Application of Punicalagin in Anti-Staphylococcus Aureus

Abstract

The present invention relates to the application of punicalagin to fight Staphylococcus aureus, such as fighting MRSA infection, including local and systemic infection, such as sepsis. β The applicant has discovered that PA has a unique mechanism against Staphylococcus aureus and is less likely to cause the bacteria to evolve or become drug-resistant.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to the anti-Staphylococcus aureus infection resistance of punicalagin. Background Art

[0002] Staphylococcus aureus (S. aureus) is a bacterial species that can cause a variety of infectious diseases in humans. It is the leading cause of bacterial infection-related deaths in approximately 135 countries and the most common bacterial pathogen causing death in people over 15 years of age worldwide. Methicillin-resistant S. aureus (MRSA), with its broad spectrum of resistance and high prevalence, is a major cause of community-acquired and hospital-associated bacteremia, with a mortality rate as high as 15%-60%. This makes the treatment of MRSA a clinically challenging problem. With the increasing prevalence of antibiotic resistance worldwide, S. aureus infection has become a global public health threat. Reports on vancomycin-resistant MRSA strains suggest that controlling and preventing MRSA infections is an urgent global public health concern. Reversing the trend of antibiotic resistance is imperative, otherwise we will face a situation where there are no available treatments. In the post-antibiotic era, alternative antibiotic therapies are emerging, and developing novel antibiotic alternatives or implementing drug combinations is a promising approach.

[0003] β-lactam antibiotics are commonly used but are prone to drug resistance, such as cefoperazone (Cef). One approach to addressing drug resistance is to combine them with β-lactamase inhibitors, such as sulbactam (Sulbactam).

[0004] Punicalagin (PA) is a novel antibacterial compound. Extensive searches of patents and other literature have revealed limited research on its anti-Staphylococcus aureus mechanism. There are also limited reports on the combined use of PA with other antibiotics against Staphylococcus aureus (MRSA), and no reports on its use against systemic S. aureus infections. Summary of the Invention

[0005] The applicants have discovered that PA has a unique mechanism of action against Staphylococcus aureus (particularly MRSA), making it less likely to cause the bacteria to evolve or develop drug resistance. Experiments have shown that PA can inhibit the proliferation of MRSA in vitro, slow the development and progression of drug resistance, and, to a certain extent, reduce the degree of damage to cells and organisms after bacterial infection. In particular, the combination of PA and β-lactam antibiotics, particularly Cef, has a significant synergistic inhibitory effect on MRSA proliferation. When the same dose of Cef is used to inhibit bacterial growth and exert cytoprotective effects, PA can reduce the amount of antibiotic used by approximately 50%. In in vivo animal studies, the combination of PA and Cef significantly improved the pathological manifestations and inflammatory response caused by drug-resistant bacterial infection, alleviating the morphological damage and functional disorders of multiple organs caused by bacterial infection. The overall therapeutic effect is similar to that of standard treatment with Cef+Sul at the same dose, and is more active than the Cef+Sul combination in improving inflammatory responses and inhibiting the development and progression of bacterial resistance. In particular, PA helps inhibit the spread of relevant β-lactam antibiotic resistance genes through the intestinal-fecal-environmental pathway. The present invention confirms the proliferation inhibitory effect of PA and Cef on multidrug-resistant Staphylococcus aureus through bacterial experiments, in vitro cell experiments and in vivo animal experiments. Because PA has the characteristics of high antibacterial activity and is not easy to induce drug resistance, it can be used as an ideal candidate drug or therapeutic adjuvant for future drug development.

[0006] Thus, the present invention discloses PA for preventing and / or treating S. aureus (especially MRSA) infections, including local and systemic infections, such as sepsis.

[0007] Specifically, the present invention provides a combination of PA and β-lactam antibiotics for antibacterial use, wherein the β-lactam antibiotics include penicillin, cephalosporin and atypical β-lactam antibiotics. Penicillin can be selected from penicillin G, amoxicillin and piperacillin. Cephalosporin can be selected from ceftriaxone, cefaclor, cefathiamidine, cefodizime, cefditoren pivoxil, cefazolin, cefdinir, cefixime, cefradine and cefoperazone. The atypical β-lactam antibiotic can be cefoxitin or aztreonam.

