Application of fludarabine in treatment of methicillin-resistant staphylococcus aureus infection

Abstract

The invention discloses application of fludarabine in treatment of methicillin-resistant staphylococcus aureus infection, and in an MRSA infected mouse model, it is verified that fludarabine can improve the survival rate of MRSA infected mice, reduce the MRSA bacterial count of lungs, livers, spleens and kidneys and relieve the injury degree of lungs, livers, spleens and kidneys caused by MRSA infection; pSMalpha3 is found to be an important virulence factor of MRSA, is closely related to high pathogenicity of MRSA, and can induce death of macrophages in a concentration-dependent manner, and fludarabine can significantly reduce cytotoxicity caused by PSMalpha3.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of fludarabine in treating methicillin-resistant Staphylococcus aureus infection. Background Art

[0002] The emergence and spread of the "superbug" methicillin-resistant Staphylococcus aureus (MRSA) caused by the abuse of antibiotics around the world has brought great difficulties and challenges to clinical anti-infection treatment and has become a major public health issue that threatens human life and health. MRSA is the main pathogen of hospital-acquired and community-acquired infections, and is also an important cause of severe suppurative infections, pneumonia, endocarditis, sepsis and septic shock. According to the Centers for Disease Control and Prevention of the United States, about 20,000 people die from MRSA infection in the United States each year, and the mortality rate caused by MRSA infection ranks first among all clinically isolated drug-resistant bacteria. In my country, the problem of MRSA infection is also serious. Its clinical detection rate is higher than 30% all year round, and it is also the main pathogen causing death from clinical severe infection. To date, the number of deaths caused by MRSA infection worldwide has exceeded that of AIDS and hepatitis B, becoming the world's top three serious infectious diseases [1,2]. At the same time, MRSA is multidrug-resistant and can resist most of the commonly used antimicrobial drugs in clinical practice, making clinical treatment strategies for MRSA extremely scarce. What is even more serious is that in recent years, vancomycin, the "last line of defense" for treating MRSA infections, has been breached, and the emergence of vancomycin-resistant Staphylococcus aureus has forced humans into a desperate situation where there are no drugs available [3].

[0003] Phenol-soluble modulin α3 (PSMα3) is an extremely important virulence factor of MRSA. It can not only significantly increase the pathogenicity of MRSA, but also help MRSA attack host immune cells and achieve immune escape. Therefore, in-depth research on the mechanism of action of PSMα3 in the MRSA infection process and clarifying its molecular mechanism and key targets for regulating the body's immunity are expected to become an important breakthrough for further revealing the pathogenic mechanism of MRSA and finding drugs to control MRSA infection [4].

[0004] As research continues to deepen, researchers have found that the pathogenicity of bacteria is closely related to the virulence factors they secrete. The strategy of reducing bacterial virulence rather than directly killing bacteria can reduce bacterial pathogenicity and enhance the host's antibacterial immune response by selectively inhibiting the transmission of virulence factor effects, biofilm formation, immune escape, etc., while not exerting selective survival pressure on bacteria. It has the unique advantage of not inducing bacterial resistance. Therefore, discovering the key virulence factors of pathogenic bacteria, revealing their unique mechanism of action, and developing new anti-virulence drugs have become a new trend in the research of new antibacterial drugs that do not induce resistance, and are also a new direction for people to effectively control MRSA infections [5].

[0005] At present, the cost of research and development of antimicrobial drugs is increasing, and the research and development cycle is getting longer and longer. In the past decade, only a very small number of antimicrobial drugs have been proven to be effective against MRSA-resistant bacteria in vitro, and the toxicity and pharmacokinetic characteristics of the drugs are still unclear. What is more worrying is that the speed of emergence of resistant strains has far exceeded the speed of development of new drugs, and after the new antimicrobial drugs are used in clinical practice, if they produce selective survival pressure on bacteria, they will inevitably cause more serious bacterial resistance. Therefore, in order to effectively combat the increasingly serious MRSA resistant bacteria infection, it is necessary to break through the traditional thinking and find new antimicrobial strategies that do not induce resistance [6].

