Application of fludarabine in the treatment of methicillin-resistant Staphylococcus aureus infection

Abstract

The present invention discloses the use of fludarabine in treating methicillin-resistant Staphylococcus aureus infection. In a MRSA infection mouse model, it was verified 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 alleviate the degree of damage to the lungs, liver, spleen and kidneys caused by MRSA infection. It was also found that PSMα3 is an important virulence factor of MRSA and 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.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of fludarabine in treating methicillin-resistant Staphylococcus aureus infection. Background Art

[0002] The global emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA), a "superbug" caused by overuse of antibiotics, has brought significant difficulties and challenges to clinical anti-infective treatment and has become a major public health issue threatening human life and health. MRSA is a major pathogen causing both hospital-acquired and community-acquired infections and a significant cause of severe suppurative infections, pneumonia, endocarditis, sepsis, and septic shock. According to the US Centers for Disease Control and Prevention, approximately 20,000 people die annually in the United States from MRSA infection, making it the leading cause of mortality among all clinically isolated drug-resistant bacteria. In my country, MRSA infection is equally a serious problem, with clinical detection rates consistently exceeding 30%, making it the leading cause of death from severe clinical infections. To date, the global death toll caused by MRSA infection has surpassed that of HIV and hepatitis B, becoming the world's top three serious infectious diseases [1,2]. Furthermore, MRSA is multidrug-resistant and resistant to most commonly used antimicrobial agents, 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 no medicine is available [3].

[0003] Phenol-soluble modulin α3 (PSMα3) is an extremely important virulence factor of MRSA. It not only significantly increases the pathogenicity of MRSA, but also helps MRSA attack host immune cells and achieve immune escape. Therefore, in-depth research on the mechanism of action of PSMα3 in the process of MRSA infection and clarifying its molecular mechanism and key targets for regulating the body's immunity is 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 discovered 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, and other processes. At the same time, it will not produce selective survival pressure on bacteria and 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 drug resistance, and are also a new direction for people to effectively control MRSA infections [5].

[0005] Currently, the cost of developing antimicrobial drugs is increasing, and the development cycle is lengthening. 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. More worryingly, the emergence of resistant strains has far exceeded the development of new drugs, and if new antimicrobial drugs are used clinically, if they exert selective survival pressure on bacteria, they will inevitably lead to more serious bacterial resistance. Therefore, in order to effectively combat the increasingly serious MRSA resistant bacterial infections, it is necessary to break through traditional thinking and find new antimicrobial strategies that do not induce drug 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, the study "Large Pericardial Effusions as a Manifestation of Graft Versus Host Disease: a Single Institution Retrospective Study"[8] published a retrospective study to explore the occurrence of 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), and the conditioning regimen mentioned in the document included fludarabine; there are no reports on the efficacy of fludarabine in treating 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:

[0009] 1. Collaborators, GBDAR, 全球细菌抗菌药物耐药负担 1990 - 2021年:一项到2050年预测的系统分析。 Lancet,2024.404(10459): p. 1199-1226.

[0010] 2. van Hal, SJ, et al., 金黄色葡萄球菌菌血症死亡率的预测因素 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.

[0011] 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.

[0012] 4. Peschel, A. and M. Otto, 酚溶性调节素与葡萄球菌感染 酚溶性调节素与葡萄球菌感染。 Nat Rev Microbiol, 2013.11(10): p. 667-73.

[0013] 5. Rasko, DA and V. Sperandio, 对抗细菌介导疾病的抗毒力策略 对抗细菌介导疾病的抗毒力策略。 Nat Rev Drug Discov, 2010.9(2): p. 117-28.

[0014] 6. Nazli, A., et al., 耐甲氧西林金黄色葡萄球菌感染的治疗:我们现在处于什么阶段? CurrMed Chem, 2024.31(28): p. 4425-4460.

[0015] 7. Ricci, F., et al., 氟达拉滨治疗慢性淋巴细胞白血病:综述 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.

[0016] 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:一项单机构回顾性研究” “大量心包积液作为移植物抗宿主病的一种表现:一项单机构回顾性研究” . Blood, 2009.114(22): p. 4659-4659.

