基于网络药理学和实验验证探究三仁颗粒治疗湿热型胃炎的作用机制

Mechanism of Sanren Granules in treating damp-heat gastritis based upon network pharmacology and experimental verification

目的:基于网络药理学结合动物体内实验探究三仁颗粒对湿热型胃炎(damp-heat gastritis,DHG)的药效及作用机制。方法:通过TCMSP、PubMed数据库筛选三仁颗粒的关键活性成分及其相应靶点;借助OMIM、GeneCards数据库收集治疗DHG相关靶点;通过STRING数据库与Cytoscape 3.9.0将交集靶点构建蛋白-蛋白互作网络(protein-protein interaction network,PPI);采用DAVID数据库对治疗DHG相关靶基因进行GO功能及KEGG通路富集分析,并建立“成分-靶点-通路”网络。采用2%水杨酸钠溶液灌胃,并饲以高脂高糖高热饮食建立DHG大鼠模型,随机分为对照组,模型组,阳性药组(奥美拉唑,4 mg·mL^(–1)),三仁颗粒低、中、高剂量组(2 250、4 500、9 000 mg·kg^(–1)),评价给药前后DHG大鼠体质量、体温和粪便含水率变化;采用苏木素-伊红(hematoxylin-eosin,HE)染色观察大鼠胃黏膜组织形态;采用酶联免疫吸附法(ELISA)和实时荧光定量法(RT-qPCR)测定血清中炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子(TNF-α)、热休克蛋白60和70 (heat shock protein,HSP60/HSP-70)的变化;利用蛋白免疫印迹法(Western blot)检测胃组织NF-κB/MAPK信号通路相关蛋白表达。结果:三仁颗粒治疗DHG的潜在活性成分有62个,交集靶点44个;肿瘤蛋白53(tumor protein 53,TP53)、IL-6、IL-1β、JUN蛋白(JUN),血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)等靶点为关键靶点;GO、KEGG分析显示NF-κB/MAPK信号通路可能是三仁颗粒治疗DHG的重要途径。动物学实验表明,中、高剂量三仁颗粒可显著增加大鼠体质量,降低体温,改善胃部炎性细胞浸润情况,显著降低血清中IL-1β、IL-6、TNF-α、HSP-60和HSP-70含量(P<0.05),下调MAPK、NF-κB蛋白表达(P<0.01)。结论:三仁颗粒可能通过NF-κB/MAPK通路抑制炎症发生治疗湿热型胃炎,为三仁颗粒的临床应用提供理论依据。

OBJECTIVE To explore the efficacy and mechanism of Sanren Granules(SG) on damp-heat gastritis(DHG) based upon network pharmacology and in vivo animal experiments.METHODS The key active components and their corresponding targets of SG were screened by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and PubMed.And the related therapeutic targets for DHG were collected by disease databases of OMIM and GeneCards.Proteinprotein interaction(PPI) network was constructed by STRING and Cytoscape 3.9.0.DAVID database was employed for performing gene ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis on the target genes related to the treatment of DHG.And a “component-target-pathway” network was established.DHG rat model was established by an intragastric injection of 2% sodium salicylate solution and feeding with a high-fat,high-sugar and high-heat diet.The animals were randomized into six groups of control,model,positive drug(omeprazole,4 mg·mL^(-1)) and low/medium/high-dose SG(2 250/4 500/9 000 mg kg^(-1)).The changes of body weight,body temperature and fecal water content pre/postdosing were evaluated.Hematoxylin-eosin(HE) stain was utilized for observing the morphologies of gastric mucosa.The serum levels of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),HSP60 and HSP-70 were quantified by enzyme-linked immunosorbent assay(ELISA) and real-time fluorescent quantitative method.Western blot was utilized for detecting the expressions of NF-κB/MAPK signaling pathway-related proteins in gastric tissue.RESULTS A total of 62potential active components were screened in SG and 44 intersection targets obtained.Among them,TP53,IL-6,IL-1β,JUN and VEGFA were the key targets.Through the analysis of GO/KEGG,NF-κB/MAPK signaling pathway might be an important way for SG for treating DHG.Animal experiments indicated that body weight of rats gained and body temperature dropped markedly in middle/high-dose SG groups.And infiltration of inflammatory cells improved.The serum levels of IL-1β,IL-6,TNF-α,HSP-60 and HSP-70 declined significantly(P<0.05) while the expression levels of MAPK and NF-κB proteins were downregulated(P<0.01).CONCLUSION SG may stunt inflammation through the NF-κB/MAPK pathway for treating damp-heat gastritis,providing theoretical rationales for clinical application of SG.