Quorum Sensing Effect Of Paeonol On Pseudomonas Aeruginosa And Mechanism Study

The communication mechanism of bacterial quorum sensing(QS)is ubiquitous in microorganisms and microbial communities,regulating the activities of microbial populations to increase their pathogenicity and drug resistance and then adapt to the external environment.In recent years,the study of using Chinese herbal medicines as quorum sensing inhibitors(QSI)has become one of the main directions to interfere or block the QS pathway.Paeonol,as a monomer component of botanicals,is a smallmolecule phenolic compound derived from Cortex Moutan.It has a variety of pharmacological effects and has broad application prospects.P.aeruginosa(PA)multifunctional metabolites,intrinsic and acquired antibiotic resistance,biofilm formation,and production of multiple virulence factors make it a major opportunistic pathogen in clinical hospitals bacteria,which can cause a variety of acute and chronic infections.Since QS system of P.aeruginosa regulates its virulence factors and antibiotic resistance,the development of new antibacterial drugs targeting the N-Acyl homoserine lactone(AHL)quorum sensing system can provide,which provides a reliable reference for the research and development of anti-infective drugs.The research targeted the QS system of P.aeruginosa,screened the traditional Chinese medicine monomers with AHL quorum sensing inhibitory activity,and further studied the effect of paeonol on the quorum sensing mechanism of P.aeruginosa.The research contents are as follows:(1)Effects of paeonol on virulence factors of P.aeruginosaThe quorum sensing inhibitory activity of paeonol was confirmed by screening the model strain C.violaceum ATCC12472;The minimum inhibitory concentration(MIC)and growth curve of paeonol on PAO1 were determined,and pyocyanin,protease,elastase and rhamnolipid production of paeonol on PAO1 was explored.The results showed that paeonol had no significant effect on its growth at 128-512 μg/mL;Paeonol significantly reduced pyocyanin,LasA hydrolase,LasB elastase,rhamnolipid of PAO1 at 128-512 μg/mL in a concentration-and time-dependent manner(p < 0.05 or p < 0.01).(2)The effect of paeonol on biofilm of P.aeruginosaThe effect of paeonol on the movement of P.aeruginosa was determined,and its effect on the biofilm formation of P.aeruginosa was evaluated by crystal violet staining and silver staining.The ultrastructural changes of biofilm were further observed by scanning electron microscope.Paeonol(128-512 μg/mL)significantly inhibited biofilm formation and cluster migration(p < 0.05 or p < 0.01)of 4 strains of P.aeruginosa(PAO1,ATCC 27853,ATCC 9027 and PAO3401),and Different strains had different inhibitory effects,and the density and structure of biofilm were significantly destroyed in a concentration-dependent manner;(3)Study on the quorum sensing effect and mechanism of paeonol on P.aeruginosaThe reporter strain CV026 was used to detect the effect of paeonol on PAO1 signal molecules,and qPCR was used to detect the expression levels of QS signal molecules,virulence genes,biofilm and movement-related genes,and the anti-infection effect was evaluated by a C.elegans infection model.The results showed that paeonol(128-512μg/mL)significantly inhibited the synthesis and activity of PAO1 AHL signaling molecules(p < 0.01),and significantly decreased the mRNA expressions of QS signaling molecules lasI/R,rhlI/R,and pqsA/R,significantly inhibited the mRNA expression of virulence genes phzA,phzM,phzH,phzS,lasA,lasB,rhlA,rhlC(p < 0.05 or p < 0.01),significantly reduced the exopolysaccharide-regulated genes pelA,pslB and motor genes fliA,pilA mRNA expression(p < 0.05 or p < 0.01);paeonol(128-256 μg/mL)increased the survival rate of PAO1-infected C.elegans;(4)The mechanism of paeonol on macrophages infected by P.aeruginosaPaeonol concentration lower than 256 μg/mL had no effect on the viability of RAW264.7 cells(p < 0.01).The macrophage model of P.aeruginosa infection was established.The adhesion and invasion efficiency of PAO1 to macrophages was significantly increased(p < 0.05 or p < 0.01);Based on the QS system,paeonol(32-128μg/mL)had a therapeutic effect on PAO1-infected macrophages and was significantly down-regulated PAO1 QS signal molecules lasI/R,rhlI/R,pqsA/R mRNA expression;significantly inhibited the virulence genes phzA,phzM,phzH,phzS,lasA,lasB,rhlA,rhlC mRNA expression(p < 0.05 or p < 0.01).Paeonol(32-128 μg/mL)significantly reduced the content of LDH released in the cell supernatant,significantly increased cell viability,decreased adhesion rate,invasion rate,and alleviated cell damage(p < 0.05 or p < 0.01);and Compared with the PAO1 model group,the paeonol treatment group(32-128 μg/mL)significantly inhibited the mRNA expression of inflammatory factors and up-regulated the mRNA expression of pro-inflammatory factors(p < 0.05 or p < 0.01);significantly downregulated TLR4/MyD88 /NF-κB signaling pathway related genes(p < 0.05 or p < 0.01).In conclusion,paeonol down-regulated QS system signaling molecules,virulence genes,and biofilm genes,thereby inhibiting virulence factors,motility,and biofilm formation.Paeonol attenuates the QS of P.aeruginosa,alleviates the cytotoxic damage induced by P.aeruginosa,improves cell viability,resists the adhesion and invasion of P.aeruginosa,mediates the TLR4/MyD88/NF-κB signaling pathway to inhibit the inflammatory response regulated by macrophages.