Pharmaceutical composition for treating bacterial infection and application thereof

Abstract

The invention relates to a pharmaceutical composition for treating bacterial infection and application thereof, in particular to a pharmaceutical composition which has an anti-inflammatory effect and inhibits biofilm formation, the pharmaceutical composition comprises malic acid, fumaric acid, glycine and hippuric acid, the mass ratio of malic acid to fumaric acid to glycine to hippuric acid is 12: 1: 1: 3. The pharmaceutical composition is a novel malic acid composition MAC4, and MAC4 has antibacterial, anti-biofilm formation and anti-inflammatory characteristics, and is particularly suitable for treating diseases in which infection and inflammation are associated with each other. The MAC4 is different from traditional antibiotics, the traditional antibiotics are often specific to specific bacterial populations, and the MAC4 provided by the invention has broad-spectrum antibacterial activity. The MAC4 has the capabilities of resisting pathogens and regulating the inflammatory response of a host at the same time, so that the MAC4 can be used as a potential candidate drug for treating bacterial infection and inflammatory lung diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and relates to a pharmaceutical composition for bacterial infection and uses thereof, and in particular to a composition having anti-inflammatory effects and inhibiting biofilm formation and uses thereof. Background Art

[0002] Bacterial infections are a major challenge in global health. Escherichia coli is a typical pathogen that can cause a range of health problems from mild gastroenteritis to severe sepsis and hemolytic uremic syndrome. E. coli infections pose a heavy burden on global healthcare systems, leading to increased morbidity and mortality. Currently, treatment strategies mainly rely on antibiotics, but these strategies face huge challenges due to the growing problem of antimicrobial resistance (AMR), with some bacterial strains showing resistance to multiple drug classes.

[0003] A particularly challenging aspect of bacterial pathogenesis is the formation of biofilms, which are structured communities of microorganisms encapsulated by a polymer matrix synthesized by the microorganisms themselves. The structure of biofilms not only provides a protective barrier for bacteria, but also facilitates cell-to-cell interactions and information exchange. Inside biofilms, bacteria display phenotypic changes that significantly increase their resistance to antimicrobial agents and host immune defense mechanisms. For species such as Escherichia coli, biofilm formation significantly increases pathogenicity and complicates treatment outcomes. Biofilms are able to resist the effects of antibiotics through multiple mechanisms, including limiting antibiotic penetration, increasing the expression of resistance genes within the bacteria, and slowing the bacterial growth rate (thus reducing the effectiveness of antibiotics). In addition, bacteria within biofilms can enter a state called the "persistent state," which makes them extremely resistant to traditional antimicrobial treatments. The presence of biofilms not only increases the persistence of infection, but may also promote horizontal gene transfer, the process of exchanging genetic material between different bacteria, which further accelerates the development of resistance. Therefore, the resistance of biofilms to conventional antibiotics highlights the need to develop novel therapeutic strategies that are not only effective in preventing biofilm formation but also have the ability to eliminate established biofilms.

[0004] Traditional Chinese medicine (TCM) provides a promising path for the discovery of novel antimicrobial compounds. Recent studies have shown that natural compounds derived from traditional medicines have significant antimicrobial and anti-inflammatory properties. For example, in a 2014 study, Rashed and Butnariu discovered potent antimicrobial components from Bauhinia racemosa (Bauhinia racemosa Lam. and chemical content. Iranian Journal of Pharmaceutical Research, 13 (4), 1073.), while studies on Eriobotrya japonica (Japanese loquat) revealed bioactive compounds with both antimicrobial and antioxidant properties (Rashed, KN, & Butnariu, M. (2014). Isolation and antimicrobial and antioxidant evaluation of bio-active compounds from Eriobotrya japonica stems. Advanced Pharmaceutical BuHetin, 4 (1), 75-81). These studies suggest that traditional medicine may be an important resource for developing new therapeutic agents to fight bacterial infections.

[0005] CN111544420A discloses an inhalable effervescent powder spray, which includes an active agent and an effervescent agent, and the mass ratio of the active agent to the effervescent agent is 1:1.2-4; the effervescent agent is composed of an acid and a base, wherein: the acid at least includes a diketopiperazine, which can be selected from one or more of citric acid, malic acid, fumaric acid, maleic acid, succinic acid or tartaric acid; the base is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate; the diketopiperazine is 2,5-diketo-3,6-di(4-fumaryl-aminobutyl)piperazine; the active agent is azithromycin; the preparation method of the inhalable effervescent powder spray is to spray-dry a mixed solution containing an active agent and an effervescent agent to obtain the inhalable effervescent powder spray. Although the pharmaceutical preparation can be used to prepare a drug for the treatment of infectious pneumonia, the technical problem it solves is how to improve the dispersibility of the drug and the deposition rate of the drug in the deep lung. However, new treatments that can effectively fight bacterial infections while minimizing the development of drug resistance remain elusive. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a pharmaceutical composition for treating bacterial infection, especially a pharmaceutical composition having anti-inflammatory effect and inhibiting biofilm formation, and comprising malic acid, fumaric acid, glycine and hippuric acid.

[0007] The present invention discloses a novel malic acid composition MAC4, which is composed of malic acid, fumaric acid, glycine and hippuric acid. MAC4 shows good antibacterial effect. The novel malic acid composition MAC4 has both strong antibacterial activity and immunomodulatory effect. This dual mechanism of action distinguishes MAC4 from traditional antibiotics, which usually only focus on eliminating specific bacteria. MAC4 can target bacterial infections accompanied by biofilm formation and inflammatory response. In particular, traditional antibiotics only target specific bacterial populations, while MAC4 provided by the present invention has broad-spectrum antibacterial activity.

