Use of isorotenol in the preparation of inhibitors of klebsiella pneumoniae capsular

Abstract

Klebsiella pneumoniae can cause respiratory infection, digestive system infection, bacteremia and other pyogenic infection in cattle, pigs, deer, chicken and human, and is an important pathogenic bacterium in clinic. Capsule, as a basic virulence factor of Klebsiella pneumoniae, can promote infection through multiple pathways such as blocking complement binding and killing, hindering phagocytosis of immune cells, and is a potential target for anti-Klebsiella pneumoniae infection. In the present application, K2 type clinical isolated Klebsiella pneumoniae K7 strain is used as a research model strain, and a compound, isoferulic acid, which can significantly inhibit capsule synthesis, is obtained through glycuronate test. It is clarified that isoferulic acid can effectively protect mice by inhibiting capsule synthesis through electron microscope observation, centrifugal resistance and Klebsiella pneumoniae pneumonia treatment experiment in mice. As a capsule inhibitor, isoferulic acid will provide a new way and lead compound for anti-Klebsiella pneumoniae infection, and has extremely high development significance and application prospect under the current situation of high drug resistance level.

Description

Technical Field

[0001] This invention belongs to the field of medical and pharmaceutical technology, and relates to the pharmaceutical use of isoflavone in the preparation of Klebsiella pneumoniae capsular inhibitors. Background Technology

[0002] Klebsiella pneumoniae (K. pneumoniae) is a major zoonotic pathogen causing hospital-acquired infections in livestock and poultry such as cattle, pigs, deer, and chickens, as well as humans. It can cause respiratory infections, urinary tract infections, bacteremia, and other purulent infections. In recent years, multidrug-resistant Klebsiella pneumoniae has become increasingly prevalent, causing significant economic losses to the livestock industry, especially dairy farming. Currently, antibiotics remain the first-line treatment for bacterial infections; however, multidrug-resistant Klebsiella pneumoniae is resistant to almost all first-line antibiotics, and carbapenems, as the last line of defense, are gradually being overcome. Epidemiological surveys show that my country has become a high-incidence area for carbapenem-resistant Klebsiella pneumoniae (CRKP), with the CRKP detection rate surging from 3% in 2005 to 25%, highlighting the urgent clinical need for treatment strategies beyond antibiotics.

[0003] The capsule, primarily composed of polysaccharides and water, is a protective structure surrounding the surface of bacteria and is also known as the "K antigen." Numerous studies have shown that Klebsiella pneumoniae can utilize its capsule to survive under adverse conditions such as dryness, extreme acidity, and starvation. After breaching natural barriers like the skin and mucous membranes and invading the host, it promotes infection through the following mechanisms: ① inhibiting phagocytosis and opsonization by immune cells; ② blocking the bactericidal effects of antimicrobial peptides such as defensins and lactoferrin by binding to molecules distal to the outer membrane; ③ blocking the interaction between complement components and the bacterial membrane, thereby preventing complement lysis and opsonization; ④ reducing reactive oxygen species (ROS), IL-8, IL-6, and TNF-α by assisting in the activation of the NOD-dependent pathway and shielding LPS from recognition by immune cell receptors, thus preventing explosive activation of the immune response. The capsule, as the most basic virulence factor, is crucial for the pathogenesis of Klebsiella pneumoniae. Screening for inhibitors targeting the capsule will provide new pathways and lead compounds for combating Klebsiella pneumoniae infection. Given the persistently high and even rising levels of drug resistance, this is of great significance for the prevention and control of Klebsiella pneumoniae infection.

[0004] Isoferric acid is a phenolic acid compound widely found in plants such as Salvia miltiorrhiza and Cimicifuga foetida. Its chemical structural formula is shown in the appendix. Figure 1 Modern pharmacological studies have shown that it possesses pharmacological activities such as antidiabetic, anti-influenza virus, and enhancement of β-endorphin secretion. Currently, there are no reports, either domestically or internationally, on the use of isoflavone in the preparation of Klebsiella pneumoniae capsular inhibitors. Summary of the Invention

[0005] The purpose of this invention is to propose the medical use of isoflavonic acid in the preparation of capsular inhibitors, providing a novel strategy and lead compound for the prevention and treatment of Klebsiella pneumoniae infection that is independent of antibiotic effects.

