Application of osmundone in preparation of medicine for treating methicillin-resistant staphylococcus aureus infectious pneumonia

Abstract

The invention discloses an application of osmundone in preparation of a medicine for treating methicillin-resistant staphylococcus aureus infectious pneumonia. Experimental results of an MRSA mouse pneumonia infection model show that osmundone can significantly reduce the bacterial load in lung tissues, alleviate excessive inflammatory response caused by infection and effectively improve the survival rate of mice. In addition, the invention discloses that osmundone reduces the adhesion and invasion ability of bacteria, reduces the toxicity of the bacteria and plays an antibacterial role by inhibiting the protein activity of staphylococcus aureus Sortase A (SrtA). Different from traditional antibiotics, osmundone does not apply survival pressure to bacteria, so that generation of drug resistance is not easily induced. On the basis, osmundone shows a good application prospect in the aspect of research and development of safe and effective MRSA infectious pneumonia treatment drugs.

Description

Technical Field

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

[0002] Staphylococcus aureus is a common Gram-positive bacterium that can cause a variety of infections. In recent years, with the abuse of antibiotics and the acceleration of bacterial evolution, methicillin-resistant Staphylococcus aureus (MRSA) has become a global public health threat. The emergence of MRSA is mainly attributed to the acquisition of resistance by bacteria carrying the mecA gene, which encodes an altered penicillin-binding protein (PBP2a) that allows the bacteria to survive in the presence of methicillin and other β-lactam antibiotics. This resistance mechanism not only makes MRSA stubbornly resistant to traditional antibiotic therapy, but also increases the difficulty of infection treatment.

[0003] Since the 1980s, the incidence of methicillin-resistant Staphylococcus aureus pneumonia (MRSA pneumonia) has continued to rise, especially cases caused by hospital-acquired and community-acquired strains have attracted widespread attention. Its clinical manifestations include fever, cough, shortness of breath and chest pain, which may progress to sepsis or multiple organ failure in severe cases. With the widespread use of antibiotics, MRSA infections are becoming increasingly resistant to existing drugs. Although new antimicrobial drugs such as linezolid and vancomycin have been developed in recent years, the problem of MRSA resistance has not been completely solved, and some strains have even shown resistance to these drugs, especially in cases of infection in medical institutions.

[0004] The problem of antibiotic resistance makes the treatment of MRSA pneumonia more complicated, leading to higher mortality and medical expenses. Therefore, there is an urgent need to develop therapeutic drugs with significant efficacy and high safety. In view of this, exploring compounds that can effectively inhibit the pathogenicity of pathogens has become an important direction in today's anti-infection treatment research.

[0005] In pneumonia caused by MRSA, the virulence factor Sortase A (SrtA) plays a key role. SrtA is a transpeptidase that is involved in a variety of important pathogenic processes, including anchoring and modifying proteins located on the cell wall. These proteins, especially adhesins, can help MRSA bind tightly to host cells and are one of the key steps for bacteria to achieve initial colonization in lung tissue and cause infection. In addition, SrtA can also anchor a variety of surface proteins, thereby interfering with the host's immune recognition, especially in the innate immune defense of the lungs, such as the role of phagocytes and the complement system. This process helps MRSA escape the immune system, allowing it to survive and spread in the host.

[0006] SrtA also significantly enhances the ability of bacteria to induce an overreaction of the host immune system by anchoring virulence factors (such as protein A, SpA), leading to increased release of inflammatory factors, which in turn causes extensive pathological damage to lung tissue and expansion of inflammation. This pathological process can cause severe damage to lung tissue and aggravate the severity of infection.

[0007] Studies have shown that inhibiting the activity of SrtA can significantly reduce the pathogenicity of MRSA, reduce its survival in the host and its ability to induce pathological inflammation. Therefore, SrtA is considered an important potential target for the treatment of MRSA infections (especially pneumonia). It is worth noting that targeted inhibition of SrtA does not directly kill bacteria or inhibit their growth. This non-bactericidal intervention strategy does not exert selection pressure on bacteria, thereby helping to reduce the risk of the emergence of drug-resistant strains.

