Dietary supplement of Acanthopanax senticosus decoction formula improves immune response via intestine flora of rabbits

Abstract

Young rabbits are sensitive to surrounding changes and conditioned pathogens in intestine which might result in slow inflammation and diarrhea after microbial invasion. Traditional medicine herbs could provide efficacious treatment on slow infection and inflammation. The present research designed an Acanthopanax senticosus (ACS) formula consisted of five types of Chinese herbs including Acanthopanax senticosus (Rupr & Maxim) Harms (Ciwujia in Chinese), Astragalus membranceus (Fisch) Bge (Huangqi in Chinese), Indigo naturalis (Qingdai in Chinese), Houttuynia cordata Thunb (Yuxingcao in Chinese), and Glycyrrhizae radix et Rhizoma (Gancao in Chinese). The effects of ACS decoction supplement were investigated via determination of cytokines and growth performances of young rabbits, and the flora in intestinal digesta from six fragments were further explored using 16S rRNA gene sequencing technology. Compared to the control group, rabbits supplied with different doses of ACS decoction possessed lower diarrhea and death rates, together with the IL-10 concentration, while the declined IL-1β and IL-12 levels and inflammatory factor gene expressions in intestinal tissues. Additionally, ACS addition changed the diversity of flora in each segments of intestine. Functional prediction on abundances of genera enriched to seven KEGG immunity pathways. Moreover, strong correlations were determined between the abundance of bacteria with interleukins contents, and the predictive immune signaling abundances, respectively. Especially, ACS exhibited anti-inflammation effects via decreasing the abundances of Bacteroides, Clostridia_vadinBB60_group, NK4A214_group, and dgA_11_gut_group in intestine of young rabbits. In conclusion, dietary supplement with ACS exerted diarrhea-reducing effects, and improved immunity homeostasis by modulating intestinal flora diversity in young rabbits.

Keywords: immunity; inflammation; intestinal flora; rabbit; traditional medicinal herbs.

Figure 1

Effects of ACS on rates of mortality (p = 0.058 between ACS and NC groups) (A) and diarrhea (B), ADG (C) and feed conversion ratio (D) (n = 15) with different letters as significant difference (p < 0.05) on the columns.

Figure 2

Changes in the numbers of white blood cells (A) and lymphocytes (B) from the first week to the fifth week (n = 3) with star as different significance compared with NC group (p < 0.05).

Figure 3

The indexes of spleen (A), thymus (B), sacculus rotumdus (C) and vermiform appendix (D) in rabbits. The different superscript letters on the columns within the same bar chart were differ significantly (p < 0.05). Data were presented as mean ± SEM (n = 15).

Figure 4

The concentrations of IL-1β (A), IL-12 (B), IL-10 (C) in each segment of intestinal tissue. Values were expressed as means ± SE (n = 3). Differences were assessed by ANOVA. *p < 0.05. **p < 0.01.

Figure 5

The relative expressions of genes Claudin-1 (A), ZO-1 (B), Occludin (C), Caspase-1 (D), NLRP3 (E), NF-κB (F), TLR2 (G), NOD1 (H) in each intestinal segment. Values were expressed as mean ± SE (n = 3). Differences were assessed by ANOVA. *p < 0.05. **p < 0.01.

Figure 6

Relative abundances of top 10 bacteria at levels of phyla (A), class (B), and genera (C) in six segments of intestinal samples of rabbits supplied with ACS at low (DL), medium (DM) and high (DH) dosages compared with control group (NC) (n = 3).

Figure 7

The indices of chao1 and Shannon (A) and PCoA (B) analysis of six intestine segments (n = 3) (p > 0.05 among groups in A,B, respectively).

Figure 8

Taxonomic differences of gut flora among different groups. Taxonomic cladogram obtained by LefSe analysis. Bacterial biomarker did not detected form colon digesta (n = 3).

Figure 9

The heat map of KEGG hierarchy pathways enriched in immune system (A) of intestinal microbes (n = 3) and flora related to immunity (B). The “Up” on the right side of the genera lists denot the increased abundance and “Down” as the decreased abundance of bacteria regulated by ACS treatment. The positive correlation was showed in red color while negative relationship was indicated in blue color (n = 3), *p < 0.05. **p < 0.01.

Figure 10

The regulation network by ACS on the innate and adaptive immune systems of young rabbits. The related six immune signaling were listed as numbers ①: Toll and lmd signaling pathway, ②: NOD-like receptor signaling, ③: FcγR-mediated phagocytosis, ④: Antigen processing and presentation, ⑤: Th17 cell differentiation, ⑥: IL-17 signaling pathway, NOD, NOD-like receptor signaling; APC, Antigen-presenting cell; PAMP, pathogen-associated molecular pattern; LPS, Lipopolysaccharide; PGN, peptidoglycan.