Application of 2-aminoquinoline in the preparation of drugs for inhibiting Salmonella typhimurium

Abstract

The present invention discloses the use of 2-aminoquinoline in the preparation of a drug for inhibiting Salmonella typhimurium. The present invention inhibits the expression of QS-related genes and virulence genes and the formation of biofilms by binding 2-aminoquinoline to an LsrB protein, thereby reducing the pathogenicity of Salmonella typhimurium. 2-aminoquinoline can also inhibit the invasion and adhesion capabilities of Salmonella typhimurium to cells. Therefore, 2-aminoquinoline can be used in the preparation of a drug for inhibiting Salmonella typhimurium.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of 2-aminoquinoline in preparing a drug for inhibiting Salmonella typhimurium. Background Art

[0002] Salmonella enterica serovar Typhimurium (S. typhimurium) is a common pathogen that causes foodborne illness and can enter the human body through multiple pathways. S. typhimurium infections are commonly treated with antibiotics, but antibiotic use inevitably induces bacterial resistance, leading to the emergence of multi-antibiotic-resistant pathogens. Therefore, new strategies are urgently needed to protect the host from pathogens without increasing the development of resistance.

[0003] Quorum sensing (QS) is a communication process between bacteria that involves the binding of signaling molecules called autoinducers (AIs) to specific receptors, enabling bacteria to sense bacterial density and regulate gene expression within the population accordingly. QS is used by bacterial pathogens to coordinate various physiological processes, such as biofilm formation, regulation of virulence factor expression, bacterial motility, production of secondary metabolites, and other microbial interactions. Therefore, inhibiting the QS system of pathogenic bacteria can reduce their pathogenicity and prevent the development of drug resistance.

[0004] 2-Aminoquinoline has been shown to be effective in treating osteoarthritis and has the potential to reduce food intake in mammals, but its inhibition of Salmonella typhimurium has not been reported. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of 2-aminoquinoline in the preparation of a drug for inhibiting Salmonella typhimurium.

[0006] The technical solution of the present invention is summarized as follows:

[0007] Application of 2-aminoquinoline in the preparation of drugs for inhibiting Salmonella typhimurium.

[0008] Advantages of the present invention:

[0009] The present invention inhibits the expression of QS-related genes and virulence genes, suppresses biofilm formation, and thus reduces the pathogenicity of Salmonella typhimurium by binding 2-aminoquinoline to the LsrB protein (PDB ID: 1TJY). 2-aminoquinoline also inhibits the invasion and adhesion of Salmonella typhimurium to cells, making it a promising drug for inhibiting Salmonella typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 These are the results of surface plasmon resonance (SPR) experiments on 2-aminoquinoline and LsrB protein.

[0011] Figure 2 The figure shows the effect of different concentrations of 2-aminoquinoline on the growth curve of LT2 strain.

[0012] Figure 3 Figure 3 The effect of 2-aminoquinoline on the biofilm formation of LT2 strain obtained by crystal violet staining. A is the 96-well plate staining results, ***: p < 0.001; ****: p < 0.0001; ns: p > 0.05, no significant difference; B is the result of optical microscopy observation after adding 2-aminoquinoline.

[0013] Figure 4 Effects of 2 mM 2-aminoquinoline on QS-related genes and virulence genes of LT2.

[0014] Figure 5 The effect of different concentrations of 2-aminoquinoline on cell viability, ****: p<0.0001; ns: p>0.05, no significant difference.

[0015] Figure 6 The effect of 2-aminoquinoline on the invasion and adhesion of LT2 cells is shown in Figure 5. A shows the effect of 2-aminoquinoline on cell adhesion, and B shows the effect of 2-aminoquinoline on cell invasion. ***: p < 0.001; ****: p < 0.0001. DETAILED DESCRIPTION

[0016] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0017] The 2-aminoquinoline (CAS: 580-22-3) involved in the following examples was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. with the product number FDB012467 and dissolved in dimethyl sulfoxide (DMSO) to prepare a 400 mM stock solution.

[0018] The strain used was the LT2 strain of Salmonella typhimurium, purchased from Beinachuanglian Biotechnology Co., Ltd. in China Mall (https: / / www.bncc.com / pro / p2 / 8 / p_316132.html), referred to as LT2.

[0019] Culture conditions were LB medium at 37°C and 220 rpm. The cells used were commercially available human normal intestinal epithelial cells NCM460, cultured in high-glucose DMEM medium (commercially available) supplemented with 1% bispecific antibiotics (100 U / mL penicillin, 0.1 mg / mL streptomycin) and 10% fetal bovine serum, i.e., complete DMEM medium, and cultured as a monolayer at 37°C and 5% CO2. The strains and cell types used are only examples and should not be used to limit the scope of application of this application.

[0020] Example 1: Surface plasmon resonance experiment of 2-aminoquinoline and LsrB protein.

