Application of piceatannol in preparation of salmonella flagellum inhibitor

Abstract

The invention provides application of piceatannol in preparation of a salmonella flagellum inhibitor, and belongs to the technical field of biological medicines. Through analysis of salmonella sliding motion test, biofilm formation test, salmonella cell invasion test and other tests, it is found that piceatannol down-regulates flagellum-related gene transcription, weakens the sliding capacity, reduces biofilm formation and weakens the invasion capacity to host cells at non-inhibitory concentration; the invention relates to a salmonella flagellum inhibitor, particularly relates to a salmonella flagellum inhibitor, supports the development value of the salmonella flagellum inhibitor as an anti-virulence drug, provides a non-bactericidal treatment strategy capable of remarkably weakening bacterial pathogenicity, can be used as a potential lead compound or a composition therapy component for treating salmonella infection, and is applied to production and preparation of the salmonella flagellum inhibitor.

Description

Technical Field

[0001] This invention relates to novel medical uses of paclitaxel, particularly disclosing its application in the preparation of Salmonella flagella inhibitors, belonging to the field of biomedical technology. Background Technology

[0002] salmonella( Salmonella Salmonella (Salmonella) is a foodborne and enteric pathogen that can spread through contaminated food, water, and animal products, causing clinical symptoms such as acute gastroenteritis and sepsis, placing a heavy burden on public health and medical systems. In recent years, with the widespread use of antibiotics and antibiotic residues in the environment, multidrug-resistant (MDR) strains of Salmonella and their resistance mechanisms (including plasmid-mediated resistance genes, target mutations, enhanced efflux pumps, and biofilm-mediated reduced drug penetration) have significantly increased, posing challenges to traditional bactericidal therapies. Flagella are important virulence factors of Salmonella, conferring motility and chemotaxis to help them reach infection sites. They also play a crucial role in initial adhesion, biofilm formation, and invasion of host cells. Their main structural protein, FliC, can be recognized by host receptors such as TLR5, triggering an innate immune response and potentially leading to excessive inflammation and tissue damage. Because flagella play a vital role in the early stages of biofilm formation and colonization, inhibiting flagellar function is believed to simultaneously reduce adhesion, invasion, biofilm tolerance, and pathogenicity.

[0003] To address drug resistance, antiviral strategies are increasingly emerging as alternative or adjunctive therapies. The underlying principle is to target and weaken bacterial pathogenicity without directly killing the bacteria, thereby reducing selective pressure and the risk of resistance. Natural products, especially plant polyphenols, represent a potential library of antiviral molecules. Many natural compounds have shown the ability to modulate bacterial virulence or interfere with biofilms. Piceatannol, a natural polyphenol, has been reported to possess antioxidant and anti-inflammatory activities, but no studies have yet been published reporting its role as an antiviral candidate for inhibiting flagellar assembly. Summary of the Invention

[0004] This invention discloses the application of paclitaxel in the preparation of Salmonella flagella inhibitors, aiming to solve the problem of Salmonella's antiviral efficacy in inhibiting flagella assembly.

[0005] The molecular structure of the leucopicrin described in this invention is as follows: ; The chemical formula is C 14 H 12 O4 has a molecular weight of 244.24.

[0006] The purpose of this invention is to provide the application of paclitaxel in the preparation of Salmonella flagella inhibitors, which has a significant inhibitory effect on the motility and gliding ability of Salmonella.

[0007] Another object of the present invention is to provide paclitaxel for the preparation of a medicament for treating flagella-associated bacterial adhesion / biofilm-associated infections.

[0008] The paclitaxel described in this invention can be used as an active ingredient to inhibit flagellar assembly and to prepare pharmaceutical inhibitors. The carrier can be any pharmaceutically acceptable solvent, excipient, or formulation.

