摘要
旨在观察三七提取物对免疫应激肉鸡炎症反应的影响,并通过网络药理学和分子对接结合体内动物试验对其机理进行探讨.根据TCMSP数据库及GeneCard等疾病数据库寻找三七及肉鸡炎症相关靶点,应用Cytoscape 3.7.1和String数据库分别进行关键化合物和靶点的筛选,并构建中药-关键成分-靶点网络关系图;通过DAVID平台进行GO功能富集分析和KEGG通路富集分析,依托于chiplot在线网站进行可视化,最后通过Pymol软件对核心聚类蛋白进行分析得到核心作用靶点,使用分子对接技术预测活性成分与核心靶点匹配程度以及动物试验进一步探索三七提取物的药效机制.动物试验将60只1日龄红羽肉鸡,随机分为3组(LPS组、CON组、PN组),试验周期为35 d.LPS组和PN组在第12、14、33和35天以250 μg/kg腹腔注射LPS,CON组腹腔注射等量的无菌生理盐水.采用ELISA法检测三七提取物对血清中炎性细胞因子的影响,同时检测各组血清中的激素含量,荧光定量PCR检测各组对STAT3、IL-6和CASP3的mRNA表达水平的影响.结果表明,在第2和5周,LPS攻毒后,血清中IFN-γ、IL-6、iNOS、TNF-α和TNF-β含量显著增加(P＜0.05),而IL-10含量显著下降(P＜0.05).而与LPS组相比,基础日粮中添加三七提取物后血清中IFN-γ、IL-6、iNOS、TNF-α和TNF-β的含量显著下降(P＜0.05),IL-10含量显著增加(P＜0.05),并且三七提取物显著降低了血清中促肾上腺皮质激素(ACTH)和皮质酮(CORT)的水平(P＜0.05),提高了生长激素(GH)的水平(P＜0.05).网络药理学共预测了 8种有效成分和123个肉鸡炎症的潜在靶点.三七对肉鸡炎症的保护机制可能与C型凝集素受体(CLR)信号通路、Toll样受体(TLR)信号通路、MAPK信号通路、NOD样受体(NLR)信号通路和FoxO信号通路有关.根据预测,三七缓解肉鸡炎症反应可能与作用于12个关键靶点有关.荧光定量PCR显示,三七提取物下调了 IL-6和CASP3的mRNA表达(P＜0.05),上调了 STAT3的mRNA表达(P＜0.05).分子对接结果也显示三七提取物中的有效成分可通过IL-6和CASP3发挥抗炎作用.结果提示,三七提取物可能通过多成分、多靶点、多途径缓解免疫应激肉鸡炎症反应,本研究有利于提出新的治疗策略并为开发以赶黄草提取物为主的饲料添加剂提供理论依据.
Abstract
This study aims to observe the effect of Panax notoginseng extracts on inflammatory re-sponse in immunosuppressed broilers and to investigate the mechanism through network pharma-cology and molecular docking combined with in vivo animal tests.Based on the TCMSP database and GeneCard and other disease databases,we searched for targets related to Panax notoginseng and broiler inflammation,screened key compounds and targets by applying Cytoscape 3.7.1 and String databases,respectively,and constructed a network relationship diagram of traditional Chi-nese medicine(TCM)-key components-targets,and carried out GO functional enrichment and KEGG pathway enrichment analyses by using the DAVID platform.The GO functional enrichment analysis and KEGG pathway enrichment analysis were carried out by the DAVID platform,visual-ized by the Chiplot online website,and finally,the core clustered proteins were analyzed by Pymol software to obtain the core targets,and molecular docking technology was used to predict the de-gree of matching between the active ingredients and the core targets as well as the animal experi-ments to further explore the pharmacological mechanism of Panax notoginseng extracts.Sixty 1-day-old red-feathered broilers were randomly divided into three groups(LPS group,CON group,and PN group),and the test period was 35 days.The LPS and PN groups were injected intraperito-neally with 250 μg/kg body weight of LPS,and the CON group was injected with an equal amount of sterile physiological saline on the 12,14,33,and 35 d.The LPS and PN groups were injected with 250 μg/kg body weight of LPS,and the CON group was injected with an equal amount of sterile physiological saline.The effect of Panax notoginseng extract on inflammatory cytokines in serum was detected by ELISA,and the hormone content in serum was also detected in each group,and fluorescence quantitative PCR was used to detect the effect of each group on the mRNA ex-pression levels of STAT3,IL-6,and CASP3.The results showed that the serum levels of IFN-γ,IL-6,iNOS,TNF-α,and TNF-β were significantly increased(P＜0.05),while the level of IL-10 was significantly decreased(P＜0.05)after LPS tapping at weeks 2 and 5.The serum levels of IFN-y,IL-6,iNOS,TNF-α,and TNF-β were significantly decreased(P＜0.05)and IL-10 was sig-nificantly increased(P＜0.05)by the addition of Panax ginseng extracts to the basal diet com-pared with the LPS group.Panax notoginseng extracts significantly decreased the serum levels of adrenocorticotropic hormone(ACTH)and corticosterone(CORT)(P＜0.05)and increased the levels of growth hormone(GH)(P＜0.05).A total of 8 active ingredients and 123 potential tar-gets for broiler inflammation were predicted by network pharmacology.The protective mechanism of Panax notoginseng against broiler inflammation may be related to the C-type lectin receptor(CLR)signaling pathway,Toll-like receptor(TLR)signaling pathway,MAPK signaling pathway,NOD-like receptor(NLR)signaling pathway,and FoxO signaling pathway.According to the pre-diction,the alleviation of inflammatory response in broiler chickens by Panax notoginseng may be related to the action on 12 key targets.Fluorescence quantitative PCR showed that Panax notogin-seng extract down-regulated the mRNA expression of IL-6 and CASP3(P＜0.05)and up-regula-ted that of STAT3(P＜0.05),and molecular docking results also showed that the active ingredi-ents in Panax notoginseng extracts could exert anti-inflammatory effects through IL-6 and CASP3.The results suggested that Panax quinquefolium extracts might alleviate the inflammatory response of immune-stressed broilers through multi-components,multi-targets,and multi-path-ways,and this study helps propose new therapeutic strategies and provides a theoretical basis for the development of feed additives based on Penthorum chinense Pursh extract.
基金项目
国家自然科学基金资助项目(32002325)
中央高校基本科研业务费基金资助项目(SWU-KT22011)
重庆市技术创新与应用发展专项基金资助项目(cstc2021jscxlyjsAX0008)
维普
万方数据