Application of 2-aminoquinoline in preparing medicine for inhibiting salmonella typhimurium

Abstract

The invention discloses an application of 2-aminoquinoline in preparation of drugs for inhibiting salmonella typhimurium. 2-aminoquinoline is combined with LsrB protein to inhibit expression of QS-related genes and virulence genes and inhibit formation of a biofilm, so that the pathogenicity of salmonella typhimurium is reduced, and the application of 2-aminoquinoline in preparation of drugs for inhibiting salmonella typhimurium is realized. And the 2-aminoquinoline can inhibit invasion and adhesion of salmonella typhimurium to cells, so that the 2-aminoquinoline can be applied to preparation of salmonella typhimurium inhibiting drugs.

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 invade the human body through multiple pathways. S. typhimurium infections are commonly treated with antibiotic therapy, but the use of antibiotics will inevitably induce 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 drug resistance.

[0003] Quorum sensing (QS) is a communication process between bacteria that allows bacteria to sense information about bacterial density and regulate gene expression within the population accordingly through signal molecules called autoinducers (AIs) that bind to specific receptors. QS is used by bacterial pathogens to coordinate a variety of physiological processes, such as biofilm formation, regulation of virulence factor expression, bacterial motility, production of secondary metabolites, and interactions between other microorganisms. Therefore, the pathogenicity of pathogens can be reduced by inhibiting their QS system to avoid their 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 application of 2-aminoquinoline in preparing 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 and inhibits the formation of biofilm by combining 2-aminoquinoline with LsrB protein (PDB ID: 1TJY), thereby reducing the pathogenicity of Salmonella typhimurium. In addition, 2-aminoquinoline can inhibit the invasion and adhesion of Salmonella typhimurium to cells, so 2-aminoquinoline can be used as a drug to inhibit Salmonella typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of surface plasmon resonance (SPR) experiment of 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 The effect of 2-aminoquinoline on the biofilm formation of LT2 strain obtained by crystal violet staining method, A is the 96-well plate staining result, ***: p<0.001; ****: p<0.0001; ns: p>0.05, no significant difference; B is the result of optical microscope observation after adding 2-aminoquinoline.

[0013] Figure 4 Effect 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, where A is the effect of 2-aminoquinoline on cell adhesion, and B is the effect of 2-aminoquinoline on cell invasion. ***: p<0.001; ****: p<0.0001. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but 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., product number: FDB012467, 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] The culture conditions are LB medium, 37°C, 220rpm; the cells used are human normal intestinal epithelial cells NCM460 (commercially available), cultured in high-glucose DMEM medium (commercially available) containing 1% double antibody (100U / mL penicillin, 0.1mg / mL streptomycin) and 10% fetal bovine serum, that is, DMEM complete medium, and monolayer cultured at 37°C, 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: The purified LsrB protein (PDB ID: 1TJY) was fixed on a CM5 chip (commercial product) by amino coupling, and an SPR experiment was performed after the coupling was completed. The LsrB protein was used as the stationary phase, and different concentrations of 2-aminoquinoline (solvent: DMSO) were used as the mobile phase to flow through the chip at a speed of 30 μL / min. When 2-aminoquinoline binds to the LsrB protein, it will generate pressure on the chip, thereby obtaining a response value. In the saturated state before stopping the injection, the KD value (equilibrium dissociation constant) between the two is calculated. This value is often used to represent its binding affinity. The smaller the KD value, the greater the 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-aminoquinoline to LT2 was determined by the doubling dilution method. The highest concentration of 2-aminoquinoline solution was 40mM, which was diluted to 0.315mM with DMSO and co-cultured with LT2 at 37°C for 24h. The lowest concentration without bacterial growth was the MIC of the 2-aminoquinoline. Three parallels were set 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 carried out in a shaker at 37°C and 220 rpm for 24 hours. 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 LT2 strain. The experimental results show that the MIC of 2-aminoquinoline to LT2 is 10mM. The growth curve shows that 6mM, 4mM and 2mM concentrations of 2-aminoquinoline solution have no effect on the growth curve of LT2, so it is obtained that when 2-aminoquinoline is at a concentration of 2mM, it will inhibit its pathogenicity but will not make Salmonella typhimurium resistant.

[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, biofilm was studied using two methods: 96-well plate observation and optical microscope observation.

