The Effects of Dietary Silybin Supplementation on the Growth Performance and Regulation of Intestinal Oxidative Injury and Microflora Dysbiosis in Weaned Piglets

Abstract

Oxidative stress is the major incentive for intestinal dysfunction in weaned piglets, which usually leads to growth retardation or even death. Silybin has caught extensive attention due to its antioxidant properties. Herein, we investigated the effect of dietary silybin supplementation on growth performance and determined its protective effect on paraquat (PQ)-induced intestinal oxidative damage and microflora dysbiosis in weaned piglets. In trial 1, a total of one hundred twenty healthy weaned piglets were randomly assigned into five treatments with six replicate pens per treatment and four piglets per pen, where they were fed basal diets supplemented with silybin at 0, 50, 100, 200, or 400 mg/kg for 42 days. In trial 2, a total of 24 piglets were randomly allocated to two dietary treatments with 12 replicates per treatment and 1 piglet per pen: a basal diet or adding 400 mg/kg silybin to a basal diet. One-half piglets in each treatment were given an intraperitoneal injection of paraquat (4 mg/kg of body weight) or sterile saline on day 18. All piglets were euthanized on day 21 for sample collection. The results showed that dietary supplementation with 400 mg/kg silybin resulted in a lower feed conversion ratio, diarrhea incidence, and greater antioxidant capacity in weaned piglets. Dietary silybin enhanced intestinal antioxidant capacity and mitochondrial function in oxidative stress piglets induced by PQ. Silybin inhibited mitochondria-associated endogenous apoptotic procedures and then improved the intestinal barrier function and morphology of PQ-challenged piglets. Moreover, silybin improved intestinal microbiota dysbiosis induced by the PQ challenge by enriching short-chain fatty-acid-producing bacteria, which augmented the production of acetate and propionate. Collectively, these findings indicated that dietary silybin supplementation linearly decreased feed conversion ratio and reduced diarrhea incidence in normal conditions, and effectively alleviated oxidative stress-induced mitochondrial dysfunction, intestinal damage, and microflora dysbiosis in weaned piglets.

Keywords: growth performance; intestinal health; microflora dysbiosis; mitochondria function; silybin; weaned piglet.

Figure 1

Dietary silybin supplementation alleviated the redox imbalance and growth retardation induced by paraquat challenges in piglets. (A) Schematic diagram of experimental design. (B) ADG on days 18–21. (C) ADFI on days 18–21. (D) F/G on days 18–21. (E–I) The activity of plasma CAT (E), SOD (F), GSH-Px (G), and the concentration of MDA (H) and H₂O₂ (I). Data are expressed as mean ± standard error. * p < 0.05. Ctrl = basal diet group treated with saline; Si = silybin diet group treated with saline; PQ = basal diet group treated with paraquat; Si + PQ = silybin diet group treated with paraquat; ns = not significant; ADG = average daily gain; ADFI = average daily feed intake; F/G = ADFI/ADG; CAT = catalase; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; MDA = malondialdehyde; and H₂O₂ = hydrogen peroxide.

Figure 2

Dietary silybin supplementation alleviated paraquat-induced intestinal oxidative stress in piglets. The activities of CAT (A), SOD (B), GSH-Px (C), and the level of MDA (D) in the jejunum. The heat maps of the mRNA abundance for antioxidant enzyme genes (E) and Nrf2/Keap1 signaling pathway genes (F), * p < 0.05 vs. Ctrl group; ## p < 0.01 vs. PQ group. Data are expressed as mean ± standard error. * p < 0.05 and ** p < 0.01. Ctrl = basal diet group treated with saline; Si = silybin diet group treated with saline; PQ = basal diet group treated with paraquat; Si + PQ = silybin diet group treated with paraquat; CAT = catalase; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; MDA = malondialdehyde; GPX = glutathione peroxidase; Nrf2 = nuclear factor-erythroid 2-related factor 2; Keap1 = kelch-like ECH-associated protein l; HO-1 = heme oxygenase-1; and NQO1 = NAD(P)H: quinone oxidoreductase 1.

