Matrine Interferes With ?-hemolysin-producing Staphylococcus Aureus Injure Bovine Mammary Epithelial Cells

Staphylococcus aureus(S.aureus)is one of the major pathogens causing mastitis in cows.It is an atypical intracellular parasite that can cause cells inflammation and aggravate cell infections by releasing multiple toxins,especially the release of a-hemolysin.During the invasion of S.aureus into the breast tissue of dairy cows,a large number of cells death were caused by disrupting the cytomembrane.It was found in many studies that neutralizing or inhibiting the secretion of virulence factors can achieve the purpose of relieving inflammation.Many traditional Chinese medicines have been shown to have good effects on neutralizing bacterial toxins,inhibiting the secretion of virulence proteins,and eliminating inflammation.This study aims to research the effects of matrine on the damage of bovine mammary epithelial cells(bMECs)caused by a-hemolysin-producing S.aureus,so as to lay a foundation for studying the mechanism of traditional Chinese medicines interference with host cells damage by the pathogens.Firstly,the drug sensitivity of the matrine to S.aureus and its effect on the growth of the bacteria were determined by the twofold dilution method and the ultraviolet spectrophotometer method;Rabbit red cell hemolysis test was used for the detection of matrine on S.aureus a-haemolysin activity;a-Hemolysin secreted by S.aureus was detected by qPCR and Western Bloting;The effect of matrine on the proliferation of bMECs and the effect of S.aureus-induced apoptosis of bMECs were detected by CCK-8 and flow cytometry.In this project,it was foused on the effect of matrine on S.aureus invasion into bMECs and confirmation of its regulatory pathway.The MIC of the matrine to S.aureus was found to be 1.25 mg/mL-5 mg/mL.Matrine at concentrations of 16?g/mL-256?g/mL can significantly down-regulate the transcription level of alpha-hemolysin-encoding gene hla,thus reducing the secretion of a-hemolysin from S.aureus.a-hemolysin-producing S.aureus had greater damage to bMECs than a-hemolysin-deficient strain.Matrine at a concentration of 75?g/mL could significantly reduce the S.aureus-induced bMECs apoptotic(p<0.05).Matrine(25?g/mL-75?g/mL)can interfere with NO release,expression of antibacterial peptides TAP,BNBD5 and cytokines TNF-a,IL-1?,IL-6 induced by S.aureus in bMECs.The 75?g/mL matrine group had the most significant intervention effect(p<0.01).Matrine was able to dose-dependently reduce the number of S.aureus invading bMECs;S.aureus inactivates NF-?B signaling pathway in bMECs,while matrine can reduce the nuclear content of p65 protein in a dose-dependent manner.After blocking of the NF-?B signaling pathway,it was revealed that matrine and NF-?B inhibitors act similarly and both inhibit the invasion of S.aureus into bMECs.The results showed that matrine can inhibit the secretion of a-hemolysin from S.aureus and interfere with the damage of S.aureus to bMECs.It was further found that this intervention was not related to the secretion of a-hemolysin from S.aureus.Matrine can down-regulate the expression of p65 protein in the NF-?B pathway and regulate downstream pathways to interfere with the invasion of S.aureus into bMECs,and this intervention is positively correlated with the matrine concentration.