早期断奶往往会造成仔猪生长性能下降、腹泻率升高、组织器官发育受损。植物提取物芦丁已被证实具有多种生物学功能，如抗氧化、抗炎、促生长及改善器官功能等，在畜牧业具有良好的应用前景。然而，目前芦丁对早期断奶仔猪的作用效果及相关机制的研究鲜有报道。鉴于此，本试验通过在饲粮中添加芦丁首先探讨其对断奶仔猪生长性能和肠道功能的影响，再从肠道微生物、炎症和抗氧化三个角度分析了芦丁改善断奶仔猪肠道屏障功能的潜在机制。在此基础上，以肝脏为靶器官，进一步探讨了其对仔猪体内组织的作用，并验证肠道中的调控机制，从而为芦丁在断奶仔猪饲粮中的应用及研究提供依据。

试验一：芦丁对断奶仔猪生长、空肠形态和功能的影响

选取16头体重相近的仔猪于28日龄断奶，随机分为CON组和Rutin组。其中CON组仔猪饲喂基础日粮，Rutin组仔猪饲喂试验日粮（基础日粮+500 mg/kg芦丁），至42日龄屠宰取样分析。结果表明：（1）日粮添加芦丁显著提高了断奶仔猪饲料转化率，降低腹泻指数（P < 0.05），但对仔猪器官指数（心脏、肝脏、脾脏、肾脏、胰腺）、平均日增重和平均日采食量无显著影响（P > 0.05），而显著提高了仔猪血清中生长激素（GH）含量（P < 0.05）。（2）日粮添加芦丁显著提高了仔猪空肠绒毛高度（VH）和绒毛高度/隐窝深度（VH/CD）（P < 0.05）。添加芦丁显著降低了仔猪空肠黏膜组织中Caspase3和Bax的mRNA表达水平（P < 0.05），显著提高了Wnt、β-catenin和Bmi1的mRNA，Wnt和β-catenin的蛋白表达水平（P < 0.05）。（3）与CON组相比，Rutin组空肠线粒体清晰完整，线粒体嵴结构紧凑且分布均匀。相比之下，CON组仔猪空肠线粒体异常形态较多，完整性较差。日粮添加芦丁还显著降低仔猪空肠肿胀的线粒体数目和线粒体相对面积（P < 0.05）。添加芦丁显著提高了仔猪空肠黏膜中PGC1α和NRF1的mRNA 表达水平和PGC1α的蛋白表达水平，显著降低了AMPK 的mRNA表达水平和pAMPK的蛋白表达水平，pAMPK/AMPK也显著降低（P < 0.05）；添加芦丁显著降低了仔猪空肠黏膜中PINK1和Parkin的mRNA和蛋白表达水平，显著提高了p62的 mRNA和蛋白表达水平，LC3-II/LC3-I也显著降低（P < 0.05）。综上所述，日粮添加500mg/kg芦丁对断奶仔猪体重、平均日增重无显著影响，但可有效降低仔猪腹泻及提高饲料转化率，后者应与芦丁通过改善线粒体功能和激活Wnt通路促进细胞增殖和抑制凋亡，从而改善空肠形态密切相关。