[0032] 96-well plate observation method: Pick a single LT2 colony and culture it overnight in a shaker at 37°C and 220rpm. 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 for 30 minutes in a dark place, discard the supernatant, wash 3 times with PBS and dry. Finally, add an equal volume of 95% ethanol, mix well by pipetting, and use an enzyme reader to measure the OD 595nm The absorbance at was detected.

[0033] Optical microscope 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, take out the cell slide and clean the remaining impurities on the cell slide with PBS. Add a 0.1% crystal violet solution to the cell slide and stain it for 30 minutes in the dark. Take out the stained cell slide, rinse it 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 inhibited the biofilm formation of LT2. Since it significantly inhibited the formation of biofilm at 2 mM, with an inhibitory effect of 20.42%, a microscopic observation was conducted on 2 mM 2-aminoquinoline. Figure 3 B. Results show 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 method: The overnight activated LT2 strain was inoculated into LB medium at a ratio of 1%, and 2-aminoquinoline was added at a final concentration of 2mM and cultured to the logarithmic phase. RNA was extracted according to the bacterial total RNA extraction kit of Tiangen, and then reverse transcribed using the reverse transcription kit of SparkJade, SPARKscript II RT Plus Kit, according to its instructions, and the concentration was measured with Nanodrop. qRT-PCR was performed on LightCycler 480Ⅱ, with a final reaction volume of 10μL, and the system and reaction procedures were carried out according to the instructions of SparkJade's 2×SYBR Green qPCR Mix.

[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's synthetic protein luxS (Gene ID: 1254340); the related virulence genes are: genes flhC (Gene ID: 1253445) and fliA (Gene ID: 1253477) related to flagella synthesis, fimD (Gene ID: 1252066) related to regulating bacterial movement, sptP (Gene ID: 1254401), sopB (GeneID: 1252609), sopE (Gene ID: 1253374) and invH (Gene ID: 1254423) related to bacterial adhesion and invasion. Figure 4 It can be seen that 2-aminoquinoline inhibited the expression of LT2's QS-related genes lsrB and lsrC, with the inhibitory effects reaching 2.98 times and 3.14 times, respectively. It also inhibited the expression of the flagellar synthesis gene flhC and the invasion and adhesion-related genes invH and sopE, with the inhibitory effects reaching 3.21 times, 5.31 times and 3.53 times, respectively.

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

[0039] Experimental method: NCM460 cells were cultured with DMEM complete medium at 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 to the wall. Cells were treated with different concentrations of 2-aminoquinoline, and then cell viability was analyzed 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 cultured for 2 hours. All experiments were repeated three times. The wells without cells were used as blank groups, and the OD values ​​were measured using an enzyme marker. 450nm The absorbance at was detected.

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

[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 / well, culture until the cells adhere to the wall before infection. Add 200μL of activated LT2 to 20ml LB medium, culture at 37℃ shaker for 3h, and infect NCM460 cells at a multiplicity of infection of 20:1. At the same time, set up a control group without adding 2-aminoquinoline and an experimental group containing 2-aminoquinoline with a final concentration of 0.5mM and 0.25mM, and culture for 2h at 37℃ and 5% CO2. After the culture is completed, the supernatant is discarded, washed with PBS three times, and lysed with 0.1% Triton X-100 at room temperature for 20min. Dilute the suspension step by step, take 100μL of each dilution and inoculate it in LB solid medium, culture at 37℃ for 16h, and count the bacteria on the plate to obtain the total number of bacteria infecting the cells. The number of adherent bacteria = the total number of bacteria infecting the cells - the number of bacteria invading the cells.

[0043] In the invasion experiment, the infection step is the same as the adhesion step. After the infection, the cells were washed with PBS, and then a DMEM solution containing 100 μg / ml gentamicin was added and incubated for 50 minutes to kill extracellular bacteria. Then, the cells were washed three times with PBS, and then washed and lysed with 0.1% Triton X-100. The suspension was diluted step by step, and 100 μL of each dilution was inoculated into LB solid medium. After culturing at 37°C for 16 hours, the plate bacteria were counted to obtain the total number of bacteria invading the cells.

[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, 0.25mM 2-aminoquinoline has an inhibitory effect of 36.89% on cell invasion, and 0.5mM 2-aminoquinoline has an inhibitory effect of 54.19%. In summary, 2-aminoquinoline will not be toxic to cells at a concentration of 0.5mM, but it will inhibit the invasion and adhesion of Salmonella typhimurium to cells, thereby inhibiting its pathogenicity, which proves 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 other 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 can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

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

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