Figure 3

Dietary silybin supplementation protected against PQ-induced mitochondrial injury. (A–E) The expression of mitochondrial biogenesis genes includes FIS1 (A), DNM1 (B), MFN1 (C), MFN2 (D), and OPA1 (E). The relative mRNA abundance of mitochondrial respiratory chain membrane protein-related genes includes NDUFS2 (F), NDUFV2 (G), SDHA (H), UQCRB (I), and ATP5H (J). The heatmap of Spearman’s correlation between the expression of mitochondrial function-related genes and Nrf2 pathway genes (K). The activities of mitochondrial complex I (L), complex V (M), and ATP level (N) in the jejunum. Data are expressed as mean ± standard error. * p < 0.05 and ** p < 0.01. Ctrl = basal diet group treated with saline; Si = silybin diet group treated with saline; PQ = basal diet group treated with paraquat; Si + PQ = silybin diet group treated with paraquat; FIS1 = fission; DNM1 = dynamin 1; MFN = mitofusin; OPA1 = mitochondrial dynamin-like GTPase; NDUFS2 = NADH ubiquinone oxidoreductase core subunit S2; NDUFV2 = NADH ubiquinone oxidoreductase core subunit V2; SDHA = succinate dehydrogenase complex flavoprotein subunit A; UQCRB = ubiquinol-cytochrome c reductase binding protein; ATP5H = ATP synthase subunit d; CAT = catalase; SOD = superoxide dismutase; GPX = glutathione peroxidase; Nrf2 = nuclear factor-erythroid 2-related factor 2; Keap1 = kelch-like ECH-associated protein l; HO-1 = heme oxygenase-1; NQO1 = NAD(P)H: quinone oxidoreductase 1; COX = mitochondrial complex; and ATP = adenosine triphosphate.

Figure 4

Dietary silybin addition mitigated PQ-induced intestinal apoptotic procedures. The activities of caspase 3 (A) and caspase 3 (B) in the jejunum. (C–G) The levels of apoptotic proteins in the jejunum were detected using Western blot. Data are expressed as mean ± standard error. * p < 0.05 and ** p < 0.01. Ctrl = basal diet group treated with saline; Si = silybin diet group treated with saline; PQ = basal diet group treated with paraquat; Si + PQ = silybin diet group treated with paraquat; Bcl-2 = B-cell lymphoma-2; Bax = Bcl-2-associated-X-protein; and Bcl-2/Bax = the ratio of Bcl-2 to Bax.

Figure 5

Dietary silybin addition ameliorated PQ-induced intestinal barrier dysfunction. (A) Representative images of H&E staining of the jejunum sections (scale bars: 40 μm). Quantification of intestinal morphology of the jejunum (B). Goblet cells in the jejunum were observed via AB-PAS staining (scale bars: 100 μm) (C) and quantification (D). The expression of MUC2 at the gene level (E) and protein level (F). (G,H) Plasma DAO activity and D-lactate content. (I,J) The levels of tight junction proteins in the jejunum were detected with Western blot. Data are expressed as mean ± standard error. * p < 0.05 and ** p < 0.01. Ctrl = basal diet group treated with saline; Si = silybin diet group treated with saline; PQ = basal diet group treated with paraquat; Si + PQ = silybin diet group treated with paraquat; VCR = the ratio of villus height-to-crypt depth; MUC2 = mucin 2; DAO = diamine oxidase; and ZO-1 = zonula occludens-1.

Figure 6

Dietary silybin addition improved PQ-induced intestinal microbiota disorders. (A–C) α-diversity index. (D) Ven diagram of ASV in cecal chyme of all samples. (E) Principal coordinate analysis. The relative abundance of gut microbiota at the phylum (F) and genus level (G). (H,I) Gut microbiota with significant differences of the comparison groups Ctrl vs. PQ and PQ vs. Si + PQ at the genus levels. (J,K) The concentration of cecal short-chain fatty acids. Data are expressed as mean ± standard error. * p < 0.05 and ** p < 0.01. Ctrl = basal diet group treated with saline; Si = silybin diet group treated with saline; PQ = basal diet group treated with paraquat; Si + PQ = silybin diet group treated with paraquat; PCoA = principal coordinates analysis; and SCFAs = short-chain fatty acids.

Figure 7

Spearman correlation analysis and RDA analysis. (A) The heatmap of Spearman’s correlation between the abundance of gut microbiota and the intestinal homeostasis phenotype indexes. (B) The RDA of the differences between the gut microbiota and SCFA levels. * p < 0.05 and ** p < 0.01. CAT = catalase; SOD = superoxide dismutase; MDA = malondialdehyde; Nrf2 = nuclear factor-erythroid 2-related factor 2; COX = mitochondrial complex; ATP = adenosine triphosphate; ATP5H = ATP synthase subunit d; VH = villus height; V/C = the ratio of villus height-to-crypt depth; DAO = diamine oxidase; ZO-1 = zonula occludens-1; SCFAs = short-chain fatty acids; and RDA = redundancy analysis.