Medical application of tectorigenin in preparation of salmonella fimbriae inhibitor

Abstract

The invention relates to medical application of tectorigenin in preparation of a salmonella fimbriae inhibitor. Tests such as a salmonella sliding motion inhibition test, a minimum inhibitory concentration determination test, a growth curve test, a test for inhibiting salmonella from being adhered to Caco-2 cells, a biofilm determination test and the like verify that the tectorigenin can inhibit bacterial pilus-mediated adhesion to host cells on the premise of not inhibiting bacterial growth; therefore, the pathogenicity of the salmonella is reduced. The tectorigenin mainly exists in tectoridae plants blackberry lily and iris tectorum, and has the characteristics of low toxicity, no residue or low residue and low drug resistance. Therefore, when the tectorigenin is used for preventing and treating salmonella infection, the drug selectivity can be improved, and the tectorigenin has a wide application prospect and is of great significance to the development of new drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medical pharmacy, and particularly relates to the medical use of irisin in preparing a Salmonella fimbriae inhibitor. Background Art

[0002] Salmonellosis is a common intestinal disease in poultry farming. It is a septicemia of chickens and turkeys caused by avian Salmonella infection. Salmonellosis is common throughout the world and is very harmful to the poultry industry. Avian Salmonella can infect chickens of any age. The clinical symptoms of infected chicks include anorexia, diarrhea, dehydration, weakness and high mortality. In addition, animal products such as meat and eggs are easily contaminated by Salmonella during processing. Humans are prone to illness after eating food contaminated by Salmonella. Clinical symptoms include diarrhea, enteric fever, and even systemic diseases. The infectivity and harmfulness of Salmonella make its treatment measures particularly important. The traditional method of treating Salmonella typhimurium is antibiotic therapy, but due to the abuse of antibiotics, multidrug-resistant Salmonella has shown extensive resistance to antibiotics such as ciprofloxacin and ceftriaxone, which makes the development of new treatment methods imminent.

[0003] Salmonella can survive, reproduce and spread in the host, which is inseparable from the assistance of many virulence factors, mainly including virulence island virulence factors, plasmid virulence factors, structural virulence factors (including pili and flagella), enterotoxin virulence factors, etc. Among them, pili play an important role in the adhesion process of Salmonella. By expressing a large number of pili, Salmonella can establish a variety of strategies for adhering to host tissues. In addition, pili are also directly related to the formation of biofilms. These characteristics of pili greatly enhance the pathogenicity of Salmonella to host animals.

[0004] Irisin is a natural flavonoid compound extracted from the Chinese medicinal material Belamcanda chinensis. It is one of the main active ingredients of Belamcanda chinensis and the main material basis for its pharmacological effects. Modern pharmacological studies have shown that irisin has multiple pharmacological effects, including anti-cancer, anti-diabetic, liver protection, anti-inflammatory, antioxidant, cardioprotective and neuroprotective. However, there is no research on the inhibition of Salmonella pili by irisin at home and abroad. Summary of the invention

[0005] The invention proposes the medical use of irisin in preparing a Salmonella fimbriae inhibitor, and provides a new choice for drugs for treating Salmonella infection.

[0006] The present invention uses Salmonella as a research model bacterium, and verifies through tests such as a sliding motion inhibition test of Salmonella, a minimum inhibitory concentration determination test, and a Salmonella adhesion cell inhibition test that irisin can significantly inhibit the sliding motion of Salmonella on a semi-solid culture medium without affecting bacterial growth, reduce the adhesion rate of Salmonella to Caco-2 cells, and inhibit the biofilm formation of Salmonella.

[0007] The molecular formula of irisin of the present invention is C 16 H 12 O6, molecular weight is 300.26. The molecular structure of irisin is as follows:

[0008] The positive effects of the present invention are: A new medical use of tectoriusin in preparing a Salmonella fimbriae inhibitor is provided, and it is disclosed that tectoriusin can inhibit the fimbriae-mediated adhesion ability to host cells, thereby reducing the pathogenicity of Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Irisin inhibits the sliding movement of Salmonella SL1344 on semi-LB solid plates; Figure 2 : Irisin does not inhibit the growth of Salmonella SL1344 within the effective concentration range; Figure 3 : Irisin has no toxic effect on Caco-2 cells within the effective concentration range; Figure 4 :Tectoriusin inhibits the adhesion of Salmonella SL1344 to Caco-2 cells; Figure 5 : Irisin inhibits the formation of biofilm of Salmonella SL1344; Figure 6 : Quantitative results of biofilm formation of Salmonella SL1344 after treatment with different concentrations of irisin. DETAILED DESCRIPTION

[0010] The present invention is further described by way of the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0011] Example 1 Irisin can be used as a Salmonella fimbriae inhibitor in any pharmaceutically acceptable carrier.

[0012] Example 2 Irisin can be used as a Salmonella fimbriae inhibitor for preparing drugs for treating infectious diseases.

[0013] Example 3 Irisin is used as a Salmonella fimbriae inhibitor to treat infectious diseases caused by bacteria.

[0014] Test Example 1 Salmonella gliding motility inhibition assay Take 10 mL of sterile LB medium (containing 0.5% agar and glucose), add irisin to the specified concentration, and pour into a sterile culture dish. Centrifuge (10000 rpm, 5 min) the overnight cultured SL1344 bacterial solution to collect the cells, wash three times with PBS and resuspend the cells to an OD of 600nm =0.1, 3 μL was aspirated and dropped to the center of the solid plate, and then placed in a 37°C constant temperature incubator for 12 hours. The sliding ability of each group of colonies was evaluated based on the sliding circle diameter. The larger the sliding circle, the stronger the sliding ability; conversely, the weaker the sliding ability.

