钩藤碱治疗炎症性肠炎可行性及机制研究

Analysis of target and mechanism of rhynchophylline in treatment of inflammatory bowel disease

目的应用网络药理学结合体内、外实验,探究钩藤碱治疗炎症性肠炎(inflammatory bowel disease,IBD)的可行性及机制。方法运用数据库获取钩藤碱-IBD交集靶点并进行GO和KEGG富集分析。通过分子对接筛选关键靶蛋白的结合情况。体内实验利用葡聚糖硫酸钠(DSS)诱导小鼠IBD模型,钩藤碱干预7 d,观察各组小鼠体征并进行疾病活动指数(DAI)评分;ELISA法检测小鼠结肠组织中白细胞介素-1β(IL-1β)、髓过氧化物酶(MPO)等炎症因子水平;检测各组小鼠肠道通透性。体外实验构建DSS诱导Caco2细胞炎症模型,明确钩藤碱对关键靶点的调控作用。结果共筛选到70个钩藤碱-IBD交集靶点,富集分析发现其与炎症反应过程、PI3K-Akt、Hippo信号通路相关;分子对接结果表明,钩藤碱与JAK2和JAK1结合最稳定。体内实验结果表明,与模型组比较,钩藤碱组体重、结肠长度及重量明显升高(P<0.05);DAI评分、结肠组织中IL-1β、MPO等炎症因子水平以及肠通透性明显降低(P<0.01)。体外实验结果表明,与模型组相比,钩藤碱组明显促进Caco2细胞增殖(P<0.05);同时显著降低细胞的TNF-α、IL-6及NO水平(P<0.05);Western blot结果表明,钩藤碱可以降低关键蛋白JAK2和JAK1的表达(P<0.05)。结论钩藤碱可能通过抑制JAK2和JAK1蛋白表达,减轻机体炎症反应,从而发挥治疗IBD的作用。

Aim To investigate the feasibility and mechanism of rhynchophylline in the treatment of inflammatory bowel disease(IBD)based on network pharmacology combined with in vivo and in vitro experiments.Methods The target of rhynchophylline-IBD intersection was obtained from the database,and GO and KEGG enrichment analysis were performed.The binding of key target proteins was screened by molecular docking.In vivo the IBD model of mice was induced by sodium dextran sulfate(DSS).After seven days of rhynchophylline intervention,the signs of mice in each group were observed and DAI scores were recorded.The levels of interleukin-1β(IL-1β),myeloperoxidase(MPO)and other inflammatory factors in colon tissue of mice were detected by ELISA.The intestinal permeability of each group was detected.In vitro experiments were conducted to establish the inflammatory model of Caco2 cells induced by DSS,and to clarify the regulatory effect of leptosinine on key targets.Results A total of 70 rhynchophylline-IBD intersection targets were screened,and enrichment analysis showed that they were related to the inflammatory process,PI3K-Akt and Hippo signaling pathways.Molecular docking results showed that rhynchophylline was most stable in binding with JAK2 and JAK1.In vivo experiment results showed that compared with model group,body weight,colon length and weight of rhynchophylline group significantly increased(P<0.05).DAI score,IL-1β,MPO and other inflammatory factors in colon tissue and intestinal permeability significantly decreased(P<0.01).In vitro experiment results showed that compared with model group,rhynchophylline group significantly promoted the proliferation of Caco2 cells(P<0.05).The levels of IL-6 and NO were significantly reduced(P<0.05).Western blot results showed that rhynchophylline could decrease the expressions of JAK2 and JAK1(P<0.05).Conclusion Rhynchophylline may play a role in the treatment of IBD by inhibiting the expression of JAK2 and JAK1 proteins and reducing inflammatory response in body.