[0008] The present invention discloses a natural compound derived from the medicinal fungus Phellinus linteus, namely, porphyrinone, whose chemical structure is as follows: Figure 1 As shown. Porphyrin is a phenolic compound that has been shown to have a variety of biological activities by pharmacological studies, including anti-tumor, anti-oxidative damage, anti-inflammatory neuroprotection and antibacterial activity. However, there are currently no reports on the use of porphyrin for the treatment of pneumonia caused by Staphylococcus aureus at home and abroad. Summary of the invention

[0009] The present invention reveals for the first time the potential application of purpurogenol in treating Staphylococcus aureus infection, especially in treating infectious diseases such as pneumonia caused by Staphylococcus aureus. By inhibiting Sortase A (SrtA), a key virulence factor of Staphylococcus aureus, purpurogenol can significantly reduce its pathogenicity, thereby enhancing the host immune system's ability to eliminate pathogens.

[0010] Specifically, the present invention discloses the use of porphyrin in the preparation of drugs for treating Staphylococcus aureus infectious diseases. Its antibacterial mechanism is mainly through targeted inhibition of SrtA, thereby weakening the colonization and invasion ability of the pathogen, providing a novel and potentially effective anti-infection treatment strategy.

[0011] Furthermore, the present invention discloses the use of porphyrin in the preparation of a medicine for treating pneumonia caused by Staphylococcus aureus infection.

[0012] Furthermore, the present invention discloses the use of porphyrin in the preparation of a medicine for treating methicillin-resistant Staphylococcus aureus pneumonia.

[0013] The invention discloses application of porphyrin in preparing a drug for inhibiting the sortase SrtA of Staphylococcus aureus.

[0014] In the present invention, porphyrin can be used as an active ingredient and a pharmaceutically acceptable excipient to prepare various pharmaceutically acceptable dosage forms, such as capsules, injections or tablets, which are well known to those skilled in the art, and the present invention is not limited thereto.

[0015] Compared with the prior art, the technical effects of the present invention are:

[0016] 1. The present invention aims to apply purpurogenol, a compound extracted from natural plants, to the treatment of Staphylococcus aureus infectious diseases, and for the first time reveals the new use of purpurogenol in the preparation of drugs for treating such diseases. The research of the present invention shows that purpurogenol can inhibit the key virulence factor Sortase A (SrtA) of Staphylococcus aureus, reduce the adhesion of pathogens to Fc fragments, significantly inhibit the formation of biofilms, reduce the anchoring of surface protein SpA, and reduce the invasive ability to human lung adenocarcinoma cells A549. In addition, the results of in vivo experiments show that purpurogenol can significantly improve the survival rate of mice with lethal MRSA-infected pneumonia.

[0017] 2. The present invention further clarifies the therapeutic effect of purpurogenol in the pneumonia animal model. The experimental results of the MRSA mouse pneumonia infection model show that purpurogenol can significantly reduce the bacterial load in the lung tissue, reduce the infiltration and aggregation of inflammatory cells, significantly reduce lung tissue damage, and effectively improve the survival rate of mice. These research results provide a scientific basis for purpurogenol as a new therapeutic drug for the treatment of Staphylococcus aureus pneumonia, and at the same time open up a new path for the development of new drugs and provide an important reference for the development of similar traditional Chinese medicines.