[0006] Fludarabine is an anti-tumor drug used to treat patients with B-cell chronic lymphocytic leukemia (CLL)[7]. Fludarabine is also used as a conditioning regimen before transplantation. For example, Large Pericardial Effusions as a Manifestation of Graft Versus Host Disease: a Single Institution Retrospective Study[8] published a retrospective study that investigated large pericardial effusions (LPE) after allogeneic hematopoietic stem cell transplantation as a manifestation of graft-versus-host disease (GVHD). The conditioning regimen included fludarabine combined with busulfan or total body irradiation (TBI); "Reduced Intensity Conditioning Matched Unrelated Donor Allograft for Kostmann Syndrome." [9] published a case report on allogeneic unrelated donor (MUD) transplantation in patients with Kostmann syndrome (congenital agranulocytosis). The conditioning regimen mentioned in the document included fludarabine; there have been no reports on the effectiveness of fludarabine against methicillin-resistant Staphylococcus aureus infection.

[0007] Faced with the severe situation of MRSA raging around the world, serious drug resistance and rising mortality rate after infection, exploring the pathogenic mechanism of MRSA and finding new drugs and new strategies to effectively control MRSA infection have become urgent issues that scientific researchers must face.

[0008] References: 1. Collaborators, GBDAR, 全球细菌负担 1990 - 2021年抗菌药物耐药性:一项系统分析及预测 2050. Lancet, 2024. 404(10459): p. 1199-1226. 2. van Hal, SJ, et al., 金黄色葡萄球菌菌血症死亡率的预测因素 。 Clin Microbiol Rev, 2012. 25(2): p. 362-86. 3. McGuinness, WA, N. Malachowa, and FR DeLeo, 万古霉素 金黄色葡萄球菌中的耐药性 Yale J Biol Med, 2017. 90(2): p. 269-281. 4. Peschel, A. and M. Otto, 酚溶性调节素与 葡萄球菌感染。 Nat Rev Microbiol, 2013. 11(10): p. 667-73. 5. Rasko, DA and V. Sperandio, 对抗细菌介导疾病的抗毒力策略 。 Nat Rev Drug Discov, 2010. 9(2): p. 117-28. 6. Nazli, A., et al., 耐甲氧西林金黄色葡萄球菌感染的治疗:我们目前的状况如何? CurrMed Chem, 2024. 31(28): p. 4425-4460. 7. Ricci, F., et al., 氟达拉滨治疗慢性 淋巴细胞白血病:综述。 Ther Clin Risk Manag, 2009. 5(1): p. 187-207. 8. Norkin, M., et al., “大量心包积液作为移植物抗宿主病的一种表现:一项单中心 回顾性研究”。 。 Blood, 2009. 114(22): p. 4659-4659. 9. Thachil, J., et al., 降低强度预处理匹配 无关供体异基因移植治疗 Kostmann 综合征。 Blood, 2005. 106(11): p.3854. Summary of the invention

[0009] The present application found that fludarabine can improve the survival rate of MRSA-infected mice, reduce the number of MRSA bacteria in the lungs, liver, spleen and kidneys, and reduce the degree of damage to the lungs, liver, spleen and kidneys caused by MRSA infection; it also found that PSMα3 is an important virulence factor of MRSA, which is closely related to the high pathogenicity of MRSA, PSMα3 can induce macrophage death in a concentration-dependent manner, and fludarabine can significantly reduce the cytotoxicity caused by PSMα3. The specific technical scheme of the present invention is as follows: First, the study of fludarabine in treating MRSA-infected mice

[0010] The MRSA infection mouse model was established by using methicillin-resistant Staphylococcus aureus USA300 (abbreviated as "MRSA USA300"). psmα3 Gene of MRSA USA300 (abbreviated as " Δpsmα3 ”)Δ psmα3 Infection mouse model; Fludarabine was injected intraperitoneally 1h and 6h after mice were infected with MRSA USA300.

[0011] Results: After 2 days of infection, all mice in the MRSA USA300 infected control group died, while all mice in the normal control group survived. psmα3 The survival rate of mice in the infection control group was 50%, the survival rate of the 10 mg / kg fludarabine treatment group was 30%, the survival rate of the 20 mg / kg fludarabine treatment group was 40%, and the survival rate of the 50 mg / kg fludarabine treatment group was 80%; After 24 hours of infection, compared with the MRSA USA300 infection control group, Δ psmα3 The logarithmic colony counts in the whole blood, liver, lungs, spleen, and kidneys of the infection control group decreased significantly. P The values ​​are all less than 0.001; At 24 hours after infection, the logarithmic colony counts in the whole blood, liver, lung, spleen, and kidney of the 50 mg / kg fludarabine-treated group were significantly decreased compared with the MRSA USA300-infected control group. P The values ​​are all less than 0.001; 24 hours after infection, HE staining results of lung, liver, spleen and kidney tissues showed that the degree of organ damage in mice was: MRSA USA300 infection control group>Δ psmα3The infection control group>50 mg / kg fludarabine treatment group>normal control group.