[0009] The positive effects of this invention are as follows: It discloses a new medical use for piracetam, for the preparation of Salmonella flagellation inhibitors and drugs for treating gastrointestinal infections caused by Salmonella; through analysis of Salmonella sliding motion assays, biofilm formation assays, and Salmonella invasion cell assays, it was found that piracetam, at non-bacterial inhibitory concentrations, regulates flagellation-related gene transcription, weakens sliding ability, reduces biofilm formation, and reduces the ability to invade host cells, supporting its development value as an antiviral drug. This intervention, which does not directly kill bacteria or inhibit their growth and is unlikely to threaten bacterial survival, is less likely to trigger strong evolutionary selection and can weaken the bacteria's defense or damage repair capabilities, making them easier for the host immune system to clear. This invention provides a non-bacterial but significantly weakens bacterial pathogenicity therapeutic strategy, which can serve as a potential lead compound or component of combination therapies for the treatment of Salmonella infections. Attached Figure Description

[0010] Figure 1 The effect of different concentrations of paclitaxel on the growth curve of Salmonella as provided in Example 1 of this invention; Figure 2 The effect of leucopicrin on bacterial motility provided in Example 2 of this invention; Figure 3 This is the transcriptional regulation of flagellar assembly-related genes by leucopicrin provided in Example 3 of the present invention; Figure 4 This is the quantitative result of paclitaxel on the formation of biofilms provided in Example 4 of the present invention; Figure 5 This is a quantitative result of the ability of paclitaxel to inhibit Salmonella adhesion, as provided in Example 5 of the present invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0012] The chemical formula of the leucopicrin used in the embodiments of this invention is C. 14 H 12 O4, with a molecular weight of 244.24, has the following molecular structure: .

[0013] The Salmonella used in this embodiment of the invention is Salmonella Typhimurium SL1344, which was donated by Professor Qiu Jiazhang of Jilin University.

[0014] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0015] Example 1: Salmonella growth curve determination: Salmonella typhimurium SL1344 was inoculated into LB medium and cultured overnight (37℃, 180 rpm). The next day, the overnight bacterial culture was expanded at a ratio of 1:100 into 20 mL of LB medium, and different concentrations of paclitaxel (0, 8, 16, 32, 64 μg / mL) were added. The culture was then continued at 37℃ with shaking at 180 rpm. The absorbance (OD) of the bacterial samples at 600 nm was measured every 1 hour. 600nm The absorbance values ​​were recorded for each treatment group at all time points until the bacteria reached the plateau phase, and growth curves were plotted. The results are shown below. Figure 1 As shown, within the measured concentration range, paclitaxel does not affect the growth of Salmonella (i.e., it does not directly kill bacteria).

[0016] Example 2, Salmonella sliding inhibition test: LB solid medium containing 0.3% agar was prepared. Paclitaxel was added to the cooled medium to final concentrations of 32 and 64 μg / mL, respectively. A positive control group (equal volume of DMSO solvent) was also established. Overnight cultured Salmonella typhimurium SL1344 was adjusted to OD using sterile PBS. 600 =0.5, take 5 μL of bacterial suspension and add it to the center of the drug-containing plate, incubate at 37℃ for 6 h, take pictures and observe, and measure the sliding diameter of the bacteria as they expand outwards. The size of the sliding circle reflects the sliding ability. The results are as follows. Figure 2 As shown, paclitaxel has an inhibitory effect on the sliding movement of Salmonella, as the diameter of the sliding ring gradually decreases with increasing concentration, and it significantly inhibits the sliding ability of bacteria compared with the solvent control.

[0017] Example 3: Effects of paclitaxel on Salmonella flagella assembly-related genes: Salmonella Typhimurium SL1344 was inoculated into 2 mL of LB medium and cultured overnight. The next day, it was expanded to fresh LB medium at a 1:20 ratio, with the addition of paclitaxel to a final concentration of 16 and 32 μg / mL, respectively. The culture was continued at 37℃ and 180 rpm for 4 h. Bacteria were collected by centrifugation at 12000 rpm for 5 min. Total RNA was extracted from Salmonella Typhimurium SL1344 using the Trizol method. 1 μg of RNA was reverse transcribed into cDNA, and detection was performed using SYBR Green RT-qPCR. flhD , fliA , flgM、fliT and fliC The relative expression levels of isogenes were measured, with 16S rRNA as an internal control, using 2... -ΔΔCt The method calculates the relative expression change. The results are as follows: Figure 3 As shown, paclitaxel treatment significantly downregulated the transcriptional expression of multiple flagella assembly-related genes, and this downregulated expression was concentration-dependent, suggesting that it could perturb the expression of flagellar master regulators and downstream structural genes at the molecular level.

