冷热结合是治疗溃疡性结肠炎的常用方法,以经典药物对为代表,黄连和干姜。本研究探讨了小檗碱和6-shogaol的协同作用,黄连和干姜的主要成分,分别,对溃疡性结肠炎小鼠肠道炎症及肠道菌群的影响,揭示冷热结合治疗溃疡性结肠炎的作用及机制。用葡聚糖硫酸钠(DSS)诱导小鼠溃疡性结肠炎模型。模型小鼠给予小檗碱(100mg·kg~(-1)),6-shogaol(100mg·kg〜(-1)),小檗碱(50mg·kg~(-1))与6-shogaol(50mg·kg~(-1))联合灌胃,每天一次。给药20天后,小鼠血清,结肠组织,和粪便取样。采用苏木精-伊红(HE)染色观察结肠组织病理学改变。采用阿尔辛蓝/高碘酸-希夫(AB/PAS)染色观察结肠组织粘液层的变化。采用酶联免疫吸附试验(ELISA)检测血清肿瘤坏死因子-α(TNF-α)含量,白细胞介素-1β(IL-1β),和白细胞介素-6(IL-6)。采用免疫组织化学方法检测结肠组织中巨噬细胞表面标志物F4/80、黏蛋白-2、claudin-1和闭塞带-1(ZO-1)的蛋白表达。采用高通量Meta-扩增子文库测序技术检测小鼠肠道菌群的变化。结果表明,6-shogaol组,小檗碱组,联合组肠损伤明显减轻,结肠组织中F4/80标记的阳性巨噬细胞数量减少,粘蛋白-2,claudin-1和ZO-1的蛋白表达增加,TNF-α的血清水平降低,IL-1β,IL-6香农,辛普森,Chao,6-shogaol组和联合组小鼠肠道菌群Ace指数显著升高,小檗碱组的Chao和Ace指数显著升高。肠道菌群测序的生物信息学分析显示,门的Verrucomicrobia的相对丰度,类,用药后,所有治疗组的订单水平均显着下降,而杆菌逐渐增加。在6-shogaol组和组合组中,Akkermansiamuciniphila完全消失了,但是产酸杆菌仍然大量存在。作为结论,6-shogaol和小檗碱都能抑制肠道炎症,减少巨噬细胞的浸润和活化,缓解肠道损伤,减少肠道通透性,改善植物的结构,促进肠道微生态平衡。黄连素与6-肖果酚联合应用具有显著的协同增效作用。
Cold-heat combination is a common method in the treatment of ulcerative colitis, which is represented by classic drug pair, Coptidis Rhizoma and Zingiberis Rhizoma.The present
study explored the synergetic effects of berberine and 6-shogaol, the primary components of Coptidis Rhizoma and Zingiberis Rhizoma, respectively, on intestinal inflammation and intestinal flora in mice with ulcerative colitis to reveal the effect and mechanism of cold-heat combination in the treatment of ulcerative colitis.The ulcerative colitis model was induced by dextran sulfate sodium(DSS) in mice.The model mice were administered with berberine(100 mg·kg~(-1)), 6-shogaol(100 mg·kg~(-1)), and berberine(50 mg·kg~(-1)) combined 6-shogaol(50 mg·kg~(-1)) by gavage, once per day.After 20 days of drug administration, mouse serum, colon tissues, and feces were sampled.Hematoxylin-eosin(HE) staining was used to observe histopathological changes in colon tissues.Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to observe the changes in the mucus layer of colon tissues.Enzyme-linked immunosorbent assay(ELISA) was employed to detect the serum content of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6).Immunohistochemical method was adopted to detect the protein expression of macrophage surface markers F4/80, mucin-2, claudin-1, and zonula occludens-1(ZO-1) in colon tissues.High-throughput Meta-amplicon library sequencing was used to detect changes in the intestinal flora of mice.The results indicated that the 6-shogaol group, the berberine group, and the combination group showed significantly relieved intestinal injury, reduced number of F4/80-labeled positive macrophages in colon tissues, increased protein expression of mucin-2, claudin-1, and ZO-1, and decreased serum le-vels of TNF-α, IL-1β, and IL-6.Shannon, Simpson, Chao, and Ace indexes of the intestinal flora of mice in the 6-shogaol group and the combination group significantly increased, and Chao and Ace indexes in the berberine group significantly increased.As revealed by the bioinformatics analysis of intestinal flora sequencing, the relative abundance of Verrucomicrobia at the phylum, class, and order levels decreased significantly in all treatment groups after drug administration, while that of Bacillibacteria gradually increased.In the 6-shogaol group and the combination group, Akkermansia muciniphila completely disappeared, but acid-producing bacillus still existed in large quantities.As concluded, both 6-shogaol and berberine can inhibit intestinal inflammation, reduce the infiltration and activation of macrophages, relieve intestinal damage, reduce intestinal permeability, improve the structure of flora, and promote intestinal microecological balance.The combined application of berberine and 6-shogaol has a significant synergistic effect.