试验二：芦丁通过Nrf2通路改善肠屏障的潜在途径

基于上述结果发现芦丁有效降低仔猪腹泻，本试验进一步研究了芦丁对肠道屏障功能的影响，并分析了其潜在机制。结果表明：（1）添加芦丁显著降低了仔猪血清中二胺氧化酶（DAO）的活性（P < 0.05），而空肠黏膜中ZO-1和Claudin-1的mRNA和蛋白表达水平，MUC2和MUC4的mRNA表达水平均显著升高（P < 0.05）。（2）添加芦丁显著降低了仔猪空肠黏膜中白介素1β（IL-1β）的含量，显著降低了仔猪空肠黏膜中TNF-α、IL-1β、IL-6、IFN-γ和NF-κB的mRNA表达水平，而NF-κB p65蛋白表达显著下调，IκBα蛋白表达显著上调（P < 0.05）。（3）添加芦丁显著降低了丙二醛（MDA）的含量，显著提高了总超氧化物歧化酶（T-SOD）和谷胱甘肽过氧化物酶（GSH-Px）的活性（P < 0.05）；添加芦丁显著提高了仔猪空肠黏膜中Nrf2、GPX1和SOD1的mRNA表达水平，Keap1的 mRNA的表达显著下降，Nrf2和GCLM的蛋白表达显著升高，Keap1的蛋白表达显著降低（P < 0.05）。（4）相关性分析结果显示，DAO活性与MDA含量呈正相关，腹泻指数与MDA和IL-1β含量呈正相关，但和T-SOD活性呈负相关（P < 0.05）；Nrf2基因表达水平与MUC2、ZO-1和MUC4基因表达水平呈显著正相关；NF-κB基因表达水平与MUC2和ZO-1基因表达水平呈显著负相关（P < 0.05）。（5）两组间仔猪空肠黏膜中ZONAB、SP1、 KLF4和STAT3的mRNA表达水平无显著差异（P > 0.05）；与CON组相比，Rutin组仔猪空肠黏膜中NKRF的mRNA表达显著上升（P < 0.05），但HDAC3、CBP、MafK、β-TrCP、GSK3β、VCAM-1、CD62E、TRAF6和NGF的mRNA表达水平无显著差异（P > 0.05）。（6）CON组和Rutin组之间盲肠微生物Chao1指数、ACE指数、Shannon指数和Simpson指数无显著差异（P > 0.05）。PCoA分析结果显示，CON组和Rutin组微生物组成没有明显分离（R² = 0.082，P = 0.642），PC1和PC2分别占总变异度的23 %和14%。在门水平上，盲肠微生物群主要由Firmicutes、Bacteroidota、Proteobacteria 和Spirochaetota组成，与CON组相比，Rutin组仔猪盲肠微生物群中Firmicutes相对丰度显著上升，Campylobacterota相对丰度显著下降（P < 0.05）。在属水平上，Clostridium.sensu.stricto.1、Alloprevotella、Blautia、Actinobacillus、Terrisporobacter、Roseburia、Lactobacillus、Prevotella-9、Ruminococcus和Subdoligranulum菌属的相对丰度占主要优势。与CON组相比，Rutin组仔猪盲肠微生物群中Clostridium sensu stricto 1、Prevotellaceae NK3B31 group、Phascolarctobacterium和Megasphaera的相对丰度显著上升（P < 0.05）。但相关性分析结果表明，肠道紧密连接蛋白和黏蛋白mRNA表达量、腹泻指数和DAO含量均与肠道菌群无相关性。综上所述，芦丁可能通过激活Nrf2/GCLM通路提高空肠抗氧化能力来抑制NF-κB通路缓解炎症反应，从而改善肠屏障。芦丁虽然可以改变盲肠微生物组成，但肠屏障的改善与微生物组成的变化无相关性。

试验三：芦丁的转运及对断奶仔猪肝脏抗氧化及炎症的影响

上述试验明确了芦丁可在肠道内通过激活Nrf2通路增强抗氧化抑制炎症发生，但其是否可至体内直接对组织器官（如肝脏）产生作用尚不明确，故本试验以肝脏为靶器官，从芦丁吸收转运及其对肝脏功能，特别是抗氧化及炎症等方面，进一步研究其在体内的作用效果。结果表明：（1）Rutin组仔猪肝脏中检测到芦丁含量为2.49 μg/g ，CON组中没有检测到芦丁的含量；添加芦丁显著提高了仔猪空肠黏膜和回肠黏膜中P-gp和MRP3的mRNA表达水平，空肠黏膜中MRP2的 mRNA的表达显著上升，血清中ALB的含量显著上升，肝脏OATP2B1的mRNA表达显著上升（P < 0.05）。分子对接结果显示芦丁与P-gp、MRP3和OATP2B1的结合能分别为-8.7、-8.7、-8.8 kcal/mol。（2）添加芦丁显著提高了仔猪肝脏中PGC1α、NRF1和ERRα的mRNA表达，AMPK 的mRNA的表达显著下降（P < 0.05），PGC1α的蛋白表达显著升高，pAMPK的蛋白表达显著降低，pAMPK/AMPK也显著降低（P < 0.05），PINK1、LC3-II和Parkin的mRNA和蛋白表达显著下降，p62的 mRNA和蛋白的表达显著升高，LC3-II/LC3I也显著降低（P < 0.05）。（3）添加芦丁显著降低了仔猪肝脏中MDA的含量，T-AOC和GSH-Px活性显著升高（P < 0.05），Nrf2、GPX1、GPX4、GCLC、GCLM、SOD1和NQO1的mRNA表达显著上升，Keap1和ALOX12的 mRNA的表达显著下降（P < 0.05），Nrf2、HO-1、GCLC和GCLM的蛋白表达显著升高，Keap1的蛋白表达显著降低（P < 0.05）；与CON组相比，Rutin组仔猪肝脏中TNFα、IL-1β和IFN-γ的mRNA表达显著下降，IL-10的mRNA表达显著上升，NF-κB p65的蛋白表达显著下降（P < 0.05）。（4）与CON组相比，Rutin组仔猪肝脏VCAM-1和CD62E的mRNA表达显著降低，NKRF的mRNA表达显著上升（P < 0.05），但HDAC3、CBP、MafK、β-TrCP、GSK3β、TRAF6和NGF的mRNA表达水平在CON和Rutin组间无显著差异（P > 0.05）。（5）与CON组相比，Rutin组仔猪盲肠内容物中乙酸、丙酸、丁酸和戊酸的含量显著升高；肝脏中FFAR2、FFAR3和HCAR2的mRNA表达显著降低（P < 0.05）。综上所述，芦丁可直接进入肝脏，并通过激活Nrf2通路来抑制NF-κB通路降低肝脏炎症发生，这与芦丁在肠道中的作用效果类似。此外，芦丁还可通过促进盲肠中SCFA的产生，进而激活肝脏SCFA受体基因的表达，提高肝脏抗氧化能力。