[0008] According to a preferred embodiment, the mass percentage of malic acid is 40-80%, and the mass percentage of malic acid is preferably 70%.

[0009] According to a preferred embodiment, the mass percentage of fumaric acid is 5-10%, and the mass percentage of fumaric acid is preferably 6%.

[0010] According to a preferred embodiment, the mass percentage of glycine is 5-10%, and the mass percentage of glycine is preferably 6%.

[0011] According to a preferred embodiment, the mass percentage of hippuric acid is 10-20%, and the mass percentage of hippuric acid is preferably 18%.

[0012] According to a preferred embodiment, the mass ratio of malic acid, fumaric acid, glycine and hippuric acid is 12:1:1:3.

[0013] The second invention of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a medicament for treating infections caused by microorganisms.

[0014] The third aspect of the present invention provides the use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a drug for treating lung infection. The fourth aspect of the present invention provides the use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a substance that inhibits the activity of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and / or Serratia marcescens.

[0015] The fifth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a substance for inhibiting the formation of bacterial biofilm.

[0016] The sixth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a substance for reducing bacterial biofilm density.

[0017] The seventh aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in preparing a substance that destroys the connection structure between bacteria.

[0018] The eighth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a substance for destroying the integrity of bacterial cells.

[0019] The ninth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in the preparation of a substance for reducing bacterial adhesion efficiency.

[0020] Preferably, the above substances include daily chemicals and medicines.

[0021] The tenth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in down-regulating the expression of ABC transporter proteins.

[0022] The eleventh aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in the preparation of anti-inflammatory substances.

[0023] The twelfth aspect of the present invention provides the use of the pharmaceutical composition of the first aspect of the present invention in down-regulating the expression of pro-inflammatory cytokines. The pro-inflammatory cytokines preferably include IL-6, IL-1β, and TNF-α.

[0024] The thirteenth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in up-regulating the expression of anti-inflammatory cytokines. The anti-inflammatory cytokines preferably include IL-10.

[0025] The fourteenth aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in enhancing the immune response of an organism.

[0026] The fifteenth aspect of the present invention provides the use of the pharmaceutical composition of the first aspect of the present invention in the preparation of daily chemical products for reducing oral pathogen colonization. Preferably, the daily chemical products include toothpaste, mouthwash or oral spray. Preferably, the pharmaceutical composition reduces the risk of lung infection caused by the migration and / or proliferation of oral pathogens by inhibiting the formation of pathogenic bacteria biofilm and / or anti-inflammatory effects.

[0027] The pharmaceutical composition provided in the first aspect of the present invention can effectively intervene in the colonization and proliferation of pathogenic microorganisms through its anti-inflammatory and biofilm-inhibiting effects. Due to the close correlation between the oral environment and respiratory tract infection (for example: the inhalation migration of oral pathogens may cause lung infection), the present invention applies the pharmaceutical composition to daily chemical products (such as toothpaste, mouthwash, etc.), which can directly act on the local environment of the oral cavity to reduce the biofilm formation and load of pathogens, thereby indirectly reducing the risk of lung infection. Specifically, the pharmaceutical composition of the present invention can destroy the bacterial quorum sensing system, inhibit the biofilm formation of pathogens (such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and / or Serratia marcescens), reduce its adhesion to the oral mucosa and tooth surface, and reduce the risk of bacteria inhaled through the respiratory tract. On the other hand, the pharmaceutical composition of the present invention can avoid the imbalance of the respiratory immune microenvironment caused by inflammatory factors (such as IL-6, TNF-α), thereby indirectly inhibiting the occurrence of lung infection.

[0028] In addition, the present invention can be compatible with conventional toothpaste excipients (such as hydrated silica and sorbitol) and avoid antagonism between components (such as the ion chelation effect of chitosan derivatives and sodium fluoride is solved by coating technology) by optimizing the component ratio and dosage form parameters (such as maintaining stable activity within the pH range of 6.5 to 7.5), that is, the pharmaceutical composition provided by the present invention can be adapted to carriers such as toothpaste through conventional dosage form optimization means (the pharmaceutical composition is uniformly dispersed and sustained-released in toothpaste).

[0029] Technical effects:

[0030] Biofilm is a major factor leading to antibiotic resistance and treatment failure. The novel malic acid composition MAC4 disclosed in the present invention can disrupt the formation of biofilm, which solves a key problem in the treatment of infection. MAC4 has a concentration-dependent bactericidal effect, and this mechanism of action helps to overcome antibiotic resistance, especially in the use of natural products to combat the growing antimicrobial resistance. Therefore, MAC4 can become an important part of future treatment strategies and help promote the global fight against antimicrobial resistance.