[0021] Experimental Method: Purified LsrB protein (PDB ID: 1TJY) was immobilized on a commercial CM5 chip via amino coupling. After coupling, an SPR experiment was performed. LsrB protein served as the stationary phase, and varying concentrations of 2-aminoquinoline (DMSO) were flowed through the chip at a rate of 30 μL / min. Binding of 2-aminoquinoline to LsrB protein exerted stress on the chip, resulting in a response. At saturation before sample injection was stopped, the KD value (equilibrium dissociation constant) was calculated. This value is often used to represent binding affinity; smaller KD values ​​indicate greater affinity.

[0022] Experimental results: Figure 1 As shown in the figure, the SPR results of 2-aminoquinoline and LsrB protein were obtained, and the KD value was 2.007×10 -7 , that is, it has a strong binding force with LsrB protein in vitro, proving that 2-aminoquinoline can bind to LsrB protein.

[0023] Example 2: Effect of 2-aminoquinoline on the growth curve of LT2.

[0024] Experimental methods:

[0025] (1) Minimum inhibitory concentration (MIC) of 2-aminoquinoline against LT2

[0026] The MIC of 2-aminoquinolines against LT2 was determined using a serial dilution method. The highest concentration of a 2-aminoquinoline solution was 40 mM, serially diluted with DMSO to 0.315 mM, and incubated with LT2 at 37°C for 24 hours. The lowest concentration at which bacterial growth was absent was defined as the MIC for that 2-aminoquinoline. Three replicates were performed for each 2-aminoquinoline.

[0027] (2) The overnight LT2 bacterial suspension was inoculated into LB medium at a 1% inoculation ratio. The concentrations of 2-aminoquinoline in each sample were MIC, 5 / 6MIC, 4 / 6MIC, 3 / 6MIC, 2 / 6MIC, and 1 / 6MIC, respectively. The positive control was free of 2-aminoquinoline. Three replicates were set for each concentration. The culture was shaken at 37°C and 220 rpm for 24 hours, and the OD was recorded every 2 hours using a spectrophotometer. 600nm value.

[0028] Experimental results: Figure 2 The figure shows the effect of different concentrations of 2-aminoquinoline on the growth curve of the LT2 strain. The experimental results show that the MIC of 2-aminoquinoline against LT2 is 10 mM. The growth curve shows that 2-aminoquinoline solutions at concentrations of 6 mM, 4 mM, and 2 mM have no effect on the growth curve of LT2. Therefore, it is concluded that 2-aminoquinoline at a concentration of 2 mM inhibits pathogenicity but does not induce drug resistance in Salmonella Typhimurium.

[0029] Example 3: Effect of 2-aminoquinoline on LT2 biofilm.

[0030] Experimental methods:

[0031] Crystal violet staining was used to determine the effect of 2-aminoquinoline on LT2 biofilm. In this study, biofilms were studied using both 96-well plate observation and optical microscope observation.

[0032] 96-well plate observation method: Pick a single LT2 colony and culture it in a shaker at 37°C and 220rpm overnight. Dilute the overnight cultured bacterial solution 100 times with LB medium, add different volumes of 2-aminoquinoline solution to the final concentration of 5mM, 4mM, 3mM, 2mM, and 1mM, mix well and add to the 96-well plate. Incubate the 96-well plate in a 37°C incubator for 24 hours, then discard the supernatant, wash 3 times with PBS, and dry in an oven. Add 2% crystal violet solution to the sample well, stain in the dark for 30 minutes, discard the supernatant, wash 3 times with PBS and dry. Finally, add an equal volume of 95% ethanol, pipette and mix well, and use an enzyme-linked microplate reader to measure the OD 595nm The absorbance at was detected.

[0033] Optical microscopy observation method: Dilute the overnight activated LT2 100 times with LB medium, place the sterilized cell slide in a 12-well plate, add bacterial solution and 2-aminoquinoline solution to a final concentration of 2mM, and incubate at 37°C for 24 hours. After the culture is completed, remove the cell slide and clean the residual impurities on the cell slide with PBS. Add a 0.1% crystal violet solution to the cell slide and stain it in the dark for 30 minutes. Remove the stained cell slide, rinse with PBS and dry it. Use an optical microscope (10x100 times) to observe the distribution of cell membranes on the cell slide.

[0034] Experimental results: Figure 3 The results of the 96-well plate stained with crystal violet showed that 5, 4, 3, and 2 mM 2-aminoquinoline all inhibited the biofilm formation of LT2. Since it significantly inhibited the biofilm formation at 2 mM, with an inhibitory effect of 20.42%, a microscopic observation was conducted on 2 mM 2-aminoquinoline. Figure 3 B. Results showed that the addition of 2 mM 2-aminoquinoline reduced the aggregation of LT2 biofilm, effectively inhibited the formation of biofilm, and reduced the pathogenicity of LT2.

[0035] Example 4: Effects of 2-aminoquinoline on QS-related genes and virulence genes of LT2.

[0036] Experimental Methods: LT2 strains activated overnight were inoculated into LB medium at a 1% inoculation ratio, and 2-aminoquinoline was added to a final concentration of 2 mM. The cells were cultured until the logarithmic phase. RNA was extracted using the Tiangen Bacterial Total RNA Extraction Kit and reverse transcribed using the SparkJade SPARKscript II RT Plus Kit according to the manufacturer's instructions. The concentration was measured using a Nanodrop. qRT-PCR was performed on a LightCycler 480 II in a final reaction volume of 10 μL. The system and reaction procedures were performed according to the SparkJade 2×SYBR Green qPCR Mix manufacturer's instructions.

