关键词: 中草药饲料添加剂, 肝, 转录组, 胆汁酸, 免疫

Abstract:

The aim of this study was to investigate the effects of herbal feed additives on bile acids metabolism and immune function in Hu sheep liver tissues by a transcriptomic methods. Eighteen healthy male lambs of Hu sheep with similar body weight ((19.57±1.56) kg) at 3 months of age were selected and randomly divided into three groups of six sheep each. The control group was fed a basal ration, and the herbal feed additives in test groups Ⅰ and Ⅱ were added at 0.5% and 1% of the concentrate supplement in the ration for a pre-test period of 10 d and a formal test period of 90 d. At the end of the experiment, 6 sheep in each group were slaughtered, liver tissue samples were collected, and RNA was extracted from the liver tissue for transcriptomic sequencing, and differentially expressed genes were obtained based on the screening criteria of |log2Fold Change|>1 and FDR < 0.05, and GO function and KEGG pathway enrichment analysis of differentially expressed genes were carried out, as well as fluorescence quantification for detecting the genes related to bile acids metabolism and immune function in the liver tissue. Corresponding kits were used to detect the content of immune indicators (IgA, IgG, IgM, C3, C4) in the liver. The results of immunological indexes showed that the liver IgA, IgG, C3 levels in test group Ⅰ were significantly higher than those in the control group (P < 0.05). Sequencing results showed that 915 differentially expressed genes (DEGs) were obtained by two-by-two comparison between the groups. There were 237 and 270 up-regulated genes and 78 and 106 down-regulated genes in test Ⅰ and test Ⅱ groups, respectively, compared with the control group. Among them, the results of GO functional annotation showed that the differentially expressed genes were mainly associated with GO terms including viral defence regulation, immunity and hormone regulation and biosynthesis. The results of KEGG enrichment analysis showed that compared with the control group, DEGs in test group Ⅰ was mainly enriched in the pathways of cholesterol synthesis, arachidonic acid metabolism which were related to the biological processes and metabolic pathways, and most of the genes in the pathways were significantly up-regulated. As the dose of the herbal feed additive was further increased, DEGs in test group Ⅱ were more enriched in metabolism-related pathways such as glutathione and caffeine metabolism. This was confirmed by RT-qPCR of bile acid-related genes (FXR, UGT2B18, ABCC4, UGT1A6, CYP7A1, HMGCR, SHP, FGFR4, GPBAR1, HSL) and immune-related genes (LOC101119773, MAPK4, XAF1, AHR, TLR2, TLR4, IL-16), which showed that the expression of SHP and FXR genes in test group Ⅰ was significantly higher than that in control group (P < 0.05), while the expression of FGFR4, CYP7A1, GPBAR1, ABCB11 and HSL genes showed an increasing trend in test group Ⅰ, but the difference with the control group was not significant (P>0.05). LOC101119773, MAPK4, XAF1, AHR, TLR4, TLR2 and IL-16 genes were significantly higher in the test Ⅰ group than in the control group (P < 0.05). The results suggest that herbal feed additives can promote the synthesis and secretion of bile acids in the liver of Hu sheep, enhance immunity, and the addition of 0.5% has the best effect under the current condition.

Key words: Chinese herbal feed additives, liver, transcriptome, bile acids, immunity