[0008] Furthermore, the present invention provides a combination of PA and β-lactam antibiotics, comprising PA and β-lactam antibiotics that are mixed or physically separated.

[0009] It should be noted that the active molecules mentioned in the present invention can be provided in pharmaceutical alternative forms, such as pharmaceutically acceptable salts, analogs, and metabolites thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 , PA, Cef and Sul intervention on bacterial growth

[0011] Figure 2 , The inhibitory effect of PA on the growth of MRSA of different generations after continuous induction and passage with different concentrations of PA Note: The results are expressed as OD 600 The values indicate that the curve of Punicalagin 256 μg / mL partially overlaps with the curve of Control.

[0012] Figure 3 , After continuous induction and passage of PA at different concentrations, the inhibition rate of PA on each generation of MRSA at different time points

[0013] Figure 4 , Inhibitory effects of different drugs on MRSA generations after continuous induction

[0014] Figure 5 , cellular inflammatory factors and macrophage typing biomarkers relative expression calorimetry (with the unintervention group as the control and GAPDH as the internal reference gene)

[0015] Figure 6 , Body weight changes of each group in intraperitoneal infection experiment

[0016] Figure 7 , HE staining of mouse liver

[0017] Figure 8 , HE staining of mouse colon (cross section)

[0018] Significance analysis symbols involved in each figure:

[0019] ns: no significant difference, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001. DETAILED DESCRIPTION

[0020] The following experimental examples are used to describe embodiments of the present invention in detail. However, the following experimental examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The MRSA bacteria used in the experiments were kept in our laboratory. Unless otherwise specified, the reagents, biological materials, culture media, and solutions used below are commonly used in the art and are publicly available or commercially available.

[0021] Experiment 1: Drug synergy detection

[0022] Experimental methods: The microdilution method was used to detect the minimum inhibitory concentration (MIC) of laboratory MRSA strains to different antibiotics; the checkerboard method was used to detect the fractional inhibitory concentration index (FICI) of PA and different antibiotics against MRSA.

[0023] 1. Determination of MIC

[0024] The antibiotic or compound PA to be tested was diluted two-fold in a 96-well plate to a final concentration of 512 μg / mL-0.125 μg / mL, with 100 μL of drug solution per well. 100 μL of MHB (Muller-Hinton broth) medium was added to each well of the control group, and 100 μL of 2×10 6 After culturing MRSA suspension at 35°C for 24 hours, the drug concentration at which there was no statistically significant difference in OD value was defined as MIC. The results are shown in Table 1.

[0025] Compound or antibiotic MIC (μg / mL)

[0026]

[0027] 2. Determination of FICI

[0028] (1) Adjust the bacterial solution concentration to 2×10 6 CFU / mL.

[0029] (2) Mark the 96-well plate as rows A to G and columns 1 to 9. Add 200 μL of MHB medium to each well in column 1 as a blank control. Add 50 μL of an antibiotic solution with a concentration of 4×MIC to each well in column 2. Dilute the solution in columns 3 to 8 by 2 times in sequence, so that the antibiotic concentrations in columns 2 to 8 are 4×MIC, 2×MIC, 1×MIC, and 50 μL of 4×MIC, 2×MIC, 1×MIC, 21×MIC were added to each well of rows A to G. and Add 100 μL of PA solution with a concentration of 2×10 6 CFU / mL of bacterial solution, at this time, rows A to G and columns 2 to 8 constitute 1×MIC to In the 9th column, 100 μL of bacterial solution and 100 μL of MHB medium were added to each well as the control group.

[0030] (3) Follow the above steps to construct a 96-well plate for the PA and antibiotic combination experiment, and measure the OD 600 The value is calculated according to the FICI calculation formula:

[0031]

[0032] In the formula It represents the ratio of the MIC value after the combination of antibiotics and PA to the MIC value measured when the antibiotics were used alone. It represents the ratio of the MIC value after the combined use of PA and antibiotics to the MIC value measured when PA is used alone. According to the standard that FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates an indifferent effect; and FICI > 2 indicates an antagonistic effect, the subsequent drugs were identified and screened.

