Application of bakuchiol in preparation of drug for resisting pseudomonas aeruginosa infection

Abstract

The invention provides an application of bakuchiol in preparation of a drug for resisting pseudomonas aeruginosa infection. A research result verifies that bakuchiol obviously inhibits gene expression related to a PQS quorum sensing system of the pseudomonas aeruginosa under the condition that normal growth of the pseudomonas aeruginosa is not hindered. Further analysis shows that the action mechanism of the bakuchiol definitely points to a transcriptional activator PqsR of a PQS quorum sensing system, in addition, the bakuchiol remarkably reduces the athletic ability of pseudomonas aeruginosa in an experiment, remarkably reduces gene expression related to a virulence factor, and has a significant effect in infection models of plants and animals. The bakuchiol shows the effect of remarkably weakening the toxicity of the pseudomonas aeruginosa on the Chinese cabbage and the caenorhabditis elegans.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical preparations, and in particular to application of bakuchiol in the preparation of drugs for resisting Pseudomonas aeruginosa infection. Background Art

[0002] Pseudomonas aeruginosa is a common conditional pathogen that is widely present in natural and artificial environments. It is often found in hospitals and is susceptible to infecting immunocompromised patients, including causing burn infections, skin infections, urinary tract infections, and lung infections in patients with cystic fibrosis (CF). Antibiotic treatment is a common means of addressing bacterial infections, but this approach is difficult for Pseudomonas aeruginosa. Pseudomonas aeruginosa can resist multiple classes of antibiotics and acquire drug resistance. In 2017, the World Health Organization (WHO) listed carbapenem-resistant Pseudomonas aeruginosa as one of the three bacterial species that urgently need to develop new antibiotics to treat infections. Given the limited antimicrobial drug library for preventing and treating Pseudomonas aeruginosa infections, finding alternative prevention and treatment strategies is a top priority.

[0003] The quorum sensing system (QS) is an important regulatory means for the survival of Pseudomonas aeruginosa populations and the virulence of infection. Currently, three classic QS systems (LAS, RHL and PQS) have been found in Pseudomonas aeruginosa. In the LAS system, LasI can synthesize 3-oxo-C 12 -HSL acts as an autoinducer of the system. When its concentration reaches a threshold, it binds to LasR and forms LasR-3-oxo-C 12-HSL complex, activating the expression of genes related to biofilm formation, protease, elastase and exotoxin A production. Similarly, RhlI synthesizes C4-HSL and binds to its cognate receptor RhlR to form RhlR-C4-HSL, thereby activating the expression of genes encoding virulence factors such as pyocyanin, hydrogen cyanide, siderophore, elastase, alkaline protease, etc. The PQS system is the third quorum sensing system of Pseudomonas aeruginosa, which uses 2-heptyl-3-hydroxy-4-quinolone (also known as Pseudomonas quinolone signal, PQS) as a signal molecule and involves an operon pqsABCDE and a pqsR gene. The PqsR protein encoded by pqsR can activate the expression of the operon pqsABCDE with the PQS signal molecule as a ligand, regulating the production of extracellular proteases, lectins, hydrogen cyanide and pyocyanin of Pseudomonas aeruginosa and the formation of biofilm. Many studies have shown that inhibiting the QS system can effectively reduce the formation of biofilm and the production of virulence factors of Pseudomonas aeruginosa. Unlike traditional antibiotics, quorum sensing inhibitors (QSI) do not interfere with the normal metabolic process of Pseudomonas aeruginosa, but target the QS system to block intra-species or inter-species information exchange to reduce the expression of virulence factors and pathogenicity of Pseudomonas aeruginosa. Therefore, developing an effective QSI can not only avoid the problem of antibiotic resistance, but also effectively solve the problem of Pseudomonas aeruginosa infection.

[0004] Bakuchiol is a monoterpene phenolic compound with the chemical name of phenol, 4-[(1E, 3S)-3-vinyl-3,7-dimethyl-1,6-octadienyl. It is mainly extracted from the seeds of Cullen corylifolium (Linnaeus) Medikus, a plant of the leguminous family. Bakuchiol has a boiling point of 145-147°C and is a light yellow slurry at room temperature. It is easily oxidized, insoluble in water, and soluble in organic reagents such as alcohols and DMSO. Studies have shown that bakuchiol has anti-QS properties. AhlamAlasiri et al. found that bakuchiol is an effective LasR antagonist, which effectively inhibits the production of pyocyanin and rhamnolipid by binding to LasR, and inhibits the formation of biofilm (AlasiriA, Soltane R, TahaMN, AbdEl-Aleam RH, Alshehri F, Sayed AM. Bakuchiol inhibits Pseudomonas aeruginosa'squorum sensing-dependent biofilm formation by selectively inhibiting its transcriptional activator protein LasR. Int J Biol Macromol. 2024Jan;255:128025.). Different from the results of this study, the present invention found that bakuchiol can bind to PqsR to inhibit the PQS system of Pseudomonas aeruginosa, and inhibit the expression of related virulence factors, such as pyocyanin, elastase, hydrogen cyanide, lectin, etc., but bakuchiol shows no inhibitory effect on the LAS system and does not affect the expression of the lasI gene. Currently, there is no report on the inhibition of Pseudomonas aeruginosa PQS quorum sensing system by bakuchiol.

[0005] The CAS number of bakuchiol is 10309-37-2, and the molecular formula is C 18 H 24 O, with a molecular weight of 256.38, has the following chemical formula:

[0006] Summary of the invention

[0007] (I) Purpose of the invention

[0008] The invention provides the application of bakuchiol in preparing a drug for resisting infection of Pseudomonas aeruginosa.

[0009] The second object of the present invention is the use of bakuchiol in the preparation of a preparation for inhibiting the movement ability of Pseudomonas aeruginosa.

[0010] The third object of the present invention is to use bakuchiol in the preparation of genes related to inhibiting the expression of virulence factors (pseudomonas aeruginosa, elastase, hydrogen cyanide, and lectin) of Pseudomonas aeruginosa.

[0011] The fourth object of the present invention is the use of bakuchiol in the preparation of drugs for reducing the pathogenicity of Pseudomonas aeruginosa.

[0012] The fifth object of the present invention is a Pseudomonas aeruginosa PQS quorum sensing system inhibitor, wherein the active ingredient of the Pseudomonas aeruginosa PQS quorum sensing system inhibitor is bakuchiol.

