool as well as for temporal BAs-targeted metabolomics.