Application of methylcatechol in preparing medicine for inhibiting salmonella typhimurium

Abstract

The invention discloses an application of methyl catechol in preparation of a medicine for inhibiting salmonella typhimurium, and the methyl catechol is 3-methyl catechol or 4-methyl catechol. 3-methylcatechol or 4-methylcatechol can be used for reducing the adhesion and invasion ability of salmonella typhimurium to human colorectal epithelial cells NCM460 and inhibiting the virulence of salmonella typhimurium.

Description

Technical Field

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

[0002] Salmonella enterica serovar Typhimurium is the most common foodborne pathogen that causes salmonellosis, including gastroenteritis, diarrhea, enteric fever, and focal infections. The main hosts of S. Typhimurium are humans, poultry, and livestock, and its serotypes are the main culprits causing the harm of Salmonella infection. S. Typhimurium can invade and proliferate host cells, including intestinal epithelial cells and macrophages. Salmonella infection is highly dependent on the activity of type III secretion system (T3SS)-related effector proteins encoded by Salmonella pathogenicity island 1 (SPI-1) and SPI-2-related genes. Conventional anti-Salmonella therapy includes antibiotic treatment, but the abuse of antibiotics and the spread of multidrug resistance have greatly troubled the prevention and treatment of Salmonella.

[0003] Quorum sensing (QS) is a communication process that regulates intercellular communication behaviors, including bioluminescence, virulence factor secretion, public metabolite production, and biofilm formation. Biofilm is a polymer matrix of proteins, extracellular DNA (eDNA), and polysaccharides produced by bacteria attached to biological or non-biological surfaces, thus forming a dense protective barrier that makes them more adaptable to harsh external environments.

[0004] Methyl catechol, especially 3-methyl catechol (also known as 3-methylcatechol, 3-MC) and 4-methyl catechol (also known as 4-methylcatechol, 4-MC), has antioxidant and potential enzyme inhibition activities. The application of 3-MC or 4-MC in inhibiting pathogens such as Salmonella has not been reported. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide application of methyl catechol in preparing a drug for inhibiting Salmonella typhimurium.

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

[0007] Application of methyl catechol in the preparation of drugs for inhibiting Salmonella typhimurium.

[0008] The methylcatechol is selected from 3-methylcatechol or 4-methylcatechol.

[0009] Advantages of the present invention:

[0010] Experiments have shown that 3-methylcatechol or 4-methylcatechol can reduce the adhesion and invasion ability of Salmonella typhimurium to human colorectal epithelial cells NCM460 and inhibit the virulence of Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Effects of different concentrations of 3-MC (a) and 4-MC (b) on the growth curve of Salmonella typhimurium LT2.

[0012] Figure 2 Effects of 1 mM 3-MC and 4-MC on biofilm formation of Salmonella typhimurium LT2. **: P < 0.01.

[0013] Figure 3 Effect of 1 mM 3-MC and 4-MC on the motility of S. typhimurium LT2.

[0014] (a) The figure shows the effect of 3-MC and 4-MC on the swarming motility of LT2.

[0015] (b) The figure shows the effect of 3-MC and 4-MC on the swimming motility of LT2.

[0016] Figure 4 Effects of 1 mM 3-MC (a) and 4-MC (b) on the virulence genes of LT2.

[0017] Figure 5 Effect of 1 mM 3-MC and 4-MC on the adhesion and invasion ability of LT2 in NCM460 cells.

[0018] (a) The figure shows the effect of 3-MC and 4-MC on the adhesion ability of LT2.

[0019] (b) The figure shows the effect of 3-MC and 4-MC on the invasion ability of LT2. ***: P<0.001; ****: P<0.0001. DETAILED DESCRIPTION

[0020] 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.

[0021] The 3-methylcatechol (CAS No: 488-17-5) involved in the following examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number M814462, referred to as 3-MC;

[0022] 4-Methylcatechol (CAS No: 452-86-8) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with the product number M797029, referred to as 4-MC.

[0023] 3-Methylcatechol and 4-methylcatechol were dissolved in dimethyl sulfoxide (DMSO) to prepare a 400 mM stock solution.

[0024] The bacterial strain used was the LT2 strain of Salmonella typhimurium, purchased from China Mall Beina Chuanglian Biotechnology Co., Ltd., hereinafter referred to as LT2. The culture conditions were LB broth medium, 37°C, and 220rpm shaking culture.

[0025] The cells used are human colorectal epithelial cells NCM460 (commercially available), cultured in a high-glucose DMEM medium (commercially available) containing 1% double-antibody (100 U / mL penicillin, 0.1 mg / mL streptomycin) and 10% fetal bovine serum, i.e., DMEM complete medium, and cultured in 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.