[0018] 3. In addition, the present invention reveals that purpurogenol reduces the adhesion and invasion ability of bacteria by inhibiting the activity of Staphylococcus aureus Sortase A (SrtA) protein, and exerts antibacterial effects by reducing its virulence, rather than directly inhibiting bacterial growth. Therefore, compared with traditional antibiotic treatment, purpurogenol does not exert selection pressure on pathogens, significantly reducing the risk of drug resistance. Based on this, purpurogenol shows good application prospects in the development of safe and effective MRSA infectious pneumonia treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0020] Figure 1 is the chemical structural formula of porphyrone;

[0021] Figure 2This is a bar graph showing the in vitro inhibitory effect of porphyrin on Staphylococcus aureus USA300 SrtA;

[0022] Figure 3 This is a curve diagram showing the effect of porphyrin on the growth of Staphylococcus aureus USA300;

[0023] Figure 4 is the minimum inhibitory concentration of porphyrin to Staphylococcus aureus;

[0024] Figure 5 The effect of porphyrin on the adhesion of Staphylococcus aureus;

[0025] Figure 6 The effect of porphyrin on the invasion of Staphylococcus aureus;

[0026] Figure 7 The appearance and section diagram of porphyrinone protecting lung tissue from Staphylococcus aureus pneumonia;

[0027] Figure 8 This is a curve diagram of the protection rate of purpurogenol against Staphylococcus aureus pneumonia in mice. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention.

[0029] The experimental methods in the following examples are all 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 the present invention was purchased from the American Type Culture Collection (ATCC BAA-1717), and is a highly pathogenic and drug-resistant Staphylococcus aureus strain originally discovered in the U.S. It is a representative strain of community-acquired MRSA (CA-MRSA) and is one of the main pathogens causing skin and soft tissue infections worldwide.

[0032] Staphylococcus aureus USA300 srtA deletion strain was purchased from.

[0033] The human lung epithelial cells A549 used in the present invention were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0034] The application of porphyrin provided by the present invention in the preparation of a drug for treating methicillin-resistant Staphylococcus aureus pneumonia is described in detail below in conjunction with the examples.

[0035] Example 1 Inhibitory effect of porphyrin on the virulence factor SrtA of Staphylococcus aureus

[0036] Using the existing technology, the expression bacteria Escherichia coli BL21 containing the pET28a-srtA vector was inoculated into LB medium and shaken at 37°C and 200 rpm overnight. The next day, it was re-inoculated into LB medium with kanamycin resistance at a ratio of 1:100 and cultured at 37°C and 200 rpm until OD 600 Reach 0.6-0.8. Take 1mL of bacterial solution as a pre-induction control. Ice bath for the rest for 30 minutes, add IPTG with a final concentration of 1mM at 16°C, 180rpm for induction overnight. The next day, take 1mL of bacterial solution as a post-induction control, and collect the bacteria by centrifugation (4000rpm, 30min). Resuspend the collected bacteria with protein buffer, use an ultrasonic disruptor to ultrasonically disrupt the resuspended bacteria, and use a centrifuge to collect the supernatant and precipitate after disruption (12000rpm, 1h). Finally, SDS-PAGE electrophoresis was used to detect the expression of proteins before and after induction, in the supernatant, and in the precipitate, and Ni-NTA affinity chromatography technology was used to separate and purify the SrtA protein with a His tag. In order to ensure that the purified protein is active, the experiment needs to be carried out at 4°C.

[0037] The activity of SrtA was determined by the fluorescence resonance energy transfer (FRET) method. The assay was performed in a 96-well black plate. The synthetic substrate peptide Abz-LPATG-Dap(Dnp)-NH2 was used as a substrate for internal quenching of fluorescence. The wells contained 10 μM purified SrtA protein, different concentrations of purpurogenol and reaction buffer in a final volume of 100 μL. The reaction was carried out at 37°C for 30 min. The substrate peptide was added and incubated at 37°C for another 20 min, and finally the fluorescence was read using a microplate reader (excitation wavelength 309 nm, emission wavelength 420 nm). Only buffer and substrate peptide were added as controls. The inhibition rate of the drug on SrtA activity was determined by 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.

