Research On The Effect And Mechanism Of Bavachinin Inhibits Staphyloxanthin

Staphylococcus aureus(S.aureus)is a common pathogenic bacterium,which can cause both community-acquired infection and hospital-acquired infection.As a conditioned pathogen,Staphylococcus aureus can cause superficial skin diseases such as skin follicular inflammation,acute skin infections such as furuncle,carbuncle and abscess,as well as many severe diseases such as pneumonia,osteomyelitis,endocarditis and toxic shock syndrome.Staphylococcus aureus infection in livestock and poultry breeding can cause mastitis in cows and respiratory system infection in pigs.However,due to the irrational use of antibiotics in production and life,the drug resistance of S.aureus increases rapidly.The emergence and widespread prevalence of Methicillin-resistant S.aureus(MRSA)and Vancomycin-resistant S.aureus(VRSA)make it increasingly urgent to explore new targets and develop new drugs for the treatment of S.aureus infection.S.aureus produces a variety of virulence factors that protect microbial pathogens from the immune system and help the pathogen obtain nutrients.Staphyloxanthin(STX)is the signature virulence factor produced by S.aureus,and also the key factor affecting the pathogenicity of S.aureus.At present,there are few researches on STX inhibitors at home and abroad,which focus on compound drugs.The development of drug inhibitors in traditional Chinese medicine resource library is not yet mature.In this paper,we screened STX inhibitors from Chinese medicinal materials and studied their activity and mechanism.In this study,multiple strains of S.aureus were analyzed by polymerase chain reaction.The results showed that the detection rate of STX synthesis pathway,upstream pathway and related regulatory genes was 100%,which was highly conservative.USA300 strain of MRSA,which produces a large amount of STX at 36 h after inoculation,was selected as the research object.The psoralea psoralea extract was screened from the existing Chinese medicine extract library in the laboratory.Bavachinin(IC50=0.529 μg /m L),coryfolin(IC50=38.66 μg /m L)and isopsoralen dihydroflavone(IC50=15.80 μg /m L)were detected by HPLC in psoralen alcohol extract.The minimum inhibitory concentrations of psoralen alcohol extract and active ingredient for 24 h were 32 μg /m L,and bavachinin,coryfolin and isopsoralen dihydroflavone were all greater than 512 μg /m L.The results of the growth curve of MRSA strain USA300 showed that the three components were within the effective concentration range of inhibiting STX.None of them affected the growth of bacteria.Bavachinin has the most significant inhibitory effect on STX,which is the main research object of STX inhibitors.Inhibiting the synthesis of lutein makes S.aureus more sensitive to oxidants such as superoxide radicals,hydrogen peroxide,subchlorides,hydroxyl radicals,etc.Therefore,through oxygen sensitivity stimulation and stress resistance tests,this study proved that bavachinin treatment can increase the sensitivity of S.aureus to oxidative stimulation and acid base,cold and heat adverse stimulation environment,and bavachinin can reduce the survival rate of USA300 against adverse stimulation.In addition,in vitro cytotoxicity tests demonstrated the safety of bavachinin in inhibiting the effective concentration of lutein.A549 cell adhesion tests showed that bavachinin could reduce the adhesion of S.aureus to cells.The staining results of live and dead cells also showed that bavachinin could protect J774 against USA300 infection.We investigated the inhibitory effect of bavachinin on STX at the transcriptional and protein levels.The results showed that 8 μg/m L bavachinin could significantly reduce the catalytic enzyme genes Crt M,Crt N,Crt O,Crt P,Crt Q and Ald H related to glutein production(p<0.05),and the upstream isoprene pathway catalytic enzyme gene Isp A(p<0.05)expression level.At the same time,the treatment of bavachinin could significantly reduce the production of total protein of S.aureus.The intracellular protein thermal migration test showed that the drug did not directly bind to the key catalytic enzymes Crt M and Crt N.In this study,it was found that the production of ROS,ATP and NADPH in bacteria was inhibited after treatment with bavachinin,suggesting that bavachinin may play a role by affecting the energy metabolism of bacteria.In order to investigate the therapeutic effect of bavachinin on S.aureus infection,a mouse model of S.aureus suppurative dermatitis was established successfully.The effects of bavachinin on the pathogenicity of USA300 infection in vivo were analyzed by investigating the changes of body weight,wound healing,skin pathological changes and bacterial load of mice with dermatitis.Compared with the control group,bavachinin treatment can make mice regain weight faster and promote wound healing in mice with dermatitis.In the course of treatment,the bacteria load in the wound and liver of mice with dermatitis was significantly decreased in the bavachinin treatment group compared with the control group on day 3,day 6 and day 9(p<0.05),it can be observed through pathological sections that bavachinin treatment can reduce the suppurative injury caused by USA300 infection,and inhibit the production of inflammatory factors.Therefore,this study proved that the traditional Chinese medicine monomer bavachinin can effectively inhibit the production of STX,reduce the virulence of S.aureus,and has a significant therapeutic effect on the skin trauma infection of mice caused by MRSA strain USA300.This study provides theoretical support and lead compound for the development of bavachinin as a new drug for the treatment of S.aureus infection through anti-virulence strategies.