Dietary Moutan Cortex Radicis Alters Serum Antioxidant Capacity, Intestinal Immunity and Colonic Microbiota in Weaned Piglets

Background: Moutan cortex radicis (MCR), as a common traditional Chinese medicine, has been widely used as antipyretic, antiseptic and anti-inammatory agent in China. However, few studies have evaluated the positive effects of MCR, as a new feed additives, on alleviating weaning stress and improving intestinal health and microbiom in pigs. This study aimed to investigate the effect of dietary MCR supplementation on serum antioxidant capacity, intestinal morphology, anti-inammatory mechanism, and microbiota in weaned piglets. Results: Supplemental 2000 mg/kg and 4000 mg/kg MCR increased (P < 0.05) the nal body weight, ADG and ADFI of weaned piglets, and 2000 mg/kg MCR diet signicantly decreased (P < 0.05) the F/G ratio and increased (P < 0.05) serum catalase activity compared with CON group. Also, the villus height and crypt depth in the ileum and the concentrations of total SCFA, acetic acid, butyric acid and valeric acid in the colonic contents were higher (P < 0.05) in the 2000 mg/kg and 4000 mg/kg MCR diets than CON group. Dietary MCR supplementation at 4000 mg/kg MCR signicantly increased (P < 0.05) total antioxidative capability and the crypt depth in the jejunum but decreased (P < 0.05) the mRNA expression levels of Interferon γ, tumor necrosis factor-α, interleukin-1β, inhibiting kappa B kinase β (IKKβ), inhibiting nuclear factor kappa-B (IκBα) and nuclear factor kappa-B (NF-κB) in the jejunum and ileum. Supplemental 8000 mg/kg MCR had the higher total antioxidative capability and catalase activity in the serum but decreased (P < 0.05) the villus height and crypt depth in the jejunum compared with the CON group. MCR addition reduced (P < 0.05) serum malondialdehyde content, and tended to increase the mRNA expression of zonula occludens-1 in


Background
Oxidative stress often causes mammalian tissues cells damage, especially the intestinal, which signi cantly affects health status and decrease performance [1,2]. Various challenges, such as changes in feed nutrition and environment, pathogenic micro-organisms as well as vaccine and drugs use, induces oxidative stress for weaning piglets [3]. Including weaning stress, these combined stresses result in a decrease in food intake, impaired intestinal barrier function, and disordered intestinal ora, which contribute to damaging immune function and increasing the susceptibility to disease [4,5]. Antioxidative enzymes forming an antioxidative defense system protect the body against reactive oxygen species (ROS) overproduction. Besides, oxidative stress and in ammation are closely related. Cytokines are activated and secreted when the systemic in ammatory incidences. The activation of nuclear factor kappa-B (NF-κB), a transcription factor, can promote the pro-in ammatory cytokines expression [6]. Antibiotics have been used as growth promoters and immune enhancers at subtherapeutic levels in feed for many years. However, in recent years, the reduction or removal of dietary antibiotics has become a developing tendency in swine production [7]. Therefore, nding effective and safe feed additives to be instead of antibiotics is a strong demand for the swine industry. Traditional Chinese medicine is a natural substance, safe, and reliable with little toxicity [8]. Due to the extensive antibacterial and synergistic effects, traditional Chinese medicine has no drug resistance and overcomes the shortcomings of antibiotics.
Moutan cortex radicis (MCR) from the tree peony (Paeonia suffruticosa), commonly known as Mu Dan Pi, is a traditional Chinese medicine commonly used for anti-in ammatory, analgesic, antispasmodic and anti-oxidation properties [9,10]. Traditionally, MCR has the clearing heat, promoting blood circulation and removing blood stasis effects on alleviating sickness in humans. Previous researches have demonstrated that MCR has potent free radicals and superoxide anion radicals scavenging capacity, and inhibits ROS production for alleviating oxidative stress [11,12]. MCR is rich in various chemical components, including paeonol, paeoni orin, oxypaeoni orin, galloylpaeoni orin, gallic acid, and so on [13]. Paeonol is known to be the main active ingredient, which is reported to inhibit blood coagulation and platelet aggregation for enhancing blood circulation [14], and reduce the production of pro-in ammatory cytokines [15]. Moreover, MCR and its bioactive components have also been reported to alleviate obesity, diabetes, and in ammation [16,17]. Previous studies showed that in neuro-in ammatory therapy, paeonol inhibited IκBα to suppress the translocation of NF-κB and decrease the release of pro-in ammatory products [18].
In addition, gut microbiota are the important contributor to animal health and growth such as nutritional conversion, immunity, and intestinal mucosal barrier function [19]. However, the positive effect of MCR on weaning stress, in ammatory response, and gut microbiota composition of weaned piglets have not been reported. In the present study, the effects of dietary MCR on growth performance, serum antioxidant indexes, intestinal morphology and anti-in ammatory response, and gut microbiota composition in weaned piglets were explored for the rst time.

