摘要
目的:基于斑马鱼模型和网络药理学探究三七总皂苷对阿司匹林致斑马鱼肠道损伤的保护作用及机制。方法:采用阿司匹林诱导斑马鱼肠道损伤,以中性红染色、阿尔新蓝染色及中性粒细胞数量变化分别评价三七总皂苷对斑马鱼肠道吞噬功能、杯状细胞分泌及中性粒细胞聚集的影响;采用ELISA法检测三七总皂苷对斑马鱼肿瘤坏死因子α(TNF-α)、前列腺素E2(PGE2)以及紧密连接蛋白ZO-1、Occludin含量;利用网络药理学技术预测三七总皂苷干预肠道损伤的潜在作用机制,并通过实时荧光定量聚合酶链式反应(RT-qPCR)验证相关靶基因表达情况。结果:与模型对照组相比,三七总皂苷能够显著增加斑马鱼肠道中性红染色面积(P<0.01),改善肠道吞噬功能;增加斑马鱼肠道阿尔新蓝染色面积(P<0.01),提高肠道杯状细胞分泌功能;降低肠道中性粒细胞数量(P<0.01),改善肠道损伤程度;降低斑马鱼肠道TNF-α、PGE2含量,增加ZO-1和Occludin的分泌,修复肠道损伤黏膜屏障。网络药理学分析得到三七总皂苷潜在靶点297个,肠道损伤靶点1 797个,三七总皂苷干预肠道损伤的潜在靶点有128个。蛋白质互作(PPI)网络分析度值排名前5的靶点有丝氨酸/苏氨酸激酶1(AKT1)、表皮生长因子受体(EGFR)、信号传导及转录激活因子3(STAT3)、热休克蛋白90AA1(HSP90AA1)、丝裂原活化蛋白激酶3(MAPK3)等。基因本体(GO)分析结果显示,涉及的生物过程主要包括磷酸化、激素响应、细胞激活和对外源刺激的响应等;涉及的细胞组分主要包括受体复合物、膜筏、细胞外基质和核内体腔等;影响的分子功能主要包括蛋白激酶活性、激酶结合、磷酸酶结合和核受体活性等。京都基因与基因组百科全书(KEGG)分析结果显示,主要涉及通路有癌症相关通路、PI3K-AKT信号通路、化学致癌-受体激活、T细胞受体信号通路、MAPK信号通路等。RT-qPCR结果表明三七总皂苷能够抑制Keap1、Akt1、Akt2、Mapk1等基因表达,并上调Nrf2、Ho1和Eefr基因表达。结论:三七总皂苷对阿司匹林致斑马鱼肠道损伤具有良好治疗作用,其保护机制可能与其调控KEAP1/Nrf2、PI3K-Akt和MAPK等信号通路有关。
Objective:To investigate the protective effect and mechanism of Panax notoginseng saponins(PNS)on aspirin-induced intestinal injury in zebrafish based on the zebrafish model and network pharmacology.Methods:Zebrafish intestinal injury was induced by aspirin,and the effect of PNS on intestinal phagocytosis,goblet cell secretion,and neutrophil aggregation was evaluated using neutral red staining,Alcian blue staining,and changes in neutrophil cell counts,respectively.ELISA was used to detect the expression levels of tumor necrosis factor-α(TNF-α),prostaglandin E2(PGE2),and tight junction proteins ZO-1 and occludin in zebrafish intestines.Network pharmacology was employed to predict the potential mechanism of PNS in intervening in intestinal injury,and the expression of related target genes was verified by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Results:Compared with the model group,PNS significantly increased the neutral red staining area in the intestines of zebrafish(P<0.01)to improve intestinal phagocytic function,increased the Alcian blue staining area(P<0.01)to enhance goblet cell secretion,reduced the number of neutrophils in the intestine(P<0.01)to improve the degree of intes-tinal injury,and decreased the content of TNF-α and PGE2 in the intestines of zebrafish to increase the secretion of ZO-1 and occludin,thus repairing the damaged intestinal mucosal barrier.Network pharmacological analysis identified 297 potential targets for PNS,1 797 targets for intestinal injury,and 128 potential targets for PNS intervention in intestinal injury.The top five ranked targets by degree value in the protein-protein interaction(PPI)network analysis were AKT serine/threonine kinase 1(AKT1),epidermal growth factor receptor(EGFR),signal transducer and activator of transcription 3(STAT3),heat shock protein 90 alpha family class A member 1(HSP90AA1),and mitogen-acti-vated protein kinase 3(MAPK3).Gene Ontology(GO)analysis revealed that the biological processes primarily included phosphorylation,response to hormones,cell activation,and response to xenobiotic stimuli;cellular components involved receptor complexes,membrane rafts,ex-tracellular matrix,and endosomal lumen;molecular functions focused on protein kinase activity,kinase binding,phosphatase binding,and nu-clear receptor activity.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis indicated that PNS treatment of intestinal injury mainly involved cancer pathways,the PI3K-Akt signaling pathway,chemical carcinogenesis-receptor activation,T cell receptor signaling pathway,and MAPK signaling pathway.RT-qPCR results showed that PNS inhibited the gene expression of Keap1,Akt1,Akt2,and Mapk1 while increasing the expression of Nrf2,Ho-1,and Egfr.Conclusion:PNS exhibits a beneficial therapeutic effect on aspirin-induced intestinal injury in ze-brafish,and its protective mechanism may be related to the regulation of the KEAP1/Nrf2,PI3K-Akt,and MAPK signaling pathways.
作者
黄英红
郭慧琼
黄海丽
覃其静
赵崇军
肖特
孙宗喜
HUANG Yinghong;GUO Huiqiong;HUANG Haili;QIN Qijing;ZHAO Chongjun;XIAO Te;SUN Zongxi(Zhuang Medicine Clinical Medical College,Guangxi University of Chinese Medicine,Nanning 530201;Beijing Key Laboratory of Quality Evaluation of Chinese Medicines,Beijing University of Chinese Medicine,Beijing 102488;Sichuan Academy of Chinese Medicine Sciences,Chengdu 610041)
出处
《中药药理与临床》
北大核心
2025年第5期43-49,共7页
Pharmacology and Clinics of Chinese Materia Medica
基金
广西科技基地和人才专项(桂科AD22035058)
全国中药特色技术传承人才培训项目(国中医药人教函[2023]96号)
广西国际壮医医院高层次人才队伍建设三年行动计划项目(编号:GZCX20231204)
全国名中医黄汉儒学术思想及临床经验传承推广中心(编号:2022V004)。
作者简介
通信作者:孙宗喜,博士,副研究员,主要从事中药制剂及合理应用研究,E-mail:sunzx@gxtcmu.edu.cn;通信作者:赵崇军,博士,助理研究员,主要从事中药品质评价与安全性研究,E-mail:1014256537@qq.com;黄英红,硕士,助理研究员,主要从事中药民族药药效及作用机制研究,E-mail:944423362@qq.com。