[0038] The results are as follows Figure 2 As shown in the figure, purpurogenol has a significant inhibitory effect on the activity of SrtA and can reduce the fluorescence signal in a dose-dependent manner. By calculation, the half maximal inhibitory concentration (IC 50 ) value was 1.29μg / mL.

[0039] Example 2 Effect of porphyrin on the growth of Staphylococcus aureus USA300

[0040] To further evaluate the effect of chloranone on the growth of Staphylococcus aureus USA300, a USA300 growth experiment in the presence of 64 μg / mL chloranone was carried out using the prior art method. Overnight cultured Staphylococcus aureus USA300 was inoculated into BHI broth medium containing or not containing 64 μg / mL chloranone at a ratio of 1:100 and cultured with shaking at 37°C for 24 hours. A culture medium containing only DMSO was used as a control. The optical density (OD) at 600 nm was measured at different time points using a UV spectrophotometer. 600 ) and draw the bacterial growth curve.

[0041] The results are as follows Figure 3 As shown in Figure 2, even when the concentration of purpurogenol reaches its IC 50 The growth rate of Staphylococcus aureus was still similar to that of the wild type (WT, USA300 treatment group) at 60 times of that of the wild type (WT, USA300 treatment group). This result shows that porphyrin not only has the potential to resist virulence factors, but also does not affect the growth of Staphylococcus aureus USA300, indicating that its application will not induce the development of drug resistance.

[0042] Example 3 Minimum inhibitory concentration of porphyrin to Staphylococcus aureus

[0043] The present invention determines the minimum inhibitory concentration (MIC) of porphyrin by the microbroth dilution method to evaluate its antibacterial activity. The specific steps are as follows: Staphylococcus aureus is cultured overnight in tryptone soy broth TSB. The next day, it is inoculated into CAMHB at a ratio of 1:100 and incubated at 37°C until an OD of 600 The bacterial suspension was then diluted into CAMHB at a ratio of 1:1000 and inoculated into a sterile 96-well plate. The concentration of purpurogenol in a sterile 96-well plate was adjusted to 8–1024 μg / mL by multiple dilution. Next, 2 μL of diluted resazurin dye (5 mg / mL) was added to each well, and the 96-well plate was incubated in a constant temperature incubator at 37°C for 16 h. Finally, the antibacterial effect of purpurogenol was evaluated by observing the color change (pink indicates live bacteria and blue-purple indicates reduced bacterial activity). In addition, we set up a positive control group containing only Staphylococcus aureus USA300 and a negative control group containing only CAMHB culture medium.

[0044] The results showed that after being treated with 512–1024 μg / mL of purpurogenol, Staphylococcus aureus showed a blue-purple color, indicating that the activity of Staphylococcus aureus decreased within this concentration range. This result indicates that the MIC of purpurogenol against Staphylococcus aureus is 512 μg / mL.

[0045] Example 4 Effect of porphyrin on adhesion of Staphylococcus aureus

[0046] In order to further evaluate the effect of purpurogenol on the adhesion of Staphylococcus aureus, 7 groups were designed in the present invention, namely, Staphylococcus aureus infection group (WT group), blank control group (control group), 64 μg / mL purpurogenol treatment group, 32 μg / mL purpurogenol treatment group, 16 μg / mL purpurogenol treatment group, 8 μg / mL purpurogenol treatment group and Staphylococcus aureus USA300srtA deletion strain infection group (ΔsrtA group).

[0047] Specifically, fibrinogen (Fn, 20 μg / mL) was added to a 96-well plate and incubated overnight at 4°C. Subsequently, the wells were washed with phosphate buffered saline (PBS) and incubated with 0.5% BSA for 2 h. Next, a mixture of USA300 bacteria incubated with different concentrations of purpurogenol (8–64 μg / mL) was added to a 96-well plate. After incubation at 37°C for 2 h, the wells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and then stained with crystal violet. The stained wells were washed with anhydrous ethanol, and the eluate was transferred to a new 96-well plate. The absorbance value at 595 nm was detected, and the adhesion rate was calculated using software.

