肝转录组揭示中草药饲料添加剂可能影响湖羊肝组织胆汁酸代谢和免疫功能

doi:10.11843/j.issn.0366-6964.2025.06.043

Abstract

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

CLC Number:

LIU Zilong, LI Qiao, WU Yi, WANG Huihui, LI Taotao, MA Youji. Transcriptomics Reveals the Effects of Chinese Herbal Feed Additives on Bile Acids Metabolism and Immune Function in Hu Sheep Liver Tissue[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 3014-3026.

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有效活性成分 Efferent active composition	粗提物 Crude extract	总多糖 Total polysaccharides	总黄酮 Total flavonoid	总皂苷 Total sponins
含量Content	33.86	27.21	0.36	1.27

组成Ingredients	含量Content
玉米Corn	40
棉籽粕Cottonseed meal	5.5
豆粕Soybean meal	12.5
花生秧Peanut seedling	29
麦麸Wheat bran	9
磷酸氢钙CaHPO₄	0.5
食盐NaCl	1
石粉Limestone	1.5
预混料Premix¹	1
合计	100
营养水平Nutrient levels²
代谢能/(MJ·kg^－1)ME	8.57
粗蛋白质CP	14.51
粗脂肪EE	2.21
中性洗涤纤维NDF	30.11
酸性洗涤纤维ADF	19.58
钙Ca	1.01
磷P	0.47

基因 Gene	序列(5′→3′) Primer sequence(5′→3′)
HRG	AGAACAAGCTGACAACGCCC ACAGCCCTCATCACTTTGTCC
IFI6	GCTCAGAGGAAAACAGCGACT GTCTGCTTTCGACTTGCTTG
GPBAR1	AGCCACACTTGTCACCTACAG GCCCATAAACTTGACGTCCG
AQP3	AAGCTGCCTGTGTACACCTT GGACGGGGTTGTTGTAGGG
HSL	AGTCCCACGAGCCTTACCTC AGTCCCACGAGCCTTACCTC
β-actin	TATTGCTGCGCTCGTGGTTG GTCAGGATGCCTCTCTTGCT
MX1	CAAGATGTCCTCTCGTGCTGA ACTGATCCCCATGCCTTCG
UGT2B7	TGCCAAACCCCTGCCTAAG AGTGATCATCGACCCCAAAGT
MYC	CTCTGAATGCATCGACCCCT CTCTGAATGCATCGACCCCT
FMO5	TGGTCACGGAATCTGAAGGG TCCTCCAGAATTCCCAATGCC
SHP	CCACCCTCCCCAACACTCTATG CTGTGGCAGGTAAGTGGGAGT
FGFR4	GCCTGTGCGGTTATGCTGTG TCGGGTAGGAAGCTGGCAAT
FXR	TGGTGGAGGAGAACGGTTTG CCCGACTTTCTATTTCAGCAGC
ABCB11	GTGGTGTCTCATCTTCTTGTGC TGACACTGAGGAAAATCTGGACA
CYP7A1	GCTACTTGACGCTCTTTGGC GTCTTGAACACGTGGATGGG
PC	TGCTGGGGTCCGTTTCATT CGGCCTTGAAGATAATGGGGA
UGT2B18	AATCATGTGGAAGTTTGGTGAC GGAGTGAAGCGGAGACTGTA
HMGCR	TCAGCCATTTTGCCCGAGTC TTGGACACATTTTCATCCAGTCC
ABCC4	TTTTGAAAGCTGAGAAAGATGCAC TTACTCAAGCGAAGGGCCTCA
LOC101119773	TACCCATGCAAAGCCCAAGA TGTAGTGATCAGCCGCCTTC
TLR2	ATCTGCGGACCCTGAAAGTA ATGTCCAGGAGTAAAACAGGCT
TLR4	TTAGCCTGGGGAGCCTTTTCT GAAAGATCCAAGTGCTCCAGG
IL-16	CTGACCCCTTCGAGAGTTCC CTTTGTGGGTCCAGTTTTCCG
AHR	CCAGTTTATTCATGCTGCTGAC CGGTTAGCGGTCTCTGAGTT
XAF1	ACAGAGAAGTGTGTCAGCGTG TGAGACTCGACGGCATCTATC

项目 Item	组别Group			P值 P-value
项目 Item	对照Control	试验Ⅰ组	试验Ⅱ组	P值 P-value
免疫球蛋白G IgG	3.14±0.31^b	4.56±0.16^a	3.52±0.38^ab	0.001
免疫球蛋白A IgA	0.16±0.02^b	0.25±0.01^a	0.22±0.04^ab	0.043
免疫球蛋白M IgM	0.13±0.02	0.16±0.01	0.14±0.02	0.237
补体C3 Complement 3	0.11±0.03^b	0.18±0.01^a	0.17±0.01^ab	0.002
补体C4 Complement 4	0.04±0.01	0.03±0.02	0.03±0.01	0.144

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Transcriptomics Reveals the Effects of Chinese Herbal Feed Additives on Bile Acids Metabolism and Immune Function in Hu Sheep Liver Tissue

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