[0031] MAC4 is a new malic acid composition based on traditional Chinese medicine theory. It not only has a significant dual antibacterial effect, but also can regulate the immune system. The experimental results of the present invention show that MAC4 has significant antibacterial activity. Specifically, the minimum inhibitory concentration (MIC) of MAC4 for Escherichia coli and Staphylococcus aureus is 0.1C, while the MIC for Pseudomonas aeruginosa and Serratia marcescens is 0.2C. In addition, the new malic acid composition can effectively inhibit the formation of bacterial biofilm, reduce the quality of biofilm, and reduce the metabolic activity of bacteria by more than 94.97% (at 2MIC and 4MIC concentrations). Transcriptome analysis showed that MAC4 reprogrammed the gene expression of bacteria, with 1,150 differentially expressed genes affecting membrane, energy metabolism and DNA replication. Transcriptome analysis showed that MAC4 acts on multiple cellular processes simultaneously, for example, gene downregulation (such as evgA, yihL, fimB, fimE) involves membrane components, DNA binding and recombination activities, and the results of the embodiment show that MAC4 can act on these key processes, resulting in impaired bacterial cell integrity, blocked DNA replication and repair, and ultimately cell death. In addition, downregulation of metabolic pathways (such as glycerophospholipid metabolism and ABC transporters) shows that MAC4 can interfere with energy production and nutrient acquisition, hindering bacterial growth and survival.

[0032] In a mouse model of E. coli lung infection, MAC4 not only reduced bacterial load but also regulated the inflammatory response, down-regulating the expression levels of inflammatory factors such as IL-6, IL-1β, and TNF-α, while maintaining the integrity of lung tissue structure. MAC4 can effectively balance the host's pro-inflammatory and anti-inflammatory responses, as evidenced by the regulation of cytokines and immune cell receptors. The upregulation of genes related to cytokine signaling (such as Csf3, 114i1, Ccl4, and Tnf) and the upregulation of genes related to immune cell receptor function (such as Cd180, Fcrl1, and Fcer1g) indicate that MAC4 promotes the host's immune response. In addition, MAC4 down-regulates the expression of genes related to cell adhesion molecules (such as Itga3 and Itga8), extracellular matrix (such as Col12a1 and Col6a1), and cell cycle / apoptosis (such as Bcl211), and these results indicate that MAC4 can inhibit excessive inflammation and tissue damage. That is, the therapeutic effect of MAC4 is not limited to direct bacterial killing but also includes strategic modulation of the host immune response to promote recovery and minimize tissue damage.

[0033] MAC4 has antibacterial, anti-biofilm and anti-inflammatory properties, making it particularly suitable for treating conditions where infection and inflammation are interrelated. Because MAC4 has the ability to both fight pathogens and modulate host inflammatory responses, it is a potential candidate for the treatment of bacterial infections and inflammatory lung diseases, especially when conventional antibiotics are ineffective. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the result of MAC4 having a broad-spectrum antibacterial activity, among which, Figure 1 A~ Figure 1 D is the minimum inhibitory concentration (MIC) of MAC4 and CIP against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens determined by the culture medium microdilution method, with an exposure time of 24 h. Figure 1 E~ Figure 1 H is the growth curve of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens treated with MAC4 and CIP at a certain concentration over time. Figure 1 I is the antibacterial activity of MAC4 against a group of Gram-positive and Gram-negative bacteria, data represent mean ± SD, n = 3 independent experiments;

[0035] Figure 2 The results showed that MAC4 inhibited the biofilm formation of Escherichia coli and Staphylococcus aureus, among which, Figure 2 A and Figure 2 B is the result of evaluating the inhibitory effect of different concentrations of MAC4 and CIP on the biofilm formation of Escherichia coli and Staphylococcus aureus by crystal violet (CV) staining. Figure 2 C is the metabolic activity of Escherichia coli and Staphylococcus aureus biofilms treated with MAC4 and CIP determined by MTT assay. Figure 2 D is a scanning electron microscope (SEM) image of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) biofilms treated with MAC4 at 0.1 times the minimum inhibitory concentration (MIC) for 24 hours, compared with the untreated control group; Figure 2 E is a transmission electron microscope (TEM) image of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) treated with MAC4 at 0.1 times the minimum inhibitory concentration (MIC) compared with the untreated control group, wherein MAC4 treatment resulted in the following phenomena: 1. uneven cytoplasm distribution; 2. blurred cell edges; 3. overflow of contents; 4. increased transparency of cell edges; 5. dehydration; 6. DNA agglutination (indicated by the red arrow), data represent mean ± standard error, n = 3 independent experiments; ** P<0.01, *** P < 0.001, ****P < 0.0001, t test);

[0036] Figure 3 The induction of MAC4 results in decreased bacterial adhesion and altered gene expression in E. coli, among which Figure 3 A is the adhesion rate of E. coli to HaCaT cells after MAC4 treatment. Figure 3 B is a volcano plot of differentially expressed genes in Escherichia coli treated with MAC4, gray dots: genes with no significant difference; red dots: up-regulated differentially expressed genes; blue dots: down-regulated differentially expressed genes, horizontal axis: log2 (fold change); vertical axis: -log 10 (q value), Figure 3 C is GO enrichment analysis, Figure 3 D is the KEGG pathway analysis of differentially expressed genes downregulated in E. coli treated with MAC4; Figure 3 E is the correlation between RNA-seq data and RT-PCR data of selected differentially expressed genes (DEGs), R 2 : Determination coefficient of linear regression, data represent mean ± standard error, n = 3 independent experiments; ** P<0.01, **** P < 0.0001, t test;

