Application of swertiflavin in preparation of medicine for treating infectious pneumonia caused by methicillin-resistant staphylococcus aureus

Abstract

The invention discloses application of swertiflavin in preparation of a medicine for treating infectious pneumonia caused by methicillin-resistant staphylococcus aureus, and relates to the technical field of medicine. It is found for the first time that swertiflavin can significantly reduce the toxicity of staphylococcus aureus by inhibiting the activity of ClpP protein in MRSA. In addition, the swertiflavin shows a remarkable protection effect in the treatment of the MRSA infectious pneumonia. Specifically, the swertiflavin not only can relieve the injury degree of lung tissues, but also can reduce infiltration and aggregation of inflammatory cells in the lung. The discovery shows that the swertiflavin has potential application value in the field of anti-infection treatment. The invention provides a novel treatment strategy, namely, swertiflavin is used as an active ingredient for preparing the medicine for treating the staphylococcus aureus infectious pneumonia.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of dapoxetine in preparing a medicine for treating infectious pneumonia caused by methicillin-resistant Staphylococcus aureus. Background Art

[0002] In the pharmaceutical field, methicillin-resistant Staphylococcus aureus (MRSA) pneumonia is an increasingly serious problem. MRSA is a type of Staphylococcus aureus that is resistant to β-lactam antibiotics, including methicillin. This resistance significantly increases the difficulty of treating infections and often leads to poor outcomes. Globally, the incidence of MRSA pneumonia continues to rise, particularly in healthcare settings, where MRSA has become a leading cause of hospital-acquired infections. However, the efficacy of traditional antimicrobial drugs in treating MRSA pneumonia has gradually declined, and the long-term and widespread use of antibiotics has further exacerbated the emergence of drug-resistant strains. Therefore, the development of new, safe, effective, and less resistant anti-MRSA drugs has become a top priority.

[0003] The pathogenicity of MRSA is closely linked to its virulence factors, particularly the casein hydrolase ClpP, secreted by Staphylococcus aureus. ClpP plays a key role in bacterial protein degradation, virulence regulation, and antibiotic resistance mechanisms. Studies have shown that mutations in the clpP gene can lead to multiple phenotypic changes in S. aureus, including increased sensitivity to environmental stress, abnormal cell morphology, cessation of cell division, and reduced virulence. Furthermore, inhibition of ClpP activity has been shown to significantly reduce MRSA pathogenicity, including its ability to survive in the host and induce pathological inflammation. Notably, targeted inhibition of ClpP does not directly inhibit the growth or kill S. aureus and, therefore, does not increase the risk of developing drug-resistant strains. This property is particularly important in the current context of increasing bacterial antibiotic resistance. With the increasingly urgent need for the development of new antimicrobial drugs, ClpP, due to its unique proteolytic function, has become a potential new target for antimicrobial drug development, offering new strategies and hope for anti-infective treatment.

[0004] In this context, the present invention aims to provide a new method for treating MRSA-infected pneumonia, namely using a drug with scutellarin as an active ingredient. Scutellarin, also known as scutellarin, scutellarin or scutellarin, has a chemical formula of C 22 H 22 O 11 , molecular weight is 462.4, molecular structure is as follows Figure 1As shown. It is a natural flavonoid compound extracted from medicinal plants such as Belamcanda chinensis, Iris truncatula, and Swertia japonica. This compound has been shown to have multiple pharmacological activities, including antioxidant, anti-diabetic, anti-depressant, anti-inflammatory, antiviral, and anti-radiation effects. However, although flavonoids have shown potential in the treatment of multiple diseases, there are currently no reports on the use of flavonoids for the treatment of MRSA pneumonia at home and abroad. This shows that further research and exploration is needed for the application of flavonoids in the field of anti-infection, especially for pneumonia caused by MRSA. Summary of the Invention

[0005] The present invention first discovered that dapoxetine can significantly reduce the virulence of Staphylococcus aureus by inhibiting the activity of ClpP protein in MRSA. In addition, the present invention's experiments demonstrated that dapoxetine exhibited a significant protective effect in the treatment of MRSA-infected pneumonia.

[0006] Specifically, in a first aspect, the present invention provides the use of dapoxetine in the preparation of a drug for resisting Staphylococcus aureus infection.

[0007] Furthermore, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0008] Furthermore, the methicillin-resistant Staphylococcus aureus is Staphylococcus aureus USA300.

[0009] The invention provides application of dapoxetine in the preparation of a drug for resisting Staphylococcus aureus infection. The drug is used for treating infectious pneumonia.

