Application of rutin as salmonella type III secretion system inhibitor

Abstract

The invention discloses a novel application of rutin in preparation of a salmonella type III secretion system (T3SS) inhibitor. By constructing a salmonella mouse infection model, the rutin is proved to be capable of remarkably improving the survival rate of infected mice, reducing the bacterial load of liver and spleen target organs and effectively relieving the tissue damage of intestinal tracts, livers and spleens. In-vitro tests show that rutin does not affect the growth of bacteria, and the mechanism of the rutin playing a protection role is that the expression of a salmonella T3SS virulence factor is specifically inhibited, so that the pathogenicity of salmonella is weakened. The invention discloses a potential application of rutin as a salmonella T3SS inhibitor, and provides a theoretical basis for development of related anti-infective drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of rutin in the preparation of a Salmonella type III secretion system inhibitor. Background Art

[0002] Salmonella is a common foodborne pathogen that can cause serious diseases such as gastroenteritis, typhoid fever, and sepsis in humans and animals. Veterinary clinics currently rely primarily on antibiotics to control Salmonella infections. While this has achieved some success, it has inevitably contributed to the emergence and spread of drug-resistant strains. While the development of new antibiotics and antimicrobial enhancers has alleviated some of the resistance challenges, such strategies still exert significant selective pressure on pathogens, making it difficult to fundamentally curb the continued development of resistance. Since July 2020, my country has explicitly banned the use of antibiotic additives in feed (except for traditional Chinese medicines). While this policy has promoted healthy farming, it has also posed new challenges to the prevention and control of bacterial diseases, impacting farming profitability. Therefore, innovative anti-infection strategies are urgently needed to effectively address clinical multidrug-resistant bacterial infections.

[0003] Among the many new strategies, anti-virulence therapy is becoming a key area of research and development for anti-infective drugs because it targets bacterial pathogenicity rather than bacterial growth itself. This reduces the selective pressure on bacterial survival, significantly lowers the risk of drug resistance, and maximizes the maintenance of the host microbiome. Currently, several anti-virulence drugs are in the development or clinical trial stages internationally, providing valuable experience and technical references for this field.

[0004] Natural products have become an important source for antivirulence drug screening due to their diverse bioactivities and favorable safety profiles. Rutin, a natural flavonoid, is widely used in antioxidant, anti-inflammatory, and antibacterial applications. However, its role in the prevention and control of Salmonella infections is limited, and its mechanism of action remains unclear. Therefore, this study systematically evaluated the anti-infective efficacy and potential mechanisms of rutin using Salmonella infection models and in vitro experiments. Summary of the Invention

[0005] The CAS registration number of the rutin of the present invention is 153-18-4, and the molecular formula is C 27 H 30 O 16 , with a molecular weight of 610.52 g / mol. The structure is as follows:

[0006] This study established a Salmonella infection model in mice and demonstrated that rutin can enhance the survival rate of lethal Salmonella-infected mice and alleviate the pathological damage caused by Salmonella. Furthermore, TCA precipitation and immunoblotting revealed that rutin can target Salmonella virulence factors to exert its antibacterial activity.

[0007] The positive effects of the present invention are: Provided is the application of rutin in developing T3SS inhibitors, disclosing that rutin can inhibit the secretion and expression of the T3SS effector protein SipA and thus inhibit the function of the Salmonella T3SS. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Rutin enhances the survival of mice infected with lethal Salmonella; Figure 2 Histopathological analysis of rutin on target organs of Salmonella-infected mice; Figure 3 Effect of rutin on colonization of target organs of Salmonella-infected mice; Figure 4 Effects of rutin on the growth of Salmonella; Figure 5 Rutin inhibits the secretion of Salmonella effector proteins; Figure 6 Rutin inhibits the expression of Salmonella effector proteins. DETAILED DESCRIPTION

[0009] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0010] Protective effects of rutin on mice 1.1 Construction of Salmonella mouse model Female BALB / c mice aged 6 to 8 weeks (weighing 18-20 g) were selected and acclimated for 3 days, during which they were allowed free access to food and water. Three days before infection, mice were pretreated with 5 g / L streptomycin in water. Twelve hours before infection, mice were fasted and deprived of water. A lethal dose of Salmonella (1×10 7 CFU / mouse), and successfully established a mouse Salmonella enteritis infection model.