Materials And Methods
The animal protocols and care standards of this experiment were accepted and approved by the Committee of Animal Care and Use of the Institute of Subtropical Agriculture, Chinese Academy of Science (Changsha, CAS20190409).

Animals, experimental design and sample collection
Thirty-two Duroc × Large White × Landrace piglets (castrated male), weaned at an age of 21 d, were allocated randomly into four dietary treatments based on an average initial body weight of 6.37 ± 0.10 kg. Each treatment had eight replicates with one piglet each, and each replicate were assigned into an individual pen. Before starting the study, all piglets were adaptated for 3-days and fed a basal diet (cornsoybean meal). Four groups included a basal diet (control, CON), the basal diet + 2000 mg/kg Moutan cortex radicis (LMC), the basal diet + 4000 mg/kg Moutan cortex radicis (MMC) and the basal diet + 8000 mg/kg Moutan cortex radicis (HMC). This experiment lasted for 21 d, and all piglets had unlimited access to feed and water. The formulation of basal diet was to meet the NRC (2012) requirements [20] for growing pigs (Table 1) without antibiotics. Moutan cortex radicis was provided by the Institute of Botany, Chinese Academy of Sciences (Beijing, China). The initial and nal body weights, and feed intake were weighted and recorded throughout the experimental stage. The average daily weight gain (ADG), average daily feed intake (ADFI) and F/G ratio were determined. On day 22, twenty-four piglets (8 piglets per treatment group) were stunned (250V, 0.5A, for 5 ~ 6 s) and killed. Blood samples were collected from precaval vein and kept into vacuum tubes at room temperature for 2 h. Serum was obtained from the supernatant of blood after centrifugation at 3500 × g for 15 minutes, and then stored at -20 °C for further analysis. Approximately 2 cm in length segments of duodenum and jejunum were stored in 4% phosphate-buffered paraformaldehyde (pH 7.6) for histological analysis. Other middle intestinal samples ushed with 0.9% ice-cold physiological saline were immediately frozen in liquid nitrogen and stored at -80℃ for molecular analysis. Colonic contents were collected for short-chain fatty acids (SCFAs) measurement; one sample of colonic content was allocated for microbiota composition determination.

Assessment of Intestinal morphology
The method of Histological hematoxylin eosin (HE) staining, as previously described, was used to evaluate intestinal histomorphological changes [22]. In brief, the middle sections of jejunum and ileum were embedded in para n after removed from xation uid and dehydrated, and made into approximately 5 µm thickness transverse sections, then stained with HE. Villus height (VH) and crypt depth (CD) were measured by a computer-assisted microscopy (Leica DMI3000B microscopy, Switzerland, Germany). Morphological indices were measured from ten microscopic elds at 100 × magni cation. The ratio of villus height to crypt depth (VH/CD) was calculated and analyzed.