[0033] The experimental results are shown in Table 2.

[0034] Table 2 Determination results of FICI for the combination of each antibiotic and PA

[0035]

[0036] Experiment 2: Determination of the in vitro antibacterial effect of the combination of PA

[0037] Experimental method:

[0038] (1) Prepare an MRSA bacterial solution with a concentration of 2×10 6 CFU / mL.

[0039] (2) Prepare solutions of Sul, PA, and Cef with a concentration of 20 μg / mL; prepare a Cef solution with a concentration of 40 μg / mL; prepare Cef + PA mixed solutions with concentrations of (5 + 20), (10 + 20), (15 + 20), and (20 + 20) μg / mL respectively; prepare a Cef + Sul mixed solution with a concentration of (20 + 20) μg / mL.

[0040] (3) Take centrifuge tubes, set up 10 groups as shown in Table 3, with the final concentration of each group of bacterial solution being 1×10 6 CFU / mL, incubate in a constant temperature shaker, take samples at 2 h, 4 h, 6 h, 8 h, and 12 h respectively, dilute with PBS and spread on plates, and perform colony counting the next day. The experimental results are as Figure 1 shown.

[0041] Table 3 Grouping and composition of the in vitro antibacterial experiment

[0042]

[0043] Note: * The concentration of the bacterial solution is 2×10 6 CFU / mL.

[0044] Experiment 3: Drug resistance induction experiment

[0045] Experimental method:

[0046] ① Prepare an MRSA bacterial solution with a concentration of 2×10 6 CFU / mL.

[0047] ② Prepare PA solutions at concentrations of 512 (2×MIC), 256 (1×MIC), 128, 64, 32, 16, and 8 μg / mL;

[0048] ③ Take centrifuge tubes and add 3 mL of PA solution of different concentrations, then add an equal amount of 2×10 6 The bacterial suspensions with the same CFU / mL were mixed, incubated on a constant temperature shaker for 12 h, and samples were taken. After the bacteria were subcultured, the above steps were repeated to obtain 9 consecutive generations of MRSA bacteria that were propagated under different concentrations of PA pressure.

[0049] ④ According to the microdilution method for determining MIC, a blank control group, a MRSA group, and different concentrations of PA intervention groups were set up in a 96-well plate, and the OD was measured after incubation at a constant temperature for 24 hours. 600 , record the absorbance value.

[0050] ⑤ According to the method of Experiment 2, the plate coating method was used to collect samples at 2h, 4h, 6h, 8h and 12h to determine the inhibition rate:

[0051]

[0052] Where ρ0 is the initial concentration of MRSA, ρ 实验组 and ρ 对照组 Represent the colony counts of the experimental group and the control group at a certain time point, respectively.

[0053] ⑥ Separately prepare 40 μg / mL Cef solution, (20+20) μg / mL Cef+PA solution, and (20+20) μg / mL Cef+Sul solution. Follow the method in step ③ above to subculture the bacteria under the intervention of each drug. Obtain 9 consecutive generations of MRSA strains grown under different drug interventions. Perform microdilution method to determine MIC and OD 600 value.

[0054] The experimental results are as follows Figure 2-4 As shown, the experimental results show that MRSA exhibits partial adaptability to continuous antibiotic intervention, but can effectively avoid the development of resistance to the natural active small molecule compound PA under continuous action.

[0055] Experiment 4: PA intervention cell experiment

[0056] Experimental methods:

[0057] ① Cell culture: Human monocytic leukemia cells (THP-1 cell line) were selected for cell passage.

[0058] ② Induction of differentiation: The cultured THP-1 suspension cells were induced with phorbol methyl parathionate (PMA) and the morphology and adhesion of THP-1 macrophages were observed.

[0059] ③ Cell intervention: THP-1 macrophages and MRSA bacterial suspension were prepared, and 8 experimental groups were set up as shown in Table 4.