[0037] Experimental results: The results are as follows Figure 4As shown, 2-aminoquinoline can inhibit the QS-related genes and virulence genes of LT2. The QS-related genes explored by qRT-PCR this time are: lsrB (Gene ID: 94541) and lsrC (Gene ID: 946105) (lsr operon-related genes) and AI-2 synthetic protein luxS (Gene ID: 1254340); related virulence genes are: genes related to flagella synthesis, flhC (Gene ID: 1253445) and fliA (Gene ID: 1253477), related to the regulation of bacterial movement, fimD (Gene ID: 1252066), related to bacterial adhesion and invasion, sptP (Gene ID: 1254401), sopB (GeneID: 1252609), sopE (Gene ID: 1253374) and invH (Gene ID: 1254423). Figure 4 As shown in the results, 2-aminoquinoline inhibited the expression of LT2's QS-related genes, lsrB and lsrC, by 2.98-fold and 3.14-fold, respectively. It also inhibited the expression of the flagellar synthesis gene flhC and the invasion and adhesion-related genes invH and sopE by 3.21-fold, 5.31-fold, and 3.53-fold, respectively.

[0038] Example 5: Effect of 2-aminoquinoline on cell activity.

[0039] Experimental method: NCM460 cells were cultured with DMEM complete medium at a rate of 2×10 4 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated at 37°C in a humidified environment with 5% CO2 until the cells adhered. Cells were treated with different concentrations of 2-aminoquinoline and then analyzed for cell viability according to the instructions of the Cell Counting Kit-8 (CCK-8) purchased from TargetMOI. After 24 hours of treatment, 10 μL of CCK-8 was added to each well and incubated for 2 hours. All experiments were repeated three times. Wells without cells were used as blank groups, and the OD values ​​were measured using a microplate reader. 450nm The absorbance at was detected.

[0040] Experimental results: The effects of different concentrations of 2-aminoquinoline on cell viability are shown in the following table: Figure 5 As shown in the figure, 2-aminoquinoline had a significant effect on cell viability when the concentration was 1 mM and 2 mM, so when performing invasion and adhesion experiments, the concentration of 2-aminoquinoline was selected as 0.5 mM.

[0041] Example 6: Effects of 2-aminoquinoline on LT2 adhesion and invasion.

[0042] Experimental method: NCM460 cells were seeded in 12-well plates with DMEM complete medium at a density of 5×10 5 Cells were cultured at 4% RT for 2 h at 37°C and infected after culturing. 200 μL of activated LT2 was added to 20 ml of LB medium and incubated at 37°C on a shaker for 3 h. NCM460 cells were then infected at a multiplicity of infection (MOI) of 20:1. A control group without 2-aminoquinoline and experimental groups containing 2-aminoquinoline at final concentrations of 0.5 mM and 0.25 mM were also established. These cells were incubated at 37°C and 5% CO2 for 2 h. After incubation, the supernatant was discarded, the cells were washed three times with PBS, and lysed with 0.1% Triton X-100 at room temperature for 20 min. The suspension was serially diluted, and 100 μL of each dilution was inoculated onto solid LB medium. After incubation at 37°C for 16 h, bacterial counts were performed on the plates to determine the total number of bacteria infecting the cells: the number of adherent bacteria = the total number of bacteria infecting the cells minus the number of bacteria invading the cells.

[0043] In the invasion assay, the infection procedure was identical to the adhesion procedure. After infection, cells were washed with PBS and then incubated in DMEM containing 100 μg / mL gentamicin for 50 minutes to kill extracellular bacteria. The cells were then washed three times with PBS and lysed with 0.1% Triton X-100. The suspension was serially diluted, and 100 μL of each dilution was inoculated into LB solid medium. After incubation at 37°C for 16 hours, the total number of bacteria invading the cells was determined by plate count.

[0044] Experimental results: The results of 2-aminoquinoline on LT2 adhesion cells are as follows Figure 6 As shown in A, 0.25mM 2-aminoquinoline has an inhibitory effect of 57.63%, while 0.5mM 2-aminoquinoline has an inhibitory effect of 69.48% on cell adhesion. Figure 6 As shown in Figure B, 0.25 mM 2-aminoquinoline inhibited cell invasion by 36.89%, while 0.5 mM 2-aminoquinoline achieved an inhibitory effect of 54.19%. In summary, 2-aminoquinoline at a concentration of 0.5 mM was not toxic to cells, but it inhibited the invasion and adhesion of Salmonella typhimurium to cells, thereby inhibiting its pathogenicity. This demonstrates that 2-aminoquinoline can be used as a drug to inhibit Salmonella typhimurium.

[0045] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0046] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

Application of 1.2-aminoquinoline in the preparation of drugs for inhibiting Salmonella typhimurium.

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