[0018] Example 4, Bacterial Biofilm Experiment: Overnight culture of Salmonella Typhimurium SL1344 was transferred to TSB diluted 1 / 20 with sterile water, and the bacterial count was adjusted to 6 × 10⁻⁶. 7 CFUs / mL were added to achieve final concentrations of 16, 32, and 64 μg / mL of paclitaxel, respectively. The cells were then seeded into 24-well plates at a concentration of 1 mL per well and incubated at 30°C for 48 h. During this incubation period, the cells adhered to the bottom of the plate and formed a biofilm. The culture medium was then discarded, and each well was washed with PBS and stained with 0.1% crystal violet for 30 min. The biofilm biomass was then assessed by measuring the optical density at 590 nm after dissolving the stained wells in 30% acetic acid.

[0019] The results are as follows Figure 4 As shown, paclitaxel can reduce the formation of Salmonella biofilm at non-antibacterial concentrations, as evidenced by a decrease in the absorbance of crystal violet staining, suggesting that it can affect the early adhesion and biofilm establishment process.

[0020] Example 5, Adhesion Test: Caco-2 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics at 37°C and 5% CO2 until over 90% confluence was achieved, then injected at 4.0 × 10⁶ cells / year. 4 Cells / well were passaged into 24-well cell culture plates; Salmonella Typhimurium SL1344 was cultured in LB broth with shaking until the logarithmic growth phase (OD200). 600 After approximately 0.6%, wash and resuspend in PBS to adjust the concentration to 4 × 10⁻⁶. 6CFUs / mL; paclitaxel was added simultaneously to achieve final concentrations of 16, 32, and 64 μg / mL, respectively. The mixture was incubated at 37°C and 5% CO2 for 30 min. The mixture was discarded, and the cells were gently washed three times with PBS. Cell lysis was performed using 1 mL of PBS containing 0.1% Triton X-100 to determine the amount of adhering bacteria (CFU count was determined by plating onto XLD agar). Results were obtained as follows: Figure 5 As shown, paclitaxel can significantly reduce the adhesion ability of Salmonella to epithelial cells. The number of adherent bacteria in the treatment group was significantly reduced compared with the control group, and it showed a certain dose-dependent effect, indicating that it can indirectly weaken the bacterial adhesion phenotype by inhibiting flagella-related functions.

[0021] Statistical analysis was performed using GraphPad Prism 9.0. All experiments were independently repeated three times. Data are expressed as mean ± standard deviation (SD). The significance of differences between groups was assessed by one-way ANOVA, and the significance level was indicated by an asterisk (*) in the results. p < 0.05、** p < 0.01).

[0022] In summary, the embodiments of this invention demonstrate that paclitaxel has no significant effect on bacterial growth kinetics within the non-bacterial inhibition concentration range (i.e., it does not directly kill bacteria), but it can significantly inhibit the motility and gliding ability of Salmonella, and downregulate, at the molecular level, the growth kinetics of Salmonella. flhD , fliA and fliC It inhibits the expression of various flagellated control and structural genes. Meanwhile, piperidine treatment significantly reduces biofilm formation and also reduces bacterial adhesion levels in in vitro epithelial cell models. Therefore, it provides a new approach and potential lead compound for the prevention and treatment of Salmonella infection, and supports the inclusion of piperidine in combination strategies to reduce antibiotic use pressure and the risk of drug resistance. Based on the above conclusions, paclitaxel can be used as an active ingredient to inhibit flagellar assembly and to prepare pharmaceutical inhibitors. The carrier can be any pharmaceutically acceptable solvent, excipient, or formulation (oral, topical, injection, etc.). It can also be used as an anti-adhesion and anti-biofilm component in the local or food protection industry to reduce the adhesion and biofilm formation of Salmonella in the environment or food. Paclitaxel can also be used to treat flagellar-related bacterial adhesion / biofilm-related infections.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of piceatannol for the preparation of a Salmonella flagellum inhibitor, characterized in that: The piceol reduces the pathogenicity of the bacteria by reducing bacterial motility, impairing the ability to adhere to host cells and inhibiting biofilm formation.

2. Use according to claim 1, characterized in that: The Salmonella flagellum inhibitor is directed against Salmonella typhimurium SL1344.

3. The use of piceatannol as an active ingredient for the preparation of a medical inhibitor of flagellum assembly, characterized in that: The carrier can be any solvent, excipient, or formulation that is pharmaceutically acceptable.

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