综上所述，断奶仔猪日粮添加500mg/kg芦丁（14天）虽未显著影响体重，但可激活Wnt信号通路、提高线粒体功能有效改善肠道形态，提高断奶仔猪饲料转化率。此外，芦丁可改善肠道屏障功能缓解仔猪腹泻，这应与芦丁激活Nrf2增强抗氧化能力进而抑制NF-κB通路降低炎症密切相关，但与芦丁导致盲肠道微生物组成的改变无关。芦丁可直接进入肝脏，激活Nrf2通路抑制NF-κB通路降低炎症，与肠道作用结果类似。

Early weaning frequently results in reduced growth performance, elevated diarrhea incidence, and impaired organ development in piglets. Rutin, a plant-derived flavonoid extract, has been demonstrated to exhibit multiple biological functions including antioxidant, anti-inflammatory, growth-promoting, and organ function-enhancing properties, demonstrating promising application potential in animal husbandry. However, to date, the effects of rutin on early-weaned piglets and the related mechanisms remain poorly understood, with limited studies systematically addressing this subject. In view of this, this experiment was conducted to explore the effects of rutin on the growth performance and intestinal function of weaned piglets by adding rutin to the diet firstly, and then analyzed the potential mechanisms of rutin to improve the intestinal barrier function of weaned piglets from three perspectives: intestinal microorganisms, inflammation and antioxidant. On this basis, the effects of rutin on piglets' internal tissues were further investigated using the liver as the target organ, and the intestinal regulatory mechanism was verified, so as to provide a basis for the application of rutin in the diets of weaned piglets and for research.

Experiment 1: Effects of rutin on growth, jejunum morphology and function of weaned piglets

Sixteen piglets of similar weight were selected and weaned at 28 days of age, and randomly divided into CON group and Rutin group. The piglets in the CON group were fed the basal diet, and the piglets in the Rutin group were fed the experimental diet (basal diet + 500 mg/kg rutin), and the samples were slaughtered and analyzed at 42 days of age. The results showed that: (1) Dietary supplementation of rutin significantly increased the feed conversion ratio and decreased the diarrhea index of weaned piglets (P < 0.05), but had no significant effect on piglet organ indices (heart, liver, spleen, kidney, and pancreas), average daily gain, and average daily feed intake(P > 0.05), while significantly increasing the serum growth hormone (GH) content of piglets(P < 0.05). (2) Dietary supplementation of rutin significantly increased jejunal villus height (VH) and villus height/crypt depth (VH/CD) in piglets (P < 0.05). Addition of rutin significantly decreased the mRNA expression levels of Caspase3 and Bax in jejunal mucosal tissues of piglets (P < 0.05), and significantly increased the mRNAs of Wnt, β-catenin and Bmi1, and the protein expression levels of Wnt and β-catenin (P < 0.05). (3) Compared with the CON group, jejunal mitochondria in the Rutin group were clear and intact, and the mitochondrial cristae were compact and evenly distributed. In contrast, piglets in the CON group had more abnormal mitochondrial morphology and poorer integrity. Dietary supplementation of rutin also significantly reduced the number of mitochondria and the relative area of mitochondria in the swollen jejunum of piglets (P < 0.05). Addition of rutin significantly increased the mRNA expression level of PGC1α and NRF1 and the protein expression level of PGC1α, significantly decreased the mRNA of expression level AMPK and the protein expression level of pAMPK in the jejunal mucosa of piglets. pAMPK/AMPK was also significantly decreased (P < 0.05); Addition of rutin significantly decreased the mRNA and protein expression levels of PINK1 and Parkin, significantly increased the mRNA and protein expression levels of p62, and significantly decreased LC3-II/LC3I in the jejunal mucosa of piglets (P < 0.05). In conclusion, dietary supplementation of 500 mg/kg rutin had no significant effect on the body weight and average daily weight gain of weaned piglets, but it could effectively reduce piglet diarrhea and improve feed conversion rate, the latter should be closely related to the fact that rutin improves jejunal morphology by improving mitochondrial function and activating the Wnt pathway to promote cell proliferation and inhibit apoptosis.