Analysis of mRNA expression
The extraction process of total RNA from intestinal tissues was followed by the description of Xiong et al.
(2015) [23]. Brie y, samples were homogenized in the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) for total RNA extraction, then further puri ed with the RNeasy kit (Eppendorf AG, Hamburg, Germany). In the 10 ul reaction systems, 1.0 µg of total RNA was incubated with DNase I for synthetising rst-strand cDNA. Then reverse-transcription using Oligo (dT) primers (Takara, Otsu, Japan) was further to synthesise the double-strand cDNA. Real-time PCR was performed with SYBR Green Master Mix reagent (Takara, Otsu, Japan) and objective gene primer pairs using the LightCycler® 480 Real-Time PCR System (Roche, Switzerland, Germany). Primer 6.0 software was used to design primers of the β-actin housekeeping gene and target genes ( Table 2). The fold changes in target genes was determined using the 2 −ΔΔCt method.  [24]. FLASH, as a very fast and accurate analysis tool, merged paired-end reads from the original DNA fragments, and then identi ed each sample based on the unique barcodes [25]. To mine deeper data of microbial diversity of the differences between the samples, signi cance test were conducted with some statistical analysis methods, including T-test, MetaStat, linear discriminant analysis effect size (LEfSe), Anosim and multi-response permutation procedure (MRPP). Evaluation of the correlation between the gut microbiota and other dimensions was frequently performed with the Spearman's rank correlation test.

Microbiological function and phenotypic prediction
Based on metagenomic 16S rRNA data, Tax4Fun, as a software package, was used for predicting functional pro les [26]. Tax4Fun could perform a mapping of 16 rRNA gene sequences reads to SILVA labeled OUT abundances. Normalized Taxonomic abundances are used to linearly combine the precomputed functional pro les of the KEGG organisms for predicting the microbial functional pro le. Bugbase is a tool for measuring high-level phenotypes in the colonic microbiota using 16S RNA datasets and mapping le [27]. Besides, the Spearman correlation analysis between colonic microbiota and metabolites was performed in R software (v3.2.1).

Short-chain fatty acids (SCFAs) composition of colonic contents
The composition of SCFAs in the colonic contents was determined according to the method described by Kong et al. (2016) [28]. About 1.0 g of the fresh colonic contents were mixed thoroughly with 5 mL distilled water in a centrifuge tube, incubated and shaken 30 min, and centrifuged at 10000 × g, 10 min at 4 °C. After transferring the supernatant into a new centrifuge tube, the precipitate was repeatedly extracted twice with 2 mL distilled water. Mix all supernatants (0.9 mL) with 25% metaphosphoric acid solution (0.1 mL) for 3 ~ 4 h at room temperature, then centrifuge at 10000 × g for 10 min at 4 °C. After ltered through a 0.45-µm polysulfone lter, the supernatant portion was subjected for analyses on Agilent 6890 gas chromatography (Agilent Technologies, Inc, Palo Alto, CA, USA). The standard solutions of acetic, propionic, butyric, isobutyric, valeric and isopentanoic acids were prepared at concentrations of 5, 10, 15, 20 and 25 mmol/L.

Statistical analysis
All statistical analysis were performed using IBM SPSS 22.0 software (SPSS Inc, Chicago, IL, USA) except for microbiome analysis. One-way ANOVA and Tukey-Kramer multiple comparison tests were used to compare the differences among experimental treatments. After nonparametric tests, the 16 rRNA sequencing data were analyzed by a Kruskal-Wallis analysis to det ermine signi cant differences. The differences were declared signi cant at P < 0.05 and a trend at 0.05 < P ≤ 0.10 in all analyses. Results are expressed as means ± standard errors (SEM) unless otherwise noted.

Growth performance
As shown in Table 3, the initial body weight of pigs had no signi cant differences among the treatments (P > 0.05). Compared to the CON and HMC groups, the nal body weight, ADG, and ADFI were signi cantly increased in LMC and MMC groups (P < 0.05). And then, LMC signi cantly reduced the F/G ratio compared with the CON group (P < 0.05). Data are expressed as means ± SEM (n = 8). Means within a row with different superscripts are signi cantly different (P < 0.05).

