Antibacterial And Antifungal Activity And Action Mechanism Of Antimicrobial Peptides Of HPRP-A1/HPRP-A2 Combined With Chlorhexidine Acetate

The impacts of gynecological vaginitis on women's health are severe.Among the negative consequences are common mucosal infections,including bacterial vaginosis(BV)and vulvovaginal candidiasis(VVC).Patients with bacterial and fungal biofilms caused by vaginitis are prone to suffering recurrences and persistent resistance to antibiotics.Gynecological vaginitis is considered to be a common cause of these diseases.Many women are often infected,and BV and VCC are often persistent because of the difficulty in successfully treating the patient.In clinical treatment,topical drugs,biguanide disinfectant,and preservative chlorhexidine acetate(CHA)are often used for antimicrobial treatment.CHA has no tissue-dissolving activity.However,high concentrations of CHA can irritate the mucosa.In addition,some patients may have allergic reactions.More and more studies show that antimicrobial peptides(AMP)have a broad spectrum,rapid bacteriostatic activity.They are not susceptible to the effects of bacteria and fungi on traditional antibiotic resistance mutations and have good synergistic effects with antibacterial drugs that have gradually become important candidates for new anti-infective medicines.Still,there are also problems,including a high cost.Therefore,this study intends to examine the issue of the combined use of antimicrobial peptides and CHA.It is hoped that it will not only exert its potential for vigorous activity,but also reduce costs,reduce the amount of drug therapy needed,and reduce the toxicity and allergic factors of the drug itself.1.Study on the antibacterial and fungal activity of HPRP-A1/HPRP-A2 and CHA in combinationThe antimicrobial activities of the synergistic effects of the ?-helical cationic antimicrobial peptides(AMPs)HPRP-A1 and its enantiomers HPRP-A2,and/or CHA,against Gram-negative and Gram-positive bacteria,and one fungus Candida albicans(C.albicans)were investigated in vitro and in mouse and rat vaginitis infection models in vivo.In vitro studies,HPRP-A1/HPRP-A2,and CHA alone have been shown to treat bacteria and fungi.HPRP-A2 has slightly higher antimicrobial activity than HPRP-A1,which may be due to its insensitivity and stable action as a D-amino acid structure to protease degradation.The antibacterial and antifungal mechanisms and chiral properties of HPRP-A1 and HPRP-A2 are independent.The antimicrobial activity depends on the influence of ?-helix and amphiphilic structure.CHA showed weak antimicrobial activity.More notably,the combination of HPRP-A1,HPRP-A2,and CHA showed more potent antibacterial activity against Gram-positive and Gram-negative bacteria,and one fungus C.albicans and a more vigorous antimicrobial activity than the drug alone,and all bacteria and fungi had synergistic effects except the additive effect on S.aureus,and the hemolytic activity of the combination reduces the toxicity of the drug.In vivo studies,HPRP-A2 and CHA,in combination,have a more effective bacteriostatic activity in the animal model of bacterial and fungal vaginitis than the drug alone,with the bacteriostatic rate of 99.9 %(P < 0.001)in the high concentration groups.There has been a very significant improvement in the symptoms of vaginitis.2.Study on the mechanism of action of HPRP-A1/HPRP-A2 and CHA in combinationThe mechanism of HPRP-A1/HPRP-A2 and CHA combined on the inhibition of bacterial and fungi was studied.We used laser confocal,flow cytometry,and turbidimetric methods to analyze the integrity of cells to membranes.The bacterial cell wall LPS,intracellularly produced reactive oxygen species,and DNA binding were all examined.In the study of binding to the main component of the Grambacteria cell wall and also the main proinflammatory endotoxin molecule LPS,HPRP-A2,and CHA alone or in combination with LPS in exogenous Gram-negative bacteria E.coli 055: B5 showed significant affinity.On the contrary,the binding ability of CHA and LPS is relatively weak.In addition,in the combined application of drugs,LPS binding capacity has not been significantly improved.Conjecture: One of the possible mechanisms of antimicrobial peptide inhibition by drugs against LPS;On the effect on the production of reactive oxygen species in fungal cells,HPRP-A1,HPRP-A2,and CHA alone did not produce ROS,the oxygen consumption of fungi did not increase.There was no significant difference between HPRP-A1,HPRP-A2,or CHA alone compared with the control group(P > 0.05).Also,in the combination,the oxygen consumption of the fungus did not increase.There was no significant difference between the HPRP-A1 and CHA,HPRP-A2 and CHA combined drug groups in synergistic concentration compared with the HPRP-A1,HPRP-A2 alone group,and the control group,respectively(P > 0.05).Also,in the combination,the oxygen consumption of the fungus did not increase.There were very significant differences between HPRP-A1 and CHA,HPRP-A2 