基于网络药理学与实验验证探讨茯苓对化疗性肠黏膜炎小鼠的作用及机制

Exploration of Effect and Mechanism of Poria Cocos on Chemotherapy Induced Intestinal Mucosal Inflammation in Mice Based on Network Pharmacology and Experimental Verification

目的观察茯苓对5-氟尿嘧啶(5-fluorouracil,5-FU)诱导的化疗性肠黏膜炎(chemotherapeutics-induced intestinal mucositis,CIM)模型小鼠的治疗作用,并结合网络药理学探讨其作用机制。方法通过TCMSP数据库获得茯苓的潜在成分与核心靶点;通过GeneCards数据库收集CIM相关基因靶点;使用Metascape数据库进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)富集分析。小鼠连续5 d腹腔注射5-FU诱导CIM模型;小鼠给予茯苓预防治疗5 d,边造模边给药治疗5 d,造模结束后治疗3 d,记录动物基本体征指标;苏木精-伊红染色(hematoxylin-eosin staining,HE)观察回肠黏膜组织病理损伤情况;免疫荧光染色观察回肠组织肠黏膜核因子(nuclear factor,NF)-κB p65、环氧化酶(cyclooxygenase,COX)-2与闭合蛋白(Occludin)的阳性表达情况。结果网络药理学结果显示,茯苓经筛选获得活性成分14个,茯苓治疗CIM的核心靶点182个;茯苓影响CIM作用机制可能是通过环磷酸腺苷(cyclic-AMP dependent protein kinase A,cAMP)信号通路、Toll样受体(toll-like receptor,TLR)信号通路等途径;动物实验结果显示,与模型组比较,柳氮磺胺吡啶组和茯苓各剂量组小鼠体质量和食物消耗量上升,结肠长度和脾脏指数增加(P<0.05);组织水平上,柳氮磺胺吡啶组和茯苓各剂量组小鼠的回肠绒毛长度与杯状细胞数增加,柳氮磺胺吡啶组和茯苓中剂量组隐窝深度减小(P<0.05);蛋白表达上,茯苓中、高剂量组和柳氮磺胺吡啶组小鼠回肠NF-κB p65、COX-2表达明显减少,Occludin的表达明显增加(P<0.01)。结论茯苓对5-FU诱导的小鼠CIM有明显的治疗作用,其机制与抑制TLR通路中NF-κB p65、COX-2蛋白表达,增加Occludin表达并提高机体免疫有关。

Objective To observe the therapeutic effect of Poria Cocos on the intestinal mucosa of 5-fluorouracil(5-FU)-induced chemotherapeutic intestinal mucositis(CIM)model mice,and explore its possible mechanism in combination with network pharmacology.Methods The potential components and core targets of Poria Cocos were obtained by TCMSP database.Gene targets related to CIM were collected through GeneCards database,and Cytoscape3.7.2 was used to replace the data related to Poria Cocos to map the"drug-actives-target-disease"network.The Metascape database was substituted into the intersection target for GO enrichment analysis and KEGG pathway analysis.The mice were given prophylactic treatment with Poria Cocos for 5 days,followed by drug administration for 5 days,and the basic signs of the animals were recorded after treatment for 3 days.The pathological injury of ileum mucosa was observed by HE staining.The positive expression of nuclear factor(NF)-κB p65,cyclooxygenase(COX-2)and Occludin in intestinal mucosa of ileal tissue was observed by immunofluorescence staining.Results The results of network pharmacology showed that 14 active ingredients of Poria Cocos were selected by database and related literature,and 182 core targets of Poria Cocos for CIM were identified.The effect of Poria Cocos on CIM may be through cAMP signaling pathway and Toll-like receptor(TLR)signaling pathway.The results of animal experiments showed that compared with the model group,the body weight and food consumption,colon length and spleen index were increased in sulfasalazine and poria groups,and the differences were statistically significant(P<0.05).At the tissue level,the villi length of ileum in sulfasalazine group and Poria Cocos groups increased,the crypt depth of medium dose Poria Cocos group and sulfasalazine group decreased,and the goblet cell number increased significantly in sulfasalazine group and Poria Cocos groups,the difference was statistically significant(P<0.05).In terms of protein expression,the expression of NF-κB p65 and COX-2 in ileum of mice in medium and high dose groups and sulfasalazine group of Poria Cocos significantly decreased,and the expression of Occludin significantly increased,with statistical significance(P<0.01).Conclusion Poria Cocos has obvious therapeutic effect on 5-FU-induced CIM in mice,and its mechanism is related to inhibiting the expression of NF-κB p65 and COX-2 proteins in TLR pathway,increasing the expression of Occludin and improving immunity.