[0048] The results are as follows Figure 5 As shown in the results, compared with the WT group, purpurogenol significantly reduced the adhesion of Staphylococcus aureus, and at the highest concentration (64 μg / mL), the adhesion was reduced by 18.15%. These findings indicate that purpurogenol disrupts the interaction with fibronectin, thereby inhibiting the ability of Staphylococcus aureus to adhere to host cells. The adhesion of the ΔsrtA group was similar to that of the control group, indicating that the virulence factor SrtA is a key virulence factor affecting the adhesion ability of Staphylococcus aureus, and purpurogenol may reduce the adhesion ability of Staphylococcus aureus by inhibiting the activity of SrtA.

[0049] Example 5 Effect of porphyrin on Staphylococcus aureus invasion

[0050] In order to further evaluate the effect of purpurogenol on the invasion of Staphylococcus aureus, the present invention designed 5 groups, namely, Staphylococcus aureus infection group (WT group), blank control group (control group), 32 μg / mL purpurogenol treatment group, 64 μg / mL purpurogenol treatment group and Staphylococcus aureus USA300srtA deletion strain infection group (ΔsrtA group).

[0051] Specifically, A549 cells were cultured at 1×10 5The cells were seeded at a density of 10 cells / well in a 24-well plate and cultured in F-12K medium containing fetal bovine serum, streptomycin and penicillin for 24 h. In addition, a suspension of Staphylococcus aureus containing different concentrations of purpurogenol (32–64 μg / mL) was shaken at 37°C and 220 rpm until the OD 600 When the concentration reached 1.0, the cells were collected by centrifugation and resuspended in F-12K medium. 5 CFU / mL concentration to infect A549 cells. Add 500 μL of bacterial suspension of the required concentration to each well of a 24-well plate and incubate at 37°C for 2 hours. After washing with PBS, incubate in F-12K medium containing gentamicin (300 μg / mL) for 1 hour to eliminate extracellular Staphylococcus aureus. Then wash twice with PBS, and finally lyse the cells with 5% (v / v) Triton X-100, spread the cell lysate on a blood plate and incubate at 37°C for bacterial count.

[0052] The results are as follows Figure 6 As shown in the figure, compared with the WT group, the number of Staphylococcus aureus invading A549 cells after purpurogenol treatment was greatly reduced, and the number of A549 cells invading ΔsrtA group was the lowest. This indicates that purpurogenol mainly reduces the ability of Staphylococcus aureus to invade host cells by inhibiting the virulence factor SrtA.

[0053] Example 6 Experimental Therapeutic Study of Staphylococcus aureus Pneumonia in Mice

[0054] 6.1 Establishment of the Staphylococcus aureus pneumonia model in mice

[0055] The experiment selected 6-8 week old SPF grade C57BL / 6J female mice as the pneumonia infection model. The mice were adapted to the standard laboratory environment for one week, during which they had free access to food and water. Staphylococcus aureus USA300 was diluted 1:100 in TSB and cultured until OD 600 nm = 1.0. After centrifugation, the bacterial pellet was washed and resuspended in PBS. The mice were anesthetized by inhalation of isoflurane gas and kept in an upright position. 30 μL (2×10 8 CFU) of bacterial suspension was dripped into the left nostril to induce natural inhalation, thereby establishing a Staphylococcus aureus pneumonia infection model. For the pathological histological observation group, 30 μL (1×10 8 CFU).