Serum antioxidant indexes
As shown in Table 4, pigs fed the MMC and HMC diets had higher T-AOC activity (P < 0.05) compared with the CON and LMC groups. The higher CAT activity (P < 0.05) was observed in LMC and HMC pigs compared with CON group. Compared to the MMC group, HMC had a higher CAT activity (P < 0.05) in the serum. Pig fed the CON diet had the highest GSH-Px activity (P < 0.05) and MDA concentration (P < 0.05) in the serum compared with that of the pigs fed the LMC, MMC, and HMC diets. There was no signi cant effect of dietary MCR on SOD activity (P > 0.05).

Jejunal and ileal morphology
The effects of MCR on jejunal and ileal morphology in the pigs are shown in Table 5 and Fig. 1.
Compared with the CON and HMC groups, MMC signi cantly increased the CD (P < 0.05) in the jejunum. The LMC diet increased the ratio of VH/CD(P < 0.05) in the jejunum compared with the MMC diet. Pig fed with the HMC diet had the shortest VH (P < 0.05) compared with that of the pigs fed other three diets. Compared to the CON and HMC diets, LMC and MMC diets markedly increased (P < 0.05) the VH and CD in the ileum. Expression of genes associated with proin ammatory factors, tight junction proteins and NF-κB signaling pathway As shown in Fig. 2, HMC group signi cantly decreased the mRNA expression level of interferon γ (IFN-γ, P < 0.05) in the jejunum and ileum compared with the CON group. Compared to the CON diet, the MMC diet Compared to the CON and HMC diets, LMC and MMC diets signi cantly inhibited (P < 0.05) the expressions of inhibiting kappa B kinase β (IKKβ) and inhibiting nuclear factor kappa-B (IκBα) in the jejunum. MMC group had a lower (P < 0.05) expression level of NF-κB mRNA in the jejunum compared with the HMC group. LMC and MMC had a decreasing tendency for the mRNA expression levels of IKKβ (P = 0.078) and NF-κB (P = 0.064) in the ileum compared with CON and HMC groups. Compared to the CON group, MMC also signi cantly down-regulated the IκBα mRNA expression (P < 0.05) in the ileum.

Concentrations of SCFA in the colonic contents
Analysis of the concentrations of SCFA in the colonic contents revealed differences among all treatments (Fig. 3). The pigs fed the enhanced The concentrations of total SCFA, acetic acid, butyric acid, and valeric acid in the colonic contents were higher in the LMC and MMC groups than in the the CON group (P < 0.05). Also, dietary supplementation of MCR showed a tendency to to increase the concentration of isobutyric acid (P = 0.062) in the colonic contents of pigs. The concentrations of propionic acid and isopentanoic acid among the four treatments had no signi cant difference (P > 0.05).

