兽研摘要
本研究探讨了富含吴茱萸碱（EVO）的功能性日粮对犬肠道健康的影响，重点关注其对微生物群组成的调节作用及对肠道健康的促进作用。经过一周的适应期后，犬被随机分为对照组和EVO组，分别饲喂相应日粮8周。在0、4和8周时评估生理和血液学参数，并在4和8周时采集新鲜粪便进行微生物分析，以评估功能性日粮对犬肠道微生物的影响。为期8周的试验显示，功能性肠道日粮调节了微生物组成，改善了肠道健康，且未影响体尺指标或常规血液指标，日粮的安全性通过正常的生化指标得到证实。对比分析表明模型犬的微生物丰度发生变化，凸显了有利于肠道屏障增强的积极变化。α多样性分析证实EVO组的物种多样性增加，反映出更健康的肠道。此外，研究证明了功能性日粮对微生物代谢途径和物种差异的调节作用，且未观察到副作用，进一步证实了其对肠道健康的积极影响。因此，含EVO的犬肠道功能性日粮对生理健康无显著影响，但对肠道微生物表现出调节作用。EVO有效调节了微生物丰度和多样性，促进肠黏膜修复和屏障保护。此外，该日粮通过调节犬肠道代谢途径改善了微生物功能。本研究为未来促进犬肠道健康的研究提供了有价值的参考。
关键词：犬；功能性肠道日粮；吴茱萸碱；微生物群
引言
肠道疾病是一组主要影响结肠的慢性疾病[1]，包括克罗恩结肠炎和溃疡性结肠炎等[2]。这些疾病导致免疫介导的胃肠道炎症，常伴有一系列症状和并发症[3]，被认为无法治愈[4]。治疗方式包括药物治疗、生活方式改变，偶尔需要手术[5,6]。虽然许多肠道疾病的确切病因仍不清楚，但遗传、环境和免疫因素被认为在其发病中起作用[7]。
肠道微生物群可能在肠道疾病的发病机制中发挥重要作用[8]，维持肠道微生物群的平衡对于炎症性肠病（IBD）的预防和治疗至关重要。肠道微生物群的平衡对宿主整体健康至关重要[9,10]，失衡可能与IBD、癌症、心血管疾病和代谢综合征等多种疾病相关[11]。因此，调节肠道微生物群的因素对疾病的预防和治疗至关重要。许多天然产物（NPs）被认为对肠道疾病患者具有免疫调节和营养作用[12]，尽管其应用仍存在争议。
在当前的IBD治疗方法中，NPs因其多样的生物活性被认为是开发新药的潜在来源[13]。吴茱萸碱是一种以抗炎和抗氧化特性著称的NP[14–16]。一些研究表明，吴茱萸碱在实验性IBD模型中表现出体内治疗活性，可能通过改善肠道微生物群来减轻肠道炎症[17,18]。犬是研究疾病的有价值动物模型，实验结果可为人类临床疾病治疗提供有价值的见解[19]。此外，宠物行业近年来经历了快速增长，因此改善犬肠道疾病的研究对全球宠物经济和动物福利具有重要意义。尽管现有研究表明吴茱萸碱具有生理活性，但其添加到犬粮后对犬肠道改善的具体影响仍不清楚。虽然吴茱萸碱在先前研究中已显示出理活性，但其添加到犬粮中能否改善犬肠道健康的问题仍未得到解答。
因此，我们提出一个科学假说：富含吴茱萸碱的功能性日粮可通过调节肠道微生物群促进犬肠道健康，从而促进犬的健康。为验证这一假说，我们制备了含吴茱萸碱的犬肠道处方粮，并进行了一系列动物实验，以评估该功能性日粮产品对肠道功能和微生物学的积极调节作用。
材料与方法
处方粮制备方法
犬肠道处方粮由赣南创新与转化医学研究院与江西汇洲宠物科技有限公司联合研发。本处方粮的配方设计参考国家标准（GB/T 31216-2014）全价宠物食品犬粮以及美国饲料管理协会（AAFCO）提供的指南，基于肠道微生物的调控机制，针对特定营养需求进行设计。在实验犬的日常日粮中添加吴茱萸碱的每日剂量，连续口服给药8周。配方中使用的原料和添加剂均符合《饲料原料目录》和《饲料添加剂目录》。肠道处方粮的粗蛋白（GB/T 6432-2018）、粗脂肪（GB/T 6433-2006）、粗纤维（GB/T 6434-2006）、灰分（GB/T 6438-2007）、水分（GB/T 6435-2014）、钙（GB/T 6436-2018）和总磷（GB/T 6437-2018）参照国家标准测定，能量采用氧弹式热量计测定。
动物
选取10只健康中华田园犬，体重7–10 kg，年龄1–5岁，无疾病史，试验前进行常规体内外驱虫和疫苗接种。动物在22°C–26°C、12 h明暗交替环境中自由采食和饮水。实验经赣南医学院生物医学研究伦理委员会批准（批准号2021335），所有动物操作均遵循赣南医学院生物医学伦理委员会的操作指南。
实验方法
将犬随机分为对照组（饲喂对照日粮，n = 5）和EVO组（饲喂富含吴茱萸碱的功能性日粮，n = 5）。所有犬在正式饲喂前接受一周的食物适应期。每日饲喂量按公式计算：维持能量需求（MER）/代谢能（ME），MER = 体重（BW）（kg）0.75 × 70 × 生命阶段系数。实验犬每日饲喂两次（上午9：00和下午4：00），持续8周。实验期间记录每日采食量。在0周、4周和8周进行体格检查，包括空腹血糖和体况评分（BCS）。在0周、4周和8周采集血样进行全血细胞计数和血液生化检查。在实验开始后第8周采集粪便样本。用无菌粪便采集器采集中间段粪便5 g，立即放入灭菌冻存管并标记，随后立即放入液氮中保存，再放入–80°C冷冻保存[20]。
体格检查与血液检测
每日早餐前测量体重，体况由两名研究人员独立评分。从远端头静脉采集全血5 mL。用血糖仪（AccU-Chek Active，Roche）以20 μL血液检测空腹血糖。使用五分类乙二胺四乙酸（EDTA）抗凝血细胞分析仪（Abaxis VetScan HM5，Zoetis），将1 mL静脉血收集于EDTA抗凝管中进行血常规检测。抽取2 mL血液保存于肝素锂抗凝管中，以1，000×g离心10分钟，取上层血清用于血液生化检测。生化检测使用全自动生化分析仪（Hitachi 3110，Hitachi）进行。全血细胞计数和血液生化检测由维尔动物检验医学实验室完成。
16S rRNA基因测序研究肠道微生物群