[0006] This invention uses K2-type clinically isolated Klebsiella pneumoniae K7 strain as the research model strain. Through uronic acid assay, isoflavonic acid, a compound that can significantly inhibit capsule synthesis at concentrations that do not affect bacterial growth, was obtained. It can significantly reduce capsule thickness and effectively weaken capsule-mediated centrifugation resistance. The mouse Klebsiella pneumoniae pneumonia treatment experiment confirmed that isoflavonic acid can effectively protect mice by inhibiting capsule synthesis. Attached Figure Description

[0007] Figure 1 : The chemical structural formula of isoflavonic acid.

[0008] Figure 2 Isoflavic acid does not affect the growth of Klebsiella pneumoniae within a certain concentration range (horizontal axis represents time, vertical axis represents OD). 600 (Measured value).

[0009] Figure 3 Isoflavic acid dose-dependently reduced the content of capsular polysaccharides on the surface of Klebsiella pneumoniae (horizontal axis represents isoflavic acid concentration, vertical axis represents uronic acid content).

[0010] Figure 4 Isoflavonic acid inhibits the capsule thickness of Klebsiella pneumoniae.

[0011] Figure 5 Isoflavonic acid dose-dependently weakens capsule-mediated centrifugation resistance (horizontal axis represents isoflavonic acid concentration, vertical axis represents OD of the supernatant after centrifugation). 600 Measured values ​​and initial bacterial culture OD 600 (Ratio of measured values).

[0012] Figure 6 The protective effect of isoflavonic acid in a mouse model of Klebsiella pneumoniae pneumonia. Detailed Implementation

[0013] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention.

[0014] Example 1

[0015] Isoflavonic acid can be used as a capsular inhibitor of Klebsiella pneumoniae in any pharmaceutically acceptable carrier.

[0016] Example 2

[0017] Isoflavonic acid can be used as a capsular inhibitor in the preparation of drugs for treating Klebsiella pneumoniae infection.

[0018] Experimental Example 1

[0019] Determination of the minimum inhibition concentration (MIC) of isoflavone against Klebsiella pneumoniae

[0020] The MIC (Minimum Inhibitory Concentration) value of isoflavone against Klebsiella pneumoniae was determined according to the standard dilution method for minimum inhibitory concentration (MIC) determination published by the Clinical and Laboratory Standards Institute (CSIS). The OD (Displacement Limiting Factor) of overnight K7 bacterial culture was measured using LB medium. 600 Adjust to 0.1 and set aside. Serially dilute isoflavone in 1.5 ml centrifuge tubes with LB medium to achieve final concentrations of 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml, 128 μg / ml, 256 μg / ml, and 512 μg / ml. Add 200 μl of medium containing different concentrations of isoflavone to a 96-well plate, then add 10 μl of the adjusted bacterial suspension to each well (to achieve an inoculum size of 5 × 10⁻⁶). 5 (CFU / ml). After mixing by shaking on a shaker, the 96-well plate was incubated at 37°C in a 5% CO2 incubator. Bacterial growth was observed on the 96-well plate under suitable light the following day. The concentration of the compound that visually inhibits bacterial growth was determined as the MIC value of that compound against Klebsiella pneumoniae.

[0021] Conclusion: The MIC value of isoflavonic acid against Klebsiella pneumoniae is greater than 512 μg / ml.

[0022] Experimental Example 2

[0023] Growth curve determination

[0024] The K7 bacterial culture that had been cultured overnight was diluted 1:100 and then transferred to fresh LB medium. OD was then calculated. 600 When the bacterial culture concentration reached approximately 0.3, it was evenly distributed into five 50ml Erlenmeyer flasks (approximately 20ml per flask), and different concentrations (0, 4, 8, 16, 32, and 64 μg / ml) of isoflavonic acid were added to each flask. The culturing was continued at 37℃ and 180 rpm, and the OD of each sample was measured every 1 hour. 600 Until it reaches a plateau in growth.