[0037] Figure 4 Results showed that MAC4 treatment reduced bacterial load and modulated inflammatory cytokine expression in a mouse model of Escherichia coli-induced pneumonia (ELI). Figure 4 A is the bacterial load in the lung tissue of ELI mice treated with MAC4 or CIP, and the data represent the mean ± SD (n = 6 mice per group; compared with the control group, t test, ### P<0.001; compared with the model group, *** P<0.001), Figure 4 B is the histopathological results of lung tissue sections stained with hematoxylin-eosin (H&E). Figure 4 C , mRNA levels of inflammatory cytokines IL-10, IL-6, IL-1β, and TNF-α in mouse lungs were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and normalized with GAPDH as a reference, and data represent mean ± SD ( n = 4 mice / group; ** P < 0.01; t test), Figure 4 D shows the protein expression levels of IL-10, IL-6, IL-1β, and TNF-α detected by Western blotting, with β-actin used as a loading control. The quantitative results of the normalized protein expression levels are shown on the right, and the data represent the mean ± SD (n = 4 mice per group; *P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001; t test);

[0038] Figure 5 The results showed that MAC4 treatment altered gene expression in the ELI mouse model, Figure 5 A is the volcano plot comparison of differentially expressed genes (DEGs) in lung tissues of ELI mice treated with MAC4 and the control group, red dots: up-regulated genes; blue dots: down-regulated genes, the top 10 up-regulated and down-regulated genes ranked by fold change are annotated; Figure 5 B is the GO enrichment analysis results of up-regulated differentially expressed genes; Figure 5 C is the GO enrichment analysis result of down-regulated differentially expressed genes; Figure 5 D is the KEGG pathway enrichment analysis result of up-regulated differentially expressed genes; Figure 5 E is the KEGG pathway enrichment analysis result of down-regulated differentially expressed genes; Figure 5 F is the correlation between RNA-Seq data and RT-qPCR data for selected differentially expressed genes (DEGs), R 2 is the coefficient of determination of linear regression. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following examples, various processes and methods not described in detail are conventional methods or technical means in the art. The sources of reagents used, trade names, and those that need to list their components are all indicated when they first appear, and the same reagents used thereafter are not specifically stated, and are all the same as the sources indicated for the first time; the reagents, materials, etc. involved are not specifically stated, and are all obtained for commercial purposes.

[0040] The present invention evaluates the dual antibacterial and immunomodulatory properties of MAC4 through comprehensive in vitro and in vivo experiments. To this end, the present invention uses a variety of methods, including minimum inhibitory concentration (MIC) determination, biofilm formation analysis, whole transcriptome sequencing, and E. coli-induced lung infection mouse model to explore the direct antibacterial effect of MAC4 and its ability to regulate host immune responses. These experiments not only tested the efficacy of MAC4 in inhibiting bacterial growth, but also evaluated its potential in reducing inflammatory damage.

[0041] The present invention was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (8th edition, 2011) and was approved by the Institutional Animal Care and Use Committee of Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (approval number: GPTAP20220208-1).

[0042] The raw data of the present invention have been shared in the NCBI public database, please refer to the accession numbers: PRJNA1050840 and PRJNA1050877.

[0043] The minimum inhibitory concentration (MIC) was defined as the lowest concentration of MAC4 that could completely inhibit visible bacterial growth.

[0044] The minimum bactericidal concentration (MBC) is the lowest drug concentration that kills 99.9% of the original inoculum of bacteria, which is different from the minimum inhibitory concentration (MIC), which only refers to the lowest concentration that inhibits bacterial growth.

[0045] In the present invention, the statistical results are presented as the mean ± standard error of three independent experiments. Statistical significance was determined by unpaired t-test (P < 0.05) using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA).

[0046] Four bacterial strains were used in this example: Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 25923, Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853, and Serratia marcescens (S. marcescens) BNCC 107931. All strains are from public culture collections and provided by Dr. Aloysius Wong and Dr. Bo Zhang of Wenzhou-Kean University. ATCC 25922, ATCC 25923, and ATCC 27853 refer to the catalog numbers of the strains in the American Type Culture Collection; BNCC 107931 refers to the catalog number of the strains in the BeNa Culture Collection of China. These numbers help researchers accurately identify and obtain specific strains, ensuring that the microorganisms used in experiments have the correct source and characteristics.

[0047] Bacterial cultures were stored in a cryopreservative containing glycerol at -80 °C. When needed for use in experiments, the strains were first revived by streaking onto solid LB plates (15 g Bacto-agar per liter of LB, provided by Qingdao Haibo Biotechnology Co., Ltd., China) and incubated at 37 °C for 10 to 14 hours. Subsequently, a single colony was selected from the plate and inoculated into LB medium (provided by Qingdao Haibo Biotechnology Co., Ltd., China), and the bacteria were cultured in a shaker at 37 °C and 200 rpm / min until they reached the logarithmic growth phase. The bacterial suspension was then standardized to about 10 in fresh LB medium. 8 The concentration of CFU / mL was used for subsequent experiments.

[0048] HaCaT cell culture: HaCaT cells were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Penicillin-Streptomycin, Gibco) solution. HaCaT cell cultures were cultured in a constant temperature incubator at 37°C and 5% carbon dioxide to maintain appropriate humidity. Cell growth was monitored using a cell counter, and the culture medium was replaced regularly to ensure optimal cell growth and viability.

[0049] Example 1

[0050] This embodiment provides a composition having anti-inflammatory effect and inhibiting biofilm formation.