[0017] 9. Thachil, J., et al., 减低剂量预处理匹配无关供体同种异体移植治疗 Kostmann 综合征。 减低剂量预处理匹配无关供体同种异体移植治疗 Kostmann 综合征。 Blood, 2005.106(11): p. 3854. Summary of the Invention

[0018] The present application discovered that fludarabine can treat MRSA-infected mice, improve their survival rate, reduce the number of MRSA bacteria in the lungs, liver, spleen, and kidneys, and alleviate the degree of damage to the lungs, liver, spleen, and kidneys caused by MRSA infection. It also discovered that PSMα3 is an important virulence factor of MRSA and is closely related to the high pathogenicity of MRSA. PSMα3 can induce macrophage cell death in a concentration-dependent manner, and fludarabine can significantly reduce the cytotoxicity caused by PSMα3. The specific technical solutions of the present invention are as follows:

[0019] First, a study on the treatment of MRSA-infected mice with fludarabine

[0020] 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; 1h and 6h after mice were infected with MRSA USA300, fludarabine was injected intraperitoneally.

[0021] 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 in the 10 mg / kg fludarabine treatment group was 30%, the survival rate in the 20 mg / kg fludarabine treatment group was 40%, and the survival rate in the 50 mg / kg fludarabine treatment group was 80%;

[0022] 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 infected control group decreased significantly. P The values ​​are all less than 0.001;

[0023] 24 hours after infection, the logarithmic colony counts in the 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;

[0024] 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>50 mg / kg fludarabine treatment group>normal control group.

[0025] 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 exerting an anti-infection effect.

[0026] Second, research on PSMα3 as a virulence factor of MRSA

[0027] (I) Evaluation of cytotoxicity of MRSA virulence factor PSMα3

[0028] After human myeloid leukemia monocytes (THP-1 cells) were differentiated into mature macrophages, a gradient solution of PSMα3 was added. PSMα3 was able to induce macrophage cell death in a concentration-dependent manner, with an IC50 of 2.496±0.0513 μM.

[0029] (II) Study on the cytotoxic mechanism of MRSA virulence factor PSMα3

[0030] Results: PSMα3 activated the downstream necroptosis classical RIP1 / RIP3 / MLKL signaling pathway in a concentration-dependent manner, significantly increasing the phosphorylation levels of RIP1, RIP3, and MLKL. These results confirmed that PSMα3 can induce macrophage necroptosis by activating the RIP1 / RIP3 / MLKL signaling pathway.

[0031] The third aspect is the evaluation of fludarabine's inhibition of PSMα3

[0032] (I) Evaluation of Fludarabine's Inhibition of PSMα3 in Vitro

[0033] After human myeloid leukemia monocytes (THP-1 cells) were differentiated into mature macrophages, fludarabine was added, followed by PSMα3;

[0034] Results: PSMα3 caused approximately 50% macrophage cell death; however, pretreatment of cells with fludarabine significantly inhibited PSMα3-induced macrophage cell death and increased cell survival to approximately 72%. Fludarabine significantly reduced PSMα3-induced macrophage cell death.

[0035] (II) Evaluation of Fludarabine's Inhibition of PSMα3 in Vivo

[0036] 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.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] This application discovered for the first time that fludarabine can significantly reduce the bacterial count in organs, alleviate the degree of organ damage in infected mice, and improve the survival rate of infected mice when treating 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

[0039] Figure 1 、 7 Day survival curve;

[0040] Figure 2 , infection 24 hours, MRSA USA300 infection control group, Δ psmα3 Comparative statistical graph of logarithmic colony counts in whole blood, liver, lung, spleen, and kidney of mice in the infection control group and the 50 mg / kg fludarabine treatment group;

[0041] Figure 3 , infection 24 hours later, 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;

[0042] Figure 4 , THP-1 cell survival rate curve after addition of PSMα3;

[0043] Figure 5 , Western-Blot detection results of MRSA virulence factor PSMα3 promoting RIP1 / RIP3 / MLKL necroptosis signaling pathway;

[0044] Figure 6, fludarabine inhibited PSMα3-induced macrophage cell death;

[0045] Figure 7 , fludarabine inhibits necroptosis of macrophages in the lungs of mice infected with MRSA.

[0046] in Figure 2 and Figure 6 middle,

[0047] *, **, *** represent: P <0.05, P <0.01, P <0.001, indicating that the difference between the two groups was statistically significant. DETAILED DESCRIPTION

[0048] 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.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, all materials and reagents in the following examples can be obtained from commercial sources.