[0015] Conclusion: Compared with the control group (without irisin treatment), treatment with different concentrations of irisin (16-64 μg / mL) can significantly inhibit the sliding of Salmonella on LB soft agar solid plates (see Appendix Figure 1 ).

[0016] Test Example 2 Minimum inhibitory concentration test The MIC value of irisin against Salmonella SL1344 was determined according to the standard dilution method of the minimum inhibitory concentration value determination test published by the American Clinical and Laboratory Standards Association. In a sterile 96-well plate, irisin was diluted in LB broth in multiple ratios to a final concentration of 512 μg / ml, 256 μg / ml, 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, and 2 μg / ml, and overnight cultured Salmonella SL1344 (adjusted to 5×10 5 CFU / ml). After oscillation and mixing, the mixture was placed in a 37°C constant temperature incubator for 20h-24h to observe the bacterial growth. The concentration of the compound that inhibited bacterial growth visible to the naked eye was determined as the MIC value of irisin against Salmonella.

[0017] Conclusion: The MIC value of irisin against Salmonella SL1344 is greater than 512 μg / ml.

[0018] Test Example 3 Growth curve of Salmonella SL1344 Salmonella SL1344 was inoculated into 2 mL LB medium and cultured overnight. The next day, the culture was expanded to 100 mL LB medium at a ratio of 1:100 and continued to be cultured until OD 600nm =0.3, the bacterial solution was divided equally into 5 conical flasks, and irisin was added to each flask to make the final concentrations of 16, 32, and 64 μg / mL, respectively. A DMSO control group was set up at 37°C and 180 rpm to continue culturing. The OD of the bacterial culture was measured at different time points. 600nm The values ​​were recorded until the bacteria grew to the plateau phase and a growth curve was drawn.

[0019] Conclusion: Within the effective concentration range (16-64 μg / mL), irisin has no effect on the growth of Salmonella SL1344 (see Appendix Figure 2 ).

[0020] Test Example 4 Cytotoxicity of irisin on Caco-2 cells Caco-2 cells were plated in 96-well culture plates (2×10 4 The cells were plated in 4% paraformaldehyde (P<0.05) and 1 mL of DMEM (containing 10% fetal bovine serum) containing different concentrations of irisin was added. After incubation in a 37°C constant temperature incubator for 6 h, the release of lactate dehydrogenase (LDH) was measured at a wavelength of 490 nm using a lactate dehydrogenase cytotoxicity assay kit and a microplate spectrophotometer to evaluate cell viability.

[0021] Conclusion: Irisin has no significant cytotoxic effect on Caco-2 cells within the effective concentration range (8-128 μg / ml) (see Appendix Figure 3 ).

[0022] Test Example 5 Irisin inhibits the adhesion of Salmonella SL1344 to Caco-2 cells Caco-2 cells were plated in 24-well culture plates (2×10 4 / well) and cultured in a 37℃ constant temperature cell culture incubator for 12h. The culture medium was replaced with DMEM (containing 10% fetal bovine serum) containing SL1344 (MOI=80) to infect the cells, and irisin was added to reach the specified concentration, and then placed in a 37℃ constant temperature incubator for incubation for 1.5h. Washed three times with PBS, 1mL of sterile distilled water was added to lyse the cells for 5min, and then diluted and plated, and the plates were placed in a 37℃ constant temperature incubator for 12h to count and statistically analyze the colonies.

[0023] Conclusion: Treatment with irisin (16-64 μg / mL) can significantly inhibit the adhesion of Salmonella SL1344 to Caco-2 cells (see Appendix Figure 4 ).

[0024] Test Example 6 Biofilm assay The overnight cultured SL1344 bacterial suspension was centrifuged (10000 rpm, 5 min) to collect the bacterial cells, washed three times with PBS and resuspended, and then the bacterial suspension was diluted to 1×10 7 CFU / mL, and irisin was added to the culture medium to reach the specified concentration, then added to a 24-well culture plate and cultured at 37°C for 24 hours. After removing the culture medium from each well, wash three times with PBS, and add 0.5% crystal violet to stain the biofilm for 30 minutes. After absorbing the excess crystal violet, wash three times with PBS, add 400μL 33% glacial acetic acid to each well, and then use a microplate reader to measure OD 570nm value.

[0025] Conclusion: Irisin (16-64 μg / mL) treatment can significantly inhibit the formation of Salmonella SL1344 biofilm (see Appendix Figure 5 -Attached Figure 6 ).

Claims

1. The medical use of irisin in the preparation of Salmonella fimbriae inhibitors.

2. The use according to claim 1, characterized in that The medical use refers to the biological function of tectoriusin in inhibiting the adhesion of Salmonella pili-mediated host cells.

3. The use according to claim 1, characterized in that The Salmonella fimbriae inhibitor can effectively inhibit the adhesion mediated by Salmonella fimbriae, thereby reducing the pathogenicity of Salmonella.

4. The use according to claim 1, characterized in that The application of the Salmonella fimbriae inhibitor tectorigenin in the preparation of a drug for treating Salmonella infection.

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