[0051] The composition is a MAC4 preparation (formula). Specifically, the ingredients of MAC4 include a variety of organic acids, which are all purchased in powder form. The initial concentration of MAC4 is marked as "C", and "4C" is the concentration of the 4C mother solution formed by adding 12 mg malic acid, 1 mg fumaric acid, 1 mg glycine, and 3 mg hippuric acid to 1 mL of solution. The specific formula of MAC4 is as follows:

[0052] Malic acid: 12 mg / mL; (Beijing Puxitang Biotechnology Co., Ltd., China, purity 98%);

[0053] Fumaric acid: 1 mg / mL; (Beijing Puxitang Biotechnology Co., Ltd., China, purity ≥99%);

[0054] Glycine: 1 mg / mL; (Beijing Puxitang Biotechnology Co., Ltd., China, purity ≥ 99%);

[0055] Hippuric acid: 3 mg / mL; (Beijing Puxitang Biotechnology Co., Ltd., China, purity 98%).

[0056] In order to reduce waste and ensure the accuracy of the experiment, a stock solution 4 times the initial concentration (4C) was first prepared, and then the required working concentrations were prepared by diluting the stock solution in sequence, including: 4C, 2C, 1C, 0.5C, 0.25C, 0.125C, 0.0625C and 0.03125C.

[0057] 1. Antimicrobial test (antibiotic sensitivity test)

[0058] The minimum inhibitory concentration (MIC) assay was performed using a sterile 96-well polystyrene microplate (NEST, China). A mixture of 180 μL LB medium and 3 μL bacterial suspension (containing 5 × 10 4 CFU of bacteria). A series of two-fold serial dilutions of MAC4 (ranging from 4C to 0.03125C) were added to columns 3 to 10, respectively. Column 1 was a negative control, containing only LB medium; column 2 was an untreated bacterial growth control, containing LB medium and bacterial inoculum. Ciprofloxacin (CIP, BIOMYC-3, Advanced Biotech Co., Ltd.) was used as a positive control, consisting of 180 μL LB medium and 20 μL CIP in a specific well.

[0059] All samples were incubated with shaking at 37°C and 200 rpm / min, and the optical density (OD) at 600 nm was measured using a Varioskan Flash microplate reader (Thermo Fisher Scientific, China) at 3, 6, 9, 12, and 24 hours. The growth inhibition rate was calculated as follows: % growth inhibition = [(test well OD-negative control well OD) / (positive control well OD-negative control well OD)] × 100%. In order to ensure the repeatability and reliability of the results, all experiments were repeated three times independently.

[0060] This embodiment also performs an evaluation of MBC.

[0061] The minimum bactericidal concentration (MBC) of MAC4 was evaluated after determining the minimum inhibitory concentration (MIC). The specific steps were as follows: 50 μL of the solution was drawn from the test tubes where no visible bacterial growth was observed (these test tubes were determined by the minimum inhibitory concentration evaluation) and evenly spread on LB agar plates (Qingdao Haibo Biotechnology Co., Ltd., China). Subsequently, these plates were incubated at 37°C for 24 hours.

[0062] The endpoint of MBC was defined as the lowest concentration of MAC4 corresponding to the absence of any bacterial colony growth on the agar plate. The entire experimental process was repeated three times independently, and the results were verified.

[0063] The results of MAC4's broad-spectrum antimicrobial activity against key pathogens are as follows Figure 1 As shown, Figure 1 A~ Figure 1 In D, the horizontal axis is the concentration of MAC4, "control" represents the negative control, and "CIP" represents the positive control; the vertical axis represents the bacterial growth (%). When the concentration of MAC4 is 0.1C, the bacterial growth of Escherichia coli and Staphylococcus aureus is significantly reduced (red arrows in the figure); when the concentration of MAC4 is 0.2C, the bacterial growth of Pseudomonas aeruginosa and Serratia marcescens is significantly reduced (red arrows in the figure). That is, at the optimal concentration (0.1C for Escherichia coli and Staphylococcus aureus; 0.2C for Pseudomonas aeruginosa and Serratia marcescens), MAC4 exhibits a significant antibacterial effect, and the growth inhibition rate shows a significant effect at MIC. Figure 1 A~ Figure 1 As shown in D, the inhibition rate of MAC4 against Escherichia coli was 80%, the inhibition rate against Staphylococcus aureus was 67%, the inhibition rate against Pseudomonas aeruginosa was 71%, and the inhibition rate against Serratia marcescens was 73%. Figure 1 E~ Figure 1 H is the result of the change of bacterial activity over time at a certain concentration, the horizontal axis is time, and the vertical axis is bacterial activity (%). In the 24-hour antibacterial test, ciprofloxacin (CIP) showed excellent antibacterial activity, with an inhibition rate of more than 97% against all four test strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens), while the inhibition rate of MAC4 was between 67% and 80% (compared with CIP). After 24 hours of longitudinal monitoring of MIC, MAC4 continued to inhibit bacterial proliferation in all test strains, which confirmed that MAC4 has a strong inhibitory effect on bacterial proliferation.

[0064] This example also carried out the minimum inhibitory concentration (MBC) determination at multiple times the MIC concentration of MAC4 (1 MIC, 2 MIC and 4 MIC respectively). Figure 1 As shown in Figure 1, for E. coli and S. aureus, the bactericidal threshold was determined to be 4 MIC (0.4C), which highlights the strong bactericidal effect of MAC4. For P. aeruginosa and S. marcescens, the effective MBC was 1 MIC (0.2C), which highlights that MAC4 has a broad-spectrum bactericidal property against a variety of bacterial strains.

[0065] The results of this example show that MAC4 has significant antibacterial activity against a series of pathogenic bacteria, and has significant antibacterial and bactericidal abilities. MAC4 can effectively inhibit and kill bacteria at a specific concentration, indicating that it has the use as a potent broad-spectrum antibacterial agent.