Colonic microbiota diversity and composition
To better understand the differences in richness, the overlaps among treatments were illustrated using a Venn diagram (Fig. 4A). This analysis showed that CON&LMC, CON&MMC and CON&HMC contained 304, 319 and 361 common OTUs, respectively. As shown in Fig. 4B, the microbial richness indices (Chao1, ACE and observed species) were signi cantly increased (P < 0.05) in the gut microbiota of piglets with Moutan cortex radicis supplementation, whereas no signi cant differences were found in the diversity indices (Shannon and Simpson) of gut microbiota. The principal coordinate analysis (PCoA, Fig. 4C) and nonmetric multidimensional scaling (NMDS, Fig. 4D) analysis of β-diversity showed a strong difference in the microbiota from the control group to Moutan cortex radicis-treated groups. An unweighted Unifrac cluster tree based on the unweighted pair-group method with arithmetic mean (UPGMA) analysis showed the similarity and phylogeny of all observed samples at the phylum level (Fig. 4E), and Firmicutes, Bacteroidetes and Proteobacteria are the dominant bacteria in pigs' colonic microbiota. Further, MetaStat analysis of the microbial community was to explore the signi cant differences in microbial composition between the MCR-treated group and the control group (Fig. 4F). MCR supplementation signi cantly elevated the relative abundance of Tenericutes, and decreased the relative abundance of Bacteroidetes in the colonic microbiota.
As shown in Fig. 5, the phylum level analysis showed that dietary supplementation of MCR signi cantly increased the relative abundance of Firmicutes (P < 0.05) and decreased the relative abundance of Bacteroidetes (P < 0.05). In the genus level, MCR treatment signi cantly decreased the relative abundances of Bacteroides, Parabacteroides, unidenti ed_Lachnospiraceae, and Enterococcus in the colonic microbiota (P < 0.05). Compared to the CON group, LMC and MMC groups increased (P < 0.05) the relative abundance of Lactobacillus.

Metabolic functions and phenotypes of colonic microbiota
Tax4Fun was performed to determine the effects on metabolic functions of gut microbiota by MCR treatment. Based on KEGG annotation results, the principal components analysis (PCA) showed that the microbiotal metabolic functions were signi cantly separated in the CON group and MCR-treated groups (Fig. 6A). As shown in Fig. 6B, KEGG pathways associated with microbial metabolism at level 3, including mismatch repair, pyruvate and purine metabolism, DNA repair and recombination protein, were upregulated by dietary MCR-treated. Galactose metabolism, oxidative phosphorylation and amino acidrelated enzymes were signi cantly down-regulated. Moreover, based on 16S OTU results to predict bacterial phenotype database, Bugbase can be analyzed the differences among groups simultaneously. Results showed that MCR diet signi cantly increased (P < 0.05) the aerobic bacterial richness, and oxidative stress tolerance and bio lm forming of colonic microbiota compared to the CON group. The richness of Gram-positive bacteria showed a marked increasing trend (P = 0.052), while Gram-negative bacteria had a signi cant decrease trend (P = 0.052) by MCR treatments. The pathogenic potential of gut microbiota was reduced (P < 0.05) by increasing dietary MCR level. LMC and HMC groups had a lower (P < 0.05) richness of anaerobic bacteria and a higher (P < 0.05) facultative anaerobic bacteria than the CON group.
Results of Spearman's correlation coe cients between major genera and growth, serum antioxidant parameters makers and colonic SCFA contents were calculated and presented with heatmap (Fig. 7). Lactobacillus and Blautia had signi cant positive relations with ADG, serum CAT activity, and the contents of total SCFAs, acetic acid, propionic acid, butyric acid and valeric acid (P < 0.05), and was negatively related with the F/G ratio (P < 0.05). Bacteroides showed signi cant positive relations with serum GSH-Px activity and MDA content, and was negatively correlated with CAT and the contents of total SCFAs, acetic acid, propionic acid, butyric acid and valeric acid (P < 0.05). Parabacteroides showed signi cant positive correlations with serum GSH-Px activity and the F/G ratio (P < 0.05), and was negatively correlated with CAT and total SCFAs, propionic acid, butyric acid and valeric acid contents (P < 0.05). Unidenti ed_Lachnospiraceae was positively correlated with (P < 0.05) the F/G ratio and GSH-Px activity, and negatively correlated with (P < 0.05) serum SOD activity.

