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 [29–31]. In the present study, addition of MCR to the diet without antibiotic firstly showed the effect of promoting growth performance in weaned piglets. The improvement may be due to protecting piglets from oxidative stress and intestinal inflammation response caused by weaning stress, which was evidenced by the enhanced antioxidant capacity, inhibition of NF-κB signaling pathway and regulation of intestinal flora structure and metabolites in piglets.
The depletion of intracellular free-radicals and antioxidants inhibited various antioxidant enzymes activities, which induced oxidative stress . 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 . 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-inflammation closely connected to the NF-κB signaling pathway in the body [36, 37]. Dynamic changes of proinflammatory cytokines levels in the intestinal tract tissue act as crucial messengers to stimulate the intestinal inflammatory process. Therefore, during anti-inflammatory therapy, it is necessary to downregulate the production of these pro-inflammatory cytokines . 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 . 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-inflammatory cytokines by blocking NF-κB pathway in the LPS-stimulated inflammatory response [30, 40]. Thus it could be suggested that MCR has potential in antioxidant and anti-inflammation therapy in weaned piglets.
Enhanced intestinal morphology and gut barrier are closely associated with nutrients absorption and intestinal integrity . Intestinal morphology significantly changes, including villous atrophy and crypt hyperplasia, which will result in diarrhea and growth retardation in pigs . 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 . 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 . 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 . 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 . The investigation of the gut microbiome has been described as a biomarker for evaluating the effect of specific 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, unidentified_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 . 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 . 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 . Bacteroides and Parabacteroides, occurring in the early stages of life, have been reported to produce gamma amino butyric acid, associated with growth . The abundance of Enterococcus correlated positively with metabolites associated with inducing oxidative stress . 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 significantly 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 inflammations and metabolic syndrome in the colon . Corrêa-Oliveira has demonstrated that the addition of SCFA increased villi height and crypt depth, enhanced the intestinal barrier, and had anti-inflammatory properties in mice . 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 influence 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 first 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 biofilm forming and oxidative stress tolerance, while the promoting effect was negatively correlated with the added dose. Biofilm formation and oxidative stress tolerance of microbial communities were found to go together with drug resistance, inflammation, and pathogenesis . Higher MCR levels significantly 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 first revealed that MCR supplementation has widely influenced the growth and health in piglets.