[0012] Conclusion: PSMα3 is an important virulence factor of MRSA and is closely related to the high pathogenicity of MRSA. Fludarabine significantly improves the survival rate of MRSA-infected mice, reduces the bacterial load in organs, and alleviates the degree of organ damage in infected mice, thus playing an anti-infection role. Second, PSMα3 is a virulence factor of MRSA

[0013] 1. Evaluation of cytotoxicity of MRSA virulence factor PSMα3 After human myeloid leukemia monocytes (THP-1 cells) were differentiated into mature macrophages, PSMα3 gradient solution was added. PSMα3 was able to induce macrophage cell death in a concentration-dependent manner, with a median lethal concentration IC50 of 2.496±0.0513 μM.

[0014] 2. Study on the mechanism of cytotoxicity of MRSA virulence factor PSMα3 Results: PSMα3 can activate the downstream necroptosis classical RIP1 / RIP3 / MLKL signaling pathway in a concentration-dependent manner, and significantly increase the phosphorylation levels of RIP1, RIP3, and MLKL. The above results confirm that PSMα3 can induce macrophage necroptosis by activating the RIP1 / RIP3 / MLKL signaling pathway. Third aspect: Evaluation of Fludarabine's inhibition of PSMα3 1. Evaluation of Fludarabine's Inhibition of PSMα3 in Vitro

[0015] After human myeloid leukemia monocytes (THP-1 cells) were differentiated into mature macrophages, fludarabine was added, and then PSMα3 was added;

[0016] Results: PSMα3 caused about 50% macrophage cell death; however, pretreatment with fludarabine significantly inhibited PSMα3-induced macrophage cell death and increased cell survival rate to about 72%. Fludarabine significantly reduced PSMα3-induced macrophage cell death. 2. Evaluation of Fludarabine's Inhibition of PSMα3 in Vivo

[0017] Treatment with fludarabine can significantly reduce the expression level of p-MLKL, a key marker of macrophage necroptosis in the lungs of mice infected with MRSA USA300, indicating that fludarabine can block MRSA-induced macrophage necroptosis in vivo, maintain macrophage function, and prevent MRSA immune escape.

[0018] Compared with the prior art, the beneficial effects of this application are: This application first discovered that fludarabine can significantly reduce the number of bacteria in organs, alleviate the degree of organ damage in infected mice, and improve the survival rate of infected mice in the treatment of MRSA-infected mice. PSMα3 is a virulence factor of MRSA. Fludarabine can significantly reduce the cytotoxicity caused by PSMα3 both in vitro and in vivo, providing a new possibility for the treatment of MRSA infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 , 7 Day survival rate curve; Figure 2 , 24 hours after infection, MRSA USA300 infected control group, Δ psmα3 Comparative statistical graph of logarithmic colony concentrations in whole blood, liver, lung, spleen, and kidney of mice in the infection control group and the 50 mg / kg fludarabine treatment group; Figure 3 , 24 hours after infection, normal control group, USA300 infection control group, 50 mg / kg fludarabine treatment group and Δ psmα3 HE staining of lung, liver, spleen, and kidney tissues of mice infected with the control group; Figure 4 , THP-1 cell survival rate curve after adding PSMα3; Figure 5 , Western-Blot detection results of MRSA virulence factor PSMα3 promoting RIP1 / RIP3 / MLKL necroptosis signaling pathway; Figure 6 , Fludarabine inhibited PSMα3-induced macrophage cell death; Figure 7 , fludarabine inhibits necroptosis of macrophages in the lungs of mice infected with MRSA.