[0060] ④ Calculation of cell viability: After setting the intervention, incubate the cells and calculate the approximate cell viability according to the standard formula. The results are shown in Table 5.

[0061] ⑤ Carry out cell intervention experiments in the same manner as experimental steps ②③. After 6 hours, extract total cell RNA to quantitatively detect inflammatory factors and typical biomarkers of macrophage typing (IL-1β, IL-6, IL-10, TNF-α, CD80, CD86, CD163 and CD206) and the internal reference gene GAPDH using a qPCR kit. The anti-inflammatory effect is as follows: Figure 5 shown.

[0062] Table 4 Cell intervention experimental groups

[0063]

[0064]

[0065] Note: * The cell density was 2×10 5 / mL, # The bacterial concentration was 2×10 6 CFU / mL.

[0066] Table 5 Cell survival rate of each intervention group after 6 hours

[0067]

[0068] Experiment 5: Antibacterial activity of PA in vivo

[0069] Experimental animals: 4-week-old ICR male mice, SPF grade.

[0070] In vivo MRSA infection systemic sepsis model: Mice injected intraperitoneally with MRSA bacterial solution showed symptoms such as eye congestion, erect hair, curled up body, reduced activity, loose stools, and significant weight loss, which can be considered a successful model.

[0071] Experimental groups: As shown in the table below, 10 mice in each group:

[0072] Table 6 In vivo animal experiment groups

[0073]

[0074] Experimental methods:

[0075] ① According to the experimental grouping: each mouse in Group A received an intraperitoneal injection of 500 μL of sterile saline, while mice in Groups B through H received an intraperitoneal injection of 500 μL of bacterial solution. One hour later, mice in Groups A and B received an injection of 500 μL of sterile saline, while mice in Groups C through H received the same amount of drug according to their experimental grouping. Intervention was repeated 25 and 49 hours after bacterial injection. Physiological activity of the mice was observed every 12 hours after bacterial injection, with controlled feed intake. Changes in body weight and physiological activity were recorded, feces were collected, and feed and water consumption were observed.

[0076] ③② 72 hours after the start of the experiment, all mice were anesthetized and sacrificed by cervical dislocation. The mice were dissected and blood, intestines, liver, spleen, kidneys, lungs, and fresh feces were collected. Morphological changes in the organs (such as size abnormalities, abscess formation, weight changes, etc.) were observed and recorded, and the organs were weighed to calculate the organ coefficient:

[0077]

[0078] ④ Bacterial load determination: Take 0.1g organ / 100μL blood and grind into a homogenate. After 10-fold isocratic dilution of the homogenate, take 100μL and evenly spread it on a Baird-Parker (Staphylococcus aureus identification medium) plate. After incubation, count the colonies. A colony count between 30 and 300 is considered valid. Calculate the bacterial load (CFU / g) in the organ tissue using the following formula:

[0079]

[0080] Experimental results (1) Basic physiological changes of experimental animals:

[0081] Weight changes such as Figure 6As shown, PA treatment effectively reduced the weight loss caused by MRSA infection within 24 hours, and the combined treatment groups (Groups G and H) were significantly more effective in reducing weight loss than the single-drug treatment groups. Physiologically, mice in the control group were active, eating, drinking, and performing other physiological activities normally, with no significant abnormalities. However, mice in the MRSA group exhibited typical pathological changes, including conjunctival congestion and occlusion, coarse fur, hunched bodies, and significantly decreased activity. Loose stools with anal adhesions and gross bloody stools were common, and some mice experienced neurological dysfunction, including convulsions and seizures. The average daily food intake of the mice was far less than 5g, suggesting a more severe multi-organ bacterial infection. PA and Cef treatment alone produced limited improvement in these conditions, while the combined treatment groups, particularly Groups G and H, significantly reduced these pathological changes, without significant conjunctival congestion, loose stools, or decreased activity, suggesting effective treatment. The disease activity index (DAI) was scored for each group of mice. The scoring criteria are shown in Table 7. The results were consistent with the changes in physiological activities of the mice observed during monitoring. The drug combination group had a significant advantage in reducing the DAI score.