[0010] Furthermore, the drug is used to treat methicillin-resistant Staphylococcus aureus pneumonia. Dangyaoflavin not only alleviates lung tissue damage but also reduces the infiltration and accumulation of inflammatory cells in the lungs. These findings suggest that Dangyaoflavin has potential application value in the field of anti-infective therapy.

[0011] In a second aspect, the present invention also provides the use of flavin in the preparation of a drug for inhibiting the activity of Staphylococcus aureus casein hydrolase ClpP.

[0012] In a third aspect, the present invention also provides a drug for treating Staphylococcus aureus infectious diseases, characterized in that the drug reduces its toxicity by inhibiting the activity of casein hydrolase ClpP in MRSA, and the drug is prepared into a pharmaceutically acceptable dosage form by using dapoxetine as an active ingredient and pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the drug includes but is not limited to tablets, capsules and injections.

[0014] Compared with the prior art, the present invention has the following beneficial effects: First, the present invention validated the effectiveness of dansaflavin in a mouse MRSA pneumonia model. The experimental results demonstrated that dansaflavin exhibited significant therapeutic potential in this mouse MRSA pneumonia model, primarily manifested in two aspects: significantly improving the survival rate of MRSA-infected mice and effectively alleviating the inflammatory response in lung tissue induced by MRSA infection. These findings provide strong experimental evidence for dansaflavin as a potential anti-MRSA pneumonia drug.

[0015] Furthermore, the present invention reveals the mechanism of action of dapoxetine, which inhibits the activity of the casein hydrolase ClpP, a key virulence factor in MRSA, effectively reducing MRSA virulence and exerting a therapeutic effect. Importantly, compared to conventional antibiotic treatment, dapoxetine does not exert survival pressure on Staphylococcus aureus and therefore does not tend to promote the development and progression of bacterial resistance.

[0016] Therefore, the dapoxetine disclosed in the present invention has potential application value in developing safe and effective drugs for treating Staphylococcus aureus pneumonia, and is expected to provide new options for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the molecular structural formula of flavin.

[0019] Figure 2 The inhibitory effect of flavin on the in vitro activity of Staphylococcus aureus USA300 ClpP in Example 1 of the present invention is represented by a bar graph.

[0020] Figure 3 The results of Example 2 of the present invention, when flavin has no inhibitory effect on the growth of Staphylococcus aureus USA300, are represented by a bar graph.

[0021] Figure 4 In Example 3 of the present invention, the minimum inhibitory concentration (MIC) of flavin against Staphylococcus aureus USA300 is represented by a bar graph.

[0022] Figure 5 This is Example 4 of the present invention, which shows the effect of flavin on the viability of human lung epithelial cells (A549).

[0023] Figure 6In Example 5 of the present invention, flavin inhibits the invasion of Staphylococcus aureus USA300 into A549 cells.

[0024] Figure 7 This is Example 6 of the present invention, when flavin protects against lung tissue of mice with infectious pneumonia caused by Staphylococcus aureus USA300, including the lung appearance and pathological evaluation results.

[0025] Figure 8 This is a graph showing the experimental results of Example 6 of the present invention, showing that flavin treatment improves the survival rate of mice with infectious pneumonia caused by Staphylococcus aureus USA300.

[0026] Figure 9 Schematic diagram of the mechanism of action of dapoxetine.

[0027] The significance analysis symbols involved in each figure are: ns: no significant difference, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001. DETAILED DESCRIPTION

[0028] To help those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0030] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0031] The Staphylococcus aureus MRSA USA300 used in this invention was purchased from the American Type Culture Collection (CAS NO: ATCC BAA-1717). MRSA USA300 is a highly pathogenic and drug-resistant strain of Staphylococcus aureus originally discovered in the United States. It is a representative strain of community-acquired MRSA (CA-MRSA) and is one of the major pathogens causing skin and soft tissue infections worldwide. dclpP strains and BL21-pET28a- clpPAll strains were previously constructed and maintained in the laboratory. The construction methods were based on prior art literature (Jing Shisong. Study on the Inhibitory Effect and Mechanism of Myricetin on Staphylococcus aureus ClpP [D]. Jilin University, 2021. DOI: 10.27162 / d.cnki.gjlin.2021.003326.). The human lung epithelial A549 cells used in this invention were purchased from Wuhan Punosai Life Science Technology Co., Ltd. Aphrodisiac (CAS NO: 6991-10-2) was purchased from Chengdu Dester Biotechnology Co., Ltd.

[0032] The following describes in detail the application of the dapoxetine provided by the present invention in the preparation of a drug for treating infectious pneumonia caused by methicillin-resistant Staphylococcus aureus in conjunction with the examples.