使用E.Z.N.A.® Soil DNA Kit（Omega Bio-tek）试剂盒进行样本DNA提取。基因组DNA提取完成后，通过1%琼脂糖凝胶监测DNA降解程度及潜在污染。使用Qubit® dsDNA Assay Kit在Qubit® 2.0 Fluorometer（Life Technologies）中测定DNA浓度。取1 μg基因组DNA，使用Covaris仪器进行超声打断。打断后的样本用磁珠进行片段选择，使样本条带集中在200–400 bp左右。然后配制反应体系，在适宜温度下反应一定时间，以修复双链cDNA末端并在3‘端添加A碱基；配制接头反应体系，在适宜温度下反应一定时间，使接头连接至DNA。随后配制聚合酶链反应（PCR）反应体系并设置反应程序，对接头连接产物进行扩增。扩增产物用磁珠纯化回收。将PCR产物变性为单链后，配制环化反应体系，充分混匀并在适宜温度下反应一定时间，获得单链环状产物，消化未环化的线性DNA分子后获得最终文库。随后对环化产物进行上机前浓度检测。检测合格的文库安排上机测序（DNBSEQ），其中单链环状DNA分子通过滚环复制形成包含多个拷贝的DNA纳米球（DNB）。利用高密度DNA纳米芯片技术将生成的DNB加载到芯片上的孔网中，并通过联合探针锚定聚合进行测序。为确保后续信息分析结果的可靠性，需要对这些低质量数据进行过滤。使用SOAPnuke软件获得高质量CleanData。质控后，使用MEGAHIT软件对样本的CleanData数据进行组装，并过滤掉300 bp以下的片段，用于统计分析和后续基因预测。
数据处理与统计方法
使用SPSS 26.0统计软件对数据进行统计分析，组间比较采用双尾t检验或Wilcoxon秩和检验。文中所有数值以mean ± SD表示，p < 0.05认为差异有统计学意义。
结果
功能性肠道日粮的营养成分
在此，我们将吴茱萸碱添加至日粮中，并系统分析了配制的肠道功能性日粮产品与对照日粮产品的化学成分。与对照食物相比，Functional组含有0.25%的吴茱萸碱，而对照组不含该成分。为探究吴茱萸碱添加至日粮后对肠道的影响，我们设计了如图1所示的实验方案。
[图：动物实验流程图。经过一周适应期后，将现场犬随机分为两组]
功能性肠道日粮对犬生理指标的影响
经过8周饲喂实验后，我们分析了犬肠道功能性日粮产品对犬BCS（图2A）和空腹血糖（图2B）的影响。结果显示，与对照组相比，EVO组BCS和空腹血糖相对稳定，各组间BCS评分和空腹血糖均无显著差异。
[图：生理指标检测结果。分别检测现场史宾格犬的空腹血糖和BCS评分，]
功能性肠道日粮对犬血液检测指标的影响
我们进一步通过血液学检测分析肠道功能性日粮对犬安全性的影响。如图3所示，EVO组血小板计数水平显著高于对照组，其他指标包括红细胞、白细胞、平均红细胞血红蛋白和淋巴细胞均无显著差异。所有指标均在正常参考范围内。
[图：血常规指标结果。分别于0、4和8周从现场史宾格犬头静脉采集血液]
功能性肠道日粮对血液生化检测指标的影响
在血液生化指标方面，包括丙氨酸氨基转移酶（ALT；图4A）、谷草转氨酶（AST；图4B）、肌酸激酶（CK；图4C）、α-淀粉酶（AMY；图4D）和肌酐（CREA；图4E），进行了统计分析，以比较犬在8周测试期前后的数值与对照组的情况。结果显示，与对照组相比，8周时ALT水平显著降低，而CREA水平显著升高。值得注意的是，这些变化虽然具有统计学意义，但仍在正常范围内。ALT水平的降低尤其重要，因为它与发生一系列肝脏代谢疾病及其相关并发症的可能性降低有关。其他血液指标包括AST、CK和AMY未观察到显著差异。这表明功能性日粮对犬的整体健康状况未产生实质性影响。这进一步印证了功能性日粮的安全性。
[图：血液生化指标结果。血液生化指标从田野猎犬的头静脉采集]
功能性肠道日粮对肠道微生物物种丰度组成的影响
在为期8周的饲喂实验期间，从新鲜粪便中段采集粪便样本进行宏基因组分析，如图5所示。分析重点检查对照组和日粮前组在门水平（图5A）和属水平（图5B）上物种组成的变化，特别是厚壁菌门（F）和拟杆菌门（B），并通过多种分析方法进行研究。
[图：肠道微生物群相对丰度和多样性分析。（A）门水平相对丰度。（]
结果显示，与对照组相比，EVO组中厚壁菌门显著增加，同时拟杆菌门显著减少（图5A和5C）。这表明厚壁菌门可能在促进肠黏膜修复、从而增强肠道屏障方面发挥主导作用。从属水平来看，日粮前组中拟杆菌属、Phocaeicola和梭菌属的相对丰度显著降低，而Blautia、巨单胞菌属和Peptacetobacter的相对丰度增加（图5B）。此外，我们计算了厚壁菌门与拟杆菌门的比值（B/F比值），观察到与对照组相比，EVO组显著降低（图5D）。
功能性肠道日粮对犬肠道菌群多样性的影响
通过分析肠道微生物群的Alpha多样性，Chao1（图6A）、Shannon（图6B）和Simpson（图6C）指数结果显示，与对照组相比，EVO组的Chao1和Shannon指数无显著变化，而Simpson指数在EVO组中较对照组显著升高。这表明肠道功能性日粮能够调节肠道微生物多样性的升高，改善肠道功能，保护肠道健康。
[图：吴茱萸碱（EVO）对肠道微生物alpha多样性的影响。细菌alpha多样性的箱线图]
功能性肠道日粮对犬肠道菌群代谢途径的影响
京都基因与基因组百科全书（KEGG）通路富集分析结果（图7）显示，生物代谢通路分为代谢、遗传信息处理、环境信息处理、细胞过程和生物体系统。其中，代谢领域基因数量最多，为161，718个，基因分布最广。环境信息处理基因数量位居第二，共22，630个。基因数量第三多的是遗传信息处理，共22，086个。基因数量第四多的是细胞过程，共12，139个。生物体系统基因数量最少，共3，896个。这表明肠道功能性日粮能够调节肠道微生物代谢途径，促进肠道健康。
[图：京都基因与基因组百科全书（KEGG）PATHWAY数据库是手绘代谢通路的集合]
功能性肠道日粮对犬肠道菌群物种差异的影响
对肠道微生物进行了线性判别效应量分析（LEfSe）（图8），分析线性判别分析（LDA）条形图，其中特定条形对应特定物种，条形颜色对应该物种所属分组，条形长度代表LDA值，LDA值越大表示差异越显著。结果显示，在对照组和EVO组之间全面筛选出22株差异菌株。其中，与对照组相比，EVO组中有14种微生物（Blautia、巨单胞菌属、Sutterella和Collinsella等）丰度较高，而8种微生物（拟杆菌属、Desulfurella、埃里希体属和Desulfobacca等）丰度较低。这表明功能性日粮能够改变肠道菌群的物种差异，保护肠道健康。
[图：属水平样本的线性判别分析效应量（LEfSe）分析。用于发现物种ch]