[0082] Table 7 DAI scoring criteria

[0083]

[0084] Experimental results (2) Organ coefficients are shown in Table 8:

[0085] Table 8 Organ coefficients of each group

[0086]

[0087] Experimental results (3) Organ bacterial load:

[0088] Table 9 Bacterial load of animal organs in each group

[0089]

[0090] Experimental results (4) Pathological changes of organs:

[0091] The monotherapy group lengthened the shortened colon caused by MRSA infection and alleviated loose stools and swelling. The combination therapy group restored colon length. There was a statistically significant difference in colon length between Groups G and E, with Group G being longer than Group E (P < 0.001). Renal morphology was observed in the MRSA infection group, with obvious abscess formation, partial ulceration, and hemorrhagic exudation. The PA group had localized small abscesses and mild hemorrhagic exudation. The other groups showed minimal differences in renal morphology. Liver morphology was observed in the control group, with brown, smooth edges, and a shiny appearance under the microscope. There was no hemorrhagic exudation or tissue structural damage. The MRSA infection group had extensive hemorrhagic exudation, darkened color, small abscess formation, and partial ulceration. The PA group had darker color, small localized tissue damage, and mild hemorrhagic exudation. The other groups showed minimal differences in morphology, with color closer to normal.

[0092] The results of liver HE staining are as follows Figure 7 As shown, the results of colon HE staining are as follows Figure 8 shown.

[0093] Experimental results (5) Determination of liver inflammatory factors:

[0094] Table 10 Mean expression levels of mouse liver genes relative to the control group (GAPDH as the internal reference gene)

[0095]

[0096] Experimental results (7) Survival analysis:

[0097] Experimental methods:

[0098] ① 4-week-old ICR male mice, SPF grade.

[0099] ② Set up the experimental groups consistent with the above infection model and increase the bacterial concentration to 2×10 9 CFU / mL, 500 μL of bacterial solution was injected intraperitoneally into mice except the control group. One hour after infection, drug treatment was given according to the experimental group. The drug was given once every 24 hours, and the mice were observed for 144 hours, and the survival and death of the mice were recorded.

[0100] Experimental results: The 7-day survival rate of the MRSA infection group was only 12.5%, and deaths were concentrated within 72 hours after the start of the experiment. The PA treatment group was 25%, and the Cef (20 mg / kg) treatment group increased to 62.5%. The survival rate of the combined treatment group F was 75%, and deaths were concentrated within 36 hours. The 36-hour protection rate was better. The 7-day survival rate of the combined treatment groups G and H was 100%.

[0101] Experiment 6: Study on the anti-MRSA mechanism of PA

[0102] 6.1Srap L-lectin module combination

[0103] Serine-rich platelet adhesion factor (SraP) is a type of cell wall-anchored protein. The L-Lectin module located in the ligand binding region of the SraP protein can specifically bind to the sialylated receptor (Neu5Ac) on the cell membrane surface.

[0104] Experimental methods:

[0105] (1) Conservative analysis of SraP L-Lectin

[0106] (2) CADD technology targets the SraP L-Lectin module: molecular docking is performed, and the binding effect of PA and L-lectin is evaluated by referring to the binding position and interaction between Neu5Ac and L-lectin.

[0107] (3) Detection of the binding between SraP L-Lectin and PA by surface plasmon resonance (SPR)

[0108] (4) SPR detection of the dissociation constant between L-Lectin and PA

[0109] Experimental results:

[0110] (1) We randomly selected 48 MRSA strains stored in the laboratory, including 24 clinical isolates, 23 food isolates, and 1 community-associated strain (MW2), and tested the conservation of the L-Lectin module they carried. The results showed that the L-Lectin module was highly conserved among S. aureus strains; the key amino acid sites that interact with Neu5Ac were completely conserved among different strains.

[0111] (2) Molecular docking revealed that PA is more likely to bind to the L-ectin module than Neu5Ac. Furthermore, PA can occupy the spatial position in L-Lectin that binds to Neu5Ac, thereby inhibiting the binding of L-Lectin to Neu5Ac.