[0013] The sixth object of the present invention is a drug for treating or assisting in the treatment of Pseudomonas aeruginosa infection, wherein the active ingredient of the drug for treating or assisting in the treatment of Pseudomonas aeruginosa infection is bakuchiol.

[0014] (II) Beneficial effects

[0015] The present invention provides the use of bakuchiol in the preparation of an anti-Pseudomonas aeruginosa infection drug. The drug has the following beneficial effects: The research results of the present invention confirm that bakuchiol can significantly inhibit the expression of genes related to the PQS quorum sensing system of Pseudomonas aeruginosa without affecting the normal growth of Pseudomonas aeruginosa, and the drug's target is the transcriptional activator PqsR of the PQS quorum sensing system. In addition, bakuchiol significantly reduces the motility of Pseudomonas aeruginosa; and reduces the expression of genes related to virulence factors. Plant and animal infection model experiments show that bakuchiol significantly weakens the toxicity of Pseudomonas aeruginosa to Chinese cabbage and Caenorhabditis elegans. Therefore, bakuchiol is a Chinese medicine preparation with great potential for anti-Pseudomonas aeruginosa infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the effect of bakuchiol provided by the present invention on the gene expression of PQS, RHL and LAS quorum sensing systems of Pseudomonas aeruginosa;

[0017] Figure 2 It is a schematic diagram of the effect of bakuchiol provided by the present invention on the growth of Pseudomonas aeruginosa;

[0018] Figure 3 It is a schematic diagram of the effect of different concentrations of bakuchiol on the gene expression of the PQS quorum sensing system of Pseudomonas aeruginosa provided by the present invention;

[0019] Figure 4 The invention provides a method for regulating the expression of pqsA by bakuchiol. 600 And the schematic diagram of the impact of the change;

[0020] Figure 5It is a schematic diagram of the effect of different concentrations of bakuchiol on the gene expression of the PQS quorum sensing system in a strain overexpressing the PqsR protein provided by the present invention;

[0021] Figure 6 It is a schematic diagram of molecular docking between bakuchiol and PqsR protein provided by the present invention;

[0022] Figure 7 It is a schematic diagram of the effect of bakuchiol provided by the present invention on the motility of Pseudomonas aeruginosa;

[0023] Figure 8 It is a schematic diagram of the effect of bakuchiol provided by the present invention on the synthesis of pyocyanin by Pseudomonas aeruginosa;

[0024] Fig. 9 It is a schematic diagram of the effect of bakuchiol on the gene expression of virulence factors of Pseudomonas aeruginosa provided by the present invention;

[0025] Fig.10 It is a schematic diagram of the effect of bakuchiol provided by the present invention on the plant pathogenicity of Pseudomonas aeruginosa;

[0026] Fig.11 It is a schematic diagram of the effect of bakuchiol provided by the present invention on the animal pathogenicity of Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The Pseudomonas aeruginosa PAO1 used in the present invention is derived from the Pathogenic Microorganism Laboratory of Yan'an University; other related strains are constructed on the basis of PAO1 and used to explain the present invention. Bakuchiol and Calycosin used in the present invention are purchased from Baoji Chenguang Biotechnology Co., Ltd., and DMSO is used as a solvent to prepare bakuchiol and Calycosin into a Chinese medicine monomer mother solution with a concentration of 5 mg / mL. Through MIC (minimum inhibitory concentration) detection, the present invention found that the MIC (minimum inhibitory concentration) of bakuchiol and Calycosin to Pseudomonas aeruginosa is greater than 256 μg / mL, which indicates that these two Chinese medicine monomers do not affect the growth of Pseudomonas aeruginosa within a specific concentration range (<256 μg / mL), nor will they cause selection pressure on Pseudomonas aeruginosa.

[0029] Example 1: Construction of a screening model for QS system gene expression in Pseudomonas aeruginosa

[0030] Method 1: Transcriptional fusion strain construction experiment

[0031] Step 1, the present invention uses Pseudomonas aeruginosa genomic DNA as a template to design primers pqsA F / pqsA R, and uses the primer pair to perform PCR amplification of the promoter sequence PpqsA of pqsA, and the DNA fragment size is 1105 bp;

[0032] Step 2: Double digest PpqsA and plasmid mini-CTX-lacZ with SalI / PstI;

[0033] Step 3, the PpqsA after digestion was purified and connected to the plasmid mini-CTX-lacZ, transformed into Escherichia coli TG1 competent cells, and coated with LB plates containing tetracycline hydrochloride (Tetracycline Tc, 200 μg / mL, referred to as Tc200) for screening;

[0034] Step 4: After the obtained colonies were tested by PCR, plasmids were extracted to obtain recombinant vectors, and the recombinant vectors were digested and verified by SalI / PstI to obtain the recombinant vector mini-CTX-PpqsA-lacZ showing a positive result;

[0035] Step 5, transforming mini-CTX-PpqsA-lacZ into S17-1 to obtain S17-1mini-CTX-PpqsA-lacZ;

[0036] Step 6, the obtained recombinant strain S17-1 mini-CTX-PpqsA-lacZ was combined with Pseudomonas aeruginosa PAO1 on an LB plate at 37°C for 48h, the combined bacterial lawn was suspended with liquid LB, and after appropriate dilution, it was coated on an LB double-resistance plate containing kanamycin (KanamycinKm, 30 μg / mL, referred to as Km30) and Tc200. The obtained colonies were streaked and cultured again on the double-resistance plate to obtain a transcriptional fusion strain (i.e., a gene expression screening model for the Pseudomonas aeruginosa PQS quorum sensing system): PAO1attB::mini-CTX-PpqsA-lacZ; the same method was used to obtain the gene expression screening models of the other two quorum sensing systems of Pseudomonas aeruginosa (RHL quorum sensing system and LAS quorum sensing system): PAO1 attB::mini-CTX-PrhlI-lacZ and PAO1 attB::mini-CTX-PlasI-lacZ;

[0037] Example 2: Effect of Bakuchiol on the Expression of QS System Genes in Pseudomonas aeruginosa

[0038] Method 2: Detection of gene expression in the quorum sensing system under the action of bakuchiol