[0056] 6.2 Protective effect of porphyrin on mouse lung tissue

[0057] Methods: Forty C57BL / 6J mice were randomly divided into four groups: Staphylococcus aureus infection group (WT group), purpurogenol treatment group (purpurogenol group for short), blank control group (control group) and Staphylococcus aureus USA300 srtA deletion strain infection group (ΔsrtA group). Mice were anesthetized with isoflurane. Mice in the Staphylococcus aureus infection group and purpurogenol treatment group were anesthetized with 30 μL of 1×10 8 Mice were infected by intranasal drops of a suspension of CFU of Staphylococcus aureus, and the blank control group (control group) was given an equal amount of saline containing 5% DMSO. During the intranasal drops, the mice were held upright by the back to allow natural inhalation of the bacterial solution. After inhalation, the mice continued to remain upright for 30 seconds to allow full absorption of the bacterial solution, and then laid flat to allow them to wake up naturally. 2 hours later, mice infected with the bacteria were subcutaneously injected with 40 mg / kg of chloranone (chloranone group) or the same volume of DMSO (WT group), and the drug was administered once every 12 hours. 24 hours after administration, the mice were killed by cervical dislocation, and the left lung was removed under sterile operation in the clean bench to observe changes in the appearance of the lungs. The results showed that the lung tissue of the mice in the USA300 infection group was dark red and severely infected; while the lung tissue of the chloranone treatment group was pink with a spongy structure, and local infection was significantly reduced (such as Figure 7 shown).

[0058] Subsequently, the lung tissues were fixed with 10% formalin, and subjected to histopathological sectioning and hematoxylin-eosin (H&E) staining. Figure 7 ), the lung tissue of the WT group mice showed significant congestion, the integrity of the alveolar structure was destroyed, a large number of inflammatory cells infiltrated in the alveolar cavity, accompanied by obvious cavitation, the alveolar structure of the control group was intact, without obvious pathological changes, the alveolar structure of the ΔsrtA group was basically intact, with a small amount of inflammatory cell infiltration, the lung tissue of the purpurogenol treatment group was less congested, the inflammatory cell infiltration in the alveolar cavity was significantly reduced, and the alveolar structure was relatively intact, indicating that the lung inflammation of the mice was significantly alleviated after treatment.

[0059] 6.3 Experiment on the protection rate of purpurogenol against Staphylococcus aureus pneumonia in mice

[0060] Use 2×10 8 Mice were infected with 100 CFU of Staphylococcus aureus USA300, and an uninfected group (control group) was set up. Mice treated with normal saline after infection were used as negative controls (WT group), mice treated with 40 mg / kg of porphyrin after infection were used as treatment groups (porphyrin group), and Staphylococcus aureus USA300 srtA deletion strain infection group (ΔsrtA group).

[0061] The results are as follows Figure 8As shown, all the uninfected group (control group) survived. The mortality rate of the WT group was 70% at 12h. The mortality rate from 24–96h was 80%. After injection of 40mg / kg of porphyrin, the mortality rate of the porphyrin group was 20% at 12h and 40% at 24–96h. Death occurred in the ΔsrtA group at 24 hours, and the mortality rate from 24–96h was 10%.

[0062] Survival analysis showed that the chloranthone group showed a significant survival advantage, especially at the early time points after infection. These results indicate that chloranthone can increase the survival time of infected mice and protect mice from dying in the early stages of infection. The results showed that chloranthone treatment can significantly reduce the mortality rate of Staphylococcus aureus pneumonia in mice (P<0.05).

[0063] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. Application of porphyrin in the preparation of drugs for treating Staphylococcus aureus infectious diseases.

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 Staphylococcus aureus infectious disease is Staphylococcus aureus infectious pneumonia.

5. The use according to claim 4, characterized in that: The Staphylococcus aureus pneumonia is methicillin-resistant Staphylococcus aureus pneumonia.

6. Application of porphyrin in the preparation of drugs for inhibiting the sortase SrtA of Staphylococcus aureus.

7. A drug for treating Staphylococcus aureus infectious diseases, characterized in that: The medicine is prepared into a pharmaceutically acceptable dosage form by using porphyrin as an active ingredient and pharmaceutically acceptable auxiliary materials.

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

Images