讨论
维持肠道健康是一项高度复杂的任务，受多种生理因素影响，如免疫系统、肠黏膜屏障和肠道微生物群之间错综复杂的相互作用[21]。肠道内栖息着多样的细菌群落，显著影响宿主的生理反应，可能破坏内部平衡[22]。因此，确保强健的肠道健康对动物福祉至关重要。动物免疫系统通过识别肠道微生物群并调节反应发挥关键作用。它适度促进先天性和适应性免疫途径，增强肠道屏障功能。这使免疫系统能够有效应对入侵病原体，同时保持对有益微生物群和食物抗原的耐受性[23–25]。这种调节作用涉及黏液产生和上皮层聚焦等机制。这些免疫反应不仅改变肠道中的营养底物，还参与塑造肠道微生物群的组成[26]。促进肠道健康的关键在于增强免疫系统功能、强化肠道屏障和改善肠道微生物群组成[27]。寻求有效的饲料添加剂以增强动物肠道健康是一种有益策略，有助于整体动物健康。
要确认日粮的功能性，必须确保其安全性。我们研究组使用的实验日粮包括添加吴茱萸碱，一种NP。大量文献支持使用吴茱萸碱因其抗炎和抗氧化特性，间接表明吴茱萸碱在一定浓度范围内的安全性[17,28,29]。例如，研究表明吴茱萸碱可显著减轻AOM/DSS诱导结肠炎的炎症，这可能与维持肠道屏障功能有关[28]。也有研究表明，吴茱萸碱可能直接作用于增加有益菌，导致有害菌减少并维持肠道自然平衡，或可能间接作用于肠道微生物群，导致有益菌和有害菌之间的相互作用发生变化，以维持肠道自然平衡[30]。此外，我们进行了实验以直接证明添加到犬粮中的吴茱萸碱浓度的安全性。
研究表明，添加吴茱萸碱显著增强了犬肠道微生物群的α多样性。具体而言，通过测量Shannon多样性指数，我们观察到在日粮中添加吴茱萸碱的EVO组相比对照组具有更高的微生物多样性。这表明吴茱萸碱有助于在犬肠道中培育更丰富多样的微生物群落。从β多样性的角度，我们进一步分析了添加吴茱萸碱后肠道微生物群的聚类。结果显示EVO组与对照组之间的微生物聚类存在显著差异，表明引入吴茱萸碱确实导致犬肠道微生物群整体组成发生明显改变。这种变化不仅体现在存在的微生物类型上，还体现在它们之间的相对丰度上，突显了吴茱萸碱对肠道微生物群整体结构的影响。类似研究也表明，其他植物提取物，如某些植物多酚或纤维相关物质[31,32]，同样有助于增加肠道微生物群的多样性。这进一步强调了植物提取物在调节肠道微生物群平衡方面的潜在益处[33–36]。这种多样性的提高被认为有利于维持肠道微生物群的稳态，从而促进整体肠道健康和免疫功能[37]。
为进一步探究吴茱萸碱诱导的微生物群落变化是否对健康有益，我们对发生改变的细菌分类群进行了详细分析。我们观察到厚壁菌门的丰度显著增加，该门以其有益成员如乳杆菌而闻名，乳杆菌可促进肠黏膜修复并改善肠道屏障功能。同样，研究也发现植物提取物显著增加了厚壁菌门的丰度[38,39]。此外，吴茱萸碱显著降低了拟杆菌门的水平。与厚壁菌门类似，拟杆菌门也包含多种益生菌种[38]。因此，吴茱萸碱改善肠道健康的机制涉及微生物群落的整体变化，而非单个菌种的改变。植物提取物也观察到类似效应，即增加厚壁菌门并降低拟杆菌门水平[12,40–42]。此外，我们的结果表明吴茱萸碱显著降低了拟杆菌门与厚壁菌门的比值（B/F比值）。B/F比值被广泛认为对维持肠道正常平衡具有关键影响。B/F比值升高或降低均被视为生态失衡的标志，前者常与IBD相关[43]。因此，吴茱萸碱可能通过降低B/F比值发挥肠道保护作用，有助于维持健康的肠道状态。短链脂肪酸（SCFAs）在维持肠道细菌平衡、肠道上皮功能完整性、肠道免疫及炎症方面至关重要[44]。吴茱萸碱通过促进产SCFAs细菌的富集来缓解肠道炎症并维持肠道屏障。同时，吴茱萸碱处理促进了肠道微生物群的功能成熟，其特征为基础代谢（碳水化合物代谢、氨基酸代谢、辅因子和维生素代谢、能量代谢及细胞运动）增强。与SCFAs相关的代谢（如丁酸代谢和丙酸代谢）与产SCFAs细菌的增加相一致[28,45]。吴茱萸碱促进产SCFAs细菌的富集并降低促炎细菌水平，这有助于微生物群代谢的改变，尤其是色氨酸代谢，这是治疗肠道疾病的新策略[45]。
为进一步分析吴茱萸碱对微生物群落的影响，我们在属水平上进行了分析。在特定属水平上，吴茱萸碱显著增加了布劳特菌属的水平。Liu等总结了布劳特菌属的益生功能，其在改善代谢性和炎症性疾病中发挥关键作用[46]。布劳特菌属的升高可能是含吴茱萸碱的日粮改善肠道健康的原因之一。吴茱萸碱还显著降低了梭菌属的水平。据报道，梭菌属内许多菌种产生毒素，对整体健康构成威胁，如艰难梭菌以产生毒素引起伪膜性结肠炎等病症而闻名。嗜酸乳杆菌是一种从人类粪便中分离的益生菌，其生理、生化和发酵特性已被广泛研究，可通过激活ERK同工型改善上皮屏障功能和转运特性[47,48]。令人惊讶的是，添加吴茱萸碱后嗜酸乳杆菌的丰度增加[30]。在本研究中，我们设计了一种功能性肠道日粮并研究了该日粮调节犬肠道微生物群的能力。功能性肠道日粮未显著影响犬的生理参数，表明该日粮产品对犬具有一定程度的安全性。同时，犬功能性日粮不仅对犬肠道微生物具有调节作用，可调节肠道微生物的丰度和多样性、增加有益菌的丰富度、促进肠黏膜和肠道屏障的修复、保护肠道健康，还能调节犬肠道的代谢途径，促进肠道功能的改善。研究表明，吴茱萸碱可能直接作用于有益菌的增加，导致有害菌减少并维持肠道自然平衡，也可能间接作用于肠道微生物群，导致有益菌与有害菌之间相互作用的改变，从而维持肠道自然平衡[30]。这与我们的发现一致。尽管仅进行了短期测试，但大多数犬接受了日粮干预后肠道菌群发生了显著变化，与对照组相比，肠道EVO组犬的肠道微生物群物种多样性显著增加，厚壁菌门显著增加，拟杆菌门显著减少。已知SCFAs可增强肠黏膜中支持免疫耐受的调节性T细胞，并作为微生物代谢物逆转失调的微生物群，而厚壁菌门丰度的降低可能减少SCFAs的产生[49,50]。一些研究表明，SCFAs的产生与厚壁菌门的增加有关[49]。
为进一步分析这些微生物变化对宿主的潜在生理和代谢影响，我们对两组之间的差异表达基因进行了KEGG通路富集分析。我们发现1718个差异表达基因富集于免疫系统信号通路。STING、NF-κB、炎症小体、TCR、BCR信号、JAK/STAT和TLR信号等关键通路在免疫系统信号转导中发挥重要作用。这些信号通路与肠道疾病尤其是炎症密切相关，表明吴茱萸碱可能通过改变肠道微生物群来调节免疫系统信号通路。这种调节可能有助于保护和维持肠道健康。
