Application of cordate houttuynia extract as salmonella type III secretion system inhibitor

Abstract

The invention provides a novel application of a cordate houttuynia extract as a salmonella type III secretion system (T3SS) inhibitor. Through construction of a mouse infection model and in-vitro experiments, the extract is proved to be capable of remarkably reducing the bacterial load of infected mouse liver and spleen tissues, relieving tissue pathological damage and reducing inflammatory cell infiltration. Mechanism research shows that the Houttuynia cordata extract can down-regulate the expression of T3SS key virulence protein SipA in a dose-dependent manner, interfere the function of a secretion system, and further block the secretion and expression of virulence factors. The potential application of the herba houttuyniae extract in the aspect of resisting salmonella toxicity is disclosed for the first time, and a development strategy and a technical basis of a T3SS inhibitor derived from a natural product are provided.

Description

Technical Field

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

[0002] Salmonella is a major foodborne pathogen that can be transmitted between humans and animals, causing diseases such as enteritis, typhoid fever, paratyphoid fever, and sepsis, which can be fatal in severe cases. Due to the diverse serotypes of Salmonella, vaccine prevention and control are difficult, and current veterinary clinical practice relies primarily on antibiotics for treatment. However, the widespread use of antibiotics has led to an increasingly serious problem of drug resistance, which has severely weakened their clinical efficacy. Furthermore, the long development cycles and high costs of new antibiotics and antimicrobial enhancers make it difficult to keep pace with the rapid evolution of drug-resistant strains. To address the challenges posed by drug resistance, countries around the world have implemented policies of "banning, restricting, and reducing antibiotic use." Therefore, the development of new anti-infection strategies is urgently needed.

[0003] In recent years, anti-virulence strategies have received increasing attention as an important direction in the development of new anti-infective drugs. This strategy effectively circumvents the resistance selection pressure brought by traditional antibiotics by interfering with the virulence factors of pathogens rather than directly killing bacteria. Studies have shown that the Salmonella type III secretion system (T3SS) plays a key role in its pathogenicity. T3SS-1, encoded by the Salmonella pathogenicity island 1 (SPI-1), is responsible for directly injecting a variety of effector proteins into host cells, mediating processes such as invasion and immune escape. Strains lacking T3SS have significantly reduced virulence, and therefore T3SS is considered an ideal target for the development of anti-virulence drugs.

[0004] Natural compounds are an important source of new drug discovery. Houttuynia cordata is a traditional Chinese medicine with both medicinal and edible properties. It is rich in volatile oils, flavonoids, phenolic acids and other active ingredients, and has good anti-inflammatory, immunomodulatory and intestinal protective effects. Although studies have shown that Houttuynia cordata extract ( Houttuynia cordata Houttuynia cordata extract has some antiviral and anti-inflammatory activity, but systematic research evidence for its in vitro and in vivo mechanisms of action and anti-infective efficacy against Salmonella infections is lacking. Therefore, this study systematically evaluated the anti-infective activity of Houttuynia cordata extract, targeting Salmonella, and further explored its potential mechanisms of action, aiming to provide theoretical support and technical expertise for the development of natural product-based anti-virulence drugs. Summary of the Invention

[0005] The Houttuynia cordata extract described in the present invention was purchased from Xi'an Xuquan Biological Co., Ltd. Its preparation process is as follows: fresh Houttuynia cordata is dried at a constant temperature and then crushed. Distilled water is added at a liquid-to-solid ratio of 100:1, and reflux extraction is performed at 90°C for 3 hours. The extract is then vacuum-dried and the dried product is sieved through an 80-mesh sieve to produce Houttuynia cordata extract powder.

[0006] This study established a Salmonella infection model in mice. The results showed that Houttuynia cordata extract could improve the survival rate of mice infected with a lethal dose of Salmonella and reduce tissue pathological damage. Trichloroacetic acid (TCA) precipitation and immunoblotting experiments further confirmed that Houttuynia cordata extract could target Salmonella virulence factors and inhibit its virulence.

