小檗碱与6-姜烯酚配伍对溃疡性结肠炎小鼠肠道炎症和菌群影响的实验研究

Experimental study on effects of berberine combined with 6-shogaol on intestinal inflammation and flora in mice with ulcerative colitis

寒热并用法是溃疡性结肠炎临床治疗的常用治法,黄连-干姜是体现寒热并用法的经典药对,该研究以黄连的有效成分小檗碱和干姜的有效成分6-姜烯酚联合应用,探究其对溃疡性结肠炎小鼠肠道炎症和肠道菌群的影响,揭示寒热并用法治疗溃疡性结肠炎的作用和机制。采用葡聚糖硫酸钠自由饮用诱导小鼠溃疡性结肠炎模型。分别给予小檗碱(100 mg·kg^(-1))、6-姜烯酚(100 mg·kg^(-1))、小檗碱(50 mg·kg^(-1))联合6-姜烯酚(50 mg·kg^(-1))灌胃,每日1次,给药20 d后取小鼠血清、结肠组织、粪便,苏木素-伊红染色法观察结肠病理组织学变化,阿利新蓝和过碘酸雪夫染色观察结肠组织黏液层变化,酶联免疫吸附法检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)含量,免疫组织化学法检测结肠组织巨噬细胞表面标记物F4/80、黏蛋白-2(mucin-2)、闭合蛋白-1(claudin-1)、带状闭合蛋白-1(zonula occludens-1,ZO-1)蛋白表达的变化,Meta扩增子文库法高通量测序技术检测小鼠肠道菌群的变化。结果显示,6-姜烯酚组、小檗碱组、6-姜烯酚联合小檗碱组小鼠肠道损伤明显减轻,结肠组织F4/80标记的阳性巨噬细胞数量减少,mucin-2蛋白表达升高,claudin-1、ZO-1表达升高,血清炎症因子TNF-α、IL-1β、IL-6含量显著降低;6-姜烯酚组、小檗碱联合6-姜烯酚组小鼠肠道菌群Shannon、Simpson、Chao、Ace指数显著升高,小檗碱组小鼠肠道菌群Chao、Ace指数显著升高。肠道菌群测序生物信息学分析发现,给药后各治疗组疣微菌门、纲、目水平数目均显著减少,芽孢杆菌纲数目则逐渐增多,6-姜烯酚组、小檗碱联合6-姜烯酚组嗜黏蛋白阿克曼菌基本完全消失,但产酸芽孢杆菌仍旧大量存在。综上结果,6-姜烯酚、小檗碱均能够抑制肠道炎症,减少巨噬细胞浸润和活化,减轻肠道损伤,降低肠道通透性,改善菌群结构,促进肠道微生态平衡,小檗碱与6-姜烯酚联合应用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.