Experiment 2: Potential pathway for rutin to improve the intestinal barrier through the Nrf2 pathway

According to the above results, we found that rutin was effective in reducing piglet diarrhea, and we further investigated the effect of rutin on intestinal barrier function and analyzed the potential mechanisms. The results showed that: (1) Addition of rutin significantly decreased the serum diamine oxidase (DAO) activity in piglets, while the mRNA and protein expression levels of ZO-1 and Claudin-1, and the mRNA expression levels of MUC2 and MUC4 in the jejunum mucosa were significantly elevated. (P < 0.05).(2) Addition of rutin significantly reduced interleukin 1β (IL-1β) levels in the jejunal mucosa of piglets, and significantly decreased the mRNA expression levels of TNF-α, IL-1β, IL-6, IFN-γ, and NF-κB in the jejunal mucosa of piglets, while the expression of the NF-κB p65 protein was significantly downregulated, and the expression of the IκBα protein was significantly upregulated (P < 0.05).(3) Addition of rutin significantly reduced the content of malondialdehyde (MDA) and significantly increased the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) (P < 0.05). Addition of rutin significantly increased the levels of mRNA expression of Nrf2, GPX1, and SOD1, and the mRNA expression of Keap1 was significantly decreased in the jejunoileal mucosa of piglets, while the protein expression of Nrf2 and GCLM was significantly increased and that of Keap1 was significantly decreased (P < 0.05). The protein expression of Nrf2 and GCLM was significantly increased, and that of Keap1 was significantly decreased (P < 0.05).(4) Correlation analysis showed that DAO activity was positively correlated with MDA content, and diarrhea index was positively correlated with MDA and IL-1β content, but negatively correlated with T-SOD activity (P < 0.05); Nrf2 gene expression level was significantly positively correlated with MUC2, ZO-1, and MUC4 gene expression levels; NF-κB gene expression level was significantly negatively correlated with MUC2 and ZO-1 gene expression levels; NF-κB gene expression levels were significantly negatively correlated with MUC2 and ZO-1 gene expression levels (P < 0.05). (5) The mRNA expression levels of ZONAB, SP1, KLF4 and STAT3 in the jejunal mucosa of piglets were not significantly different between the two groups (P > 0.05); compared with the CON group, the mRNA expression of NKRF in the jejunal mucosa of piglets in the Rutin group was significantly elevated (P < 0.05), but the mRNA expression levels of HDAC3, CBP, MafK, β-TrCP, GSK3β, VCAM-1, CD62E, TRAF6 and NGF mRNA expression levels were not significantly different (P > 0.05). (6) There were no significant differences in cecum microbial Chao1 index, ACE index, Shannon index and Simpson index between CON and Rutin groups (P > 0.05). PCoA analysis showed that there was no significant separation in microbial composition between CON and Rutin groups (R2 = 0.082, P = 0.642), with PC1 and PC2 accounting for 23 % and 14 % of the total variability. At the portal level, the cecum microbiota was mainly composed of Firmicutes, Bacteroidota, Proteobacteria and Spirochaetota. Compared with the CON group, the relative abundance of Firmicutes in the cecum microbiota of piglets in the Rutin group was significantly higher, and that of Campylobacterota was significantly lower (P < 0.05). At the genus level, Clostridium.sensu.stricto.1, Alloprevotella, Blautia, Actinobacillus, Terrisporobacter, Roseburia, Lactobacillus, Prevotella-9, Ruminococcus and Subdoligranulum dominated the relative abundance. The relative abundance of Clostridium sensu stricto 1, Prevotellaceae NK3B31 group, Phascolarctobacterium and Megasphaera in the cecum microbiota of piglets in the Rutin group was significantly higher compared to the CON group (P < 0.05). However, the results of correlation analysis showed that the mRNA expression of intestinal tight junction proteins and mucins, diarrhea index and DAO content were not correlated with intestinal flora. In conclusion, rutin could improve the intestinal barrier by inhibiting the NF-κB pathway to alleviate the inflammatory response by activating the Nrf2/GCLM pathway to increase the antioxidant capacity of the jejunum. Although rutin could change the microbial composition of the cecum, the improvement of the intestinal barrier did not correlate with the changes in microbial composition.

