Research On Effect And Mechanism Of A Novel Indolylbenzoquinone Compound HL-J6 And Emodin Against MRSA And Its Biofilm

Staphylococcus aureus（SA）is a Gram-positive bacterium which can colonize skin and mucosa and lead to a variety of human diseases.Due to its strong pathogenicity,Staphylococcus aureus has become one of the main pathogens of hospital acquired infection（HAI）and community acquired infection（CAI）all over the world.With the wide use of antibiotics,the problem of multidrug resistance of Staphylococcus aureus is becoming increasingly serious.Especially the emergence of Methicillin-resistant Staphylococcus aureus（MRSA）has caused high incidence rate and high mortality worldwide.The emergence of MRSA biofilm not only provides a barrier for bacteria,but also further restricts the scavenging effect of antibiotics on bacteria.The research and development of new antibiotics is one of the most effective ways to fight against bacteria.Due to the increasingly severe bottleneck of MRSA treatment,the development of new therapeutic compounds is imminent.In the previous work,a series of indolylbenzoquinone compounds were synthesized by Professor Ling He from West China School of Pharmacy,Sichuan University.A novel indolylbenzoquinone compound,named as HL-J6,was found with significant anti-MRSA activity in vitro.In this study,we investigated its safety,anti-MRSA activity and biofilm inhibition activity in vitro and vivo,and preliminarily explored its mechanism.In our other previous work,we also found that the natural compound emodin has significant anti MRSA activity,and then we studied its anti MRSA biofilm activity and preliminary action mechanism.Objectives:To further investigate its medicinal safety and anti-MRSA activity in vivo,systematically study its inhibition of MRSA biofilm,preliminarily clarify the target of HL-J6 based on clarifying the antibacterial activity of HL-J6 in vitro and provide new ideas for the research and development of new anti-MRSA-infection drugs.To clarify the inhibitory effect of emodin on MRSA biofilm.Methods:1.The research on effect and mechanism of HL-J6 against MRSA and biofilm.（1）Effect of HL-J6 on other strains of Staphylococcus aureus and other common pathogenic bacteria in vitro.ATCC25923 and 11 clinical isolates from Beijing,Shijiazhuang,Guangzhou,Jinan,Kunming,and Chongqing were selected to determine MICs and MBCs of HL-J6 against bacteria by double broth dilution method.Pseudomonas aeruginosa（PAO1）,Acinetobacter baumannii（Lac-4）and Escherichia coli（O157:H7）were selected for antibacterial specificity study to determine MICs and MBC by the same method.（2）Safety assayIt was evaluated by hemolysis test in vitro.The safety in vivo was evaluated by acute toxicity test,determination of biochemical indexes of liver and kidney function and weight change.（3）Study on multi-passage resistance in vitro.Multi-passage resistance selection assay was carried out to confirm whether HL-J6induced MRSA drug-resistance easily in 20 consecutive passages.（4）Evaluation of antibacterial activity in vivo.Skin infection model（local infection）:Mice had their dorsal skin removed and wounds infected.Then the drug was administered 24 hours after infection,twice a day for 7consecutive days.The wounds of the mice were monitored and photographed to observe the healing,and Image J calculated the healing rate.Take the exudate for bacterial culture and determine the bacterial colonization of the wound.Sublethal infection model（systemic infection）:Mice were given sublethal dose（1×10⁸CFU per mouse）bacterial solution by tail vein injection.And mice were given drugs by intraperitoneal injection,24 hours after infection,twice a day for 3 days.The effect of HL-J6 on systemic infection was evaluated by the amount of bacterial colonization in mouse related tissues and the results of pathological sections.Lethal infection model（systemic infection）:Mice were given lethal dose（6.36×10⁸CFU per mouse）bacterial solution by tail vein injection.And mice were treated with corresponding drugs by intraperitoneal injection,twice a day for 7 consecutive days.The effect of HL-J6 on systemic infection was evaluated by survival curve of mice in 7 days.