此外，犬只血常规和血液生化指标分析显示，实验前后犬只血常规指标中的红细胞指数、白细胞指数、平均血红蛋白指数和淋巴细胞指数均无统计学意义。吴茱萸碱组第0周的血小板指数显著高于对照组，但结果均在正常范围内；实验前后犬只血液生化指标中的天冬氨酸氨基转移酶指数、肌酸激酶指数和淀粉酶指数均无统计学意义。实验第八周，吴茱萸碱组的天冬氨酸氨基转移酶指数显著低于对照组，吴茱萸碱组的肌酐指数显著高于对照组，但结果均在正常范围内波动。因此，结果表明吴茱萸碱对犬只的生理生化指标无显著影响。
尽管对吴茱萸碱已有大量研究，但通常用于研究其减肥[51]、抗炎[52]、抗肿瘤[53]和治疗心血管疾病[54]的作用。目前市场上尚无添加吴茱萸碱用于调节犬肠道微生物群多样性和肠道功能的处方食品。在本研究中，我们重点关注所设计的肠道功能日粮的功能性和安全性。我们还以完全创新的方式研究了犬肠道功能日粮的配方、组成和功能性。在此，我们观察到饲喂肠道功能日粮的犬只生理指标无显著变化，但有益菌群物种多样性增加、有害菌群丰度降低，并共同调节了肠道微生物代谢物的增加，从而改善肠道屏障、保护肠道健康。本研究的局限性在于我们仅在单一品种的实验犬中测试了肠道功能日粮对肠道微生物的影响，该日粮的效果尚未在宠物犬中进一步验证。未来将在多种临床动物中开展进一步的临床研究，并对配制肠道功能日粮的效果进行广泛验证。
结论
在日粮中添加吴茱萸碱显著增加了犬肠道微生物群的多样性并调节了关键微生物群的水平。这表明吴茱萸碱对肠道健康具有潜在的积极作用。这些发现为进一步研究吴茱萸碱在改善犬肠道健康和预防相关疾病中的应用提供了基础。
致谢
我们衷心感谢动物实验中心的技术人员在动物饲养、血液样本采集和实验室检测方面提供的协助。
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Abstract
This study investigated the effects of an evodiamine (EVO)-enriched functional diet on canine intestinal health, with a focus on its modulation of microbiota composition and promotion of intestinal well-being. After a one-week acclimatization period, dogs were randomly assigned to either the control group or the EVO group, receiving their respective diets for 8 weeks. Physiological and hematological parameters were assessed at 0, 4, and 8 weeks, and fresh feces were collected at 4 and 8 weeks for microbial analysis to evaluate the impact of the functional diet on canine intestinal microorganisms. The 8-week trial revealed that the functional gut diet regulated microbial composition, improving intestinal health without affecting body metrics or routine blood indicators, and the diet’s safety was affirmed by normal biochemical indices. Comparative analysis indicated altered microbial abundance in model dogs, highlighting positive changes favoring intestinal barrier enhancement. Alpha diversity analysis confirmed increased species diversity in the EVO group, reflecting a healthier gut. Moreover, the study demonstrated the functional diet’s regulatory impact on microbial metabolic pathways and species differences without observed side effects, reinforcing its positive influence on gut health. Therefore, the canine intestinal functional diet containing EVO showed no significant impact on physiological health but exhibited regulatory effects on intestinal microorganisms. EVO effectively modulated microbial abundance and diversity, fostering intestinal mucosal repair and barrier protection. Additionally, the diet improved microbial function by regulating canine intestinal metabolic pathways. This study serves as a valuable reference for future research in promoting canine intestinal health.
