基于血清代谢组学探讨三七总皂苷对阿司匹林致小肠损伤的预防作用

Investigation on Preventive Effect of Total Saponins of Notoginseng Radix et Rhizoma on Aspirin-induced Small Intestine Injury Based on Serum Metabolomics

摘要:

目的 利用代谢组学探究三七总皂苷（NTS）对阿司匹林（ASA）所致大鼠小肠损伤的预防作用。方法 雄性SD大鼠50只，随机分为正常组，模型组，NTS高、低剂量组（62.5、31.25 mg·kg^-1）及阳性药组（奥美拉唑2.08 mg·kg^-1+瑞巴派特31.25 mg·kg^-1），每组10只。除正常组外，各组大鼠每日给予ASA肠溶微丸10.41 mg·kg^-1建立小肠损伤模型。各给药组在此基础上每日分别灌胃相应剂量药物，正常组及模型组灌胃等体积饮用水。期间记录大鼠体质量变化情况、粪便性状变化情况并进行评分。给药14周后，取各组小肠组织进行苏木素-伊红（HE）染色，扫描电镜观察损伤情况，并对小肠表观损伤情况进行评分。取正常组、模型组及NTS高剂量组大鼠血清，采用超高效液相色谱-四级杆-静电场轨道阱高分辨质谱法（UPLC-Q-Exactive Orbitrap MS）进行代谢组学分析，并采用多元统计分析进行数据处理，以变量投影重要性（VIP）值≥1.0，差异倍数（FC）≥1.5或≤0.6且t检验P<0.05为标准筛选潜在生物标志物，结合人类代谢组数据库（HMDB）和京都基因与基因组百科全书（KEGG）对差异代谢物进行通路富集分析。结果 给药14周后，模型组平均体质量增量低于正常组，NTS高剂量组接近正常组；与正常组比较，模型组大鼠粪便性状评分显著增高（P<0.01），与模型组比较，阳性药组与NTS高剂量组评分降低，但差异无统计学意义；HE染色及扫描电镜结果显示，NTS能显著改善ASA所致的小肠损伤，与正常组比较，模型组小肠损伤评分显著升高（P<0.01），与模型组比较，NTS低、高剂量组小肠损伤评分明显降低（P<0.05，P<0.01）；血清代谢组学共筛选出正常组与模型组间差异代谢物75个，其中表达上调化合物55个，表达下调20个；模型组与NTS组间差异代谢物76个，其中表达上调化合物14个，表达下调62个；NTS可回调水杨酸、3-羟基苯甲酸与4-羟基苯甲酸3个差异代谢物，涉及胆汁分泌，叶酸生物合成，苯丙氨酸、酪氨酸和色氨酸的生物合成3条代谢通路。结论 NTS可预防ASA所致小肠损伤，经由血清代谢组学分析其潜在机制可能与调节大鼠胆汁分泌与氨基酸代谢途径有关。

Abstract:

Objective Metabolomics was utilized to investigate the preventive effect of notoginseng total saponins（NTS） on aspirin（acetyl salicylic acid， ASA）-induced small bowel injury in rats.Method Fifty male SD rats were randomly divided into normal and model groups， NTS high-dose and low-dose groups（62.5， 31.25 mg·kg^-1）， and positive drug group（omeprazole 2.08 mg·kg^-1+rebamipide 31.25 mg·kg^-1）， with 10 rats in each group. Except for the normal group， rats in other groups were given ASA enteric-coated pellets 10.41 mg·kg^-1 daily to establish a small intestine injury model. On this basis， each medication group was gavaged daily with the corresponding dose of drug， and the normal group and the model group were gavaged with an equal amount of drinking water. Changes in body mass and fecal characteristics of rats were recorded and scored during the period. After 14 weeks of administration， small intestinal tissues of each group were taken for hematoxylin-eosin（HE） staining， scanning electron microscopy to observe the damage， and the apparent damage of small intestine was scored. Serum from rats in the normal group， the model group， and the NTS high-dose group was taken and analyzed for metabolomics by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS）， and the data were processed by multivariate statistical analysis， the potential biomarkers were screened by variable importance in the projection（VIP） value≥1.0， fold change（FC）≥1.5 or ≤0.6 and t-test P<0.05， and pathway enrichment analysis of differential metabolites was performed in conjunction with Human Metabolome Database（HMDB） and Kyoto Encyclopedia of Genes and Genomes（KEGG）.Result After 14 weeks of administration， the average body mass gain of the model group was lower than that of the normal group， and the NTS high-dose group was close to that of the normal group. Compared with the normal group， the fecal character score of rats in the model group was significantly increased（P<0.05）， and compared with the model group， the scores of the positive drug group and the NTS high-dose group were reduced， but the difference was not statistically significant. HE staining and scanning electron microscopy results showed that NTS could significantly improve ASA-induced small intestinal injury， compared with the normal group， the small bowel injury score of the model group was significantly increased（P<0.01）， compared with the model group， the small bowel injury scores of the NTS low and high dose groups were significantly reduced（P<0.05， P<0.01）. Serum metabolomics screened a total of 75 differential metabolites between the normal group and the model group， of which 55 were up-regulated and 20 were down-regulated， 76 differential metabolites between the model group and the NTS groups， of which 14 were up-regulated and 62 were down-regulated. NTS could modulate three differential metabolites（salicylic acid， 3-hydroxybenzoic acid and 4-hydroxybenzoic acid）， which were involved in 3 metabolic pathways， namely， the bile secretion， the biosynthesis of folic acid， and the biosynthesis of phenylalanine， tyrosine and tryptophan.Conclusion NTS can prevent ASA-induced small bowel injury， and the underlying mechanism may be related to the regulation of bile secretion and amino acid metabolic pathways in rats.