[0039] Step 1. Using the Chinese medicine monomer calycosin and DMSO solvent, which have no effect on the quorum sensing system of Pseudomonas aeruginosa, as negative controls, a bakuchiol solution with a final concentration of 100 μg / mL was co-incubated with transcriptional fusion strains PAO1 attB::mini-CTX-PpqsA-lacZ, PAO1 attB::mini-CTX-PrhlI-lacZ, and PAO1 attB::mini-CTX-PlasI-lacZ, and cultured at 37°C and 200 rpm until OD 600 is 2.0;

[0040] Step 2. In order to detect the β-galactosidase activity of the reporter strain, o-nitrophenyl-β-D-galactoside (ONPG) was used as the substrate for determination: 20-100 μL of bacterial solution cultured to the logarithmic phase was taken, and 420 μL of Zbuffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform and 10 μL of 0.1% sodium dodecyl sulfate (SDS) were added to the bacterial solution in sequence. The mixed solution was quickly mixed for 20 seconds and incubated at 30°C for 1 hour. After incubation, 100 μL of ONPG at a concentration of 4 mg / mL was added to the mixed solution for reaction. The reaction was terminated by adding 250 μL of 1M Na2CO3, and the reaction time was recorded. Finally, the mixed solution was centrifuged at 14000×g for 3 minutes and the OD of the mixed solution was detected. 420 and OD 550 , and then the β-galactosidase activity was calculated in Miller units (MU) according to the following equation:

[0041] MU=1,000×(OD 420 -1.75×OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0042] Experimental Results

[0043] The results are as follows Figure 1 As shown in the figure, compared with the DMSO solvent and calycosin treatment groups, bakuchiol at a concentration of 100 μg / mL significantly inhibited the promoter activity of pqsA, and the inhibition effect exceeded 50%, and this difference reached a significant level of p<0.0001. In addition, compared with pqsA, bakuchiol did not have a significant effect on the expression of rhlI and lasI, indicating that bakuchiol only had a significant inhibitory effect on the PQS quorum sensing system of Pseudomonas aeruginosa;

[0044] Example 3: Effect of Bakuchiol on the Growth of Pseudomonas aeruginosa

[0045] Method 3: Growth curve analysis experiment under the action of bakuchiol

[0046] Step 1, pick a single colony of Pseudomonas aeruginosa PAO1 in 5 mL LB liquid medium containing Km30, and culture at 37°C and 200 rpm for 16-20 hours until the stable phase;

[0047] Step 2: Add the Pseudomonas aeruginosa culture medium to 5 mL LB liquid medium containing Km30 and bakuchiol at a final concentration of 100 μg / mL at a ratio of 1:100, and detect the initial OD 600 DMSO solvent and calycosin were used as controls;

[0048] Step 3: Incubate the transferred bacterial solution at 37°C and 200 rpm, and measure and record the OD of the bacterial solution every 2 hours. 600 , total culture for 16h.

[0049] Experimental Results

[0050] The wild strain PAO1 of Pseudomonas aeruginosa was used as the test strain. Four experimental treatments were set up. The first group was a blank control, the second group was a medium with a final concentration of 100 μg / mL of bakuchiol added to the medium, the third group was a medium with a final concentration of 100 μg / mL of calycosin, and the fourth group was a medium with the same volume of DMSO solvent added to the medium. Three biological replicates were set up for each group. The results are shown in Figure 2. Figure 2 As shown, bakuchiol had no significant effect on the growth of Pseudomonas aeruginosa compared with the control group.

[0051] Example 4: Effects of different concentrations of bakuchiol on gene expression of Pseudomonas aeruginosa PQS quorum sensing system

[0052] Method 4: PQS quorum sensing system gene expression detection experiment under different concentrations of bakuchiol

[0053] In order to detect the effect of different concentrations of bakuchiol on the PQS system, the present invention co-incubated different concentrations of bakuchiol with the PAO1::mini-CTX-PpqsA-lacZ strain according to the following steps, and analyzed the expression of the pqsA promoter by detecting β-galactosidase activity;

[0054] Step 1: Using the Chinese medicine monomer calycosin and DMSO solvent, which have no effect on the PQS quorum sensing system of Pseudomonas aeruginosa, as negative controls, different concentrations of bakuchiol solution were co-incubated with the transcriptional fusion strain PAO1 attB::mini-CTX-PpqsA-lacZ and cultured at 37°C and 200 rpm until OD 600 is 2.0;

[0055] Step 2. To detect the β-galactosidase activity of the reporter strain, o-nitrophenyl-β-D-galactoside (ONPG) was used as a substrate for determination: 20-100 μL of bacterial solution cultured to the logarithmic phase was taken, and 420 μL of Zbuffer (60 mM / L Na2HPO4, 40 mM / L NaH2PO4, 10 mM / L KCl, 1 mM / L MgSO4, pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform and 10 μL of 0.1% sodium dodecyl sulfate (SDS) were added to the bacterial solution in sequence. The mixed solution was quickly mixed for 20 seconds and incubated at 30°C for 1 hour. After incubation, 100 μL of ONPG at a concentration of 4 mg / mL was added to the mixed solution for reaction. The reaction was terminated by adding 250 μL of 1M Na2CO3, and the reaction time was recorded. Finally, the mixture was centrifuged at 14000×g for 3 min and the OD of the mixture was measured. 420 and OD 550 , and then the β-galactosidase activity was calculated in Miller units (MU) according to the following equation:

[0056] MU=1,000×(OD 420 -1.75×OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0057] Experimental Results

[0058] The results are as follows Figure 3 As shown in Figure 2, the pqsA promoter activity decreased with the increase of bakuchiol concentration, showing a concentration-dependent inhibition, and the IC 50 The inhibitory efficiency of bakuchiol on pqsA expression reached the maximum at 70 μg / mL. To explore the optimal detection point (OD) of gene expression of PQS quorum sensing system of Pseudomonas aeruginosa when the concentration of bakuchiol was 80 μg / mL, 600 The present invention detects OD values ​​according to method 4. 600 The expression of pqsA when the values ​​are 1, 1.5, 2, 2.5, 3, 3.5, and 4. The results are as follows Figure 4As shown in Figure 2, compared with the DMSO control, the treatment with bakuchiol significantly inhibited the expression of pqsA during the entire growth period. 600 After reaching 2.0, the expression of pqsA in both DMSO and bakuchiol treatment groups tended to be stable. 600 After reaching 3.0, the difference with the DMSO control group gradually increased, which may be due to the large-scale synthesis of the target of bakuchiol or the corresponding competitive ligand in Pseudomonas aeruginosa in the stable period, which interfered with its effect. Therefore, in order to ensure that bakuchiol inhibits the activity of Pseudomonas aeruginosa PQS system, the following studies used a bakuchiol concentration of 80 μg / mL and an OD 600 Conduct follow-up research for 2.0.