[0020] in Figure 2 and Figure 6 middle, *, **, *** represent: P <0.05, P <0.01, P <0.001, indicating that the difference between the two groups was statistically significant. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0024] Reagents and Materials

[0025]

[0026]

[0027] instrument Example 1: Evaluation of the efficacy of fludarabine in treating MRSA in vivo 1. Preparation of bacterial solution

[0028] Take methicillin-resistant Staphylococcus aureus MRSA USA300 (hereinafter referred to as "MRSA USA300") and knock out psm α3 Gene of MRSA USA300 (hereinafter referred to as "Δ psmα3 ”), inoculated into 10 mL of TSB medium, and cultured overnight at 37°C and 220 rpm in a shaking incubator; the absorbance of the bacterial solution was detected at a wavelength of 630 nm the next day. According to OD630 = 0.1, the bacterial solution concentration was approximately 1 × 10 8 CFU / mL conversion, adjust the bacterial solution concentration to 1.5×10 9 CFU / mL; the adjusted bacterial solution was centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant was discarded; the bacteria were rinsed twice with sterile 1× PBS buffer, each time at 12000 rpm and 4℃ for 5 min, and the supernatant was discarded; finally, an equal amount of PBS buffer was added to resuspend the bacteria to maintain a bacterial solution concentration of about 1.5×10 9 CFU / mL, that is, MRSA USA300 bacterial solution and Δ psmα3 Bacterial liquid. 2. Preparation of Fludarabine Solution

[0029] (1) Fludarabine working solution Weigh about 80 mg of fludarabine powder on a balance, dissolve the powder in 1.6 mL of DMSO in an ultra-clean workbench, add 12.8 mL of PEG300 after it is fully dissolved, add 1.6 mL of Tween80 after mixing, add 16 mL of sterile deionized water after mixing again, and mix by inverting to prepare a 2.5 mg / mL fludarabine working solution.

[0030] (2) Fludarabine dosing solution Pipette 10 mL of fludarabine working solution respectively, dilute and mix with 15 mL of DMSO-PEG300-Tween80-sterile deionized water (5:40:5:50) to obtain 1 mg / mL fludarabine dosing solution.

[0031] 3. Construction of MRSA infection and Δ psmα3 Mouse model of infection Male BALB / c mice weighing 18-22 g were selected and randomly divided into 6 groups, with 16 mice in each group, labeled as: normal control group, MRSA USA300 infection control group, 10 mg / kg fludarabine treatment group, 20 mg / kg fludarabine treatment group, 50 mg / kg fludarabine treatment group, Δ psmα3 Infected control group.

[0032] MRSA USA300 infection mouse model: 4 groups of mice were intraperitoneally injected with 200 μL of MRSA USA300 bacterial solution (concentration of about 1.5×10 9 CFU / mL), to construct a MRSA USA300 infection mouse model; Δ psmα3 Infection mouse model: 1 group of mice were intraperitoneally injected with 200 μL of Δ psmα3 Bacterial solution (concentration of about 1.5×10 9 CFU / mL), construct Δ psmα3 Mouse models of infection; Normal control group: Group 1 mice were not injected with any bacterial solution, which was the normal control group. 4. Drug administration

[0033] The three fludarabine treatment groups were given fludarabine dosing solution twice, at 1h and 6h after mice were infected with MRSAUSA300. According to the weight of mice, 1.0 mg / mL fludarabine dosing solution was injected intraperitoneally, so that the final dose of fludarabine was 10 mg / kg (injection of about 200 μL), 20 mg / kg (injection of about 400 μL), and 50 mg / kg (injection of about 1000 μL), respectively.

[0034] The recommended dose of fludarabine phosphate for adults is 25 mg / m2 per day. 2 Body surface area, 5 consecutive days, one intravenous course every 28 days; according to the body surface area conversion formula, the dose for mice is about 12.3 times the human dose. Therefore, the converted therapeutic dose of fludarabine in mice is about 307.5 mg / kg, and the median lethal dose (LD50) of fludarabine for mice is 375 mg / kg. The above-mentioned mice were given doses of 10 mg / kg, 20 mg / kg, and 50 mg / kg, which are far less than the converted therapeutic dose and median lethal dose of fludarabine in mice.

[0035] MRSA USA300 infection control group, Δ psmα3 The infected control group and the normal control group were injected with 200 μL of DMSO-PEG300-Tween80-sterile deionized water (5:40:5:50) 5. Evaluation of the therapeutic effect of fludarabine in vivo

[0036] (1) Methods The in vivo therapeutic effect of fludarabine was evaluated by organ CFU determination, tissue HE staining, and survival rate curve determination experiments.

[0037] Determination of survival rate curve: After drug administration, the 7-day survival of 10 BALB / c mice in each group was observed, and the survival curve was drawn and the survival rate was calculated.

