Combined Proteomic and Metabolomic Analysis Reveals Comprehensive Regulation of Somatostatin DNA Vaccine in Goats

Abstract

Somatostatin (SS) plays crucial regulatory roles in animal growth and reproduction by affecting the synthesis and secretion of growth hormone (GH). However, the mechanism by which SS regulates growth and development in goats is still unclear. In order to investigate the regulatory networks of the hypothalamus and pituitary in goats affected by SS DNA vaccines, in this study, we used a previously established oral attenuated Salmonella typhimurium SS DNA vaccine, X9241 (ptCS/2SS-asd), to treat wethers. We analyzed the protein changes in hypothalamic and pituitary tissues using a TMT-based proteomics approach. Additionally, we examined the metabolic profiles of the serum of control and immunized wethers through untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS). Key signaling pathways were identified based on differentially expressed metabolites (DEMs) and differentially expressed proteins (DEPs). Furthermore, the effect of critical DEPs on signaling pathways was confirmed through Western blotting (WB) experiments, which elucidated the mechanism of active SS immunization in wethers. A proteomics analysis revealed that the expression of 58 proteins in the hypothalamus and 124 in the pituitary gland was significantly altered following SS vaccine treatment (fold change > 1.2 or < 0.83, p < 0.05). In the hypothalamus, many DEPs were associated with gene ontology (GO) terms related to neuronal signaling. In contrast, most DEPs were associated with metabolic pathways. In the pituitary gland, the DEPs were largely related to immune and nutrient metabolism functions, with significant enrichment in KEGG pathways, particularly those involving the metabolic pathway, sphingolipid signaling, and the cGMP-PKG signaling pathway. A metabolomic analysis further showed that active SS immunization in wethers led to significant alterations in seven serum metabolites. Notably, the sphingolipid signaling pathway, secondary bile acid synthesis, sphingolipid metabolism, and lysine synthesis were significantly disrupted. SS vaccines induced marked changes in hypothalamic-pituitary proteins in wethers, facilitating alterations in their growth processes. This study not only provides insights into the mechanism of the SS gene in regulating GH secretion in wethers but also establishes a basis for hormone immunoregulation technology to enhance livestock production performance.

Keywords: DNA vaccine; dazu black goats; metabolomics; proteomics; somatostatin.

Figure 1

Results of principal component analysis of identified proteins in the proteome and heat map of DEPs in each treatment group. (A,B) show the PCA of proteins identified in each sample of hypothalamus (B2_vs_B1) and pituitary gland (P2_vs_P1) from goats in the immunized group and negative control group, respectively. B2-1, B2-2, and B2-3 represent hypothalamus of 3 goats in the immunized group; B1-1, B1-2, and B1-3 represent hypothalamus of 3 goats in the negative control group; P2-1, P2-2, P2-3 and P1-1, P1-2, P1-3 represent pituitary glands of three goats in the immunized and negative control groups, respectively. (C,D) show the heatmap analysis of the two groups of screened differential proteins, B2_vs_B1 and P2_vs_P1, respectively, with red and blue color indicating up- and down-regulated proteins, respectively.

Figure 2

GO and KEGG enrichment analysis in hypothalamus and pituitary group proteomics. (A,B) The top 20 GO entries and KEGG signaling pathways based on the differentially expressed proteins enriched to the B2_vs_B1 group. (C,D) The top 20 GO entries and KEGG pathways based on the differentially expressed proteins enriched to the P2_vs_P1 group.

Figure 3

Combined analysis and validation of hypothalamus and pituitary group proteomics. (A) Venn plot based on the differential proteins identified in B2_vs_B1 and P2_vs_P1. (B) Dynamic Venn plot based on the top 20 KEGG signaling pathways enriched in the hypothalamus and pituitary, respectively. (C–F) Western blot validation of the proteomics results for both hypothalamus and pituitary groups and the relative quantification results, respectively. Error lines indicate standard deviation (*** p < 0.001, **** p < 0.0001).

Figure 4

Integrated analysis of metabolomics and proteomics. (A,B) OPLS-DA score plots for the positive and negative ion modes, respectively. (C) Bubble diagram of KEGG pathways enriched for significantly different metabolites. (D) Interaction analysis of common KEGG pathways and amino acid metabolism-related pathways enriched for DEPs in the hypothalamus and pituitary and DEMs in serum. (E) Mulberry diagram of key KEGG pathways in hypothalamic and pituitary proteomics and serum metabolomics.