Experiment 3: Translocation of rutin and effects on antioxidant and inflammation in the liver of weaned piglets

The above trials clearly demonstrated that rutin could enhance antioxidant and inhibit inflammation by activating the Nrf2 pathway in the intestinal tract, but it was not clear whether it could directly exert its effects on tissues and organs (e.g., liver) in vivo. Therefore, in this trial, we took the liver as the target organ to further investigate the effects of rutin in vivo from the aspects of its absorption and transportation and its effects on liver functions, especially antioxidant and inflammation.The results showed that: (1) The liver of piglets in the Rutin group contained 2.49 μg/g of rutin, and no rutin was detected in the CON group. Addition of rutin significantly increased the mRNA expression of P-gp and MRP3 in the jejunal and ileal mucosa of piglets, and the mRNA expression of MRP2 in the jejunal mucosa was significantly elevated, and the serum content of ALB was significantly elevated, and the mRNA expression of hepatic OATP2B1 was significantly elevated (P < 0 05). mRNA expression was significantly increased (P < 0.05). The molecular docking results showed that the binding energies of rutin to P-gp, MRP3 and OATP2B1 were -8.7, -8.7 and -8.8 kcal/mol, respectively.(2) Addition of rutin significantly increased the mRNA expression of PGC1α, NRF1 and ERRα in piglet liver, the mRNA expression of AMPK was significantly decreased (P < 0.05), the protein expression of PGC1α was significantly elevated, the protein expression of pAMPK was significantly decreased, and pAMPK/AMPK was also significantly decreased (P < 0.05), the mRNA and protein expression of PINK1, LC3-II and Parkin mRNA and protein expression were significantly decreased, mRNA and protein expression of p62 was significantly elevated, and LC3-II/LC3I was also significantly decreased (P < 0.05). (3) Addition of rutin significantly decreased the MDA content, T-AOC and GSH-Px activities in piglet livers (P < 0.05), the mRNA expression of Nrf2, GPX1, GPX4, GCLC, GCLM, SOD1 and NQO1 was significantly increased, and mRNA expression of Keap1 and ALOX12 was significantly decreased (P < 0.05). The protein expression of Nrf2, HO-1, GCLC and GCLM was significantly higher, and the protein Keap1 was significantly lower (P < 0.05). Compared with the CON group, the mRNA expression of TNFα, IL-1β and IFN-γ in the livers of piglets in the Rutin group was significantly lower, the mRNA expression of IL-10 was significantly higher, and the protein expression of NF-κB p65 was significantly lower (P < 0.05). (4) Compared with the CON group, the mRNA expression of VCAM-1 and CD62E in piglet livers of the Rutin group was significantly lower, and the mRNA expression of NKRF was significantly higher (P < 0.05), but the mRNA expression levels of HDAC3, CBP, MafK, β-TrCP, GSK3β, TRAF6, and NGF did not differ significantly between the CON and Rutin groups (P > 0.05). (5) Compared with the CON group, the levels of acetic acid, propionic acid, butyric acid and valeric acid in the cecum contents of piglets in the Rutin group were significantly higher; and the mRNA expression of FFAR2, FFAR3 and HCAR2 in the liver was significantly lower (P < 0.05). In conclusion, rutin could directly enter the liver and inhibit the NF-κB pathway to reduce liver inflammation occurrence by activating the Nrf2 pathway, which is similar to the effect of rutin in the intestine. In addition, rutin could improve liver antioxidant capacity by promoting the production of SCFA in the cecum, which in turn activated the expression of liver SCFA receptor genes.

In conclusion, although the addition of 500 mg/kg rutin to the diet of weaned piglets (14 days) did not significantly affect body weight, it could activate the Wnt signaling pathway, enhance mitochondrial function, effectively improve intestinal morphology, and increase the feed conversion rate of weaned piglets. In addition, rutin improved the intestinal barrier function and alleviated piglet diarrhea, which was closely related to the activation of Nrf2 by rutin to enhance antioxidant and inhibit the NF-κB pathway to reduce inflammation, but was not related to the alteration of microbial composition of the cecum caused by rutin. Furthermore, rutin could directly enter the liver and activate the Nrf2 pathway to inhibit the NF-κB pathway to reduce inflammation, similar to the results of intestinal effects.