Sulfur Amino Acid Metabolism and the Role of Endogenous Cystathionine-γ-lyase/H2S in Holstein Cows with Clinical Mastitis

Abstract

H₂S plays an important role in various inflammatory diseases. However, the role of H₂S and synthetic enzymes in Holstein cows with CM is unknown. The aim of this study was to identify DEPs associated with sulfide metabolism and further investigate their roles in dairy cows with CM. From 3739 DEPs generated by data-independent acquisition proteomics, we identified a total of 17 DEPs included in 44 GO terms and five KEGG pathways related to sulfide metabolism, including CTH and cystathionine-β-synthase (CBS). Immunohistochemical and immunofluorescence staining results showed that CTH and CBS proteins were present mainly in the cytoplasm of mammary epithelial cells. Endogenous H₂S production in the serum of the CM group was significantly lower than that of the healthy Holstein cows. CTH and CBS mRNA and protein levels in the mammary glands of the CM group were significantly downregulated compared to those of the healthy group. These results indicate that CTH and H₂S were correlated with the occurrence and development of CM in Holstein cows, which provides important insights into the function and regulatory mechanism of CTH/H₂S in Holstein cows.

Keywords: clinical mastitis; cystathionine-γ-lyase; hydrogen sulfide; inflammation.

Figure 1

Identification of candidate DEPs and GO terms related to sulfur metabolism. (A–C) Candidate DEPs and GO terms including biological process (A), molecular function (B) and cellular component(C) related to sulfur metabolism; x-axis represents the number of DEPs. y-axis represents the GO terms. (D) Venn diagram of candidate DEPs in the BP, MF, and CC groups. (E) PPI network analysis of 17 DEPs and nine GO terms related to sulfur metabolism. (F) Relative expression levels of 17 DEPs quantified by DIA proteomics; x-axis represents the log2(FC) values. (G) Heat map of 17 DEPs related to sulfur metabolism.

Figure 2

Identification of candidate DEPs and KEGG pathways related to sulfur metabolism. (A) Enrichment circle diagram of KEGG pathways related to sulfur metabolism. Color of the outermost layer represents the different KEGG classifications related to sulfur metabolism. Second layer contains information on the number of DEPs and the degree of significant enrichment. Third layer shows the upregulation and downregulation of DEPs. Fourth layer represents the enrichment factor for each pathway. (B) Upset-Venn diagram of five KEGG classifications related to sulfur metabolism. (C) Volcano plots of the 24 DEPs, including eight downregulated and 16 upregulated DEPs. (D) PPI network and heat map analysis of amino acid biosynthesis and cysteine and methionine metabolism.

Figure 3

Location analysis of CBS and CTH proteins and measurement of endogenous H₂S in the mammary glands. (A) Pathological variation of the mammary glands of the C (A1) and CM (A2) groups (400×). (B,C) Intracellular location analysis of CBS and CTH proteins in the mammary glands of the C (B1,C1) and CM (B2,C2) groups (400×). (D) Negative control of the mammary glands of the C (D1) and CM (D2) groups. (E,F) The gray value of positive expression of CTH and CBS proteins about IHC sections were scanned and quantified. (G) Endogenous H₂S concentrations in the serum of the C and CM groups. CT, connective tissue. MA, mammary alveoli. MEC, mammary epithelial cells. PN, phagocytic neutrophils. C represents control, CM represents clinical mastitis. H₂S concentration and IHC data were analyzed by Student’s t-test (between two groups) or one-way ANOVA analysis (within multiple groups), and expressed as mean ± SEM. * represents p < 0.05 and ** represents p < 0.01.

Figure 4

Co-location analysis of CBS, CTH, and CK-18 proteins in the mammary glands (400×). (A–C) Cellular localization of CK-18 (red), CTH (green), and CBS (orange) proteins in the mammary glands of the C (A1,B1,C1) and CM (A2,B2,C2) groups, respectively (400×). (D) Nuclei stained with DAPI (blue) in the mammary glands of the C (D1) and CM (D2) groups (400×). (E) Merged colocalization of CK-18, CTH, and CBS proteins in the mammary glands of the C (E1) and CM (E2) groups (400×). C, represents control. CM, represents clinical mastitis.

Figure 5

Relative expression levels of CBS and CTH mRNA and proteins in the mammary glands. (A,B) Expression levels of CBS and CTH mRNA in the mammary glands of the C and CM groups. GAPDH mRNA was used as the internal control. (C) Western blot analysis of CBS, CTH, and β-actin proteins in the mammary glands of the C and CM groups. (D,E) Relative integral optical density of CBS and CTH in the mammary glands of the C and CM groups. C, represents control. CM, represents clinical mastitis. The data were analyzed by Student’s t-test (between two groups) or one-way ANOVA analysis (within multiple groups), and expressed as mean ± SEM. * represents p < 0.05 and ** represents p < 0.01.