CTH/H₂S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

Hydrogen sulfide (H₂S), as an endogenous gaseous signaling molecule, plays an important role in the inflammatory process. Our previous study found that Cystathionine-γ-lyase (CTH) and H₂S are correlated with the occurrence and development of Clinical Mastitis (CM) in Holstein cows. However, the functions and regulatory mechanisms of CTH/H₂S are still unknown. In this study, the inflammatory mammary cell model based on the MAC-T cell line was established by Lipopolysaccharide (LPS)-induced manner to further explore the function and regulatory mechanism of CTH/H₂S in cows with CM. In the inflammatory MAC-T cell, the CTH expression and H₂S production were both repressed in an LPS-dose dependent manner, which demonstrated that CTH/H₂S is related to the progression of inflammation. The inhibition of CTH/H₂S using a selective CTH inhibitor, β-cyano-l-Alanine (BCA), promoted LPS-induced inflammation response and the expression of inflammatory cytokines. However, this was reversed by the H₂S donor NaHS, demonstrating that H₂S can protect cells from inflammatory damage. Intriguingly, interleukin-8 (IL-8) showed an inverse expression pattern correlated with the H₂S-mediated cell protection effect during the inflammation process, and the inhibition test using a selective IL-8 receptor antagonist, SB225002, showed that IL-8 signaling plays a critical role in mediating endogenous H₂S synthesis, and CTH/H₂S exerts its anti-inflammation via IL-8-mediated signaling. This study provided support for the prevention and treatment of CM and the development of a novel anti-inflammatory strategy.