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

Abstract

Background: Moutan cortex radicis (MCR), as a common traditional Chinese medicine, has been widely used as an antipyretic, antiseptic, and anti-inflammatory agent in China. Objectives: This study aimed to investigate the effects of dietary MCR supplementation on the antioxidant capacity and intestinal health of the pigs and to explore whether MCR exerts positive effects on intestinal health via regulating nuclear factor kappa-B (NF-κB) signaling pathway and intestinal microbiota. Methods: MCR powder was identified by LC-MS analysis. Selected 32 weaned piglets (21 d of age, 6.37 ± 0.10 kg average BW) were assigned (8 pens/diet, 1 pig/pen) to 4 groups and fed with a corn-soybean basal diet supplemented with 0, 2,000, 4,000, and 8,000 mg/kg MCR for 21 d. After the piglets were sacrificed, antioxidant indices, histomorphology examination, and inflammatory signaling pathway expression were assessed. The 16s RNA sequencing was used to analyze the effects of MCR on the intestinal microbiota structure of piglets. Results: Supplemental 4,000 mg/kg MCR significantly increased (P < 0.05) the average daily weight gain (ADG), average daily feed intake (ADFI), total antioxidative capability, colonic short-chain fatty acids (SCFA) concentrations, 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 NF-κB in the jejunum and ileum. Microbiota sequencing identified that MCR supplementation significantly increased the microbial richness indices (Chao1, ACE, and observed species, P < 0.05) and the relative abundances of Firmicutes and Lactobacillus (P < 0.05), decreased the relative abundances of Bacteroides, Parabacteroides, unidentified_Lachnospiraceae, and Enterococcus (P < 0.05) and had no significant effects on the diversity indices (Shannon and Simpson, P > 0.05). Microbial metabolic phenotypes analysis also showed that the richness of aerobic bacteria and facultative anaerobic bacteria, oxidative stress tolerance, and biofilm forming were significantly increased (P < 0.05), and the richness of anaerobic bacteria and pathogenic potential of gut microbiota were reduced (P < 0.05) by MCR treatment. Regression analysis showed that the optimal MCR supplemental level for growth performance, serum antioxidant capacity, and intestinal health of weaned piglets was 3,420 ~ 4,237 mg/kg. Conclusions: MCR supplementation improved growth performance and serum antioxidant capacity, and alleviated intestinal inflammation by inhibiting the IKKβ/IκBα/NF-κB signaling pathway and affecting intestinal microbiota in weaned piglets.

Keywords: Moutan cortex radicis; NF-κB signaling; antioxidant capacity; intestinal microbiota; weaned piglets.

Figure 1

The intestinal morphology was histologically analyzed by H&E (500 μm). (A) Jejunum, (B) Ileum. CON, control group, basal diet without antibiotics; LMC, the control diet + 2,000 mg/kg Moutan cortex radicis; MMC, the control diet + 4,000 mg/kg Moutan cortex radicis; HMC, the control diet + 8,000 mg/kg Moutan cortex radicis.

Figure 2

Gene expression levels associated with the proinflammatory factors (IFN-γ, TNF-α, IL-1ß, and IL-6) and tight junction proteins (ZO-1, occludin, and claudin-1) of jejunum (A) and ileum (B), and NF-κB signaling pathway in the jejunum and ileum tissues (C) of pigs fed the Moutan cortex radicis diet. CON, control group, basal diet without antibiotics; LMC, the control diet + 2,000 mg/kg Moutan cortex radicis; MMC, the control diet + 4,000 mg/kg Moutan cortex radicis; HMC, the control diet + 8,000 mg/kg Moutan cortex radicis. Data are expressed as means ± SEM (n = 6). Means with different superscripts in the columns are significantly different (P < 0.05).

Figure 3

Total SCFAs (A), acetic acid (B), propionic acid (C), butyric acid (D), valeric acid (E), isobutyric acid (F), and isopentanoic acid (G) concentrations in colonic contents of pigs fed the Moutan cortex radicis diet for 3 weeks. CON, control group, basal diet without antibiotics; LMC, the control diet + 2,000 mg/kg Moutan cortex radicis; MMC, the control diet + 4,000 mg/kg Moutan cortex radicis; HMC, the control diet + 8,000 mg/kg Moutan cortex radicis. Data are expressed as means ± SEM (n = 8). Means with different superscripts in the columns are significantly different (P < 0.05).

Figure 4

Effect of dietary Moutan cortex radicis on the colonic microbiota diversity and composition in the pigs. (A) A Venn diagram illustrating the overlaps of OTUs in the gut microbiota; (B) The microbial alpha diversity indices (Observed-species, Chao1, Shannon, Simpson, ACE) were calculated using the mothur program; (C) Principal coordinate analysis (PCoA); (D) non-metric multidimensional scaling (NMDS) analysis; (E) unweighted UniFrac cluster tree based on Unweighted Pair-group Method with Arithmetic Mean (UPGMA) analysis; (F) The significant different species among groups based on MetaStat analysis. CON, control group, basal diet without antibiotics; LMC, the control diet + 2,000 mg/kg Moutan cortex radicis; MMC, the control diet + 4,000 mg/kg Moutan cortex radicis; HMC, the control diet + 8,000 mg/kg Moutan cortex radicis. Data are expressed as means ± SEM (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

Effects of dietary Moutan cortex radicis on microbial composition at the phylum and genus levels of growing pigs. (A) Relative contribution of the top 10 phylum in each group (left) and the relative abundance of significantly different microorganisms (right); (B) Relative contribution of the top 10 genus in each group (left) and the relative abundance of significantly different microorganisms (right). CON, control group, basal diet without antibiotics; LMC, the control diet + 2,000 mg/kg Moutan cortex radicis; MMC, the control diet + 4,000 mg/kg Moutan cortex radicis; HMC, the control diet + 8,000 mg/kg Moutan cortex radicis. Data are expressed as means ± SEM (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6

Dietary supplementation of Moutan cortex radicis altered the metabolic functions and phenotypes of colonic microbiota in the pigs. (A) Principal components analysis (PCA) of functional profiles in the gut microbiota; (B) The heatmap tree based on different metabolism-related pathways at KEGG level 3; (C) The metabolic phenotypes prediction were compared using BugBase online (https://bugbase.cs.umn.edu/). The relative abundances of discrete phenotype were performed using pair-wise Mann-Whitney U-tests. Data are expressed as means ± SEM (n = 6). CON, control group, basal diet without antibiotics; LMC, the control diet + 2,000 mg/kg Moutan cortex radicis; MMC, the control diet + 4,000 mg/kg Moutan cortex radicis; HMC, the control diet + 8,000 mg/kg Moutan cortex radicis. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7

Heatmap of the Spearman's r correlations analysis. Spearman's correlation coefficients between colonic microbiota and growth and serum antioxidant parameters (A), and colonic SCFA contents (B). *P < 0.05 and **P < 0.01.