[0066] 2. Biofilm Assay

[0067] The ability of MAC4 to inhibit biofilm formation was evaluated using sterile 96-well polystyrene microplates as described previously. In each well, 180 μL of tryptic soy broth (TSB, Qingdao Hope Biotechnology Co., Ltd., China) was mixed with different concentrations of ciprofloxacin (CIP) or MAC4, ranging from C to 4C. Then, 3 μL of bacterial suspension in logarithmic growth phase (approximately 5 × 10 4 CFU) and incubated at 37°C for 24 hours. Sterile water was used as a control.

[0068] After the incubation, the wells were gently washed with 0.1 M PBS (pH 7.3) (Shanghai Sangon Biotechnology Co., Ltd., China) to remove unattached planktonic cells. The formed biofilm was fixed with 99% methanol (Sinopharm Group Co., Ltd., China) for 20 min, then washed once with PBS and stained with 1% crystal violet (CV, Bio-Technology Co., Ltd., China) for 20 min. After staining, the wells were rinsed with PBS to remove unbound crystal violet, and the microplate was then air-dried for 1 h.

[0069] Finally, the optical density (OD) of each well was measured at a wavelength of 570 nm using a Varioskan Flash spectrophotometer (Thermo Fisher Scientific, China) to quantify biofilm formation. All experiments included three biological replicates.

[0070] After treatment with MAC4, the biofilm quality was evaluated by measuring the absorbance at 570 nm in this example, and the inhibition rate was calculated as a percentage of the untreated control group. Figure 2 shown. Figure 2 A and Figure 2 B shows the biofilm formation of Escherichia coli and Staphylococcus aureus, respectively. The horizontal axis is the concentration of MAC4, "control" is the negative control (untreated control group), "CIP" is the positive control, and the vertical axis is the biofilm formation (%). Compared with the untreated control group, the biofilm formation of Escherichia coli and Staphylococcus aureus was inhibited at all tested concentrations of MIC, as shown in Figure 2. Figure 2 A and Figure 2B. In particular, the treatment groups with MAC4 concentrations of 2 and 4 times the MIC significantly inhibited the biofilm formation of these two bacteria. According to the results of the present embodiment, for Escherichia coli and Staphylococcus aureus, there is a positive correlation between the higher concentration of MAC4 and the larger biofilm inhibition rate. This result is consistent with the metabolic activity results in the biofilm determined by the MTT assay, which assesses the metabolic activity of living cells in the biofilm. After the MAC4 treatment with 2MIC and 4MIC concentrations, the metabolic activity of the biofilm was reduced by more than 94.97%. Figure 2 C results indicate that bacterial viability or biofilm density (biofilm metabolic activity) is reduced. Figure 2 A~ Figure 2 In C, at a concentration of 2 MIC, the efficacy of MAC4 was comparable to that of ciprofloxacin, especially in the assays of inhibiting biofilm formation and reducing metabolic activity. Both ciprofloxacin and MAC4 provided in this example achieved a reduction in biofilm metabolic activity of more than 90%, indicating that MAC4 effectively inhibited the formation of bacterial biofilms.

[0071] Furthermore, in order to evaluate the effect of MAC4 on E. coli biofilm, scanning electron microscopy (SEM) analysis was also performed in this example.

[0072] First, without any treatment, the Escherichia coli culture was cultured under suitable conditions for 6 hours to promote the formation of biofilm on the test tube wall. The biofilm formed was then treated with MAC4 at the minimum inhibitory concentration (MIC, 0.1C) of MAC4 and continued to be cultured at 37°C for 24 hours. After the culture was completed, the sample was carefully poured and fixed with 25% glutaraldehyde (China National Pharmaceutical Group Co., Ltd.). Subsequently, an ethanol gradient was used for continuous dehydration, and 30%, 50%, 70%, 80%, 90% and 95% ethanol were used for each treatment for 15 minutes, and finally treated twice in 100% ethanol for 20 minutes each time. The sample was fixed in 1% osmic acid for 2 hours and then rinsed three times with 0.1M PBS (pH 7.3). The sample was dried using supercritical carbon dioxide drying (Quorum Technologies Ltd., K850). The surface of the dried sample was sputtered with gold (Hitachi Co., Ltd., Japan, MC1000) to improve conductivity and ensure high-quality SEM imaging. Under high vacuum conditions (accelerating voltage 3.0 kV), field emission scanning electron microscopy (SU8010, Hitachi, Japan) was used to obtain SEM images to observe in detail the changes in biofilm structure and biomass after MAC4 treatment. Two biological replicates were set for each condition, and several areas of each sample were randomly selected for testing to avoid selection bias and ensure the reliability of the results.

[0073] Compared with the control group, the biofilm density of Escherichia coli and Staphylococcus aureus was reduced and the cell structure was destroyed after MAC4 treatment. Figure 2 D Figure 2 The scale bars from left to right in D are 50 μm, 5 μm, and 1 μm, respectively. In the E. coli control group, bacterial cells were connected to each other by filamentous structures, which may be composed of extracellular DNA (eDNA) or matrix fibers, which are components of the biofilm matrix and provide a framework for bacterial survival and movement. After MAC4 treatment, this example observed a significant decrease in the connectivity of these structures in E. coli and S. aureus, as shown in Figure 2. Figure 2 D. The results indicate that MAC4 can directly act on eDNA, causing its denaturation and inactivation, and affecting the synthesis of matrix fibers, thereby weakening their connectivity.