[0038] Organ CFU determination: 24 h after infection, 6 BALB / c mice were taken from each group and anesthetized with sodium pentobarbital at a dose of 60 mg / kg intraperitoneally. The left eyeball of the mice was removed to collect blood, and a portion of the blood was collected using an anticoagulant tube and placed on ice for later use. The mice were killed by dislocation of the neck, and part of the liver, lung, and spleen were taken out on an ice box, weighed, and ground into homogenate. The blood and tissue homogenate were then diluted 10 times in a sterile TSB medium. 100 μL of the solution was taken for each dilution and evenly spread on the MHA agar plate. After incubation in a 37°C constant temperature incubator overnight, the number of bacterial colonies (CFU) on the plates of each group was counted, and the number of original colonies per gram of tissue (CFU / g) was calculated based on the dilution multiple.

[0039] Tissue HE staining: another part of the liver, lung, and spleen was cut off, and after being rinsed with sterile PBS buffer, the tissue was immersed in 10% paraformaldehyde fixative for 24 h, and then dehydrated with gradient alcohol 70%, 80%, 95%, and 100% for 30 min each, and repeatedly infiltrated in xylene for 20 min and paraffin for 12 min; after paraffin embedding, the sections were sliced ​​at a thickness of 3 μm, and then dewaxed for HE staining; the tissue characteristics were observed using a biological microscope.

[0040] (2) Results like Figure 1 As shown, 2 days after infection, all mice in the MRSA USA300 infected control group died (survival rate 0%), while all mice in the normal control group survived (survival rate 100%). psmα3 The survival rate of mice in the infection control group was 50%, the survival rate of the 10 mg / kg fludarabine treatment group was 30%, the survival rate of the 20 mg / kg fludarabine treatment group was 40%, and the survival rate of the 50 mg / kg fludarabine treatment group was 80%; like Figure 2As shown, after 24 hours of infection, Δ psmα3 The logarithmic colony counts in the whole blood, liver, lungs, spleen, and kidneys of the infection control group decreased significantly. P The values ​​are all less than 0.001; At 24 hours after infection, the logarithmic colony counts in the whole blood, liver, lung, spleen, and kidney of the 50 mg / kg fludarabine-treated group were significantly decreased compared with the MRSA USA300-infected control group. P The values ​​are all less than 0.001; like Figure 3 As shown, 24 hours after infection, HE staining results of lung, liver, spleen and kidney tissues showed that the degree of organ damage in mice was: MRSA USA300 infection control group>Δ psmα3 The infection control group>50mg / kg fludarabine treatment group>normal control group.

[0041] (3) Conclusion PSMα3 is an important virulence factor of MRSA and is closely related to the high pathogenicity of MRSA. Fludarabine significantly improves the survival rate of MRSA-infected mice, reduces the number of bacteria in organs, and alleviates the degree of organ damage in infected mice, thus playing an anti-infection role. Example 2: Evaluation of cytotoxicity of MRSA virulence factor PSMα3 1. Culture of human myeloid leukemia mononuclear cells (THP-1 cells)

[0042] THP-1 cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS), and the medium was supplemented with 0.05 mM β-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin; the cells were cultured in a 37°C, 5% CO2 incubator. 2. Evaluation of PSMα3 cytotoxicity

[0043] (1) Methods The cultured THP-1 cells were made into 1×10 5Cell suspension of 100 μL / well was inoculated into a 96-well culture plate, and a final concentration of 100 μL was added to the well. ng / mL of phorbol ester was induced in an incubator at 37°C and 5% CO2 for 24 hours to differentiate THP-1 cells into mature macrophages; after the induction, the culture supernatant was aspirated, the cells were washed twice with serum-free 1640 medium, and 100 μL of PSMα3 gradient solution (diluted with serum-free 1640 medium, the final concentrations were 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 μmol / L, 3 replicates for each concentration) was added to the wells, respectively, and stimulated in an incubator at 37°C and 5% CO2 for 24 hours; after the treatment, the culture supernatant was aspirated, 100 μL of 1640 medium containing 10% CCK-8 reagent was added to each well, and incubated at 37°C for 1 hour; then the absorbance of each well was measured at 450 nm using a full-wavelength multifunctional microplate reader; Cell survival rate = [(experimental well - blank well) / (negative control well - blank well)] × 100%, Negative control wells (cells, 1640 medium, CCK-8), Blank wells (1640 culture medium, CCK-8).