[0033] Example 1 Determination of the half effective inhibitory concentration (IC 50 )value (1) Purification of ClpP protein BL21-pET28a- clpP The strain was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C overnight. The next day, the strain was re-inoculated into LB medium containing 50 μg / mL kanamycin resistance at a ratio of 1:100 and cultured at 37°C, 200 rpm until the OD 600 Reaching 0.8. Isopropylthiogalactopyranoside (IPTG) was added to the bacterial solution at a final concentration of 0.25 mM, and the cells were induced overnight at 16°C and 180 rpm. The next day, the cells were collected by centrifugation and resuspended in protein buffer (40 mM Tris, pH = 8.5). The resuspended cells were ultrasonically disrupted using an ultrasonic disruptor, and the supernatant was collected by centrifugation (10,000 rpm, 50 minutes). Finally, the His-tagged ClpP protein was isolated and purified using Ni-NTA affinity chromatography.

[0034] (2) Determination of ClpP activity ClpP activity was measured using the fluorescence resonance energy transfer (FRET) method. The assay was performed in 96-well black plates. The substrate peptide Suc-LY-AMC (0.5 mg / mL) was used as a substrate for internal fluorescence quenching. Each well contained 10 μM purified ClpP protein, various concentrations of sucralose (final concentrations: 0, 4, 8, 16, 21, 64, 128, and 256 μg / mL), and reaction buffer in a final volume of 99 μL. The reaction was incubated at 37°C for 30 minutes. Then, 1 μL of the substrate peptide was added, and the cells were incubated at 37°C for 30 minutes. Fluorescence signals were read using a microplate reader (excitation wavelength: 360 nm, emission wavelength: 465 nm). A control containing only the buffer and substrate peptide was used. The inhibitory percentage of ClpP protein activity by the drug was determined using the following formula: 100% × (CT) / C, where C is the fluorescence value of the untreated group and T is the fluorescence value of the experimental group.

[0035] The results are as follows Figure 2 As shown in the figure, flavin has a significant inhibitory effect on ClpP protein activity, and its fluorescence signal decreases in a dose-dependent manner. GraphPad Prism software was used for nonlinear regression analysis, and the experimental data were curve fitted to calculate the IC 50 The value is 32.91μg / mL.

[0036] Example 2 Evaluation of the growth of Staphylococcus aureus To evaluate the effect of 128 μg / mL safflower on the growth of MRSA (USA300 strain), overnight cultures of USA300 were added to sterile TSB medium with or without 128 μg / mL safflower at a ratio of 1:100 and grown at 37°C with shaking for 24 hours. The group supplemented with 128 μg / mL safflower served as the observation group (USA300 + safflower (128 μg / mL) group), while the group without safflower served as the control group (USA300 group). The absorbance at 600 nm was measured using a UV spectrophotometer at 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours, and growth curves were plotted.

[0037] The results are as follows Figure 3 As shown, the growth rate of USA300 treated with 128 μg / mL dapoxetine (USA300 + dapoxetine (128 μg / mL) group) was similar to that of the untreated USA300 group. These results indicate that 128 μg / mL dapoxetine does not affect the growth of USA300 and that treatment with 128 μg / mL dapoxetine is unlikely to induce drug resistance in Staphylococcus aureus.

[0038] Example 3 Determination of Minimum Inhibitory Concentration (MIC) The MIC of different concentrations of dapoxetine (final concentrations of 0 to 1024 μg / mL) against USA300 was determined by the broth microdilution method to evaluate its antibacterial activity. The specific steps are as follows: USA300 was revived in TSB medium. The next day, it was inoculated into CAMHB medium at a ratio of 1:1000 and incubated at 37°C until the OD 600 The bacterial suspension was diluted 1:100 in CAMHB medium and inoculated into a sterile 96-well plate. The 96-well plate was incubated in a 37°C incubator for 16 hours. A group of USA300 cells untreated with dapoxetine served as a negative control. The lowest concentration of dapoxetine that inhibited bacterial growth to a statistically significant level compared to the negative control was defined as its minimum inhibitory concentration (MIC).

[0039] The results are as follows Figure 4 As shown, the MIC value of flavin against Staphylococcus aureus USA300 was 512 μg / mL.