and CHA groups compared with the CHA group alone,respectively(P < 0.01);The antimicrobial activity of antimicrobial peptides depends not only on the rapid penetration of cell membranes but also on their ability to bind to bacterial DNA.The study found that the AMPs HPRP-A1/HPRP-A2 can bind to DNA in the nucleus of Gram-negative bacteria at higher concentrations,and CHA cannot bind to DNA at higher concentrations.3.HPRP-A1/ HPRP-A2,and CHA combined antibacterial and fungal biofilm activityThe activity of HPRP-A1/HPRP-A2 and CHA in combination treatment of biofilms produced by bacteria and fungi was studied.The detection of the secondary structure,the minimal inhibitory biofilm concentration,the synergy index detection,the prevention of biofilm formation,the metabolic activity of the formed biofilm,and the in vivo experiments in rats and mice were all addressed.The inhibitory minimal biofilm concentration(MBIC)values of HPRP-A1,HPRP-A2,and CHA alone for different bacteria and fungi,respectively,were higher than those previously determined to inhibit bacterial and fungal minimal inhibitory concentration(MIC).The combined drug has a synergistic effect in the treatment of bacteria and fungi;AMPs or CHA alone prevent the biofilm,bacterial,or fungal biofilm from gradually decreasing in biomass.When the concentration of AMPs or CHA reached MIC,the protective effect on biofilm was the strongest,showing that both AMPs and CHA had dose-dependent inhibitory effects on the prevention of bacterial and fungal adhesion.The biomass of bacterial and fungal biofilms was significantly decreased compared with that of the drug alone when HPRP-A2 and CHA combined to inhibit the biofilm formation ability of E.coli,S.aureus and C.albicans;The combined use of AMPs and CHA can inhibit the metabolic activity of the formed biofilm.Both can inhibit the proliferation of mature biofilms.The combination of HPRP-A2 and CHA has a stronger inhibitory effect on biofilm formation than the drug alone;In the low and high dose groups of mouse and rat models of biofilm vaginitis,the combination of HPRP-A2 and CHA has a higher rate of inhibition of bacteria and fungi in vaginitis caused by biofilms of E.coli,S.aureus and C.albicans than the agents alone.In the high-dose group,the bacteriostatic rate reached 99.9 %(P < 0.001),and the treatment effect was obvious.4.Mechanism of HPRP-A1/HPRP-A2,and CHA in combination against bacterial and fungal biofilmsThe antibacterial peptides and CHA were combined to study the mechanism of bacterial and fungal biofilms.The integrity of membranes was observed and analyzed,and it was noted that membranes were quickly broken.The morphology was examined and understood with the combined use of AMPs and CHA by laser confocal,scanning electron microscopy,and atomic force microscopy techniques.The phenol-sulfuric acid method was used to extract the EPS of the main components of the biofilm,and the laser confocal observation was used to analyze the EPS biomass.Analysis of relative expression of related cell adhesion factors and regulatory factors in S.aureus biofilm by q-PCR technology.The results show that the combined use of AMPs and CHA affects the integrity of biofilm cells and the rate of biofilm reduction.The combined use of AMPs and CHA can more quickly destroy the integrity of the bacterial membrane than the agent alone,effect the fluorescence permeability and reduce the biomass of the biofilm.The combination of HPRP-A2 and CHA on the bacterial and fungal biofilms has a morphological change after treatment,which significantly reduces the area and biomass of biofilms.It has been observed that it is difficult to distinguish the initial dense aggregates from loose aggregates and even to the absence of any visible aggregation,and finally to deformation of the cell membrane,development of wrinkles,and even significant damage.The combined use of HPRP-A2 and CHA for EPS in the main components of the biofilm significantly reduced the EPS biomass compared with AMPs or CHA alone.The analysis of EPS by laser confocal analysis was consistent with the quantitative analysis of EPS extracted by the phenol-sulfuric acid method;the inhibition of cell adhesion factor expression is up-regulated,and expression of key biofilm regulators is down-regulated in S.aureus.The mechanism is mainly to quickly break the biofilm of bacteria and fungi,to inhibit the EPS of biofilm,to regulate the inhibition of adhesion factors and important regulatory factors that inhibit biofilm.In a conclusion,in this study,the ?-helix cationic AMPs HPRP-A1/HPRP-A2 and CHA were selected as the model agents.HPRP-A1/HPRP-A2 combined with CHA at synergistic concentrations could significantly increase the antimicrobial activity,anti-biofilm in vitro/in vivo as well as the targeting ability.This study would provide theoretical foundation for the targeted modification to AMPs in clinical use,May be an effective way to treat gynecological vaginitis in clinical practice in the future.