Discussion
In recent years, the misuse of feed antibiotics in the swine industry has seriously threatened human health and food safety, and China has banned the application of antibiotics in feeds in 2020. Therefore, exploring an alternative to antibiotics is necessary for the sustainable development of the livestock industry. Many previous studies have found the positive results of MCR in various animal models of disease [29][30][31]. In the present study, addition of MCR to the diet without antibiotic rstly showed the effect of promoting growth performance in weaned piglets. The improvement may be due to protecting piglets from oxidative stress and intestinal in ammation response caused by weaning stress, which was evidenced by the enhanced antioxidant capacity, inhibition of NF-κB signaling pathway and regulation of intestinal ora structure and metabolites in piglets.
The depletion of intracellular free-radicals and antioxidants inhibited various antioxidant enzymes activities, which induced oxidative stress [32]. The antioxidant mechanism of polyphenols mainly through increasing antioxidant protective barrier and eliminating intracellular ROS to maintain oxidative balance [33,34]. Previous studies demonstrated more than 50 ug/mL of MCR enhanced the antioxidant defense system by improving the activities of GSH and SOD in glucose-induced oxidative damage [35]. In this study, the activities of T-AOC and CAT were improved, and the GSH-Px activity was decreased in weaned piglets supplemented with MCR. Overall, MCR can play an antioxidant role by increasing antioxidant activity. The mechanism of antioxidative stress and anti-in ammation closely connected to the NF-κB signaling pathway in the body [36,37]. Dynamic changes of proin ammatory cytokines levels in the intestinal tract tissue act as crucial messengers to stimulate the intestinal in ammatory process. Therefore, during anti-in ammatory therapy, it is necessary to downregulate the production of these proin ammatory cytokines [38]. The phosphorylation and degradation of the NF-κB bound protein IκB, activated by the IKK signaling phosphorylation, are directly involved in the activating NF-κB [39]. As demonstrated in the present study MCR has effectively decreased the cytokine productions in jejunum and ileum via inhibiting IKKβ/IκBα/NF-κB signaling pathway. At the same time, evidences also found that MCR or paeonol could suppress the gene and protein expression of pro-in ammatory cytokines by blocking NF-κB pathway in the LPS-stimulated in ammatory response [30,40]. Thus it could be suggested that MCR has potential in antioxidant and anti-in ammation therapy in weaned piglets.
Enhanced intestinal morphology and gut barrier are closely associated with nutrients absorption and intestinal integrity [41]. Intestinal morphology signi cantly changes, including villous atrophy and crypt hyperplasia, which will result in diarrhea and growth retardation in pigs [42]. An increasing villus height/crypt depth ratio is one of the most important indexes of intestinal morphology in evaluating the improvement of intestinal function and enhancement of absorption capacity [43]. A recent study found that dietary supplemented with MCR at 2000 mg/kg improved the ratio of villus height to crypt depth in the jejunum, and increased the villus height and crypt depth in the ileum of weaned piglets. 4000 mg/kg MCR increased the villus height and crypt depth in the ileum, whereas 8000 mg/kg MCR decreased the villus height and crypt depth in the jejunum and ileum compared with 2000 mg/kg and 4000 mg/kg MCR groups. Therefore, we speculated that a high dosage (8000 mg/kg) of MCR does not promote the improvement of intestinal villi and intestinal digestive ability. Tight junctions protein, as the mechanical barrier, constitutes intestinal barrier function and prevents pathogenic antigen invasion [44]. Occludin, claudin-1 and ZO-1 are the main cytoplasmic transmembrane and adaptor protein and jointly constitute the tight intercellular junctions. Improved expression of three crucial proteins can enhance the intestinal barrier function for decreasing permeability of the intestinal wall [45]. Several studies have found that traditional Chinese medicine can alter intestinal permeability dependent on tight junctions protein changes [46,47]. Our results also demonstrated that ZO-1 and occludin mRNA expression in jejunum and ileum were increased in piglets fed MCR (8000 mg/kg feed) diet. This suggests that a high dosage of MCR contributed to improving the intestinal barrier integrity in weaned piglets.