[0040] Example 4 Evaluation of the cytotoxicity of dapoxetine on A549 cells The MTT assay was used to evaluate the potential toxic effects of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (trade name: thiazolyl blue) on human lung epithelial cell line A549 (CAS NO: CL-0016, Wuhan Punosai Life Science Technology Co., Ltd.). This reagent is widely used to assess cell viability. A549 cells in logarithmic growth phase were cultured at 5×10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate and cultured for an additional 24 hours. Subsequently, the same concentration of anaphylactin (final concentration ranged from 0 to 128 μg / mL) was added to each well and cultured for an additional 24 hours. After the incubation period, the culture medium was removed, and 100 μL of culture medium and 50 μg of MTT solution were added to the wells, followed by an additional 4 hours of incubation. Following incubation, the resulting purple crystalline MTT metabolite was dissolved in 200 μL of dimethyl sulfoxide (DMSO), and cell viability was quantified by measuring absorbance at 570 nm.

[0041] The results are as follows Figure 5 As shown, there was no significant change in the viability of A549 cells treated with dapoxetine, which indicated that dapoxetine had no obvious toxic effect on A549 cells within the concentration range tested in the experiment.

[0042] Example 5: Pharmacoflavin inhibits MRSA invasion of A549 cells The experiment was divided into four groups: control group, USA300 infection group, flavin (128 μg / mL) treatment group, USA300- dclpPInfection group. Briefly, A549 cells were cultured at 1×10 5 The cells were carefully seeded at a density of 100 cells / well in a 24-well plate and cultured in F-12K medium containing fetal bovine serum, streptomycin and penicillin for 24 hours. The USA300 infection group and the dapoxetine treatment group were treated with 0 μg / mL and 128 μg / mL dapoxetine, respectively, and cultured at 37°C and 220 rpm until the OD of the bacterial solution reached 0. 600 The value reaches 1.0. USA300-dclpP The strain was cultured to OD 600 The value reached 1.0. After the culture was completed, the bacteria were collected by centrifugation and resuspended in F-12K medium. Except for the control group, 500 μL of F-12K medium was directly administered to the other groups. The corresponding 500 μL bacterial suspension (1×10 5 CFU / mL) was added to each well of a 24-well plate containing A549 cells and incubated at 37°C for 2 hours to allow bacterial invasion. After invasion, the cells were washed with PBS to remove uninvaded bacteria and then incubated in F-12K medium containing gentamicin (300 μg / mL) for 1 hour to eliminate extracellular S. aureus. The cells were then washed twice with PBS and lysed with 5% Triton X-100. The cell lysate was plated on TSB solid medium supplemented with 5% defibrinated rabbit blood and cultured overnight. The number of colonies formed was observed to assess the number of bacteria invading A549 cells, and the results were photographed and recorded.

[0043] The results are as follows Figure 6 As shown, no bacteria were detected in the A549 cells in the control group. In the untreated USA300 infection group, the number of bacteria in the A549 cells increased significantly, indicating that Staphylococcus aureus had successfully invaded the A549 cells. Compared with the untreated USA300 infection group, the number of bacteria in the A549 cells of the USA300 infection group treated with dapoxetine was significantly reduced. Moreover, compared with the untreated USA300 infection group, the USA300- dclpP The number of bacteria that invaded A549 cells was the lowest in the infection group. These results indicate that medicamentosaminidase can significantly inhibit the invasion of MRSA into A549 cells, and its effect is similar to that of inhibiting the virulence factor ClpP.

[0044] Example 6 Experimental Therapeutic Study of Gluconolactone on MRSA Infectious Pneumonia Six to eight-week-old SPF-grade C57BL / 6J male mice were selected to establish a pneumonia infection model. The specific procedures were as follows: the mice were anesthetized with isoflurane. 30 μL of the prepared bacterial solution was then inoculated into the left nasal cavity of the mouse. The mouse was kept upright for 30 seconds to allow for full inhalation of the bacterial solution. The mouse was then laid flat and allowed to wake up naturally. In the survival evaluation experiment, 30 μL of a resuspended Staphylococcus aureus solution (concentration of 2×10 8 CFU); in the pathological histological evaluation experiment, 30 μL of Staphylococcus aureus resuspension (concentration of 1×10 8 CFU). Two hours later, mice infected with the bacteria were subcutaneously injected with 50 mg / kg of dansaflavin or the same volume of DMSO twice a day.

[0045] (1) Pathological tissue examination The mice were randomly divided into four groups (control group, USA300 infection group, 50 mg / kg danshin group and USA300- dclpP In addition to the control group, which was given an equal volume of PBS, the USA300 infection group and the flavin (50 mg / kg) group were given OD 600 USA300 bacterial solution with a value of 1.0, USA300- dclpP Group OD 600 USA300 with a value of 1.0- dclpP Bacterial liquid (USA300- dclpP The strain was USA300 with the clpP gene knocked down. Twenty-four hours after treatment with dapoxetine, mice were euthanized. Left lung tissue was obtained, and changes in the appearance of the lung tissue were observed visually and recorded. The tissue was then fixed with 4% paraformaldehyde, and histopathological sections were prepared and stained with hematoxylin and eosin (H&E). Finally, the lung tissue was observed and images were captured using a light microscope for subsequent analysis.