[0011] 1.2 Protection rate test Mice were randomly divided into four groups: a blank control group, an antibiotic group (florfenicol 100 mg / L), a Salmonella infection group, and a rutin treatment group (100 mg / kg). Following infection, mice in the treatment groups received oral rutin twice daily for four consecutive days. During this period, the mental state and survival of the mice in each group were closely observed and recorded for 10 days.

[0012] Conclusion: All the mice in the Salmonella infection group died on the 7th day, while the survival rate of the mice in the rutin treatment group reached 60% on the 7th day. This result shows that rutin has a significant protective effect on the Salmonella enteritis model mice (see Appendix Figure 1 ).

[0013] 1.3 Organ bacterial load and histopathological analysis After the experiment, the organ tissues of the euthanized mice were collected aseptically. A portion of the tissues were fixed, dehydrated, embedded, and prepared into paraffin sections. They were then stained with hematoxylin-eosin (HE) for histopathological analysis (see Appendix). Figure 2 The other part of the tissue was weighed and ground to prepare a tissue homogenate. The tissue homogenate was diluted with PBS in a gradient manner and spread on LB agar plates containing 40 μg / mL streptomycin. The plates were incubated at 37°C for 12 hours, and the bacterial load of each organ was recorded (see Appendix). Figure 3 ).

[0014] Conclusion: Rutin can significantly reduce the pathological damage of Salmonella to mouse organs and reduce the bacterial load in the organs.

[0015] 2. Growth Curve Test Transfer the overnight cultured Salmonella typhimurium SL1344 into new LB medium and wait for OD 600nm The concentration of rutin was adjusted to 0.1-0.3, and the tubes were divided into 50 mL centrifuge tubes. At the same time, different concentrations of rutin were added to make the final concentrations of 0, 4, 8, 16, 32 and 64 μg / mL. The tubes were cultured at 37 °C with shaking, and the OD values were measured every 1 hour. 600nm The absorbance value was used to draw a growth curve.

[0016] Conclusion: Rutin below 0-150 μg / mL has no significant effect on the growth of Salmonella SL1344 (see Appendix Figure 4 ).

[0017] 3. Minimum inhibitory concentration According to the standards published by the American Society for Clinical and Laboratory Standards (CLS), the minimum inhibitory concentration (MIC) of rutin against Salmonella was determined using a 96-well plate. First, 100 μL of LB liquid medium containing different concentrations of rutin was added to each well of the 96-well plate. The concentration of rutin ranged from 0 to 2048 μg / mL. Subsequently, 100 μL of bacterial solution (bacterial solution concentration of 1×10 8 CFUs / mL). After incubating the 96-well plate at 37°C for 24 hours, observe the color changes in each well. The lowest rutin concentration corresponding to the wells that did not change color is the minimum inhibitory concentration (MIC) of rutin.

[0018] Conclusion: The MIC of rutin against Salmonella typhimurium SL1344 is 1024 μg / mL 4. Effects of Rutin on the Secretion and Expression of Salmonella Effector Proteins Salmonella was first inoculated into LB liquid medium containing 0.3 M NaCl and cultured overnight at 37°C and 200 rpm. The next day, the culture solution was diluted at a ratio of 1:20 and adjusted to OD 600nm The value was approximately 0.2. Subsequently, the bacterial suspension was divided into several groups, namely a control group without drug addition and experimental groups treated with different concentrations of rutin. Each group of bacterial suspension was cultured at 37°C for 4 hours with shaking. After the incubation period, the supernatant and bacterial precipitate were separated by centrifugation. The supernatant was treated with trichloroacetic acid for 8 hours and then added with SDS-loading buffer (attached). Figure 5 ); and the bacterial precipitate was directly added with an appropriate amount of SDS-loading buffer and boiled for 8 minutes (attached Figure 6 Finally, SDS-PAGE and Western blot were used to analyze the expression of T3SS (type III secretion system) effector proteins.

[0019] Conclusion: Rutin pretreatment significantly reduced the secretion and expression levels of SipA, a key effector protein of the Salmonella T3SS. This result suggests that rutin can affect the function of the Salmonella T3SS by inhibiting the expression of T3SS effector proteins.

Claims

1. Application of rutin in the preparation of Salmonella type III secretion system inhibitors.

2. The use according to claim 1, characterized in that The rutin inhibits the function of the Salmonella type III secretion system by inhibiting the expression of Salmonella effector proteins.

3. The application according to claim 2, characterized in that The Salmonella effector protein includes at least SipA.

Images