基于代谢组学的延胡索对DSS诱导溃疡性结肠炎的治疗作用及机制解析

Therapeutic effect of Rhizoma Corydalis on ulcerative colitis induced by dextran sodium sulfate and its mechanism： a study based on metabolomics

摘要

目的：探讨延胡索（Corydalis Rhizoma,CR）对葡聚糖硫酸钠（dextran sulfate,DSS）诱导的小鼠溃疡性结肠炎（ulcerative colitis,UC）模型的干预效果，通过代谢组学及炎症相关生物标志物检测，系统解析CR治疗UC的潜在机制。方法：构建小鼠UC模型，分为5组，模型组、高剂量组（1.517 g/kg生药）、中剂量组（0.986 g/kg生药）、低剂量组（0.455 g/kg生药）和阳性药物组（718.8 mg/kg,5-氨基水杨酸）。另设未造模小鼠作为空白组（灌胃0.9%NaCl）。各组小鼠连续给药7 d，动态监测实验动物体质量变化、疾病活动指数（disease activity index,DAI）、日均摄食量及饮水量等表型参数，7 d后处死动物，获取血清和结肠组织；ELISA法定量分析血清促炎因子白细胞介素-6(interleukin-6,IL-6)、白细胞介素-17A(interleukin-17A,IL-17A)、C-反应蛋白（C-reactive protein,CRP）及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平；通过超高效液相色谱-四极杆飞行时间质谱联用（ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry,UPLC-Q-TOF/MS）进行非靶向代谢组学分析，比对各组血清代谢物谱差异；小鼠相同方法造模并进行验证，阳性药物选择谷胱甘肽（glutathione,GSH），随后评估结肠长度和黏膜损伤，实时荧光定量PCR(quantitative real-time polymerase chain reaction,qPCR)检测结肠组织中GSH合成通路关键基因，即γ-谷氨酰半胱氨酸合成酶（γ-glutamylcysteine synthetase,γ-GCS）、氧化应激调控因子（yap1p、skn7）及线粒体GSH转运蛋白（Slc25a39）的mRNA相对表达量。结果：CR可明显改善模型动物质量减轻，DAI增加和结肠长度缩短等相关评测指标，且呈剂量依赖性；血清中IL-6、CRP和TNF-α水平降低，而IL-17A无明显影响；代谢组学分析显示，有21种潜在的与氨基酸和脂质代谢相关的生物标志物，在CR作用下明显调节；验证实验证实CR和GSH治疗对结肠黏膜损伤表现出明显保护作用，但不影响结肠长度；CR上调了GSH合成相关基因的表达，特别是γ-GCS，表明其可增强肠的抗氧化防御作用。结论：CR在DSS诱导的UC小鼠模型中具有治疗潜力，可缓解症状、降低血清炎症标志物和调节代谢途径；GSH合成相关基因的上调，提示药物可增强肠抗氧化防御。

Abstract

Objective To investigate the interventional effect of Rhizoma Corydalis on mice with ulcerative colitis（UC） induced by dextran sulfate sodium（DSS），as well as the potential mechanism of Rhizoma Corydalis in the treatment of UC based on metabolomics and inflammation biomarkers. Methods A mouse model of UC was established，and then the mice were divided into model group，high-dose group（1.517 g/kg crude drug），middle-dose group（0.986 g/kg crude drug），low-dose group（0.455 g/kg crude drug），and positive drug group（5-aminosalicylic acid at a dose of 718.8 mg/kg），while the mice without modeling were selected as normal group （0.9% NaCl by gavage）. The mice in each group were administered for 7 consecutive days，and phenotypic parameters were dynamically monitored，such as body weight change，disease activity index（DAI），mean daily food intake，and daily water intake. The mice were sacrificed after 7 days to collect serum and colon tissue samples； ELISA was used to measure the serum levels of the proinflammatory factors interleukin-6（IL-6），interleukin-17A（IL-17A），C-reactive protein（CRP），and tumor necrosis factor-α（TNF-α），and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF/MS） was used to perform the non-targeted metabolomics analysis and compare the differences in serum metabolite profiles between groups. The mice were selected for modeling and validation with the same method，and glutathione（GSH） was selected as the positive drug. Colon length and mucosal damage were assessed，and quantitative real-time PCR was used to measure the relative mRNA expression levels of the key genes in the glutathione synthesis pathway（γ-glutamylcysteine synthetase［γ-GCS］ and oxidative stress regulators yap1p and skn7） and mitochondrial GSH transporter protein（Slc25a39） in colonic tissue. Results Rhizoma Corydalis significantly improved weight loss，DAI，and colon length in a dose-dependent manner in the model animals，and there were reductions in the serum levels of IL-6，CRP，and TNF-α，while it had no significant effect on IL-17A. The metabolomics analysis revealed 21 potential biomarkers associated with amino acid and lipid metabolism，which were significantly regulated by Rhizoma Corydalis. In the verification experiment，both Rhizoma Corydalis and GSH exerted a significant protective effect against colonic mucosal damage without affecting colon length. Rhizoma Corydalis upregulated the expression of genes associated with glutathione synthesis，especially γ-GCS，suggesting that Rhizoma Corydalis could enhance intestinal antioxidant defenses. Conclusion Rhizoma Corydalis has a therapeutic potential in a mouse model of DSS-induced UC and can alleviate symptoms，reduce the serum levels of inflammatory markers，and regulate metabolic pathways，and upregulation of the genes associated with glutathione synthesis suggests that the drug can enhance intestinal antioxidant defenses.

Graphical abstract

关键词

延胡索 / 溃疡性结肠炎 / 代谢组学 / 谷胱甘肽代谢 / 炎症标志物

Key words

Rhizoma Corydalis / ulcerative colitis / metabolomics / glutathione metabolism / inflammatory markers

基金资助

成都中医药大学青基人才专项资助项目(QJRC202)

成都中医药大学青基人才专项资助项目(0012)