Keywords: Canines, Functional intestinal diets, Evodiamine, Microbiota
INTRODUCTION
Intestinal disease comprises a group of chronic conditions primarily affecting the colon [1], including Crohn’s Colitis and Ulcerative Colitis et al [2]. These diseases result in immune-mediated gastrointestinal inflammation, often accompanied by a range of symptoms and complications [3], deemed incurable [4]. Treatment modalities involve medication, lifestyle changes, and occasionally surgery [5,6]. While the exact causes of many intestinal diseases remain unclear, genetic, environmental, and immune factors are believed to play a role in its development [7].
The gut microbiota may play a significant role in the pathogenesis of intestinal diseases [8], and maintaining a balanced gut microbiome is crucial for the prevention and treatment of Inflammatory bowel disease (IBD). The balance of the gut microbiota is vital for overall host health [9,10], and imbalance may be associated with various diseases such as IBD, cancer, cardiovascular diseases, and metabolic syndrome [11]. Therefore, factors regulating the gut microbiome are crucial for disease prevention and treatment. Many natural products (NPs) are considered to have immune-regulatory and nutritional effects on patients with intestinal diseases [12], although their application remains controversial.
In the current approaches to treating IBD, NPs are considered potential sources for developing new drugs due to their diverse biological activities [13]. Evodiamine is a NP known for its anti-inflammatory and antioxidant properties [14–16]. Some studies suggest that evodiamine exhibits in vivo therapeutic activity in experimental IBD models, possibly by improving the gut microbiota to alleviate intestinal inflammation [17,18]. Canines serve as valuable animal models for studying diseases, with experimental results providing valuable insights into clinical disease treatment for humans [19]. Additionally, the pet industry has experienced rapid growth in recent years, making research on the improvement of canine intestinal diseases significant for the global pet economy and animal welfare. Although existing studies indicate the physiological activity of evodiamine, its specific impact on canine intestinal improvement after being added to dog food remains unknown. While evodiamine has demonstrated physiological activity in previous research, the question of whether its addition to canine food can lead to improvements in canine intestinal health remains unanswered.
Therefore, we propose a scientific hypothesis that a functional diet enriched with evodiamine could promote canine intestinal health by modulating the gut microbiota, thus promoting the canine health. To validate this hypothesis, we prepared a canine intestinal prescription food containing evodiamine and conducted a series of animal experiments to assess the positive regulatory effects of this functional dietary product on intestinal function and microbiology.
MATERIALS AND METHODS
Prescription food preparation method
The canine intestinal prescription food is jointly developed by the Gannan Institute of Innovation and Translational Medicine and Jiangxi Huizhou Pet Technology Co. The formulation of this prescription food is designed with reference to national standards (GB/T 31216-2014), full-priced pet food specifically formulated for dogs, and guidelines provided by the American Association of Feed Control Officials (AAFCO). It is based on the regulatory mechanism of intestinal microorganisms and tailored to meet specific nutritional requirements. The daily dose of evodiamine was added to the daily food of the experimental dogs, which was continuously administered orally for 8 weeks. The ingredients and additives used in the formulations are in accordance with the Feed Ingredients Catalogue and the Feed Additives Catalogue. The crude protein (GB/T 6432-2018), crude fat (GB/T 6433-2006), crude fiber (GB/T 6434-2006), ash (GB/T 6438-2007), moisture (GB/T 6435-2014), calcium (GB/T 6436-2018) and total phosphorus (GB/T 6437-2018) of the intestinal tract prescription diet were determined referring to national standard and the energy was determined by Oxygen-type calorimeter.
Animals
Ten healthy Chinese field dogs, weighing 7–10 kg, aged 1–5 years, with no history of disease, were selected for the test and were routinely dewormed and vaccinated in vitro and in vivo before the test. The animals were fed and watered ad libitum in a 12 h alternating light and dark environment at 22°C–26°C. The experiment was approved by the Biomedical Research Ethics Committee of Gannan Medical College (Approval number 2021335), and all animal manipulations were in accordance with the operational guidelines of the Biomedical Ethics Committee of Gannan Medical College.
Experimental method
Dogs were randomly divided into a control group feed with control diet (n = 5) and EVO group feed with evodiamine-enriched functional diet (n = 5). All dogs were received a week food adaptation period before formal feeding. The daily feeding amount was calculated according to the formula: maintenance energy requirement (MER) / metabolizable energy (ME), MER = body weight (BW) (kg)0.75 × 70 × life stage factor. The experimental dogs were fed twice a day (at 9:00 am and 4:00 pm) for 8 weeks. Their daily food intake was recorded during the experiment. Physical examinations including fasting blood glucose and body condition score (BCS) were performed at 0 w, 4 w, and 8 w. The blood samples were collected for complete blood count and blood biochemistry examination at 0 w, 4 w, and 8 w. Fecal samples were taken at the 8th week after the start of the experiment. The mid-range feces 5 g were collected with a sterile fecal collector and immediately put into sterilized freezing tubes and labeled, then immediately put into liquid nitrogen for storage, and then put into –80°C for freezing and storage [20].
Physical examination and blood test
Measwere measured daily before breakfast, and body condition was scored independently by two researchers. Five milliliters whole blood was collected from the distal cephalic vein. The fasting blood glucose was tested on Blood glucose meter (AccU-Chek Active, Roche) with 20 μL blood. Using a 5-part ethylenediaminetetraacetic acid (EDTA) anticoagulant hematology analyzer (Abaxis VetScan HM5, Zoetis), 1 mL of venous blood was collected in an EDTA anticoagulant tube for routine blood tests. Draw 2 mL of blood and preserve it in heparin lithium anticoagulant tubes. Centrifuge at 1,000×g for 10 minutes to obtain the upper serum layer for blood biochemical testing. The biochemical detection was applied on Automatic biochemical analyzer (Hitachi 3110, Hitachi). Complete blood count and blood biochemical tests were performed by the medical Laboratory of Well Animal Test.