[0059] Example 5: Effects of different concentrations of bakuchiol on the expression of PQS quorum sensing system genes in PqsR protein overexpression strains

[0060] Method 5: Detection of PQS quorum sensing system gene expression in PqsR protein overexpression strains by different concentrations of bakuchiol

[0061] Step 1, using Pseudomonas aeruginosa genomic DNA as a template to design primers pqsR F / pqsR R, and using the primers to PCR amplify a 1000 bp sequence of the gene pqsR;

[0062] Step 2, double digest the pqsR gene fragment and plasmid pBBR1MCS-5 with Xho I / Pst I;

[0063] Step 3, the pqsR gene fragment after enzyme digestion was purified and connected to the plasmid pBBR1MCS-5, transformed into Escherichia coli TG1 competent cells, and coated with LB plates containing gentamicin Gm (100 μg / mL, referred to as Gm100) for screening;

[0064] Step 4: The positive clone strains were screened by colony PCR, and the recombinant vectors were extracted. The recombinant vectors were digested and verified by Xho I / Pst I to obtain the recombinant vector pBBR1MCS-5-pqsR;

[0065] Step 5, pick a single colony of PAO1 attB::mini-CTX-PpqsA-lacZ and place it in 5 mL LB liquid medium, and culture it overnight at 37°C and 200 rpm;

[0066] Step 6: Transfer the bacterial solution obtained in step 5 to 50 mL of fresh LB liquid medium at a ratio of 1:100 and culture at 37°C and 200 rpm until OD 600= 0.75-1.25, centrifuge at 8100 g for 8 min at 4°C to collect the cells;

[0067] Step 7: Resuspend the cells with 50 mL of pre-cooled 300 mM sucrose solution, centrifuge at 8100 g for 8 min at 4 °C to collect the cells;

[0068] Step 8: Resuspend the cells with 25 mL of pre-cooled 300 mM sucrose solution, centrifuge at 8100 g for 8 min at 4 °C to collect the cells;

[0069] Step 9: Resuspend the cells with 0.5 mL of pre-cooled 300 mM sucrose solution and place on ice for 30 min to obtain electroporation competent cells. Aliquot 100 μL into each tube for later use and store the rest in a refrigerator at 4 °C.

[0070] Step 10, take an appropriate amount of recombinant vector pBBR1MCS-5-pqsR and add it to the electroporation competent cells of PAO1attB:mini-CTX-PpqsA-lacZ, mix well and transfer to a sterile and pre-cooled electroporation cup for electroporation transformation. The electroporation conditions are set as: 25μF, 200Ω, 1900V, 3-5ms;

[0071] Step 11, transfer the electroporated cells into 5 mL of fresh LB liquid medium and culture at 37°C, 200 rpm for 3-5 h;

[0072] Step 12, taking an appropriate amount of bacterial liquid and coating it on TSB medium containing Km30, Tc200 and Gm100 for resistance screening, culturing at 37°C for 2 days, and then streaking the obtained colonies on new TSB medium containing Km30, Tc200 and Gm100 to obtain the pqsR gene overexpression strain PAO1attB::mini-CTX-PpqsA-lacZ (pBBR1MCS-5-pqsR);

[0073] Step 13, using the same method to obtain the empty vector control strain PAO1attB::mini-CTX-PpqsA-lacZ (pBBR1MCS-5);

[0074] Step 14: In order to detect the effect of different concentrations of bakuchiol on the gene expression of the PQS quorum sensing system in the PqsR protein overexpression strain, the present invention uses the Chinese medicine monomer calycosin and DMSO solvent, which have no effect on the PQS quorum sensing system of Pseudomonas aeruginosa, as negative controls, and incubates different concentrations of bakuchiol with PAO1 attB::mini-CTX-PpqsA-lacZ (pBBR1MCS-5-pqsR) and its empty vector control strain, respectively, and cultures them at 37°C and 200 rpm until OD 600 is 2.0;

[0075] Step 15, according to method 4 step 2, detect the β-galactosidase activity of the reporter strain: In order to detect the β-galactosidase activity of the reporter strain, o-nitrophenyl-β-D-galactoside (ONPG) was used as the substrate for determination: 20-100 μL of bacterial solution cultured to the logarithmic phase was taken, and 420 μL of Z buffer (60 mM / L Na2HPO4, 40 mM / L NaH2PO4, 10 mM / L KCl, 1 mM / L MgSO4, pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform and 10 μL of 0.1% sodium dodecyl sulfate (SDS) were added to the bacterial solution in sequence. The mixed solution was quickly mixed for 20 seconds and incubated at 30°C for 1 hour. After incubation, 100 μL of ONPG with a concentration of 4 mg / mL was added to the mixed solution for reaction. The reaction was terminated by adding 250 μL of 1M Na2CO3, and the reaction time was recorded. Finally, the mixture was centrifuged at 14000×g for 3 min and the OD of the mixture was measured. 420 and OD 550 The β-galactosidase activity was then calculated in Miller units (MU) according to the following equation: MU = 1,000 × (OD 420 -1.75×OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0076] Experimental Results

[0077] Since the PQS system is jointly determined by the biosynthetic gene cluster of the PQS signal molecule (pqsABCDE and pqsH) and the PQS receptor protein PqsR. And in the presence of PQS signal molecules and HHQ signal molecules, the PqsR regulatory protein will bind to the pqsA promoter site, thereby promoting the synthesis of PQS signal molecules. Therefore, the present invention speculates that bakuchiol may inhibit the expression of pqsA by targeted inhibition of PqsR. In order to verify this speculation, the present invention amplified the pqsR gene by method five, further overexpressed pqsR on the basis of PAO1attB::mini-CTX-PpqsA-lacZ, and then detected the effect of bakuchiol on the pqsA promoter activity in the pqsR overexpression strain by method four. The results are as follows Figure 5 As shown in the figure, in the pqsR normal expression strain, as the concentration of bakuchiol gradually increased, the pqsA promoter activity gradually decreased, and the maximum inhibition efficiency was reached at a concentration of 70 μg / mL. When pqsR was overexpressed, as the concentration of bakuchiol increased, the pqsA promoter activity did not change, and the IC 50From 41.43 μg / mL to 566271 μg / mL. However, the pqsA promoter activity in the control group did not change significantly, whether it was normal expression or overexpression of pqsR. The above results confirm the speculation of the present invention, that is, bakuchiol reduces the expression of the pqsA gene by targeting and inhibiting PqsR;