[0112] (3) The KD of PA and L-Lectin module measured by SPR was 5.656×10 -7 M, which is much smaller than that of Neu5Ac and L-Lectin.

[0113] 6.2 RNA-seq sequencing result mining and verification

[0114] Experimental methods:

[0115] MW2 bacteria were induced with PA antibiotics according to conventional methods, a control group was set up, and RNA was extracted for transcriptomic sequencing and analysis.

[0116] Experimental results:

[0117] (1) After PA treatment, the expression levels of MW2 genes changed significantly, of which 286 genes were significantly downregulated and 264 genes were significantly upregulated.

[0118] (2) The genes in this transcriptome were annotated with KEGG, and 100 KEGG pathways were obtained. Among them, the pyrimidine metabolism pathway and the Caulobacter cell cycle pathway were significantly enriched (P<0.05), while the enrichment of other pathways was not significant (P>0.05).

[0119] (3) After PA intervention, the expression levels of many virulence genes of MW2, including genes corresponding to surface proteins and exotoxins, changed significantly. For example, the expression levels of adhesion-related cell wall anchor protein genes such as spa, clfB, eap, sraP, and fnbB were significantly downregulated, as were the expression levels of exotoxin genes such as the α-toxin gene hly and the leukocidin-related gene lukS-PV.

[0120] (4) According to transcriptomic results, the expression levels of multiple genes in the pyrimidine metabolic pathway of MW2 were downregulated after PA treatment, especially the de novo pyrimidine nucleotide synthesis pathway. These differentially expressed genes were verified by qRT-PCR. The results showed that after PA treatment, genes such as pyrB, pyrC, pyrD, pyrE, pyrF, and carA involved in the de novo pyrimidine nucleotide synthesis pathway were simultaneously downregulated. The transcriptomic and qRT-PCR detection results were highly consistent.

[0121] (5) After PA treatment, the expression levels of surface protein genes such as sraP, fnbB, sdrD, spa, and clfB were significantly downregulated, and the expression of the auxiliary regulatory factor agrB was significantly upregulated. The qRT-PCR verification results were consistent with the transcriptome results.

[0122] (6) After PA treatment, the expression levels of penicillin-binding protein genes pbp2, mecA, fmt1, penicillin-binding protein-related factor reuC, and peptidoglycan biosynthesis-related genes murF and fmhC were significantly downregulated.

[0123] 6.3: Transmission of antibiotic resistance genes in intestinal flora

[0124] Experimental methods:

[0125] The feces of mice in Experiment 5 were collected, DNA was extracted, and the expression of drug-resistant genes was detected using qPCR technology.

[0126] Experimental results:

[0127] As shown in Table 11, taking the MRSA infection group as the control, after PA intervention, the structural genes mecA, pbpA, and pbp2 encoding the penicillin-binding protein PBP2a and the related regulatory genes mecR1 and femA all decreased, and the expression level of the β-lactam antibiotic-binding protein fmt1 also decreased, indicating that PA intervention reduced resistance to β-lactam antibiotics represented by methicillin and also inhibited the development of potential resistance. The expression levels of related resistance genes in the Cef intervention group were higher than those in the MRSA infection group and the PA intervention group, and thus showed a positive correlation with the progression of resistance. The expression levels in the drug combination group and whether PA was involved were consistent with the above results. For example, after the combined intervention of PA and Cef, the expression levels of resistance genes were lower than those in the group without PA, indicating that PA intervention inhibited the spread of antibiotic resistance genes.

[0128] Table 11 Mean expression levels of genes relative to MRSA infection group (with 16S rRNA as internal reference gene)

[0129]

Claims

1. Combined use of punicalagin β -Application of beta-lactam antibiotics in the preparation of anti-Staphylococcus aureus drugs, β The -lactam antibiotic is selected from cefoperazone, and the Staphylococcus aureus is MRSA.

2. The use according to claim 1, characterized in that: The medicament is used to treat sepsis.

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