背景:Si-Ni-San(SNS),一种源自伤寒论的传统中药配方,根据数千年的临床实践,被认为在治疗炎症性肠病方面是有效的。然而,生物活性成分和潜在机制尚不清楚,需要进一步研究.
目的:本研究旨在评估效果,探讨SNS改善葡聚糖硫酸钠(DSS)诱导小鼠结肠炎模型溃疡性结肠炎(UC)及相关肝损伤的生物活性成分和潜在机制。
方法:通过疾病活动指数(DAI)评估SNS(1.5、3、6g/kg)对3%DSS诱导的急性小鼠结肠炎的影响,结肠长度,炎性细胞因子,苏木精-伊红(H&E)染色,紧密连接蛋白表达,ALT,AST,和氧化应激指标。HPLC-ESI-IT/TOFMS用于分析口服SNS后UC小鼠结肠中SNS的化学成分和主要的外源性物质。然后基于主要的外源性物质进行网络药理学研究。流式细胞术和免疫组织化学技术用于证明SNS对Th17细胞分化的抑制作用和Th17/Treg细胞失衡的改善。LC-MS/MS,实时定量聚合酶链反应(RT-qPCR),和蛋白质印迹技术用于研究结肠中氧固醇-肝X受体(LXRs)信号传导活性。通过靶向胆汁酸代谢组学研究揭示了肝脏胆汁酸合成的两条主要途径的变化,并通过RT-qPCR和Western印迹技术表征胆汁酸合成关键代谢酶的表达。
结果:SNS(1.5、3、6g/kg)降低了DAI评分,保护肠粘膜屏障,抑制促炎细胞因子的产生,以剂量依赖性方式改善肝和脾肿大并减轻肝损伤。在SNS(6g/kg)治疗的结肠炎小鼠的结肠中总共鉴定出22种成分,按相对含量排名前10位的成分被视为SNS的潜在有效化学成分,并用于进行网络药理学研究。SNS的功效是由Th17细胞分化的减少介导的,结肠和脾脏中Th17/Treg细胞稳态的恢复,实验结果与我们的假设和网络药理学预测的生物学机制一致。机械上,SNS通过上调CH25H调节25-OHC和27-OHC的浓度,CYP27A1蛋白在结肠中的表达,从而影响LXR的表达和活性,最终影响Th17分化和Th17/Treg平衡。还发现SNS抑制了UC小鼠肝脏胆固醇的增加,并逆转了BA合成向酸性途径的转变,降低了总胆汁酸(TBA)中非12-OHBAs的比例,并进一步改善了结肠炎和伴随的肝损伤。
结论:这项研究为将SNS视为一种多器官受益的抗结肠炎处方奠定了基础,其具有改善肠道和肝脏损伤的显著效果,并揭示了胆固醇的衍生物,即氧固醇和胆汁酸,均与SNS抗结肠炎作用机制密切相关。
BACKGROUND: Si-Ni-San (SNS), a traditional Chinese medicinal formula derived from Treatise on Febrile Diseases, is considered effective in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice. However, the bioactive ingredients and underlying mechanisms are still unclear and need further investigation.
OBJECTIVE: This study aimed to evaluate the effect, explore the bioactive ingredients and the underlying mechanisms of SNS in ameliorating ulcerative colitis (UC) and associated liver injury in dextran sodium sulphate (DSS)-induced mouse colitis models.
METHODS: The effect of SNS (1.5, 3, 6 g/kg) on 3% DSS-induced acute murine colitis was evaluated by disease activity index (DAI), colon length, inflammatory cytokines, hematoxylin-eosin (H&E) staining, tight junction proteins expression, ALT, AST, and oxidative stress indicators. HPLC-ESI-IT/TOF MS was used to analyze the chemical components of SNS and the main xenobiotics in the colon of UC mice after oral administration of SNS. Network pharmacological study was then conducted based on the main xenobiotics. Flow cytometry and immunohistochemistry techniques were used to demonstrate the inhibitory effect of SNS on Th17 cells differentiation and the amelioration of Th17/Treg cell imbalance. LC-MS/MS, Real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting techniques were performed to investigate the oxysterol-Liver X receptor (LXRs) signaling activity in colon. Targeted bile acids metabolomics was conducted to reveal the change of the two major pathways of bile acid synthesis in the liver, and the expression of key metabolic enzymes of bile acids synthesis was characterized by RT-qPCR and western blotting techniques.
RESULTS: SNS (1.5, 3, 6 g/kg) decreased the DAI scores, protected intestinal mucosa barrier, suppressed the production of pro-inflammatory cytokines, improved hepatic and splenic enlargement and alleviated liver injury in a dose-dependent manner. A total of 22 components were identified in the colon of SNS (6 g/kg) treated colitis mice, and the top 10 components ranked by relative content were regarded as the potential effective chemical components of SNS, and used to conduct network pharmacology research. The efficacy of SNS was mediated by a reduction of Th17 cell differentiation, restoration of Th17/Treg cell homeostasis in the colon and spleen, and the experimental results were consistent with our hypothesis and the biological mechanism predicted by network pharmacology. Mechanistically, SNS regulated the concentration of 25-OHC and 27-OHC by up-regulated CH25H, CYP27A1 protein expression in colon, thus affected the expression and activity of LXR, ultimately impacted Th17 differentiation and Th17/Treg balance. It was also found that SNS repressed the increase of hepatic cholesterol and reversed the shift of BA synthesis to the acidic pathway in UC mice, which decreased the proportion of non-12-OH BAs in total bile acids (TBAs) and further ameliorated colitis and concomitant liver injury.
CONCLUSIONS: This study set the stage for considering SNS as a multi-organ benefited anti-colitis prescription based on the significant effect of ameliorating intestinal and liver damage, and revealed that derivatives of cholesterol, namely
oxysterols and bile acids, were closely involved in the mechanism of SNS anti-colitis effect.