[0078] Example 6: Molecular docking experiment of bakuchiol and PqsR protein

[0079] Method 6: Molecular docking experiment

[0080] PubChem website was used to search for the molecular structure and Compound CID of bakuchiol, and its Compound CID was 5468522. The protein information of PqsR was downloaded from the PDB database, and the PDB ID was 4jvd. The present invention performed a molecular docking experiment of bakuchiol and PqsR protein by using AutoDockVina 1.1.2 software.

[0081] Experimental Results

[0082] The docking results are as follows Figure 6 As shown, bakuchiol molecules can interact with PqsR protein. Figure 6 A shows that bakuchiol binds to the middle groove of the PqsR protein. Figure 6 The interaction details of BC show that the small molecule bakuchiol has hydrophobic interactions with MET-224, LEU-207, LEU-197, ALA-102, ALA-168, ILE-149, PRO-238, ILE-263, and LEU-208 on the PqsR protein. In addition, bakuchiol and THR-265 on the PqsR protein have hydrogen bonding. Hydrogen bonding is one of the strongest non-covalent interactions, indicating that THR-265 is an important amino acid residue for the binding of bakuchiol and PqsR protein. According to relevant literature, a negative binding affinity indicates that the small molecule has the possibility of binding to the protein, and a value less than -6kcal / mol indicates a high possibility of binding. In this result, the docking software shows that the binding affinity score of bakuchiol and PqsR is -7.5kcal / mol, which means that bakuchiol has a high binding affinity with the PqsR protein. Combined with the results of Example 5, it is shown that the target of bakuchiol is the protein PqsR.

[0083] Example 7: Effect of Bakuchiol on the Motility of Pseudomonas aeruginosa

[0084] Method 7: Pseudomonas aeruginosa Swarming Movement Detection Experiment

[0085] Step 1. Add bakuchiol or calycosin at a final concentration of 80 μg / ml or DMSO of the same volume to the swarming medium to prepare a swarming plate. Dry the plate on a clean bench for 2 minutes before use so that no water droplets can be seen on the surface.

[0086] Swarming medium: agarose 6 g / L, glucose 5 g / L, nutrient broth (NB) 8 g / L.

[0087] 121℃, sterilize for 20min;

[0088] Step 2: Pick a single PAO1 colony and place it in 5 mL of fresh LB liquid medium containing Km30, and culture it at 37°C and 200 rpm for 16-20 h;

[0089] Step 3: Measure OD 600 , dilute the bacterial solution until OD 600 =2.0;

[0090] Step 4: Take 1 μL of bacterial solution and drop it in the center of the plate;

[0091] Step 5. After the bacterial solution has penetrated the plate, slowly place the plate in a 30°C incubator and incubate for 24 hours. Observe the diffusion of the bacterial solution and measure its diameter. All experiments were set up with 3 sets of biological replicates;

[0092] Method 8: Pseudomonas aeruginosa Swimming Movement Detection Experiment

[0093] The method is the same as the Swarming plate experiment, only the plate is replaced with a Swimming plate;

[0094] Swimming medium: agarose 3g / L, NaCl 5g / L and peptone 10g / L. 121℃, sterilize for 20min;

[0095] Method 9: Pseudomonas aeruginosa Twitching Movement Detection Experiment

[0096] Step 1, add bakuchiol or calycosin at a final concentration of 80 μg / ml or DMSO of the same volume to the Twitching medium to prepare a Twitching plate, and dry the plate on a clean bench for 30 minutes before use;

[0097] Twitching medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L. 121℃, sterilize for 20min;

[0098] Step 2: Pick up a single colony of Pseudomonas aeruginosa PAO1 with the tip of a toothpick, pierce the Twitching plate, and incubate it upside down at 37°C for 24 hours;

[0099] Step 3: After the incubation, the plate was completely soaked with Coomassie Brilliant Blue staining solution (0.05 g Coomassie Brilliant Blue R250, 40 mL methanol, 10 mL acetic acid, mixed evenly, and fixed to 100 mL with deionized water);

[0100] Step 4: Decolorize with industrial alcohol until the diffusion zone can be seen, and measure the diameter of the diffusion zone. Three biological replicates were set for all experiments;

[0101] Experimental Results

[0102] In order to explore the effect of bakuchiol on the motility of Pseudomonas aeruginosa, the present invention conducted detection by the operation procedures of methods seven, eight, and nine. The results are as follows Figure 7 Compared with the control group (DMSO), the swarming diameter of Pseudomonas aeruginosa in the bakuchiol-treated group decreased by about 64.3%. Figure 7 As shown in A, this difference reached a significant level of p<0.0001, indicating that 80 μg / ml bakuchiol can significantly inhibit the Swarming movement of Pseudomonas aeruginosa; compared with the control group (DMSO), the Swimming movement diameter of Pseudomonas aeruginosa in the bakuchiol-treated group was reduced by about 38.6%. Figure 7 As shown in B, this difference reached a significant level of p < 0.0001, indicating that 80 μg / ml bakuchiol can significantly inhibit the Swimming movement of Pseudomonas aeruginosa; compared with the control group (DMSO), the Twitching movement diameter of Pseudomonas aeruginosa in the bakuchiol-treated group was reduced by about 25.8%. Figure 7 As shown in Figure C, this difference reached a significant level of p<0.0001, indicating that 80μg / ml bakuchiol can significantly inhibit the Twitching movement of Pseudomonas aeruginosa; the Swimming movement of Pseudomonas aeruginosa depends on the normal flagella function, the Swarming movement also depends on rhamnolipids on this basis, and the Twitching movement depends on the normal pili function. The motility mediated by flagella, pili and rhamnolipids is an important cause of the pathogenicity of Pseudomonas aeruginosa, and they are all regulated by the PQS quorum sensing system;

[0103] Example 8: Effect of Bakuchiol on Pseudomonas aeruginosa Pyocyanin Synthesis

[0104] Method 10: Pyocyanin production detection experiment

[0105] Step 1: Pick a single colony and place it in 5 mL of fresh LB liquid medium containing Km30, and culture it at 37°C and 200 rpm for 16-20 hours;

[0106] Step 2, adding the Pseudomonas aeruginosa bacterial solution to 5 mL PB liquid culture medium containing Km30 and bakuchiol with a final concentration of 80 μg / mL or the same volume of DMSO at a ratio of 1:100, and culturing at 37° C. and 200 rpm with shaking;

[0107] PB liquid culture medium: NaCl 6g / L, K2SO4 10 g / L, tryptone 20g / L. Sterilize at 121℃ for 20min.