Investigation of intestinal microbiota by 16S rRNA gene sequencing
The E.Z.N.A.® Soil DNA Kit (Omega Bio-tek) reagent kit was used for sample DNA extraction. After completion of genomic DNA extraction, DNA degradation degree and potential contamination were monitored on a 1% agarose gel. DNA concentration was measured using the Qubit® dsDNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies). Genomic DNA 1 μg was taken and ultrasonically interrupted using a Covaris instrument. The interrupted samples were fragment-selected with magnetic beads so that the sample bands were concentrated around 200–400 bp. Then formulate the reaction system and react for a certain period of time at the right temperature to repair the ends of double-stranded cDNA and add the A base at the 3’ end; formulate the junction reaction system and react for a certain period of time at the right temperature to make the junction connected to the DNA. Then the polymerase chain reaction (PCR) reaction system was prepared and the reaction program was set up to amplify the ligated products. The amplified products were purified and recovered using magnetic beads. After denaturing the PCR product into single-stranded, the cyclization reaction system is prepared, and the single-stranded cyclic product can be obtained by mixing well and reacting at the right temperature for a certain period of time, and the final library can be obtained after digesting the linear DNA molecules that have not been cyclized. The cyclized product was then subjected to a pre-loading concentration test. The tested libraries were arranged for on-board sequencing (DNBSEQ), where single-stranded cyclic DNA molecules were replicated by rolling rings to form a DNA nanoball (DNB) containing multiple copies. The resulting DNBs are added to a mesh of holes on the chip using high-density DNA nano-chip technology and sequenced by co-probe anchored polymerization. To ensure the reliability of the subsequent information analysis results, it is necessary to filter these low-quality data. The high-quality CleanData was obtained using the software SOAPnuke. After quality control, CleanData data were assembled using the software MEGAHIT on the samples, and fragments below 300 bp were filtered out for statistical analysis and subsequent gene prediction.
Data processing and statistical methods
The data were statistically analysed using SPSS 26.0 statistical software, and the two-tailed t-test or Wilcoxon rank sum test was used for statistical analysis between groups. All values in the text are expressed as mean ± SD, and p < 0.05 was considered a statistically significant difference.
RESULTS
Nutritional composition of functional intestinal diets
Here, we added evodiamine to a diet and systematically analyzed the chemical composition and of the formulated gut-functional dietary products and the control dietary products. Compared with the control food, the Functional group contained 0.25% of evodiamine, while the control group did not contain this ingredient. To investigate the effect of evodiamine on the intestinal tract after its addition to the diet, we designed an experimental program as shown in Fig. 1.
[图：Flow chart of animal experiment. After a one-week acclimatization phase, the field dogs were randomly divided into two g]
The effect of the functional intestinal diet on physiologic indices in dogs
After an 8-week feeding experiment, we analyzed the effects of canine intestinal functional dietary products on canine BCS (Fig. 2A) and fasting blood glucose (Fig. 2B). The results showed that the BCS and fasting blood glucose were relatively stable in the EVO group compared with the control group, and there were no significant differences in BCS score and fasting blood glucose among all groups.
[图：Physiological indicators test results. Fasting blood glucose and BCS scores were tested separately in field spaniels, an]
The effect of the functional intestinal diet on canine blood test indices
We further analyzed the effect of enteral functional diet on the safety of dogs by hematological tests. As shown in Fig. 3, the platelet count level of EVO group was significantly higher than control group, and no significant differences in the other indices including red blood cells, white blood cells, mean corpuscular hemoglobin and lymphocyte. All of the indices were within the normal reference ranges.
[图：Results of routine blood indicators. Blood was collected from the cephalic vein of field spaniels at 0, 4 and 8 weeks fo]
The effect of the functional intestinal diet on the indices of blood biochemical tests
In the context of blood biochemical indices, including alanine aminotransferase (ALT; Fig. 4A), Glutamic oxaloacetic transferase (AST; Fig. 4B), creatine kinase (CK; Fig. 4C), alpha-amylase (AMY; Fig. 4D) and creatinine (CREA; Fig. 4E), a statistical analysis was conducted to compare values in dogs before and after an 8-week testing period with those of the control group. The results revealed that ALT exhibited significantly lower level, while CREA demonstrated a significantly higher level compared to the control group at 8-week. It is noteworthy that these alterations, although statistically significant, remained within the normal range. The reduced levels of ALT are particularly significant as they are associated with a decreased likelihood of developing a spectrum of liver metabolic diseases and their associated complications. No significant differences were observed in the other blood indices, including AST, CK, and AMY. This indicating that the functional diet did not exert a substantial impact on the overall health status of dogs. This further underscored the safety profile of the functional diet.
[图：Results of blood biochemical indicators. Blood biochemical indexes were collected from the cephalic vein of field spanie]
The effect of the functional intestinal diet on the composition of intestinal microbial species abundance
During the 8-week feeding experiment, fecal samples were collected from the mid-section of fresh feces for metagenomic analysis, as shown in Fig. 5. The analysis focused on examining alterations in the species composition of ccontrol and prediet groups, specifically Firmicutes (F) and Bacteroides (B) at phylum (Fig. 5A) and genus (Fig. 5B) level and through diverse analytical method.
[图：Analysis of relative abundance and diversity of the intestinal microbiota. (A) Relative abundance at the phylum level. (]
The results revealed a noteworthy increase of the Firmicutes in the EVO group compared to the control group, accompanied by a significant decrease in the Bacteroidetes phylum (Figs. 5A and 5C). This suggests that Firmicutes might play a predominant role in facilitating intestinal mucosal repair, thereby enhancing the intestinal barrier. From a genus level perspective, there is a significant decrease in the relative abundance of Bacteroides, Phocaeicola, and Clostridium in the Prediet group, while Blautia, Megamonas, and Peptacetobacter show an increase in relative abundance (Fig. 5B). Additionally, we computed the ratio of Firmicutes to Bacteroidetes (B/F ratio) and observed a significant reduction in the EVO group compared with control group (Fig. 5D).
The effect of the functional intestinal diet on the diversity of canine intestinal flora
By analyzing the Alpha diversity of intestinal microbiota, Chao1 (Fig. 6A), Shannon (Fig. 6B), and Simpson (Fig. 6C) indices results showed that compared with the control group, Chao1 and Shannon indices of the EVO group did not change significantly, while the Simpson index was significantly elevated in EVO group when compared with control group. It indicated that the functional diet of the intestine can regulate the elevated diversity of intestinal microorganisms, improve intestinal function, and protect intestinal health.