[0051] Reagents and materials

[0052]

[0053]

[0054]

[0055] instrument

[0056]

[0057] Example 1: Evaluation of the efficacy of fludarabine in treating MRSA in vivo

[0058] 1. Prepare bacterial solution

[0059] Take methicillin-resistant Staphylococcus aureus MRSA USA300 (hereinafter referred to as "MRSA USA300") and knockout psm α3 Gene of MRSA USA300 (hereinafter referred to as "Δ psmα3 ”), were 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 × 108 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°C for 10 min, and the supernatant was discarded; the bacteria were rinsed twice with sterile 1× PBS buffer, each time centrifuged at 12000 rpm and 4°C 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 approximately 1.5×10 9 CFU / mL, that is, MRSA USA300 bacterial solution and Δ psmα3 Bacterial liquid.

[0060] 2. Preparation of Fludarabine Solution

[0061] (1) Fludarabine working solution

[0062] Weigh approximately 80 mg of fludarabine powder using a balance and dissolve the powder in 1.6 mL of DMSO in a clean bench. After complete dissolution, add 12.8 mL of PEG300 and mix thoroughly. Then add 1.6 mL of Tween80. Mix thoroughly again and add 16 mL of sterile deionized water. Invert the solution to mix thoroughly to prepare a 2.5 mg / mL fludarabine working solution.

[0063] (2) Fludarabine dosing solution

[0064] Pipette 10 mL of fludarabine working solution separately, 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.

[0065] 3. Construction of MRSA infection and Δ psmα3 Infection mouse model

[0066] 96 male BALB / c mice weighing 18-22 g were 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 infection control group.

[0067] MRSA USA300 infection mouse model: 4 groups of mice were intraperitoneally injected with 200 μL of MRSA USA300 bacterial solution (concentration of approximately 1.5×10 9 CFU / mL), to construct a MRSA USA300 infection mouse model;

[0068] Δ psmα3 Infected mouse model: 1 group of mice were intraperitoneally injected with 200 μL of Δpsmα3 Bacterial solution (concentration of about 1.5×10 9 CFU / mL), construct Δ psmα3 Mouse models of infection;

[0069] Normal control group: Group 1 mice were not injected with any bacterial solution, which served as the normal control group.

[0070] 4. Administer medication

[0071] The three fludarabine treatment groups were given fludarabine solution twice, 1 hour and 6 hours after mice were infected with MRSAUSA300. According to the weight of the mice, 1.0 mg / mL fludarabine solution was injected intraperitoneally, so that the final fludarabine dose 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.

[0072] The recommended dose of fludarabine phosphate for adults is 25 mg / m2 per day. 2 Body surface area, 5 consecutive days of intravenous treatment, with each 28-day course constituting one course. Based on the body surface area conversion formula, the mouse dose is approximately 12.3 times the human dose. Therefore, the converted therapeutic dose of fludarabine in mice is approximately 307.5 mg / kg, and the median lethal dose (LD50) of fludarabine in mice is 375 mg / kg. The doses of 10 mg / kg, 20 mg / kg, and 50 mg / kg administered to the mice are significantly lower than the converted therapeutic and LD50 doses of fludarabine in mice.

[0073] 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).

[0074] 5. Evaluation of the therapeutic effect of fludarabine in vivo

[0075] (1) Method

[0076] The in vivo therapeutic effect of fludarabine was evaluated by organ CFU determination, tissue HE staining, and survival rate curve determination experiments.

[0077] Survival rate curve determination: After drug administration, the survival of 10 BALB / c mice in each group was observed for 7 days, and the survival curve was drawn and the survival rate was calculated.

[0078] Organ CFU assay: 24 h after infection, six BALB / c mice were selected from each group and anesthetized with sodium pentobarbital (60 mg / kg) intraperitoneally. The left eyeball of the mice was then removed and blood was collected using anticoagulant tubes, and a portion was placed on ice for later use. The mice were killed by cervical dislocation, and portions of the liver, lung, and spleen were removed on an ice box, weighed, and ground into homogenates. The blood and tissue homogenates were then serially diluted 10-fold in sterile TSB medium. 100 μL of each dilution level was evenly spread on MHA agar plates. After incubation in a 37°C incubator overnight, the bacterial colony counts (CFU) on each plate were counted, and the original colony count per gram of tissue (CFU / g) was calculated based on the dilution factor.

[0079] Tissue HE staining: Another part of the liver, lung, and spleen was removed and rinsed with sterile PBS buffer. The tissues were then immersed in 10% paraformaldehyde fixative for 24 h, followed by gradient dehydration with 70%, 80%, 95%, and 100% alcohol for 30 min each, repeated infiltration in xylene for 20 min, and repeated infiltration in paraffin for 12 min. After paraffin embedding, the tissues were sectioned at a thickness of 3 μm, dewaxed, and stained with HE. Tissue characteristics were observed using a biological microscope.