[0046] (2) Survival rate experiment The grouping and treatment methods for this part of the experiment were the same as those for "Pathological Histological Examination." The mortality of mice was observed and recorded every 12 hours, and the survival rate was calculated after 96 hours.

[0047] The results of the protective effect of flavin on the lung tissue of mice infected with Staphylococcus aureus USA300 were as follows: Figure 7 As shown, the lungs of mice in the uninfected group (control group) appeared light pink and spongy, while the lungs of mice in the USA300-infected group appeared dark red and showed obvious congestion. Compared with the untreated infected group (USA300-infected group), the lung congestion of the fusaflavin-treated group (fusaflavin (50 mg / kg) group) was improved.

[0048] Pathological section results Figure 7 As shown, lung pathological sections of mice infected with USA300 showed a large accumulation of inflammatory cells in the alveolar cavity. Compared with the untreated infection group (USA300 infection group), the inflammatory cell infiltration in the alveolar cavity of mice treated with dapoxetine (dapoxetine (50 mg / kg) group) was significantly reduced. These results fully confirm that dapoxetine can alleviate lung damage in mice infected with USA300.

[0049] When flavin was used to treat Staphylococcus aureus USA300-induced pneumonia in mice, the results were as follows Figure 8 As shown, the survival rate of mice in the USA300 infection group was only 10% at 96 hours. dclpP The survival rate of mice in the infection group was 70%. Compared with the USA300 infection group, the survival rate of mice increased to 50% after 96 hours of treatment with 50 mg / kg of dansaflavin, which was similar to the USA300- dclpP These results fully confirm that dapoxetine can significantly improve the survival rate of mice infected with Staphylococcus aureus.

[0050] In summary, the present invention verifies the effectiveness of dapoxetine through a mouse MRSA infectious pneumonia model. The experimental results show that dapoxetine has significant therapeutic potential in the mouse MRSA infectious pneumonia model, which is mainly reflected in two aspects: one is to significantly improve the survival rate of MRSA-infected mice, and the other is to effectively reduce the inflammatory response of lung tissue induced by MRSA infection. These findings provide a strong experimental basis for dapoxetine as a potential anti-MRSA infectious pneumonia treatment drug. Furthermore, the present invention reveals the mechanism of action of dapoxetine, such as Figure 9 As shown, dapoxetine can inhibit the activity of casein hydrolase ClpP, a key virulence factor in MRSA, effectively reduce the virulence of MRSA, and exert a therapeutic effect. Dapoxetine can not only reduce the degree of damage to lung tissue, but also reduce the infiltration and aggregation of inflammatory cells in the lungs. Importantly, compared with conventional antibiotic treatment, dapoxetine does not put survival pressure on Staphylococcus aureus, so it does not tend to promote the occurrence and development of bacterial resistance. Therefore, the present invention provides a new treatment strategy, namely, using dapoxetine as an active ingredient for preparing drugs for treating Staphylococcus aureus pneumonia. The dapoxetine disclosed in the present invention has potential application value in the development of safe and effective drugs for the treatment of Staphylococcus aureus pneumonia, and is expected to provide new options for clinical treatment.

[0051] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. The application of flavin in the preparation of anti-Staphylococcus aureus infection drugs.

2. The use according to claim 1, characterized in that The Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

3. The use according to claim 2, characterized in that The methicillin-resistant Staphylococcus aureus is Staphylococcus aureus USA300.

4. The use according to claim 1, characterized in that The medicine is used for treating infectious pneumonia.

5. The use according to claim 4, characterized in that The medicine is used for treating methicillin-resistant Staphylococcus aureus infectious pneumonia.

6. The use of flavin in the preparation of drugs for inhibiting the activity of Staphylococcus aureus casein hydrolase ClpP.

7. A drug for treating Staphylococcus aureus infectious diseases, characterized in that: The drug reduces the toxicity of MRSA by inhibiting the activity of casein hydrolase ClpP in MRSA. The drug is prepared into a pharmaceutically acceptable dosage form by using dapoxetine as an active ingredient and pharmaceutically acceptable excipients.

8. The drug for treating Staphylococcus aureus infectious diseases according to claim 7, characterized in that: The dosage forms of the drug include but are not limited to tablets, capsules and injections.

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