[图：The effect of evodiamine (EVO) on the alpha diversity of intestinal microorganisms. Box plots of bacterial alpha diversi]
The effect of the functional intestinal diet on metabolic pathways in canine intestinal flora
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis results (Fig. 7) showed that the classification of biometabolic pathways into Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, and Organismal Systems. Among them, the metabolic domain has the highest number of genes of 161,718 and the widest distribution of genes. The number of genes in environmental information processing was the second highest, with a total of 22,630. The third largest number of genes is processed for genetic information, with a total of 22,086. The fourth largest number of genes is Cellular Processes, with a total of 12,139. And the Organismal Systems has the smallest number of genes, with a total of 3,896. It is shown that gut functional diets can regulate gut microbial metabolic pathways and promote intestinal health.
[图：Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database is a collection of hand-drawn metabolic pathways present]
The effect of the functional intestinal diet on species differences in canine intestinal flora
A linear discriminant effect size analysis (LEfSe) was performed on the gut microorganisms (Fig. 8), analyzing the linear discriminant analysis (LDA) bar graphs, where the specific bar corresponded to the specific species, the color of the bar corresponded to the group corresponding to that species, and the length of the bar represented the LDA value, and the larger the LDA value represented the more significant difference. The results revealed a comprehensive screening of 22 distinct strains between the control group and the EVO group. Among these, 14 microorganisms (Blautia, Megamonas, Sutterella, and Collinsella et al.) exhibited higher abundance, while 8 microorganisms (Bacteroides, Desulfurella, Ehrlichia, and Desulfobacca et al.) displayed lower abundance in the EVO group compared to the control group. This indicated that the functional diets were able to modify the species differences in the intestinal flora and protect the intestinal health.
[图：Linear discriminant analysis effect size (LEfSe) analysis of samples at the genus level. Used to discover the species ch]
DISCUSSION
Maintaining gut health is a highly complex task influenced by various physiological factors, such as the intricate interplay among the immune system, intestinal mucosal barrier, and gut microbiota [21]. The gut, housing a diverse bacterial community, significantly impacts the host’s physiological responses, potentially disrupting internal equilibrium [22]. Thus, ensuring robust gut health is crucial for animal well-being. The animal’s immune system plays a pivotal role by recognizing gut microbiota and modulating responses. It moderately promotes innate and adaptive immune pathways, enhancing gut barrier function. This enables the immune system to effectively respond to invasive pathogens while maintaining tolerance to beneficial microbiota and food antigens [23–25]. This regulatory role involves mechanisms like mucus production and epithelial layer focus. These immune responses not only alter nutrient substrates in the gut but also contribute to shaping the gut microbiota composition [26]. The key to promoting gut health lies in enhancing immune system functionality, reinforcing the intestinal barrier, and improving gut microbiota composition [27]. Seeking effective feed additives to enhance animal gut health is a beneficial strategy, contributing to overall animal health.
To confirm the functionality of a diet, it is essential to ensure its safety. The experimental diet utilized by our research group includes the addition of evodiamine, a NP. Extensive literature supports the use of evodiamine for its anti-inflammatory and antioxidant properties, indirectly indicating the safety of evodiamine within certain concentration ranges [17,28,29]. For example, studies have shown that evodiamine could significantly reduce the inflammation of AOM/DSS-induced colitis, which may be related to the maintenance of intestinal barrier function [28]. There are also studies that show that evodiamine may act directly on the increase of beneficial bacteria, leading to the reduction of harmful bacteria and the maintenance of intestinal balance of nature, or it may act indirectly on the gut microbiota, leading to a change in the interaction between beneficial bacteria and harmful bacteria, to maintain the intestinal balance of nature [30]. Furthermore, we conducted experiments to directly demonstrate the safety of the Evodiamine concentration added to the food for dogs.
Research indicates that the addition of evodiamine significantly enhances the α-diversity of canine gut microbiota. Specifically, by measuring the Shannon diversity index, we observed a higher microbial diversity in the EVO group, where evodiamine was added to the diet, compared to the control group. This suggests that evodiamine contributes to fostering a more abundant and diverse microbial community in the canine gut. From the perspective of β-diversity, we further analyzed the clustering of gut microbiota after the addition of evodiamine. The results revealed a significant difference in microbial clustering between the EVO group and the control group, indicating that the introduction of evodiamine indeed leads to a noticeable alteration in the overall composition of the canine gut microbiota. This change is evident not only in the types of microorganisms present but also in the relative abundance among them, highlighting the impact of evodiamine on the overall structure of the gut microbiota. Similar studies also suggest that other plant extracts, such as certain plant polyphenols or fiber-related substances [31,32], similarly contribute to increased diversity in gut microbiota. This further emphasizes the potential benefits of plant extracts in modulating the balance of gut microbiota [33–36]. The elevation of this diversity is considered conducive to maintaining the homeostasis of gut microbiota, thereby promoting overall gut health and immune function [37].
To further explore whether the changes in the microbial community induced by evodiamine are beneficial to health, we conducted a detailed analysis of the altered bacterial taxa. We observed a significant increase in the abundance of Firmicutes, a phylum known for its beneficial members such as Lactobacillus, which can promote intestinal mucosal repair and improve gut barrier function. Similarly, it was also found that plant extracts significantly increased the abundance of Firmicutes [38,39]. Additionally, evodiamine markedly reduced the levels of Bacteroidetes. Similar to Firmicutes, Bacteroidetes includes several probiotic species [38]. Therefore, the mechanism through which evodiamine improves gut health involves overall changes in the microbial community rather than alterations in individual species. A similar effect was observed with plant extracts, increasing Firmicutes and decreasing Bacteroidetes levels [12,40–42]. Furthermore, our results indicated that Evodiamine significantly lowered the ratio of Bacteroidetes to Firmicutes (B/F ratio). The B/F ratio is widely recognized as having a crucial impact on maintaining the normal equilibrium of the gut. An elevated or reduced B/F ratio is considered indicative of ecological imbalance, with the former often associated with IBD [43]. Therefore, evodiamine may exert intestinal protective effects by lowering the B/F ratio, contributing to maintaining a healthy gut state. Short-chain fatty acids (SCFAs) are essential in maintaining gut bacterial balance, gut epithelial functional integrity, gut immunology, and inflammation [44]. Evodiamine alleviates intestinal inflammation and maintains the intestinal barrier by promoting bacterial enrichment that produce SCFA. At the same time, evodiamine treatment promotes the functional maturation of the gut microbiota, which is characterized by increased basal metabolism (carbohydrate metabolism, amino acid metabolism, cofactor and vitamin metabolism, energy metabolism, and cell motility). The metabolism associated with SCFAs (e. g. butyrate metabolism and propionate metabolism) is consistent with the increase of bacteria producing SCFAs [28,45]. Evodiamine promotes the enrichment of bacteria that produce SCFAs and reduces the level of pro-inflammatory bacteria, which contributes to changes in microbiota metabolism, especially tryptophan metabolism, which is a new strategy for the treatment of intestinal disease [45].
