基于网络药理学和动物实验探究穿心莲内酯对5-Fu所致大鼠化疗性肠黏膜炎的预防作用

Mechanism of improvement effect of andrographolide on 5-Fu-induced chemotherapeutic intestinal mucositis in rats based on network pharmacology and animal experiment

目的基于网络药理学及动物实验探究穿心莲内酯对5-氟尿嘧啶(5-Fu)所致大鼠化疗性肠黏膜炎(CIM)的预防治疗机制,为其临床应用提供依据。方法检索Swiss Target Prediction、SuperPred、Genecards和OMIM等数据库,获得穿心莲内酯作用靶点及CIM疾病相关靶的交集并作为潜在靶点,通过String平台构建其蛋白-蛋白互作网络,利用R语言编程软件包对其进行GO功能及KEGG富集分析,Cytoscape分析其网络拓扑结构,最终筛选出核心靶点并进行分子对接验证。将雄性SD大鼠随机分为空白组、模型组、阳性对照组(金双歧三联活菌片,700 mg/kg)、穿心莲内酯低剂量组(30 mg/kg)、高剂量组(100 mg/kg)。各给药组分别灌胃给予对应药物,空白组和模型组灌胃给予相应体积生理盐水,持续给药10 d。给药第6天时,除空白组腹腔注射相应体积生理盐水外,其余各组分别注射5-Fu 30 mg/kg,持续5 d。每日观察记录大鼠体质量、腹泻情况;末次给药后第2天处死动物,摘取肝脏和脾脏,计算脏器指数;HE染色观察大鼠小肠组织病理变化;ELISA检测血清中TNF-α、IL-6炎症因子的水平。结果穿心莲内酯治疗CIM的潜在靶点有190个。在PPI网络中,筛出STAT3、TNF、IL6等20个核心靶点。GO富集分析得到生物学过程相关条目2315条、分子功能相关条目184条及细胞组分相关条目101条,主要涉及氧化应激反应、MAPK级联的正调控和细胞对化学应激的反应等。KEGG通路富集分析得到177条相关信号通路,主要涉及HIF-1、TNF、Toll样受体和T细胞受体信号通路等。分子对接结果表明穿心莲内酯与关键核心靶点STAT3、TNF、IL-6之间均具有较好的结合活性。动物实验结果显示,与空白组相比,模型组大鼠体质量下降,腹泻严重,脏器指数降低,肠道组织损害严重,血清TNF-α、IL-6水平显著升高;与模型组相比,穿心莲内酯高剂量组大鼠体质量下降率减少,腹泻评分减少,脏器指数升高,末端回肠组织损害程度较小,且血清中的TNF-α、IL-6含量显著降低(P<0.05)。结论穿心莲内酯能通过多个靶点对5-Fu所致大鼠化疗性肠黏膜炎发挥预防作用,其作用机制可能与抑制炎症相关因子TNF-α、IL-6等有关。

Objective To evaluate the prevention and treatment efficacy of andrographolide in 5-Fu induced chemotherapeutic intestinal mucositis(CIM)in rats based on network pharmacology and animal experiment,so that provide reference for its clinical application.Methods The Swiss Target Prediction,SuperPred,Genecards and OMIM databases were searched to obtain the action targets and corresponding disease targets of andrographolide,and the intersection of action targets and obtained disease targets was taken as the potential targets.The String platform was used to construct a protein-protein interaction network(PPI)for potential targets.The R language software package was used to analyze the function of Gene Ontology(GO)and the enrichment of Kyoto Encyclopedia of Genes and Genomes(KEGG).Cytoscape software was used to analyze its network topology,which was applied to screen the core targets.The key core targets and andrographolide were verified by molecular docking.Thirty-five SD rats were randomly divided into five groups:blank group,model group,positive drug group(bifidobacterium aureus,700 mg/kg),low dose group of andrographolide(30 mg/kg)and high dose group of andrographolide(100 mg/kg).Each group was given intragastric administration of corresponding drugs for 10 days,while blank and model group were given corresponding volume of normal saline.Starting from day 6,each group except the blank group was injected intraperitoneally with 30 mg/kg 5-fluorouracil for 5 days,and the blank group was injected with corresponding volume of normal saline.Body weight and diarrhea of rats were observed and recorded daily.Liver and spleen were collected after the animals were killed,and organ index was calculated.The pathological changes of intestinal tissue in rats were observed by HE staining.ELISA was used to measure the level of serum inflammatory factors IL-6 and TNF-α.Results There are 190 potential targets for the treatment of CIM with andrographolide.In the PPI network,20 core targets such as STAT3,TNF,and IL6 were screened.GO enrichment analysis yielded 2315 biological process related entries,184 molecular function related entries,and 101 cell component related entries,mainly involving oxidative stress response,positive regulation of MAPK cascade,and cell response to chemical stress.KEGG pathway enrichment analysis revealed 177 related signaling pathways,mainly involving HIF-1,TNF,Toll like receptors,and T cell receptor signaling pathways.The molecular docking results indicated that andrographolide has good binding activity with key core targets STAT3,TNF,and IL-6.The animal experiment results showed that compared with the blank group,the model group rats had a decrease in body mass,severe diarrhea,decreased organ index,severe intestinal tissue damage,and significantly increased serum TNF-αand IL-6 levels.Compared with the model group,the high-dose group of andrographolide reduced the rate of body mass decline,decreased diarrhea score,increased organ index,and reduced damage to the distal ileum tissue.The serum TNF-αand IL-6 content was significantly reduced(P<0.05).Conclusion Andrographolide exerts its effect on the treatment of CIM through multi-target and multi-pathway,and its mechanism may be related to the inhibition of TNF-αand IL-6 proinflammatory cytokines.