[0108] Step 3: After the culture reaches the stable period, centrifuge and take the supernatant;

[0109] Step 4: Add 3 mL of chloroform to 5 mL of the supernatant, stir vigorously for 2 min, and centrifuge at 8000 rpm for 10 min;

[0110] Step 5. Transfer the chloroform layer to a new test tube and mix with 1 mL of 0.2 M hydrochloric acid;

[0111] Step 6: Centrifuge again at 8000 rpm for 10 min, aspirate the top red solution, and measure OD 520 . Calculate the content of pyocyanin according to the formula: OD 520 / OD 600 ×17.072;

[0112] Method 11: Detection of pyocyanin synthesis gene expression under the action of bakuchiol

[0113] Step 1, construct the transcriptional fusion strains PAO1attB::mini-CTX-PphzA1-lacZ; PAO1attB::mini-CTX-PphzA2-lacZ of the pyocyanin synthesis gene operons phzA1B1C1D1E1F1G1 (phzA1) and phzA2B2C2D2E2F2G2 (phzA2) by method 1;

[0114] Step 2, according to method 4 step 2 to detect the β-galactosidase activity of the reporter strain: In order to detect the β-galactosidase activity of the reporter strain, o-nitrophenyl-β-D-galactoside (ONPG) was used as the substrate for determination: 20-100 μL of bacterial solution cultured to the logarithmic phase was taken, and 420 μL of Z buffer (60 mM / L Na2HPO4, 40 mM / L NaH2PO4, 10 mM / L KCl, 1 mM / L MgSO4, pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform and 10 μL of 0.1% sodium dodecyl sulfate (SDS) were added to the bacterial solution in sequence. The mixed solution was quickly mixed for 20 seconds and incubated at 30°C for 1 hour. After incubation, 100 μL of ONPG with a concentration of 4 mg / mL was added to the mixed solution for reaction. The reaction was terminated by adding 250 μL of 1M Na2CO3, and the reaction time was recorded. Finally, the mixture was centrifuged at 14000×g for 3 min and the OD of the mixture was measured. 420 and OD 550 The β-galactosidase activity was then calculated in Miller units (MU) according to the following equation: MU = 1,000 × (OD 420 -1.75×OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0115] Experimental Results

[0116] Pyocyanin plays an important role in the infection of Pseudomonas aeruginosa. Its synthesis is regulated by the PQS quorum sensing system and is usually used as a marker for evaluating the PQS quorum sensing system. The present invention first explored the effect of bakuchiol on the pyocyanin production of Pseudomonas aeruginosa PAO1 according to method 10. The results are as follows Figure 8 A and Figure 8 As shown in Figure B, the pyocyanin production in the bakuchiol-treated group was reduced by 61.4% relative to that in the control group (DMSO), and this difference reached a significant level of p<0.0001. In addition, the present invention detected the expression of the pyocyanin synthesis gene operons phzA1 and phzA2 according to method 11. The results are shown in Figure 10. Figure 8 C and Figure 8 As shown in D, compared with the control group (DMSO), the expression level of phzA1 in the bakuchiol-treated group decreased by 66.8%, and the expression level of phzA2 in the bakuchiol-treated group decreased by 28.4% compared with the control group (DMSO), and this difference reached significant levels p<0.0001 and p<0.001, respectively. These results indicate that 80 μg / mL bakuchiol can inhibit the production of pyocyanin by Pseudomonas aeruginosa by inhibiting the PQS system;

[0117] Example 9: Effect of Bakuchiol on the Expression of Virulence Factor Genes of Pseudomonas aeruginosa

[0118] Method 12: Detection of virulence factor gene expression under the action of bakuchiol

[0119] The hydrogen cyanide (hcnA), elastase (lasB), and lectin (lecA) transcriptional fusion strains PAO1 attB::mini-CTX-PhcnA-lacZ; PAO1attB::mini-CTX-PlasB-lacZ; PAO1 attB::mini-CTX-PlecA-lacZ were constructed by method 1;

[0120] According to method 4, step 2, the β-galactosidase activity of the reporter strain was detected: In order to detect the β-galactosidase activity of the reporter strain, o-nitrophenyl-β-D-galactoside (ONPG) was used as the substrate for determination: 20-100 μL of bacterial solution cultured to the logarithmic phase was taken, and 420 μL of Zbuffer (60 mM / L Na2HPO4, 40 mM / L NaH2PO4, 10 mM / L KCl, 1 mM / LM MgSO4, pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform and 10 μL of 0.1% sodium dodecyl sulfate (SDS) were added to the bacterial solution in sequence. The mixed solution was quickly mixed for 20 seconds and incubated at 30°C for 1 hour. After incubation, 100 μL of ONPG with a concentration of 4 mg / mL was added to the mixed solution for reaction. The reaction was terminated by adding 250 μL of 1M Na2CO3, and the reaction time was recorded. Finally, the mixture was centrifuged at 14000×g for 3 min and the OD of the mixture was measured. 420 and OD 550 The β-galactosidase activity was then calculated in Miller units (MU) according to the following equation: MU = 1,000 × (OD 420 -1.75×OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0121] Experimental Results