（5）Anti-biofilm activity assay in vitro.The anti-biofilm activity of HL-J6 was observed by crystal violet staining.The effect of HL-J6 on biofilm structure was observed by Scanning electron microscope（SEM）and Confocal laser scanning microscope（CLSM）.We tried to find the possible anti-biofilm mechanism of HL-J6 by Real-Time RT-PCR.The secretion level of key virulence factor was determined by western blot.（6）Research on anti-MRSA-biofilm activity in vivoThe biofilm model was constructed on mouse skin wound,and the morphological structure of biofilm were measured by SEM after corresponding treatment to evaluate anti-biofilm activity of HL-J6 in vivo.2.The research on effect and mechanism of emodin against MRSA and biofilm.（1）Effects of emodin on different strains of Staphylococcus aureusMRSA252 and 5 MRSA clinical isolates from Beijing,Guangzhou,Jinan,Kunming,and Chongqing were selected to determine MIC and MBC of emodin in vitro.（2）Effect of emodin on biofilm of MRSA.The anti MRSA biofilm activity of Rhein was observed by crystal violet staining.The effect of emodin on the morphology of MRSA biofilm was studied by SEM and CLSM.The possible targets of HL-J6 anti-biofilm activity were found by Real-Time RT-PCR.Results1.The research on effect and mechanism of HL-J6 against MRSA and its biofilm.（1）The MIC of HL-J6 against different strains of MRSA were 2-8μg/m L,while its MBCs were 4-32μg/m L.There were inhibitory effects on each strain of MRSA to a certain extent,but no inhibitory effects on Escherichia coli,Acinetobacter baumannii and Pseudomonas aeruginosa,indicating that HL-J6 has a specific inhibitory effect on Staphylococcus aureus.After continuous multi-passage culture to induce drug-resistance,the MIC of MRSA doubled to 4μg/m L at the 17^thgeneration with HL-J6 treated,yet that doubled at the 4^thgeneration in vancomycin group.（2）HL-J6 did not cause significant erythrocyte rupture in hemolysis experiment in vitro.The hemolysis rates were less than 5%at 4-,8-and 16-fold MIC.Intraperitoneal injection of HL-J6 did not obviously affect the weight change,liver,and kidney biochemical function of mice.LD₅₀value is 427.73 mg/kg.（3）HL-J6 reduced MRSA colonization on infected skin wounds and in organs of sublethal dose infected mice.It also improved the survival rate of lethal dose infected mice.（4）HL-J6 inhibited the formation of biofilm at sub-MIC.The inhibition effect was concentration-dependent manner,and the compound has killing effect on bacteria in biofilm.The average dead/living bacteria ratio was calculated,and it was 0.98%in control group,30.50%in 0.25×MIC group,71.24%in 0.5×MIC group.（5）Real-Time RT-PCR showed that HL-J6 significantly down regulated the expression of ica operon,agr,sar A,and atl,the genes associated with biofilm formation,and it was concentration-dependent manner.The secretion ofα-hemolysin（hla）,the main virulence factor of MRSA,was decreased by HL-J6.2.The research on effect and mechanism of emodin against MRSA and its biofilm.（1）The MICs of emodin against different strains of MRSA were range from 2 to 8μg/m L,while MBCs were range from 4 to 16μg/m L.（2）Emodin obviously inhibited formation of biofilm and its inhibitory effect displayed concentration-dependent manner.Besides,it destroyed biofilm structure and increase the ratio of dead bacterial under biofilm.Fluorescence quantitative PCR showed that emodin significantly down regulated the expression of ica operon gene.Conclusion1.HL-J6 showed significant anti-MRSA,anti-biofilm activity and good safety in vitro and vivo,and it is not easy to induce bacterial drug resistance.2.HL-J6 can down-regulate the expression of genes related to EPS synthesis during biofilm formation and down-regulate the expression ofα-hemolysin（hla）,a MRSA virulence factor,to reduce the secondary damage of bacterial infection to the host.3.HL-J6 has the research and development potential as a new anti MRSA and bacterial biofilm drug.4.Emodin has a good inhibitory effect on MRSA biofilm.