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

[0008] Figure 1 Houttuynia cordata extract enhances the survival rate of mice infected with lethal Salmonella; Figure 2 Histopathological analysis of the effects of Houttuynia cordata extract on target organs of Salmonella-infected mice; Figure 3 Effect of Houttuynia cordata extract on colonization of target organs in Salmonella-infected mice; Figure 4 Effects of Houttuynia cordata extract on the growth of Salmonella; Figure 5 Effects of Houttuynia cordata extract on the secretion of Salmonella effector proteins; Figure 6 Effects of Houttuynia cordata extract on 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] 1. Protective effects of Houttuynia cordata on mice 1.1 Construction of Salmonella mouse model Female BALB / c mice aged 6 to 8 weeks, weighing 18 to 20 grams, were selected and acclimated for 3 days with free access to food and water. Streptomycin (5 g / L) was then added to the drinking water for 3 days of pretreatment. Food and water were withheld for 12 hours before infection. Salmonella (1 × 10 7 CFU / mouse), establish a mouse Salmonella enteritis infection model.

[0011] 1.2 Protection rate test Mice were randomly divided into three groups: a blank control group, a Salmonella infection group, and a Houttuynia cordata extract-treated group (100 mg / kg). Following infection, mice in the Houttuynia cordata extract-treated group were gavage-treated twice daily for four consecutive days. Simultaneously, mice in the blank control and Salmonella infection groups were given an equal volume of PBS. Throughout the experiment, the mental state and survival of the mice in each group were closely observed and recorded for eight days.

[0012] Conclusion: The survival rate of the healthy control group was 100%, the mortality rate of the infected group on the 6th day was 100%, and the survival rate of the houttuynia cordata treatment group on the 6th day was 50%, indicating that houttuynia cordata extract has a certain protective effect on the Salmonella enteritis model mice (Appendix Figure 1 ).

[0013] 1.3 Target organ bacterial load and histopathological analysis The liver and spleen of euthanized mice were collected aseptically and embedded in paraffin to prepare sections for hematoxylin-eosin (HE) staining (see attached). Figure 2 The other part of the tissue was weighed and ground to prepare a tissue homogenate, which was then graded diluted with PBS and spread on LB agar plates containing streptomycin (40 μg / mL). The colonies were counted after incubation at 37°C for 12 hours (see attached). Figure 3 ).

[0014] Conclusion: Houttuynia cordata extract can significantly reduce the bacterial load of Salmonella in mouse tissues and alleviate its pathological damage.

[0015] 2. Growth Curve Test Transfer the overnight culture of Salmonella typhimurium SL1344 into new LB medium and dilute to OD 600nm =0.1, and dispensed into 50 mL centrifuge tubes. Different concentrations of Houttuynia cordata extract were added to make the final concentrations of 0, 25, 50, 100 and 150 μg / mL. The cells were cultured continuously at 37 °C and 180 rpm / min. The OD values were measured every 1 hour. 600nm The absorbance values were recorded and plotted into a curve.

[0016] Conclusion: 0-150 μg / mL of Houttuynia cordata extract had 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 Association for Clinical and Laboratory Standards, the Houttuynia cordata extract was diluted in LB medium in series, and 100 μL was added to a 96-well plate. At the same time, 100 μL of bacterial solution (1×10 7After incubation at 37°C for 16-24 hours, observe the experimental results. The minimum inhibitory concentration (MIC) of Houttuynia cordata extract is the concentration without turbidity.

[0018] Conclusion: The MIC of Houttuynia cordata extract against Salmonella typhimurium SL1344 is greater than 2048 μg / mL.

[0019] 4. Effects of Houttuynia cordata extract on the secretion and expression of Salmonella effector proteins Salmonella was inoculated into LB liquid medium containing 0.3M NaCl and cultured overnight at 37°C and 200 rpm. The next day, the culture was expanded at a ratio of 1:20 to an OD600nm of approximately 0.3. Different concentrations of Houttuynia cordata treatment groups (0, 25, 50, 100, and 150 μg / mL) were set up and cultured at 37°C and 200 rpm for 5 hours. The supernatant after centrifugation was treated with trichloroacetic acid for 8 hours, then boiled in SDS-loading buffer, and the secretion of effector proteins was analyzed by SDS-PAGE and Western blotting (see Appendix). Figure 5 After centrifugation of the bacterial solution, take an appropriate amount of cells and add SDS-loading buffer and boil for 10 minutes (attached Figure 6 ).

[0020] Conclusion: After pretreatment with Houttuynia cordata, the secretion and expression of Salmonella T3SS invasion-related effector protein SipA were both decreased. This result indicates that Houttuynia cordata extract affects the function of Salmonella T3SS by inhibiting the expression of Salmonella type III secretion system effector proteins.

Claims

1. Application of Houttuynia cordata extract in the preparation of Salmonella type III secretion system inhibitors.

2. The use according to claim 1, characterized in that The houttuynia cordata extract 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.

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