痛泻要方对腹泻型肠易激综合征大鼠粪便代谢组学的影响

Effect of Prescription of Importance for Diarrhea with Pain on fecal metabonomics of rats with diarrhea-predominant irritable bowel syndrome

目的探索痛泻要方对腹泻型肠易激综合征(IBS-D)肝郁脾虚证大鼠粪便中内源性代谢产物的影响,以寻找潜在的生物标志物及相关代谢通路。方法将40只大鼠随机分为4组,每组10只。除空白组外,其余组大鼠采用慢性束缚应激加番泻叶灌服法建立IBS-D肝郁脾虚证模型。造模成功后,空白组及模型组大鼠给予1 mL/100 g蒸馏水灌胃,痛泻要方组及匹维溴铵组给予1 mL/100 g相应混悬液(0.675 g/mL、1.35 mg/mL)灌胃,均每日1次,连续灌胃14 d。运用LC-MS/MS技术检测各组大鼠粪便中内源性代谢产物,对差异代谢物进行KEGG通路富集分析及MetPA分析。结果模型组与空白组中差异代谢物共47种,痛泻要方组与模型组中差异代谢物共31种,匹维溴铵组与模型组差异代谢物共25种。其中Deoxycholic acid(脱氧胆酸)、Gamma-Tocotrienol(γ-生育三烯醇)、Carboxyifosfamide(羧异环磷酰胺)、25-Acetylvulgaroside(25-乙酰基葡萄糖苷)、N’-Formylkynurenine(N’-甲酰犬尿氨酸)和Hypoxanthine(次黄嘌呤)等代谢物经痛泻要方干预后明显改善。差异代谢物KEGG通路富集分析结果显示痛泻要方富集的通路有13条,Purine metabolism(嘌呤代谢)、Cholesterol metabolism(胆固醇代谢)、Caffeine metabolism(咖啡因代谢)、Arginine biosynthesis(精氨酸生物合成)和Vitamin B_(6) metabolism(维生素B_(6)代谢)富集率差异均有统计学意义(P均<0.05)。MetPA分析显示这些差异代谢物与精氨酸生物合成、色氨酸代谢、次级胆汁酸生物合成、泛醌和其他萜类醌生物合成、胆汁分泌、嘌呤代谢等多种代谢途径相关。结论痛泻要方对IBS-D肝郁脾虚型大鼠的作用可能是通过调节脱氧胆酸、γ-生育三烯醇、羧异环磷酰胺、25-乙酰基葡萄糖苷、次黄嘌呤、黄嘌呤等代谢物质水平,影响精氨酸生物合成、色氨酸代谢、次级胆汁酸生物合成、泛醌和其他萜类醌生物合成、胆汁分泌、嘌呤代谢等相关代谢途径实现的。

Objective It is to explore the effects of Prescription of Importance for Diarrhea with Pain(PIDP)on endogenous metabolites in the feces of rats with diarrhea-predominant irritable bowel syndrome(IBS-D)with syndrome of liver depression and spleen deficiency,in order to search for potential biomarkers and related metabolic pathways.Methods Forty rats were randomly divided into 4 groups,10 rats in each group.Except for the blank group,the rats in the other groups were used to establish the models of IBS-D with syndrome of liver-depression and spleen-deficiency by chronic restraint stress plus senna leaf by gavage.After successful modeling,the rats in the blank group and model group were given 1 mL/100 g distilled water by gavage,and the rats in the PIDP group and pinaverium bromide group were given 1 mL/100 g corresponding suspension(0.675 g/mL,1.35 mg/mL)by gavage,all once a day,continuously gavaged for 14 days.The endogenous metabolites in the feces of rats in each group were detected by LC-MS/MS technique,and the differential metabolites were analyzed by KEGG pathway enrichment analysis and MetPA analysis.Results There were 47 kinds of differential metabolites in the model group and blank group,31 kinds of differential metabolites in the PIDP group and model group,and 25 kinds of differential metabolites in the pinaverium bromide group and model group.The metabolites such as Deoxycholic acid,gamma-tocotrienol,Carboxyifosfamide,25-Acetylvulgaroside,N’-Formylkynurenine and Hypoxanthine were significantly improved after treatment with PIDP.The enrichment analysis of differential metabolite KEGG pathway showed that there were 13 pathways for PIDP enrichment,and the enrichment rate of Purine metabolism,Cholesterol metabolism,Caffeine metabolism,Arginine biosynthesis and Vitamin B6 metabolism were statistically different(all P<0.05).Further MetPA analysis showed that these differential metabolites were related to arginine biosynthesis,tryptophan metabolism,secondary bile acid biosynthesis,ubiquinone and other terpenoid quinones biosynthesis,bile secretion,purine metabolism and other metabolic pathways.Conclusion The mechanism of Prescription of Importance for Diarrhea with Pain on IBS-D rats with syndrome of liver depression and spleen deficiency may be achieved by regulating the levels of metabolites such as deoxycholic acid,γ-tocotrienol,carboxyifosfamide,25-acetyl glucoside,hypoxanthine,xanthine,influencing related metabolic pathways such as arginine biosynthesis,tryptophan metabolism,secondary bile acid biosynthesis,ubiquinone and other terpenoid quinones biosynthesis,bile secretion,purine metabolism and so on.