基于网络药理学和体内实验研究桂枝解热的作用机制

Antipyretic effect of Guizhi based on network pharmacology and in vivo experiments and its mechanism

目的:运用生物信息技术分析探讨广西“桂十味”之一桂枝解热的关键靶点及相关信号通路作用机制,同时采用动物实验加以验证。方法:利用TCMSP筛选桂枝的主要活性成分,PharmMapper反向对接预测活性成分的作用靶点;通过Gene Cards、CTD和OMIM数据库预测发热相关靶点;通过交集映射得到桂枝和发热的共同靶点,利用Cytoscape 3.8.0软件建立调控网络和蛋白互作网络;对交集靶点进行GO生物功能注释和KEGG通路富集分析。最后通过整体动物、生化、ELISA法验证网络药理学富集分析结果。结果:筛选出桂枝8个活性成分及其228个预测靶点,发热相关靶点12 882个,共同靶点213个,包括ALB、AKT1、SRC、EGFR、HSP90AA1等,KEGG分析结果富集出花生四烯酸代谢信号通路、PPAR信号通路、NF-κB信号通路等。验证性实验结果表明,桂枝对酵母诱导发热6~8 h大鼠的体温变化值(△T)、最大发热净增值(△Tmax)及8 h体温反应指数(TRI8h)降低(均P<0.05);呈剂量依赖性显著降低因酵母诱导发热大鼠血清中IL-1β、TNF-α、IL-6和PGE2等外周内源性致热致炎因子水平异常升高(P<0.01);高剂量桂枝能显著降低发热大鼠脑组织中IL-1β和PGE2等中枢内源性致热致炎因子水平异常升高(P<0.01),而中、低剂量桂枝仅能明显降低异常升高的IL-1β水平(均P<0.05),对异常升高的PGE2无显著影响(P>0.05);高、中剂量桂枝能显著降低发热大鼠脑组织中COX-1、COX-2、PGDH1/15-PGDH活性异常升高(均P<0.05),而低剂量桂枝能明显降低异常升高的COX-1、COX-2活性(均P<0.05),对异常升高的PGDH1/15-PGDH活性无显著影响(P>0.05)。结论:桂枝对酵母诱导的发热具有一定解热作用,其作用机制与其减弱花生四烯酸-PGE2合成和分解途径中COX-1、COX-2、PGDH1/15-PGDH等关键酶的活性,以及降低中枢和外周的IL-1β、TNF-α、IL-6和PGE2水平有关。

Objective: To analyze the key target and related signal pathway mechanism of antipyretic of Guizhi,one of the“ten herbs of Guangxi”by bioinformatics, and verify them by animal experiments. Methods: TCMSP was used to screen the main active components of Guizhi, and PharmMapper reverse docking was used to predict the target of the active components. The febrile related targets were predicted by the Gene Cards, CTD and OMIM databases. The common targets of Guizhi and febrile were obtained by intersection mapping, and the regulatory network and protein interaction network were established using Cytoscape 3.8.0 software. GO biological function annotation and KEGG pathway enrichment analysis were performed on intersection targets. Finally, the results of network pharmacology enrichment analysis were verified by the whole animal, biochemical and ELISA assays. Results: Eight active components of Guizhi and their 228 predicted targets as well as 12,882 fever-related targets were screened out. There were 213 common genes between the targets of Guizhi and febrile, including ALB, AKT1, SRC, EGFR, HSP90AA1, etc,which were enriched into the arachidonic acid metabolic signaling pathway, PPAR signaling pathway and NF-κB signal pathway, etc, as was indicated by the KEGG analysis. The results of confirmatory experiments showed that Guizhi could alleviate febrile rats induced by yeast for 6-8 h, which was evidenced by a significant decrease in the changes of body temperature( △ T), the maximum net increment of febrile( △ Tmax) and the body temperature response index(TRI8h)(all P<0.05). Guizhi administration significantly decreased the yeast-induced high levels of the peripheral endogenous pyrogen and inflammatory factors, including IL-1β, TNF-α, IL-6,PGE2in serum of rats in a dose-dependent manner(P<0.01). High dose could significantly reduce the abnormal levels of central endogenous thermogenic factors such as IL-1β and PGE2in the brain tissue of febrile rats(P<0.01), while medium and low dose of Guizhi could only significantly reduce the abnormal levels of IL-1β(all P<0.05), and did not exert a significant effect on PGE2(P>0.05);the high and medium doses of Guizhi could significantly decrease COX-1, COX-2 and PGDH1/15-PGDH activities in the brain tissue of febrile rats(all P<0.05),while the low dose of Guizhi could significantly reduce COX-1 and COX-2 activities(all P<0.05), and had no significant effect on abnormally increased PGDH1/15-PGDH activities(P>0.05). Conclusion: Guizhi has a certain antipyretic effect on yeast-induced febrile;its mechanism is related to the weakened activity of key enzymes such as COX-1, COX-2, PGDH1/15-PGDH in the arachidonic acid-pGE2synthesis and decomposition pathway,and the reduction of the levels of IL-1β, TNF-α, IL-6 and PGE2in central and peripheral areas.