[0080] (2) Results

[0081] 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 in the 10 mg / kg fludarabine treatment group was 30%, the survival rate in the 20 mg / kg fludarabine treatment group was 40%, and the survival rate in the 50 mg / kg fludarabine treatment group was 80%;

[0082] like Figure 2 As shown, after 24 hours of infection, Δ psmα3 The logarithmic colony counts in the whole blood, liver, lungs, spleen, and kidneys of the infected control group decreased significantly. P The values ​​are all less than 0.001;

[0083] 24 hours after infection, the logarithmic colony counts in the 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;

[0084] like Figure 3As 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 infected control group was >50mg / kg fludarabine treatment group >normal control group.

[0085] (3) Conclusion

[0086] 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, thereby exerting an anti-infection effect.

[0087] Example 2: Evaluation of cytotoxicity of MRSA virulence factor PSMα3

[0088] 1. Culture of human myeloid leukemia mononuclear cells (THP-1 cells)

[0089] THP-1 cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS) 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.

[0090] 2. Evaluation of PSMα3 Cytotoxicity

[0091] (1) Method

[0092] The cultured THP-1 cells were made into 1×10 5 Cell suspension of 100 μL / well was inoculated into 96-well culture plates, and a final concentration of 100 μL was added into the wells. ng / mL 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, and 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, and 100 μmol / L, 3 replicates for each concentration) was added to the wells, and stimulated in an incubator at 37°C and 5% CO2 for 24 hours; after the treatment, the culture supernatant was aspirated, and 100 μL of 1640 medium containing 10% CCK-8 reagent was added to each well, and incubated at 37°C for 1 hour; the absorbance of each well was then measured at 450 nm using a full-wavelength multi-function microplate reader;

[0093] Cell survival rate = [(experimental well - blank well) / (negative control well - blank well)] × 100%,

[0094] Negative control wells (cells, 1640 culture medium, CCK-8),

[0095] Blank wells (1640 culture medium, CCK-8).

[0096] (2) Results

[0097] The results are as follows Figure 4 As shown, PSMα3 could induce macrophage cell death in a concentration-dependent manner, with an IC50 of 2.496±0.0513 μM.

[0098] Example 3: Mechanism of action of MRSA virulence factor PSMα3 cytotoxicity

[0099] The cultured THP-1 cells were made into 1×10 6 A cell suspension of 100 μg / mL was inoculated into a 6-well culture plate at 1 mL / well, and phorbol ester was added to the wells at a final concentration of 100 ng / mL to induce THP-1 cells in a 37°C, 5% CO2 incubator for 24 h to differentiate them into mature macrophages. After the induction, the culture supernatant was removed, and the cells were washed twice with serum-free 1640 medium, and 1 mL of PSMα3 gradient solution (diluted with serum-free 1640 medium, with final concentrations of 0, 1, and 3 μmol / L, respectively, with 3 replicates for each concentration) was added to each well and incubated at 37°C for 24 h. , and 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, and 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.

[0100] Then, Western-Blot technology was used to detect the expression and phosphorylation levels of RIP1, RIP3, and MLKL, key molecules in the macrophage immune regulation pathway. Take 60 μL of protein sample 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 later use. Use SDS-PAGE precast gel (Tris-Gly, 4-15%, 15 holes) to assemble the electrophoresis system. Add protein samples and protein markers to the corresponding gel holes in sequence. Each group of samples is added to the corresponding volume at 25 μg / well. Turn on the power supply and set the voltage to 90V. 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 gel. The membrane was transferred to a PVDF membrane and transfer system with a power supply of 100 V for 100 min. The PVDF membrane was removed and blocked with 5% skim milk powder at room temperature for 4 h, 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 1:1000 in 1×TBST) at 4°C overnight. The next day, the membrane was rinsed three times with 1×TBST for 10 min 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 1 h. After incubation, the membrane was rinsed three times with 1×TBST for 10 min each time. Finally, the Abclonal chemiluminescence kit and ProteinSimple gel imaging system were used for luminescence development and photography.

