Caffeic acid and chlorogenic acid mediate the ADPN-AMPK-PPARα pathway to improve fatty liver and production performance in laying hens

Abstract

Background: Caffeic acid (CA) and its derivative, chlorogenic acid (CGA), have shown promise in preventing and alleviating fatty liver disease. CA, compared to CGA, has much lower production costs and higher bioavailability, making it a potentially superior feed additive. However, the efficacy, mechanistic differences, and comparative impacts of CA and CGA on fatty liver disease in laying hens remain unclear. This study aimed to evaluate and compare the effects of CA and CGA on production performance, egg quality, and fatty liver disease in laying hens.

Results: A total of 1,440 61-week-old Hyline Brown laying hens were randomly divided into 8 groups and fed diets supplemented with basal diet, 25, 50, 100 and 200 mg/kg of CA, and 100, 200 and 400 mg/kg of CGA (CON, CA25, CA50, CA100, CA200, CGA100, CGA200 and CGA400, respectively) for 12 weeks. Both CA and CGA improved production performance and egg quality, while reducing markers of hepatic damage and lipid accumulation. CA and CGA significantly decreased TG, TC, and LDL-C levels and increased T-SOD activity. Transcriptomic and proteomic analyses revealed that CA and CGA reduced hepatic lipid accumulation through downregulation of lipid biosynthesis-related genes (ACLY, ACACA, FASN, and SCD1) and enhanced lipid transport and oxidation genes (FABPs, CD36, CPT1A, ACOX1, and SCP2). Of note, low-dose CA25 exhibited equivalent efficacy to the higher dose CGA100 group in alleviating fatty liver conditions. Mechanistically, CA and CGA alleviated lipid accumulation via activation of the ADPN-AMPK-PPARα signaling pathway.

Conclusions: This study demonstrates that dietary CA and CGA effectively improve laying performance, egg quality, and hepatic lipid metabolism in laying hens, with CA potentially being more economical and efficient. Transcriptomic and proteomic evidence highlight shared mechanisms between CA25 and CGA100. These findings provide a foundation for CA and CGA as therapeutic agents for fatty liver disease and related metabolic diseases in hens, and also offer insights into the targeted modification of CGA (including the isomer of CGA) into CA, thereby providing novel strategies for the efficient utilization of CGA.

Keywords: Absorptivity; Caffeic acid; Chlorogenic acid; Fatty liver; Laying hens.

Fig. 1

Effects of CA and CGA on serum lipid metabolism levels and antioxidant activity. A and D Serum TG, TC, HDL-C, LDL-C and VLDL-C levels (n = 10). B and E Serum GSH-PX, T-SOD and T-AOC activity levels (n = 10). C and F Serum MDA content (n = 10). Data are presented as the mean ± SEM. ^a−cMeans with different letter differ significantly (P < 0.05)

Fig. 2

CA and CGA alleviated hepatic lipid accumulation and liver damage. A and D liver morphology (n = 6). B and E liver H&E staining (n = 6). C and F Hepatic Oil Red O staining (n = 6). G and J (Liver index = liver weight/body weight) × 100 (n = 10). H and K Serum ALT activity level (n = 10). I and L Serum AST activity level (n = 10). M and Q Liver TG, TC, HDL-C, LDL-C and VLDL-C levels (n = 10). O and R Hepatic GSH-PX, T-SOD and T-AOC activity levels (n = 10). P and S Liver MDA content (n = 10). ^a−cMeans with different letters differ significantly (P < 0.05)

Fig. 3

CA and CGA regulated hepatic gene expression profiles. A PCA analysis. B–D Volcano plot of DGEs (n = 6). E–G GO term enrichment analysis of DEGs. BP: Biological Process, MF: Molecular Function, CC: Cellular Component (n = 6). H–J KEGG pathway enrichment analysis of DEGs. n = 6 hens per group

Fig. 4

CA and CGA regulated hepatic protein expression profiles. A PCA analysis. B–D Volcano plot of DGPs (n = 6). E–G GO term enrichment analysis of DEPs. BP: Biological Process, MF: Molecular Function, CC: Cellular Component (n = 6). H–J KEGG pathway enrichment analysis of DEPs. n = 4 hens per group

Fig. 5

GO term and KEGG pathway of GSEA (Gene Set Enrichment Analysis) enrichment analysis using all proteins. A GO term of GSEA enrichment analysis related to lipid metabolism. B KEGG pathway of GSEA enrichment analysis related to lipid metabolism. C–E GO term of GSEA enrichment analysis in fatty acid biosynthetic, lipid droplet, tricarboxylic acid cycle and mitochondrion. F KEGG pathway of GSEA enrichment analysis in pyruvate metabolism. A positive ES (enrichment score) value means that the term or pathway is up-regulated, whereas a negative ES value means that term or pathway is down-regulated. n = 4 hens per group

Fig. 6

Comprehensive analysis of liver gene transcription and protein. A The relative genes transcription and protein expression levels of lipid synthesis, lipid transport and oxidation. B–E The relative mRNA expression levels of lipid synthesis, lipid transport and oxidation (n = 6). F Protein interaction analysis of target genes by STRING. ^a−cMeans with different letters differ significantly (P < 0.05)