[0122] The present invention detected the effect of adding 80 μg / mL bakuchiol on the β-galactosidase activity of these gene promoters according to method 12. First, adding 80 μg / mL bakuchiol significantly reduced the expression of hcnA in Pseudomonas aeruginosa, and its expression was reduced by 38.0% compared with the control group (DMSO). Fig. 9As shown in Figure A, this difference reached a significant level of p < 0.0001. Second, the addition of 80 μg / mL bakuchiol significantly reduced the expression of lasB in Pseudomonas aeruginosa, and its expression level was reduced by 34.1% compared with the control group (DMSO). Fig. 9 As shown in Figure B, this difference reached a significant level of p < 0.0001. Third, the addition of 80 μg / mL bakuchiol significantly reduced the expression of lecA in Pseudomonas aeruginosa, and its expression level was reduced by 27.2% compared with the control group (DMSO). Fig. 9 As shown in C, this difference reached a significant level of p < 0.001. These results indicate that 80 μg / mL bakuchiol can inhibit the synthesis of hydrogen cyanide, elastase and lectin by inhibiting the PQS system;

[0123] Example 10: Effect of Bakuchiol on the Phytopathogenicity of Pseudomonas aeruginosa

[0124] Method 13: Chinese cabbage infection experiment

[0125] Step 1, pick a single colony of Pseudomonas aeruginosa PAO1 and culture it in 5 mL of fresh LB liquid medium containing Km30, 80 μg / mL bakuchiol or 80 μg / mL calycosin or the same volume of DMSO, and culture it at 37°C with shaking at 200 rpm for 16-20 h;

[0126] Step 2: aspirate the bacterial solution, centrifuge at 5500 rpm for 5 min, discard the supernatant, then add an equal volume of 10 mM MgSO4 to wash the bacteria, repeat twice;

[0127] Step 3: Dilute the bacterial solution to OD 600 =2.0;

[0128] Step 4, select fresh Chinese cabbage leaves, sterilize the leaves with 1% H2O2, and dry them;

[0129] Step 5: Inoculate 10 μL of the treated bacterial solution at the same position of the cabbage stem, incubate at 30°C for 3 days, and observe the rotten area of ​​the cabbage. Calculate it using Image J software;

[0130] Experimental Results

[0131] In order to explore the effect of bakuchiol on the plant pathogenicity of Pseudomonas aeruginosa, the present invention used DMSO as a solvent control and calycosin as a control Chinese medicine, and detected the infection ability of Pseudomonas aeruginosa on Chinese cabbage leaves after adding bakuchiol according to method 13. Fig.10As shown, compared with the DMSO treatment group, the rot area caused by Pseudomonas aeruginosa infection on cabbage was reduced by about 51.7% after bakuchiol treatment. This difference reached a significant level p<0.05, indicating that bakuchiol can reduce the pathogenicity of Pseudomonas aeruginosa to Chinese cabbage.

[0132] Example 11: Effect of Bakuchiol on the Animal Pathogenicity of Pseudomonas aeruginosa

[0133] Method 14: Caenorhabditis elegans infection experiment

[0134] Step 1: Take out the frozen tube of nematodes from -80°C, thaw it at room temperature, inoculate it into NGM medium, culture it at 20°C for 24 hours, observe it under a microscope to see whether the nematode eggs are hatched, and continue to culture them at 20°C after hatching until the nematodes can be transferred;

[0135] NGM medium: NaCl 3g / L, Bacto peptone 3.5g / L, Agar powder 17g / L. 121℃, sterilize for 20min. Before use, add 100μL 5mg / mL cholesterol solution, 100μL 1M MgSO4, 100μL 1M CaCl2, 250μL 1M KH2PO4 to every 100mL NGM medium;

[0136] 5 mg / mL cholesterol solution: Weigh 0.5 g of cholesterol and dissolve it in 100 mL of anhydrous ethanol. Filter it with an organic filter membrane and store it at 4°C.

[0137] Step 2: Pick a single colony of Escherichia coli OP50 and place it in 5 mL of fresh LB liquid medium containing ampicillin (Amp, 100 μg / mL), and culture at 37°C and 200 rpm for 16-20 h;

[0138] Step 3: Mix the OP50 bacteria cultured overnight and drop them on the center of the NGM plate. Rotate the plate to spread the bacterial solution on the plate. After the plate is dry, place it in a 37°C incubator for 24 hours. Then store the plate at 4°C and take it out and place it at room temperature for 2 hours before use.

[0139] Step 4: Pick up Caenorhabditis elegans and place them on NGM plates containing OP50. Culture them in a 20°C incubator. Transfer the nematodes to new NGM medium containing OP50 every 4 to 5 days and continue culturing to ensure that their offspring can continue to reproduce.

[0140] Usually, most of the nematodes on a plate are in different growth stages, which will affect the research results. Therefore, in order to study the effect of a single variable on nematodes and eliminate the influence of individual differences on the experiment, this experiment synchronized the culture of nematodes to ensure that all nematodes on a plate are in the same life cycle;

[0141] Step 5: Observe under a microscope to see if the nematodes have reached the egg-laying stage. Select a large number of NGM nematode plates that are in the egg-laying stage, rinse the NGM plates several times with M9 buffer, transfer all the nematodes into a 1.5 mL centrifuge tube, centrifuge at 1300 g for 1 min, discard the supernatant, and retain the worm bodies;

[0142] M9 buffer: Na2HPO4.12H2O 8.55g / L, KH2PO41.5 g / L, NaCl 0.25g / L, NH4Cl 0.5g / L, dilute to 1L with deionized water, sterilize at 121℃ for 20min. Before use, add 200μL 1M MgSO4 and 2mL 20% glucose per 100mL;

[0143] Step 6: Add M9 buffer and wash repeatedly until the supernatant is clear and transparent, discard the supernatant and keep the insect body;

[0144] Step 7, add about 500 μL of nematode lysis solution, place on a vortex shaker and shake for lysis (this process generally does not exceed 5 minutes to prevent the nematode lysis solution from damaging the nematode eggs);

[0145] Nematode lysis solution: 1M NaOH / NaClO / M9 buffer volume ratio = 2:1:2;

[0146] Step 8: After the nematode lysis solution has lysed the nematodes, M9 buffer is immediately added to the centrifuge tube to dilute the lysis buffer, centrifuge, and discard the supernatant;

[0147] Step 9: Wash repeatedly with M9 buffer until the lysate is rinsed clean. Centrifuge, discard the supernatant, add a small amount of M9 buffer, suspend the nematode eggs, and place in a shaker at 20°C for 24 hours;

[0148] Step 10: Take a small amount of nematode liquid and observe under a microscope to see whether the nematode eggs are hatched. If they are, the nematode synchronization is complete. Transfer the nematodes to new NGM medium and continue to culture at 20°C until the L4 stage (nematode larval stage) for later use;