To further analyze the impact of evodiamine on the microbial community, we conducted an analysis at the genus level. At the specific genus level, evodiamine significantly increased the levels of Blautia. Liu et al. have summarized the probiotic functions of the Blautia genus, playing a crucial role in improving metabolic and inflammatory diseases [46]. The elevation of Blautia may be one of the reasons for the dietary improvement in gut health containing evodiamine. Evodiamine also markedly reduced the levels of the Clostridium genus. Many species within Clostridium are reported to produce toxins, posing a threat to overall health, such as Clostridium difficile, known for producing toxins causing conditions like pseudomembranous colitis. L. acidophilu is a probiotic isolated from human feces and its physiological, biochemical and fermentative properties have been widely investigated, which can improve the epithelial barrier function and transport properties by activating the ERK isoforms [47,48]. Surprisingly, the abundance of L. acidophilus was increased after the addition of evodiamine [30]. In this study, we designed a functional gut diet and investigated the ability of this diet to regulate the gut microbiota in dogs. The functional intestinal diet did not significantly affect the physiological parameters of dogs, indicating that this dietary product has a certain degree of safety for dogs. At the same time, the functional diet for dogs not only has a regulatory effect on canine intestinal microorganisms, which can regulate the abundance and diversity of intestinal microorganisms, increase the proficiency of beneficial bacteria, promote the repair of intestinal mucosa and intestinal barriers, and protect the health of the intestinal tract, but also regulate the metabolic pathway of the canine intestine, and promote the improvement of intestinal function. Studies have shown, evodiamine may act directly on the increase of beneficial bacteria, leading to the reduction of harmful bacteria and the maintenance of intestinal balance of nature, or it may act indirectly on the gut microbiota, leading to a change in the interaction between beneficial bacteria and harmful bacteria, to maintain the intestinal balance of nature [30]. This is consistent with our findings. Although only tested for a short period of time, most of the dogs underwent a dietary intervention that produced significant changes in gut flora, with a significant increase in the species diversity of the gut microbiota Firmicutes and a significant decrease in Bacteroides in the dogs in the gut EVO group compared to the control group. SCFAs are known to enhance regulatory T cells in the intestinal mucosa that support immune tolerance and act as microbial metabolites to reverse the dysbiotic microbiota, while a decrease in the abundance of Firmicutes may reduce the production of SCFAs [49,50]. Some studies have shown that the production of SCFAs was related to the increase of Firmicutes [49].
To further analyze the potential physiological and metabolic impacts of these microbial changes on the host, we conducted KEGG pathway enrichment analysis on differentially expressed genes between the two groups. We found that 1718 differentially expressed genes were enriched in the immune system signaling pathway. Key pathways such as STING, NF-κB, inflammasome, TCR, BCR signaling, JAK/STAT, and TLR signaling play vital roles in immune system signal transduction. These signaling pathways are closely associated with intestinal diseases, especially inflammation, indicating that Evodiamine may regulate the immune system signaling pathways by altering the gut microbiota. This modulation could contribute to the protection and maintenance of intestinal health.
In addition, the analysis of routine blood and blood biochemical indicators of dogs showed that the red blood cell index, white blood cell index, mean hemoglobin index, and lymphocyte index in the dog’s routine blood indicators were not statistically significant before and after the experiment. The platelet index of the Evodiamine group at week 0 was significantly higher than that of the control group, but the results were all within the normal range; the aspartate aminotransferase index, creatine kinase index, and amylase index in the dog’s blood biochemical indicators were not statistically significant before and after the experiment. In the eighth week of the experiment, the aspartate aminotransferase index of the Evodiamine group was significantly lower than that of the control group, and the creatinine index of the Evodiamine group was significantly higher than that of the control group, but the results fluctuated within the normal range. Therefore, the results showed that Evodiamine had no significant effect on the physiological and biochemical indicators of dogs.
Although much research has been done on evodiamine, it is often used to study its effects on weight loss [51], anti-inflammation [52], anti-tumor [53], and treatment of cardiovascular disease [54]. Currently, there are no prescription foods on the market with added evodiamine for modulating canine gut microflora diversity and gut function. In this study, we focused on the functionality and safety of the designed gut functional diets. The formulation, composition and functionality of the canine gut functional diets were also studied in a completely innovative manner. Here, we observed no significant changes in physiological indices in dogs fed with enteric functional diets, but increased species diversity of beneficial flora, decreased abundance of harmful flora, and co-regulated an increase in intestinal microbial metabolites, which served to improve the intestinal barrier and protect intestinal health. A limitation of this study is that we only tested the effects of a gut-functional diet on gut microbes in a single breed of experimental dog, and the effects of this diet have not been further validated in pet dogs. Further clinical studies will be conducted in many clinical animals and the effects of a formulated gut-functional diet will be extensively validated.
CONCLUSION
The addition of evodiamine to the diet significantly increased the diversity of canine gut microbiota and regulated the levels of key microbial groups. This suggests that evodiamine has potential positive effects on intestinal health. These findings provide a foundation for further research on the application of evodiamine in improving canine intestinal health and preventing related diseases.
Acknowledgements
We extend our sincere gratitude to the technical staff at the Animal Experiment Center for their assistance with animal husbandry, blood sample collection, and laboratory testing.
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REFERENCES
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