Phosphoproteome Reveals the Role of Baicalin in Alleviating rPVL-Induced Cell Cycle Arrest in BMECs

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by <i>Staphylococcus aureus</i> (<i>S. aureus</i>) and a significant virulence factor that plays a crucial role in the pathogenesis of dairy mastitis. Previous studies by our research group demonstrated that baicalin inhibits the apoptosis and hyperphosphorylation of cytoskeletal proteins induced by recombinant Panton-Valentine leukocidin (rPVL) in bovine mammary epithelial cells (BMECs). However, the effects of baicalin on the proliferation of BMECs and the underlying mechanism remain unclear. Consequently, this study aimed to explore this underlying mechanism through an LC-MS/MS analysis performed in 4D data-independent acquisition (DIA) mode. Quantitative analysis identified 757 differentially expressed phosphoproteins, among which phosphorylation levels of proteins involved in BMEC proliferation and cell cycle regulation exhibited significant alterations (<i>p</i> < 0.05). rPVL inhibited BMEC proliferation in a dose-dependent manner and induced G0/G1 phase arrest and dephosphorylation of the cell-cycle-related proteins BCLAF1^S285, CDK7^T170, NF2^S518, and PKM2S37. Preintervention with baicalin significantly upregulated the expression and phosphorylation of these proteins and alleviated the G0/G1 phase arrest induced by rPVL in BMECs <i>in vitro</i>. The establishment of the mitotic state in BMECs due to the effect of baicalin appears to be closely related to the regulation of the phosphorylation of CDK7, PKM2, BCLAF1, and NF2. Moreover, <i>in vivo</i> analysis revealed that <i>S. aureus</i> ATCC49775 and rPVL induced dramatic structural destruction and pathological impairment of mammary gland tissues in mice and that these histopathological changes were ameliorated after baicalin intervention. Quantitative immunohistochemical analysis revealed that baicalin mitigated the rPVL-induced dephosphorylation of the aforementioned cell-cycle-related proteins and increased their phosphorylation. Both <i>in vitro</i> and <i>in vivo</i> experimental evidence demonstrated that baicalin effectively reversed rPVL-induced G0/G1 phase arrest in BMECs (<i>p</i> < 0.01) by significantly increasing the phosphorylation levels of cell cycle regulatory proteins (<i>p</i> < 0.05). Additionally, baicalin alleviates pathological damage to mammary gland tissues in mouse models. These data suggest that baicalin possesses antibacterial and antitoxin effects, indicating that it is an effective preventive agent against bovine mastitis.