[0026] Embodiment 1:

[0027] Effects of different concentrations of 3-MC (a) or 4-MC (b) on the growth curve of Salmonella typhimurium LT2.

[0028] Experimental methods:

[0029] Effects of 3-MC or 4-MC on the growth curve of LT2.

[0030] The activated LT2 culture was inoculated into fresh LB medium at a 1% inoculation ratio. Six groups were selected and 3-MC was added to make the concentrations of 0mM (negative control), 1mM, 2mM, 3mM, 4mM, and 5mM, respectively. Three replicates were set for each sample concentration. The culture was shaken at 37°C and 220rpm for 24h, and the OD was measured every 1h. 600 value.

[0031] The activated LT2 culture was inoculated into fresh LB medium at a 1% inoculation ratio. Six groups were selected and 4-MC was added to make the concentrations of 0mM (negative control), 1mM, 2mM, 3mM, 4mM, and 5mM, respectively. Three replicates were set for each sample concentration. The culture was shaken at 37°C and 220rpm for 24h, and the OD was measured every 1h. 600 value.

[0032] Experimental results: Figure 1 As shown, Figure 1(a): The growth curve shows that LT2 grows slowly under the action of 1-5mM 3-MC, and 1mM 3-MC has almost no effect on the growth curve of LT2 in the late growth stage. Therefore, the lowest concentration of 1mM 3-MC was selected as the concentration for subsequent experiments.

[0033] Figure 1 (b): The growth curve shows that LT2 grows slowly under the action of 1-4mM 4-MC, and 1mM 4-MC has almost no effect on the growth curve of LT2 in the late growth stage. Therefore, the lowest concentration of 1mM 4-MC was selected as the concentration for subsequent experiments.

[0034] Embodiment 2:

[0035] Effect of 1 mM 3-MC or 4-MC on biofilm formation of Salmonella typhimurium LT2.

[0036] Experimental method: The activated LT2 bacterial solution cultured overnight was inoculated into fresh LB medium at a 1% inoculation ratio. The experimental group was added with 3-MC or 4-MC molecular stock solution to a final concentration of 1mM (the control group was not added with 3-MC or 4-MC). After pipetting to mix, the solution was added to a 96-well plate, 200μL per well. The 96-well plate was cultured in a 37°C incubator for 24h, the supernatant was discarded, and the plate was washed 3 times with PBS and then dried. A 1% crystal violet solution was added to the wells of the 96-well plate, and the plate was stained in a dark place for 30min. The plate was washed 3 times with PBS and then dried. Finally, 200μL of a 95% ethanol aqueous solution was added to each well. After pipetting to mix, the OD was detected with an enzyme-labeled instrument. 590 value.

[0037] Experimental results: Figure 2 The results showed that 3-MC and 4-MC inhibited the biofilm formation of LT2 by 84.2% and 82.91% respectively. The results showed that 1mM 3-MC and 4-MC inhibited the biofilm formation of Salmonella typhimurium LT2.

[0038] Embodiment 3:

[0039] Effect of 1 mM 3-MC or 4-MC on the motility of S. typhimurium LT2.

[0040] Experimental methods:

[0041] Swarming movement: Inoculate the activated LT2 bacterial solution from overnight culture into fresh LB medium at a 1% inoculation ratio, incubate at 37°C, 220rpm for 4h, collect the bacteria by centrifugation, wash three times with PBS solution, and then add an equal volume of PBS solution to obtain a suspension; prepare 30mL of 0.5% agar (w / v) LB solid medium, add 3-MC or 4-MC molecules to a final concentration of 1mM, and pour the plate. Pipette 5μL of the suspension and drop it vertically in the center of the plate. Make three parallels for each group. After standing for 10min, place it upright in a 37°C incubator for 10h, and observe the plate using a gel imaging instrument.

[0042] Swimming test: Inoculate the activated LT2 bacterial solution overnight into fresh LB medium at a 1% inoculation ratio, incubate at 37°C, 220rpm for 4h, collect the bacteria by centrifugation, wash three times with PBS solution, and then add an equal volume of PBS solution to obtain a suspension; prepare 30mL LB solid medium with 0.3% agar (w / v), add 3-MC or 4-MC molecules to a final concentration of 1mM, and pour the plate. Dip the suspension with a sterile white gun tip and insert it vertically in the center of the plate. Make three parallels for each group. After standing for 10min, place it upright in a 30°C incubator for 10h, and observe the plate using a gel imaging instrument.

[0043] Experimental results:

[0044] like Figure 3 As shown, (a) shows the effect of 3-MC and 4-MC molecules on the clustering movement of LT2, and (b) shows the effect of 3-MC and 4-MC molecules on the swimming mobility of LT2. The results show that 3-MC and 4-MC significantly inhibited the clustering movement and swimming mobility of Salmonella typhimurium LT2 and reduced the mobility of LT2 on the surface of the culture medium.