The gut microbiome is a complex microbial ecosystem, whose activities and reciprocal relationship has been essential to the host health and disease [48]. The investigation of the gut microbiome has been described as a biomarker for evaluating the effect of speci c dietary components on the host. In the current research, MCR shapes intestinal microbiota in weaned piglets, including increases in the microbial richness, the abundances of the phyla Firmicutes and the genera Lactobacillus, and a decrease in the abundances of the phyla Bacteroidetes, and the genera Bacteroides, Parabacteroides, unidenti ed_Lachnospiraceae and Enterococcus. Piglets fed MCR diets had a higher observed Chao1, ACE and species number for gut microbiota, which indicates that MCR supplementation contributes to improving microbial diversity. Firmicutes and Bacteroidetes, as two main communities, are associated with the energy metabolism homeostasis [49]. Many previous studies reported that increased Firmicutes and reduced Bacteroidetes are most common in the obesity phenotype, which leaded to effectively absorb the calories from food [50]. The abundance of Lactobacillus in the intestine is closely related to activating the production of secretory IgA for improving intestinal mucosal immunity, which acts an important role in maintaining intestinal barrier function [51]. Bacteroides and Parabacteroides, occurring in the early stages of life, have been reported to produce gamma amino butyric acid, associated with growth [52]. The abundance of Enterococcus correlated positively with metabolites associated with inducing oxidative stress [53]. Moreover, changed microbial composition has been linked to the production and composition of SCFA in the colon. In the present study, we found that colonic contents of SCFA, including acetic acid, propionic acid, butyric acid, and valeric acid, were increased signi cantly in piglets fed the MCR diet at 2000 and 4000 mg/kg. SCFA, as an important metabolite of gut microbiota, could favor the energy homeostasis, and relieve in ammations and metabolic syndrome in the colon [54].
Corrêa-Oliveira has demonstrated that the addition of SCFA increased villi height and crypt depth, enhanced the intestinal barrier, and had anti-in ammatory properties in mice [55]. In summary, MCR addition regulates piglets' intestinal microbiota and microbial metabolites for improving intestinal health.
And it would be interesting to further investigate whether MCR has a marked in uence on lipid metabolism through regulating intestinal microbiota in weaned piglets.
Based on microbial function prediction, results demonstrated that MCR addition increased the pyruvate metabolism, DNA repair and purine metabolism, and decreased oxidative phosphorylation and amino acid-related enzymes. MCR may inhibit the amino acid metabolism and promote the nucleotide metabolism and multi-drug resistance in gut microbial communities. Moreover, the changes of microbial metabolic phenotypes in weaned piglets treated with different doses of MCR, were rst revealed. Dietary supplementation of MCR has a strong antimicrobial property against Gram-negative and anaerobic bacteria, but promotes the proliferation of Gram-positive and aerobic bacteria. MCR supplementation also increased bio lm forming and oxidative stress tolerance, while the promoting effect was negatively correlated with the added dose. Bio lm formation and oxidative stress tolerance of microbial communities were found to go together with drug resistance, in ammation, and pathogenesis [56]. Higher MCR levels signi cantly reduced the pathogenic potential of microbial communities. However, these metabolic phenotypes changes need to further explore the mechanism. Further, association analysis of growth performance, serum antioxidants, colonic SCFA contents and microbiota rst revealed that MCR supplementation has widely in uenced the growth and health in piglets.

Conclusions
In conclusion, dietary supplemented with Moutan cortex radicis was able to signi cantly alleviate weaning stress in piglets, as demonstrated by improving antioxidant capacity and regulating gut microbial communities. Moutan cortex radicis increased serum antioxidant capacity, improved intestinal barrier function and inhibited the NF-κB signaling pathway. Additionally, besides improving the richness indices, MCR signi cantly increased the microbial metabolic phenotypes and functions, and metabolites, which is bene t weaned piglets with better intestinal status and growth potential. The present study contributes to provide theoretical support in applicating Moutan cortex radicis at 4000 mg/kg for antioxidation and regulating intestinal health in livestock production.  Gene expression levels associated with the proin ammatory factors (IFN-γ, TNF-α, IL-1ß and IL-6) and