[0044] (2) Results The results are as follows Figure 4 As shown, PSMα3 could induce macrophage cell death in a concentration-dependent manner, with the median lethal concentration IC50=2.496±0.0513 μM. Example 3: Mechanism of action of MRSA virulence factor PSMα3 cytotoxicity

[0045] The cultured THP-1 cells were made into 1×10 6The cell suspension of 100 ng / mL was inoculated in a 6-well culture plate at 1 mL / well, and phorbol ester with a final concentration of 100 ng / mL was added to the wells to induce in a 37°C, 5% CO2 incubator for 24 h to differentiate THP-1 cells into mature macrophages; after the induction, the culture supernatant was removed, the cells were washed twice with serum-free 1640 medium, and 1 mL of PSMα3 gradient solution (diluted with serum-free 1640 medium, the final concentrations were 0, 1, and 3 μmol / L, and 3 replicates for each concentration) was added to the wells, respectively, and incubated at 37°C for 24 h. , treated in a 5% CO2 incubator for 24 hours; after the treatment, the culture supernatant was aspirated, the cells were rinsed twice with 1×PBS solution, 400 μL of cell lysis buffer (RIPA lysis buffer: PMSF: phosphatase inhibitor = 100:1:1) was added to each well, and the cells were allowed to stand in a 4°C refrigerator for 30 minutes for lysis; after sufficient lysis, the cell lysate was aspirated into a 1.5 mL EP tube, centrifuged at 12000 rpm and 4°C for 20 minutes, and the supernatant was collected as the protein extract; the protein concentration of each group of samples was detected using a BCA protein quantification kit.

[0046] Western-Blot technology was then used to detect the expression and phosphorylation levels of RIP1, RIP3, and MLKL, key pathway molecules for macrophage immune regulation. Take 60 μL of protein samples from each group, add 15 μL of 5× loading buffer (5× loading buffer) and mix well, boil in a water bath for 10 minutes, and cool for use; use SDS-PAGE precast gel (Tris-Gly, 4-15%, 15 holes), assemble the electrophoresis system, add protein samples and protein markers to the corresponding gel wells in sequence, and add the corresponding volume of each group of samples at 25 μg / well; turn on the power supply, set the voltage to 90V first, and after the sample runs out of the concentrated gel, increase the voltage to 120V until the end of electrophoresis; then use methanol to activate The PVDF membrane and transfer system were used for transfer, and the power supply was set to 100V for 100min. The PVDF membrane was taken out and blocked with 5% skim milk powder at room temperature for 4h, and then incubated with rabbit anti-human p-RIP1, rabbit anti-human p-RIP3, rabbit anti-human p-MLKL, rabbit anti-human RIP1, rabbit anti-human RIP3, rabbit anti-human MLKL, and GAPDH primary antibodies (diluted in 1×TBST at a ratio of 1:1000) at 4℃ overnight; the next day, the membrane was rinsed three times with 1×TBST for 10min each time, and then incubated with HRP-labeled goat anti-rabbit secondary antibody and Alexa Fluor 647-labeled goat anti-rabbit secondary antibody at room temperature for 1h; after the incubation, the membrane was rinsed three times with 1×TBST for 10min each time; finally, the Abclonal chemiluminescence kit and ProteinSimple gel imaging system were used for luminescence development and photography.

[0047] Results Figure 5, PSMα3 can activate the downstream necroptosis classical RIP1 / RIP3 / MLKL signaling pathway in a concentration-dependent manner, significantly increasing the phosphorylation levels of RIP1, RIP3, and MLKL. The above results confirm that PSMα3 can induce macrophage necroptosis by activating the RIP1 / RIP3 / MLKL signaling pathway. Example 4: Evaluation of Fludarabine's Inhibition of PSMα3 in Vitro 1. THP-1 cell culture (same as Example 2) 2. Fludarabine inhibits PSMα3 cytotoxicity in vitro