[0074] Figure 2 The results showed that MAC4 has the ability to effectively inhibit biofilm formation and disrupt the interactions between bacterial cells within the biofilm.

[0075] This example also evaluates the ultrastructural changes of bacteria by transmission electron microscopy (TEM), such as Figure 2 As shown in E Figure 2 The scale bars from left to right in E are 1 μm, 500 nm, and 200 nm, respectively).

[0076] The ultrastructure of the bacteria was examined by transmission electron microscopy (TEM) after treatment with MAC4 (0.1 times the minimum inhibitory concentration) or sterile water control. The specific steps are as follows:

[0077] The bacterial suspension was centrifuged at 4000 × g for 5 min and fixed with 25% glutaraldehyde in 0.1 M PBS (pH 7.3) at 4°C for 24 h. The fixed cells were centrifuged again at 4000 × g for 5 min and rinsed three times with 0.1 M PBS (pH 7.3). The samples were then dehydrated through a series of ethanol gradients: 30%, 50%, 70%, 90%, and 100% ethanol for 15 min each. The dehydrated samples were fixed in grade acetone (Sinopharm Group Co., Ltd., China) for 20 min. Subsequently, Spurr low-viscosity epoxy resin was mixed with acetone in a ratio of 1:1 (1 h), 3:1 (3 h), and finally soaked in pure resin overnight. The resin-embedded samples were polymerized at 70°C for 12 h. After polymerization, ultrathin sections with a thickness of 70 to 90 nm were prepared. Each tissue section was stained with a mixture of 10% lead chloride and uranyl acetate in 50% ethanol (Sinopharm Group Co., Ltd., China) for 10 minutes. Finally, the samples were imaged under high vacuum conditions using a Hitachi HT7800 transmission electron microscope (Hitachi, Japan) at an accelerating voltage of 100 kV, with a very short exposure time to prevent sample damage. Two biological replicates were set for each condition, and several areas of each sample were randomly selected for testing.

[0078] This example compares the untreated control group with the MAC4-treated bacteria, revealing the significant morphological differences caused by MAC4 treatment on the bacteria. Figure 2 As shown in E, in the control group, E. coli and S. aureus maintained their characteristic rod-shaped and spherical morphologies, respectively, with intact cell walls, clearly defined cell membranes, and uniformly dense cytoplasm. However, MAC4 treatment induced significant morphological disruption, including uneven cytoplasm distribution, blurred edges, overflow of contents, increased transparency of cell edges, plasmolysis, and DNA agglutination. These structural changes indicate that the integrity of the cell is impaired, which can hinder key bacterial functions, reduce its survival rate, and lead to cell death.

[0079] Example 2

[0080] This example explores the effect of MAC4 on the adhesion ability of Escherichia coli, and the specific steps are as follows:

[0081] After E. coli was treated with MAC4 at a concentration of 0.1C for 24 hours, the bacterial cells were collected by centrifugation at 3000 rpm / min for 10 minutes. The collected bacterial cells were resuspended in PBS and incubated in a FITC solution (Solabo Technology Co., Ltd.) with a final concentration of 100 μg / mL for 2 hours in a dark environment. After the incubation, the bacterial cells were collected by centrifugation again and washed three times with PBS to remove excess FITC. The washed bacterial cells were resuspended in PBS and appropriately diluted for subsequent experiments. HaCaT cells were plated at approximately 2×10 per well. 5 The cells were inoculated into a 24-well plate at a density of 100 μL and incubated at 37°C and 5% CO2 for 24 hours. 100 μL of E. coli suspension stained with FITC was added to each well and incubated in the dark for 2 hours to allow the bacteria enough time to adhere to the HaCaT cells. After the incubation, each well was washed with PBS to remove the unattached bacteria. The HaCaT cell suspension was digested with trypsin-EDTA (0.25%, v / v, Gibco, USA). The bacterial suspensions of the control group and the treatment group were mixed separately, and 200 μL of the suspension was transferred to a black 96-well plate. The fluorescence intensity was measured using a Varioskan Flash microplate reader (Thermo Scientific, China) to quantify the number of attached bacteria. The E. coli adhesion rate of each group was calculated based on the fluorescence intensity. The entire experiment was repeated three times.

[0082] The results of this example are as follows Figure 3 The adhesion rate of untreated E. coli to HaCaT cells was 6.85%, which dropped significantly to 0.81% after MAC4 treatment. The adhesion efficiency of treated E. coli to HaCaT cells was significantly reduced ( Figure 3 A) The significant decrease in adhesion efficiency after MAC4 treatment for 24 h indicates significant damage to cells during bacterial adhesion.

[0083] Example 3

[0084] This example was conducted by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. All mouse experiments were conducted using six-week-old female ICR mice (provided by China Weitong Lihua Company) and were conducted in strict compliance with animal ethics guidelines and approved research protocols. Before the formal experiment began, all mice underwent a one-week adaptive feeding period to ensure their adaptation to the new environment.

[0085] After the adaptation period, the mice were randomly divided into six experimental groups, each containing six mice, namely: control group: no treatment; model group (pre-treatment group): used to establish the disease model, but not treated; ciprofloxacin treatment group (CIP group): given ciprofloxacin (Zhejiang Jingxin Pharmaceutical Co., Ltd., which is commonly used in the clinical treatment of pneumonia caused by Escherichia coli infection); low-dose, medium-dose and high-dose MAC4 treatment groups (respectively marked as low MAC4 group, medium MAC4 group, high MAC4 group): given different concentrations of MAC4 drug treatment. All MAC4 and ciprofloxacin solutions were prepared with 0.9% saline.