[0101] See the results Figure 5 PSMα3 can activate the downstream necroptosis canonical RIP1 / RIP3 / MLKL signaling pathway in a concentration-dependent manner, significantly increasing the phosphorylation levels of RIP1, RIP3, and MLKL. These results confirm that PSMα3 can induce macrophage necroptosis by activating the RIP1 / RIP3 / MLKL signaling pathway.

[0102] Example 4: Evaluation of Fludarabine's Inhibition of PSMα3 in Vitro

[0103] 1. THP-1 cell culture (same as Example 2)

[0104] 2. Fludarabine inhibits PSMα3 cytotoxicity in vitro

[0105] The cultured THP-1 cells were made into 1×10 5A cell suspension of 100 μL / well was inoculated into a 96-well culture plate, and phorbol ester at a final concentration of 100 ng / mL was added to the wells for induction at 37°C, 5% CO2 incubator for 24 hours to allow THP-1 cells to differentiate into mature macrophages; after the induction was completed, the culture supernatant was aspirated, 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 at a final concentration of 3 μmol / L was added and stimulated at 37°C, 5% CO2 incubator for 24 hours; after the treatment was completed, the culture supernatant was aspirated, and 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;

[0106] Cell survival rate = [(experimental well - blank well) / (negative control well - blank well)] × 100%,

[0107] Negative control wells (no treated cells, 1640 culture medium, CCK-8),

[0108] Blank wells (1640 culture medium, CCK-8).

[0109] See the results Figure 6 3 μmol / L PSMα3 caused approximately 50% macrophage cell death; however, pretreatment of cells with fludarabine significantly inhibited PSMα3-induced macrophage cell death and increased cell survival to approximately 72%. Fludarabine significantly reduced PSMα3-induced macrophage cell death.

[0110] Example 5: Evaluation of Fludarabine's Inhibition of PSMα3 in Vivo

[0111] Paraffin sections of the lungs of each group (Example 2) were taken, dewaxed, dehydrated with graded alcohol, and then subjected to antigen retrieval. The sections were then rinsed three times with a mixture of phosphate buffered saline and Tween 20 (1×PBST), each for 5 minutes. The sections were blocked with 10% goat serum in a 37°C wet box for 30 minutes, and then rinsed three times with 1×PBST, each for 5 minutes. Alexa Fluor 488-F4 / 80 fluorescent antibody for macrophage labeling (1:50 dilution) was added to the tissue sections, and the sections were placed in a humidified box and incubated overnight at 4°C. The sections were rinsed three times with 1×PBST, each for 5 minutes. Rabbit anti-human p-MLKL primary antibody (1:500 dilution) was added, and the sections were incubated at room temperature for 2 hours. The sections were rinsed three times with 1×PBST, each for 5 minutes. Alexa Fluor 488-F4 / 80 fluorescent antibody for macrophage labeling (1:50 dilution) was then added. The sections were then incubated overnight at 4°C. The sections were incubated with 647-labeled fluorescent secondary antibody at room temperature for 30 min, rinsed with 1×PBST, stained with DAPI solution at room temperature for 30 min, rinsed with 1×PBST, and mounted with antifade-containing mounting solution, and scanned using a microscope slide scanner.

[0112] See the 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. These results indicate that after MRSA USA300 infection, macrophages are recruited to the lesion site in large numbers, but MRSA USA300 can induce macrophage necroptosis through the virulence factor PSMα3, thereby destroying the macrophage bactericidal function and achieving immune escape.

[0113] Treatment with fludarabine can significantly reduce the expression level of p-MLKL, a key marker 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.

[0114] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Use of fludarabine in the preparation of a drug for resisting methicillin-resistant Staphylococcus aureus (MRSA), wherein the methicillin-resistant Staphylococcus aureus (MRSA) is MRSA USA300.

2. The use according to claim 1, characterized in that The fludarabine can improve the survival rate of mice infected with MRSA USA300.

3. The use according to claim 1, characterized in that The fludarabine can reduce the MRSA USA300 bacterial count in the lungs, liver, spleen and kidneys.

4. The use according to claim 1, wherein The fludarabine can reduce the degree of damage to the lungs, liver, spleen and kidneys caused by MRSA USA300 infection.

5. The use according to claim 1, characterized in that The important virulence factor of MRSA USA300 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 induces macrophage necroptosis by activating the RIP1 / RIP3 / MLKL signaling pathway.

8. The use according to claim 5, characterized in that The fludarabine can reduce the expression level of p-MLKL, a key marker molecule of necroptosis in the lungs of mice infected with MRSA USA300.

Images