[0149] During nematode synchronization, prepare nematode infection plates (nematode infection plates should generally be prepared before nematode synchronization);

[0150] Step 11. Use a culture dish with a diameter of 35 mm to conduct the experiment. Add bakuchiol or calycosin at a final concentration of 80 μg / mL or the same volume of DMSO to the dissolved NGM medium. After the plate solidifies, add 40 μL of 100 μg / mL FURD (5-fluorouridine, a nematode development inhibitor) on the surface of the plate, spread evenly, and set aside;

[0151] Step 12: Pick a single colony of Pseudomonas aeruginosa and culture it in 5 mL LB medium containing Km30 antibiotic at 37°C until OD 600 is 2.0;

[0152] Step 13, apply 10 μL on the NGM medium plate prepared in step 11;

[0153] Step 14: After the surface of the plate is dry, place it in a 37°C incubator and incubate for 12 hours before taking it out;

[0154] Step 15, placing the plate of step 14 in a 25° C. incubator and continuing to culture for 12 hours (the purpose of this step is to reduce the toxicity of Pseudomonas aeruginosa to prevent the nematodes from being killed by Pseudomonas aeruginosa in a short period of time);

[0155] Next, the nematode infection experiment was conducted;

[0156] Step 16, rinse all the L4 nematodes on the NGM medium into a 1.5 mL centrifuge tube with M9 buffer;

[0157] Step 17: After centrifugation at 1300 rpm for 1 min, remove the supernatant and add fresh M9 buffer to wash away OP50;

[0158] Step 18, transferring the washed L4 stage nematodes to the prepared nematode infection plates, with 30 nematodes transferred to each plate;

[0159] Step 19: Place the plate with transferred nematodes in a 25°C incubator and observe under a microscope every 12 hours to record the number of surviving nematodes for 5 days. Set up 3 replicates for each experiment;

[0160] Experimental Results

[0161] In order to explore the effect of bakuchiol on the animal pathogenicity of Pseudomonas aeruginosa, the present invention used DMSO as a solvent control and calycosin as a control Chinese medicine, and detected the effect of adding bakuchiol on the infection ability of Pseudomonas aeruginosa to Caenorhabditis elegans according to method 14. The results are as follows Fig.11As shown, compared with the DMSO and calycosin treatment groups, the virulence of Pseudomonas aeruginosa to Caenorhabditis elegans was significantly weakened after treatment with bakuchiol, and this difference reached a significant level of p<0.01, indicating that bakuchiol can reduce the pathogenicity of Pseudomonas aeruginosa to Caenorhabditis elegans.

[0162] From the above research results, it can be seen that the Chinese medicine monomer bakuchiol screened by the present invention does not affect the normal growth of Pseudomonas aeruginosa. However, preliminary experiments show that bakuchiol has a significant inhibitory effect on the PQS quorum sensing system of Pseudomonas aeruginosa. Further research results show that the inhibitory effect of bakuchiol on the PQS quorum sensing system of Pseudomonas aeruginosa is concentration-dependent, and the inhibitory effect reaches the maximum at 70 μg / mL. Through gene overexpression and molecular docking experiments, it was found that the drug target of bakuchiol is the transcriptional activator protein PqsR of the PQS quorum sensing system. Bakuchiol can bind to the PqsR protein to inhibit the activity of the pqsA promoter, thereby inhibiting the PQS system of Pseudomonas aeruginosa. In addition, bakuchiol can also inhibit the expression of virulence factors including pyocyanin, elastase, hydrogen cyanide and lectin. Moreover, the Chinese medicine monomer can also significantly reduce the motility of Pseudomonas aeruginosa, including group swimming, swimming and twitching. Finally, the study also showed that bakuchiol could reduce the pathogenicity of Pseudomonas aeruginosa to Chinese cabbage and Caenorhabditis elegans.

[0163] It can be seen from the review that bakuchiol effectively inhibits the PQS quorum sensing system without affecting the normal growth of Pseudomonas aeruginosa, and can reduce the pathogenicity of the bacteria to plant and animal models, and has a protective effect on both hosts. Chemical antibacterial agents easily lead to drug resistance in Pseudomonas aeruginosa. Plant natural products have attracted people's attention because of their wide sources and high safety. At present, only a few QSI targets are relatively clear. The present invention also proves that the target of bakuchiol is PqsR protein, which greatly supports the application value of bakuchiol. Therefore, bakuchiol is a very promising quorum sensing inhibitor for solving the problem of Pseudomonas aeruginosa infection, and has the potential to be developed into a therapeutic drug or auxiliary drug for solving Pseudomonas aeruginosa infection.

[0164] The primers and related restriction enzyme cutting site information used in the present invention are shown in Table 1.

[0165] Table 1 Primers used in the present invention and related restriction site information

[0166]

[0167]

[0168] The underlined sequence is the restriction site

[0169] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of bakuchiol in the preparation of a drug for treating Pseudomonas aeruginosa infection; characterized in that: The active ingredient of the drug for treating or assisting in treating Pseudomonas aeruginosa infection includes bakuchiol.

2. Use of bakuchiol in the preparation of a composition for inhibiting the motility of Pseudomonas aeruginosa; characterized in that: The movement ability of Pseudomonas aeruginosa includes: group movement, swimming movement and twitching movement.

3. Use of bakuchiol in inhibiting the expression of virulence factor genes of Pseudomonas aeruginosa; characterized in that: The virulence factor genes of Pseudomonas aeruginosa include pyocyanin synthesis genes (phzA1 and phzA2), hydrogen cyanide synthesis gene (hcnA), elastase synthesis gene (lasB) and lectin synthesis gene (lecA).

4. Use of bakuchiol in the preparation of drugs for reducing the pathogenicity of Pseudomonas aeruginosa.

5. Use of bakuchiol in inhibiting the expression of genes related to the PQS quorum sensing system of Pseudomonas aeruginosa; characterized in that: The PQS quorum sensing related gene is the operon pqsABCDE, and the target of bakuchiol is the PqsR protein.

6. Use of bakuchiol in the preparation of a Pseudomonas aeruginosa PQS quorum sensing inhibitor; characterized in that: The active ingredient of the Pseudomonas aeruginosa PQS quorum sensing inhibitor includes bakuchiol.

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