[0025] Conclusion: Isoferric acid did not affect the growth of Klebsiella pneumoniae within the tested range (see Appendix). Figure 2 .

[0026] Experimental Example 3

[0027] Glucuronic acid analysis experiment

[0028] The K7 bacterial culture, after being cultured overnight, was expanded at a ratio of 1:50 to 2 ml of fresh LB medium containing different concentrations of isoflavonic acid and cultured for another 4 hours. After 4 hours, 500 μl of the bacterial culture was mixed thoroughly with 100 μl of 1% zwitergent (w / v, 100 mM citric acid) and incubated at 50°C for 20 minutes. The mixture was then centrifuged at 12000 × g for 5 minutes, and 300 μl of the supernatant was mixed with 1.2 ml of 100% ethanol and incubated at 4°C for 20 minutes to precipitate. The mixture was centrifuged again, and the supernatant was discarded. After the precipitate dried, it was dissolved in distilled water, and sodium tetraborate-concentrated sulfuric acid was added and vortexed. The mixture was boiled for 5 minutes and then cooled on ice. Finally, 0.15% 3-phenylphenol (w / v, 0.5% NaOH) was added, and the absorbance at 520 nm was measured. The uronic acid content was calculated based on a standard curve prepared using galactoside.

[0029] Conclusion: Isoferric acid dose-dependently reduced the content of capsular polysaccharides on the surface of Klebsiella pneumoniae, achieving a statistically significant difference at 16 μg / ml (see Appendix). Figure 2 .

[0030] Test Example 4

[0031] Transmission electron microscopy observation of the capsule structure of Klebsiella pneumoniae

[0032] Klebsiella pneumoniae was co-cultured with 32 μg / ml isoflavic acid until OD200. 600 = Approximately 0.6, take 1 ml, centrifuge to collect bacterial clumps, wash twice with sterile PBS, add 1 ml of 4% glutaraldehyde solution and fix overnight at 4℃; the next day, embed the sample in LR white resin, prepare ultrathin sections, and negatively stain with 2% uranyl acetate aqueous solution, and use transmission electron microscopy to detect capsule thickness.

[0033] Conclusion: Treatment with 32 μg / ml isoflavic acid significantly reduced the thickness of the capsule structure on the surface of Klebsiella pneumoniae (see Appendix). Figure 4 .

[0034] Experimental Example 5

[0035] Klebsiella pneumoniae centrifugation resistance test

[0036] Klebsiella pneumoniae was co-cultured with different concentrations (0, 4, 8, 16, 32, 32 μg / ml) of isoflavonic acid for 4 h. 1 ml of culture was centrifuged at 1000 × g for 5 min, the supernatant was collected, and the OD was measured using a UV spectrophotometer. 600 Statistical analysis of OD in the supernatant after centrifugation 600 Compared with the initial OD of bacterial culture 600 The ratio of .

[0037] Conclusion: In the solvent control group, Klebsiella pneumoniae was difficult to precipitate by centrifugation due to its high capsule content. Isoferric acid treatment dose-dependently weakened the capsule-mediated centrifugation resistance, achieving a significant difference at 4 μg / ml (see Appendix). Figure 5 .

[0038] Experimental Example 6

[0039] Establish a mouse model of Klebsiella pneumoniae pneumonia to evaluate the in vivo therapeutic effect of isoflavone.

[0040] 1×10 7 CFU K7 Klebsiella pneumoniae was used to infect 6-8 week old female C57BL / 6 mice via nasal drops. One hour after infection, mice were treated with isoflavonic acid (50 mg / kg) via subcutaneous injection every 8 hours for 3 consecutive days. The number of survivors in each group was observed and recorded at each time point, and a survival curve was plotted after 5 days.

[0041] Conclusion: 1×10 7 K7 infection resulted in the death of all mice in the solvent control group within 72 hours, while treatment with 50 mg / kg isoflavic acid increased the survival rate of mice with Klebsiella pneumoniae pneumonia to 50.0% (see attached image). Figure 6 .

Claims

1. Isoferric acid is used as a capsular inhibitor in the preparation of drugs for the treatment of Klebsiella pneumoniae infection.

Images