基于胆汁酸调控“肠-肝”对话探讨乳香醋炙对治疗溃疡性结肠炎的增效作用机制

Exploring Synergistic Mechanism of Vinegar-processed Olibanum in Treatment of Ulcerative Colitis Based on Bile Acid-regulated"Gut-liver"Crosstalk

目的:通过比较乳香醋炙前后对胆汁酸的干预作用,从胆汁酸调控“肠-肝”对话的角度探讨乳香醋炙对治疗溃疡性结肠炎(UC)的增效作用机制。方法:随机将24只SD大鼠分为正常组、模型组、乳香组和醋乳香组,采用2,4,6-三硝基苯磺酸(TNBS)结肠内灌注建立UC大鼠模型;利用超高效液相色谱-三重四级杆-串联质谱法(UPLC-QQQ-MS)测定大鼠结肠中30种胆汁酸含量;采用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)从mRNA和蛋白水平检测大鼠“肠-肝”对话分子法尼酯衍生物X受体(FXR)、成纤维细胞生长因子(FGF)15和FGF受体4(FGFR4)的表达变化;借助结合型胆汁酸干预的HcoEpiC细胞模型,模拟UC状态,并根据干预方式的不同,分为空白组、结合型胆汁酸组、乳香组、醋乳香组及主要差异成分3-O-乙酰基-9,11-去氢-β-乳香酸(ADHBA)组,基于Real-time PCR探究乳香醋炙前后及ADHBA对FXR和FGF19 mRNA表达的影响。结果:与正常组比较,模型组大鼠结合型胆汁酸总量显著上升(P<0.01),“肠-肝”对话因子FXR、FGF15和FGFR4的mRNA和蛋白表达均明显降低(P<0.05,P<0.01);与模型组比较,乳香和醋乳香组大鼠结合型胆汁酸总量显著下降(P<0.01),FXR、FGF15和FGFR4的mRNA和蛋白表达均明显升高(P<0.05,P<0.01),醋乳香调节强度优于乳香。在细胞实验中,与空白组比较,结合型胆汁酸组HcoEpiC细胞FXR和FGF19的mRNA表达显著降低(P<0.01);与结合型胆汁酸组比较,乳香组、醋乳香组及ADHBA组FXR和FGF19的mRNA表达明显升高(P<0.05,P<0.01),醋乳香效果更优。结论:乳香醋炙后,可能通过加剧UC大鼠结肠中结合型胆汁酸水平的下降,进一步增强FXR-FGF15/19-FGFR4“肠-肝”对话因子表达量的上调,从而增强对UC的治疗作用,醋炙前后差异成分ADHBA可能是增效的主要物质基础。

Objective:To investigate the synergistic mechanism of vinegar-processed Olibanum on ulcerative colitis(UC)via the bile acids regulating"gut-liver"crosstalk.Method:Rats were randomly divided into normal group,model group,Olibanum group and vinegar-processed Olibanum group.UC model of rats was induced by intracolonic instillation of 2,4,6-trinitrobenzenesulfonic acid(TNBS).Ultra high performance liquid chromatography-triple quadrupole-mass spectrometry(UPLC-QQQ-MS)was used to perform the qualitative analysis of 30 bile acids in the colon of rats.Real-time fluorescence quantitative polymerase chain reaction(Realtime PCR)and Western blot were used to detect changes in the expression of farnesoid X receptor(FXR),fibroblast growth factor 15(FGF15)and FGF receptor 4(FGFR4)in"gut-liver"crosstalk at mRNA and protein levels.And with the help of HcoEpiC cell model intervened by conjugated bile acids,simulating the UC state,and according to the different modes of intervention,they were divided into the blank group,conjugated bile acid group,Olibanum group,vinegar-processed Olibanum group and 3-O-acetyl-9,11-dehydro-β-boswellic acid(ADHBA)group.The effect of Olibanum before and after processing with vinegar and the main differential component ADHBA on the mRNA expression of FXR and FGF19 were explored by Real-time PCR.Result:Compared with the normal group,the levels of conjugated bile acids in the model group increased significantly(P<0.01),and the mRNA and protein expressions of"gut-liver"crosstalk factors FXR,FGF15 and FGFR4 decreased significantly(P<0.05,P<0.01).Compared with the model group,the content of conjugated bile acids in the Olibanum group and vinegar-processed Olibanum group was significantly decreased(P<0.01),the mRNA and protein expressions of FXR,FGF15 and FGFR4 were significantly elevated(P<0.05,P<0.01),and vinegarprocessed Olibanum exhibited superior effects than Olibanum.In cellular experiments,a significant decrease in mRNA expression of FXR and FGF19 was observed in the conjugated bile acid group when compared with the blank group(P<0.01).Compared with the conjugated bile acid group,the mRNA expressions of FXR and FGF19 were significantly higher in the Olibanum,vinegar-processed Olibanum and ADHBA groups(P<0.05,P<0.01),and the effect of vinegar-processed Olibanum was more favorable.Conclusion:Vinegar-processed Olibanum may enhance the ameliorating effect on UC by enhancing the down-regulation of conjugated bile acids in the colon and the up-regulation of FXR-FGF15/19-FGFR4"gut-liver"crosstalk pathway,and ADHBA may be the main material basis for the synergy.