[0045] Embodiment 4:

[0046] Effect of 1 mM 3-MC or 4-MC on virulence genes of LT2.

[0047] Experimental method: The overnight cultured activated LT2 bacterial solution was inoculated into fresh LB medium at a 1% inoculation ratio, and 3-MC or 4-MC with a final concentration of 1 mM was added, and cultured for 4 hours until the logarithmic phase. LT2 RNA was extracted according to the bacterial total RNA extraction kit (commercially available) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and then reverse transcribed using SparkJade's reverse transcription kit (SPARKscript II RT Plus Kit) (commercially available) to obtain cDNA. qRT-PCR was performed on LightCycler 480Ⅱ, and the system and reaction procedures were performed according to the instructions of SparkJade's 2×SYBR Green qPCR Mix (commercially available). The primers for qRT-PCR were synthesized from Suzhou Jinweizhi Biotechnology Co., Ltd., and the primers for the corresponding genes are listed in Table 1.

[0048] Table 1

[0049]

[0050] Experimental results: Figure 4 The results showed that 1 mM 3-MC or 4-MC could inhibit the expression of virulence genes of LT2.

[0051] The virulence genes studied by qRT-PCR were sopB (Gene ID: 1252609), sopE (Gene ID: 1253374), sptP (Gene ID: 1254401), invH (Gene ID: 1254423) and 16S rRNA (Gene ID: 1251767), which are related to bacterial adhesion and invasion.

[0052] Figure 4 (a) The results showed that 3-MC inhibited the invasion and adhesion-related gene sopB of LT2 (2 2.3 times), sopE(2 2.75 times), sptP(2 4.20 times) and invH(2 2.52 times) expression. Figure 4 (b) The results showed that 4-MC inhibited the gene sopB (2 1.48 times), sopE(2 2.08 times), sptP(2 3.56 times) and invH(2 2.04 In conclusion, 3-MC and 4-MC inhibited the expression of virulence genes related to adhesion and invasion of Salmonella typhimurium LT2.

[0053] Embodiment 5:

[0054] Effect of 1 mM 3-MC or 4-MC on the adhesion and invasion ability of LT2 in NCM460 cells.

[0055] Experimental methods:

[0056] Adhesion assay: Activated NCM460 cells were seeded in 12-well plates with DMEM complete medium at a density of 5 × 10 5 200 μL of activated LT2 bacterial solution was added to 20 ml of LB medium, and after culturing at 37°C in a shaking incubator for 4 h, NCM460 cells were infected at an infection ratio of 20:1 (number of bacteria: number of cells). A blank group without LT2 bacterial solution, a control group without molecules, and an experimental group with 1 mM 3-MC or 4-MC were set up and incubated at 37°C, 5% CO 2 The cells were cultured for 1 hour under the same conditions. After the culture was completed, the supernatant was discarded, and the cells were washed with PBS for 3 times. Then, the cells were lysed by blowing with 0.1% Triton X-100 in PBS for 20 minutes. The suspension was diluted step by step, and 100 μL of each dilution was spread on the LB solid plate. After culturing at 37°C for 12 hours, the bacteria were counted to obtain the total number of bacteria infecting the cells. The number of adherent bacteria = the total number of bacteria infecting the cells - the total number of bacteria invading the cells.

[0057] Invasion assay: The infection procedure was the same as that of the adhesion assay. After infection, the cells were washed three times with PBS, and then DMEM medium 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 the cells were lysed by pipetting with a 0.1% Triton X-100 PBS solution. The suspension was diluted step by step, and 100 μL of each dilution was spread on an LB solid plate. After culturing at 37°C for 12 hours, the bacteria were counted to obtain the total number of bacteria invading the cells.

[0058] Experimental results: Effects of 3-MC or 4-MC molecules on the adhesion and invasion of Salmonella typhimurium LT2 in intestinal epithelial cells NCM460 Figure 5 shown. Figure 5 (a) shows that 1 mM 3-MC and 4-MC inhibited the adhesion of LT2 to NCM460 cells, and the inhibitory effects were 78.24% and 68.47%, respectively; Figure 5 (b) shows that 1mM 3-MC and 4-MC also inhibited the invasion of LT2 to NCM460 cells, with the inhibition effect of 51.19% and 47.16% respectively. In conclusion, 3-MC and 4-MC inhibited the adhesion and invasion of Salmonella typhimurium LT2 to epithelial cells NCM460.

Claims

1. The application of methyl catechol in the preparation of drugs for inhibiting Salmonella typhimurium.

2. The use according to claim 1, characterized in that The methyl catechol is 3-methyl catechol or 4-methyl catechol.

Images