[0048] The cultured THP-1 cells were made into 1×10 5 A cell suspension of 100 μL / well was inoculated in a 96-well culture plate, and phorbol ester with a final concentration of 100 ng / mL was added to the wells for induction in an incubator at 37°C and 5% CO2 for 24 hours to differentiate THP-1 cells into mature macrophages; after the induction, the culture supernatant was removed, the cells were washed twice with serum-free 1640 medium, and 100 μL of 1640 medium containing 0 or 5 μmol / L fludarabine was added to the wells for pretreatment of the cells for 2 hours; then PSMα3 with a final concentration of 3 μmol / L was added, and the cells were stimulated in an incubator at 37°C and 5% CO2 for 24 hours; after the treatment, the culture supernatant was removed, 100 μL of 1640 medium containing 10% CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 1 hour; then the absorbance of each well was measured at 450 nm using a full-wavelength multifunctional microplate reader; Cell survival rate = [(experimental well - blank well) / (negative control well - blank well)] × 100%,

[0049] Negative control wells (no treated cells, 1640 medium, CCK-8), Blank wells (1640 culture medium, CCK-8).

[0050] Results Figure 6 , 3μmol / L PSMα3 can cause about 50% macrophage cell death; after pretreatment of cells with fludarabine, PSMα3-induced macrophage cell death can be significantly inhibited, and the cell survival rate is increased to about 72%. Fludarabine can significantly reduce PSMα3-induced macrophage cell death. Example 5: Evaluation of Fludarabine's Inhibition of PSMα3 in Vivo

[0051] The paraffin sections of the lungs of each group (Example 2) were taken, dewaxed, dehydrated with gradient alcohol, and then antigen repaired. Then, the sections were rinsed with a mixture of phosphate buffer and Tween 20 (1×PBST) for 3 times, each time for 5 minutes; after blocking with 10% goat serum in a 37°C wet box for 30 minutes, the sections were rinsed with 1×PBST for 3 times, each time for 5 minutes; AlexaFluor 488-F4 / 80 fluorescent antibody (1:50 dilution) for labeling macrophages was added to the tissue sections, placed in a wet box, and incubated overnight at 4°C; rinsed with 1×PBST for 3 times, each time for 5 minutes; rabbit anti-human p-MLKL primary antibody (1:500 dilution) was added, and incubated at room temperature for 2 hours; rinsed with 1×PBST for 3 times, each time for 5 minutes, and then Alexa Flu ... The sections were incubated with fluorescent secondary antibody labeled with 647 at room temperature for 30 min; rinsed with 1×PBST; incubated with DAPI solution at room temperature for 30 min, rinsed with 1×PBST, and then sealed with sealing solution containing antifade agent and scanned using a microscope slide scanner.

[0052] Results Figure 7 Compared with the normal control group, the number of macrophages (F4 / 80 marker) in the lungs of mice in the MRSA USA300 control group was significantly increased, and the expression level of p-MLKL, a key marker of necroptosis, was significantly increased. psmα3 In the infection control group, the expression level of p-MLKL was significantly lower than that in the MRSA USA300 control group. The above results indicate that macrophages will be recruited to the lesion site in large numbers after MRSA USA300 infection, but MRSA USA300 can induce macrophage necroptosis through the virulence factor PSMα3, destroy the bactericidal function of macrophages, and achieve immune escape.

[0053] Treatment with fludarabine can significantly reduce the expression level of p-MLKL, a key marker molecule of macrophage necroptosis in the organs of mice infected with MRSA USA300, indicating that fludarabine can block MRSA-induced macrophage necroptosis in vivo, maintain macrophage function, and prevent MRSA immune escape.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of fludarabine in the preparation of drugs against methicillin-resistant Staphylococcus aureus (MRSA).

2. The use according to claim 1, characterized in that The fludarabine can improve the survival rate of mice infected with MRSA.

3. The use according to claim 1, characterized in that The fludarabine can reduce the MRSA bacterial count in the lungs, liver, spleen and kidneys.

4. The use according to claim 1, characterized in that The fludarabine can reduce the damage degree of the lungs, liver, spleen and kidneys caused by MRSA infection.

5. The use according to claim 1, characterized in that The important virulence factor of MRSA is PSMα3.

6. The use according to claim 5, characterized in that The PSMα3 can induce macrophage cell death in a concentration-dependent manner.

7. The use according to claim 5, characterized in that The PSMα3 can induce macrophage necroptosis by activating the RIP1 / RIP3 / MLKL signaling pathway.

8. The use according to claim 5, characterized in that The fludarabine can significantly reduce the expression level of p-MLKL, a key marker molecule of necroptosis of macrophages in the lungs of MRSA-infected mice, indicating that fludarabine can block MRSA-induced macrophage necroptosis in vivo and prevent immune escape caused by MRSA infection.

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