The Research Of Curcumin/Shikonin/Melittin Combined With Ebselen Synergistically Target The Thioredoxin Reductase To Mediate The Death Of Staphylococcus Aureus

Background The thiol-dependent redox system（TDRS）exists in a variety of organisms and maintains the redox homeostasis in the organism.Thioredoxin reductase（TrxR）,the only key enzyme in the thioredoxin（Trx）system of TDRS in Staphylococcus aureus（S.aureus）,is naturally different from mammals in structure and function.This difference leads to the opposite oxidation-reduction reaction of some drugs on bacteria and mammalian cells under the same conditions,making TrxR a potential antibacterial target.Ebselen（EbSe）independently screened by our research group can target TrxR and kill S.aureus by inducing a large number of reactive oxygen species（ROS）to produce,revealing its antibacterial mechanism.For the TrxR target,we have also screened some natural compounds with antibacterial activity from the natural ingredient library,and studies have confirmed that these natural ingredients can target the Trx system,or have the same antibacterial mechanism as EbSe and have synergy with EbSe.Objective To explore the effect and mechanism of curcumin（Cur）,shikonin（SKN）,melittin（Mel）combined with EbSe synergistically against S.aureus.Methods（1）Spectrophotometry was used to determine the antibacterial effect of EbSe combined with Cur/SKN/Mel（EbSe-Cur/SKN/Mel）on S.aureus;PI staining was used to detect the bactericidal effect of EbSe-Cur/SKN/Mel;transmission electron microscope was used to observe the S.aureus morphology after the treatment of EbSe-Cur/SKN/Mel.（2）Detecting the effect of EbSe-Cur/SKN/Mel on bacterial ROS production by ROS fluorescent probe H₂DCFDA;DTNB was used to detect the effect of EbSe-Cur/SKN/Mel on key enzyme activity of Trx system.（3）ROS was eliminated by adding ROS scavenger dithiothreitol（DTT）,and the relevant indicators were retested by the replenishment experiment.（4）Isolating clinical multidrug-resistant Staphylococcus aureus（MDRS）,and the visible spectrum was used to determine the antibacterial effect of EbSe-Cur/SKN/Mel on MDRS.Results（1）EbSe-Cur/SKN/Mel had a significant synergistic inhibitory effect on S.aureus.（2）EbSe-Cur/SKN/Mel could significantly inhibit the activity of TrxR,without affecting the Trx.EbSe-Cur/SKN/Mel could obviously induce the production of ROS in S.aureus.（3）It was found through the supplementary experiment that DTT could save the S.aureus from the killing effect of the drugs and protect the bacteria from oxidative stress damage caused by ROS.（4）EbSe-Cur/SKN/Mel also had a significant antibacterial effect on MDRS.Conclusion EbSe-Cur/SKN/Mel could execute antibacterial effect on S.aureus through inducing a large amount of ROS by co-targeting the TrxR.