[0086] This example further provides an in vivo antibacterial activity assay of MAC4.

[0087] Specifically, this example conducted a bacterial lung infection experiment on mice, and the specific steps were as follows:

[0088] An overnight culture of E. coli ATCC 25922 was washed and resuspended in 0.9% sodium chloride solution to adjust the concentration to 1 × 10 9 CFU / mL. Starting from the first day after infection, each group of mice received corresponding treatment. Except for the control group, the remaining mice received intranasal injection of 50 μL containing 1×10 9 CFU / mL Escherichia coli suspension. The control group received the same volume of 0.9% sodium chloride solution as a placebo. The control group and model group mice were orally administered with 0.9% sodium chloride solution; the CIP group was given ciprofloxacin (dose of 0.1 g kg -1 ·d -1 ); the low-dose MAC4 group was given MAC4 (dose of 0.5 g kg -1 ·d -1 ); the medium-dose MAC4 group was given MAC4 (1.0 g kg -1 ·d -1 ); the high-dose MAC4 group was given MAC4 (dose of 1.5 g kg -1 ·d -1 ). The treatment cycle was 10 consecutive days. All mice were euthanized 24 hours after the last administration, and lung tissues were quickly removed. Lung tissues were immediately frozen in liquid nitrogen and stored in a -80°C freezer for further analysis.

[0089] This example evaluates the bacterial load in lung infection caused by E. coli, and specifically performs the following colony forming unit (CFU) assay.

[0090] Each lung tissue sample collected by the above method was accurately weighed and homogenized in 0.8 mL of sterile saline to obtain a uniform suspension. The above homogenate was carefully spread on MacConkey agar plates (Qingdao Haibo Biotechnology Co., Ltd., China). The coated plates were then incubated at 37°C for 24 hours. After the incubation, the colonies formed by Escherichia coli were counted and the number of CFU per gram of lung tissue was calculated accordingly.

[0091] like Figure 4 As shown, lung bacterial counts performed three days after infection showed a significant reduction in bacterial loads in mice treated with MAC4 compared with controls ( Figure 4 A). These results suggest that MAC4 has potent antibacterial activity in living animal models, providing good prospects for its therapeutic application.

[0092] This example also provides histopathological test content, and the operating steps of histopathological analysis are as follows:

[0093] First, lung tissue was carefully excised and immediately fixed with 4% paraformaldehyde solution (Biyuntian Biotechnology, China). Fixation was performed at room temperature for 24 h. After fixation, the tissue was dehydrated through a series of ethanol solutions with increasing concentrations; the dehydrated tissue was then transparentized with xylene; and finally, the transparent tissue was immersed in molten paraffin for embedding. Paraffin-embedded blocks were prepared into tissue sections with a thickness of approximately 5 μm using a microtome (McIlwain tissue chopper; Ted Pella, Inc., USA). Tissue sections were flattened on slides and firmly attached by heating to prepare for subsequent staining steps. Next, hematoxylin-eosin (H&E) staining kit (Biyuntian Biotechnology, China) was used for staining, and the sections were stained according to the instructions provided by the manufacturer. The stained sections were examined under an optical microscope (CX-40, Olympus America, Inc., Melville, NY, USA) for histopathological evaluation.

[0094] Figure 4B shows that the lung tissue of the control group showed normal structural characteristics, the alveolar cavity remained intact, and the degree of inflammatory cell infiltration was extremely low. In contrast, the model group showed signs of infection-induced changes, such as increased inflammatory cell infiltration (clearly visible by the dense dark purple cell clusters shown by the blue arrows), blurred alveolar cavities (indicating edema or exudate), and thickening of alveolar walls (reflecting inflammation). Treatment with MAC4 was similar to the effect of ciprofloxacin, a known drug for the treatment of pneumonia, and both significantly improved the condition of lung tissue. The number of cells in all treatment groups decreased, and the MAC4-treated group showed a dose-dependent effect. The degree of inflammatory cell infiltration was determined by the presence and density of dark purple cell clusters, which was significantly reduced in the treatment groups, especially in the medium and high dose groups of MAC4. The lung structure of all test groups became clearer, and the thickening of blood vessels and bronchiolar walls was also reduced. Among them, the high dose group of MAC4 significantly reduced inflammation (as evidenced by the phenomenon that dark cell clusters were almost invisible) and preserved the integrity of the lungs in the mouse model of E. coli-induced pneumonia.

[0095] Furthermore, this embodiment provides transcriptomic analysis.

[0096] The specific steps for RNA extraction are as follows:

[0097] Tissue or bacterial samples were lysed in 2 mL tubes with 1.5 mL TRIzol Reagent (Qiagen Life Sciences). For tissue samples, they were ground with a small amount of liquid nitrogen before adding lysis buffer and incubating for 5 min. Samples were centrifuged (12,000 × g, 5 min, 4 °C), and the supernatant obtained after centrifugation was mixed with 300 μL of dichloromethane / isoamyl alcohol (24:1, Sinopharm Group Co., Ltd.) by gentle inversion. After centrifugation (12,000 × g, 8 min, 4 °C), the aqueous phase (the aqueous phase containing RNA) was precipitated with 2 / 3 volume of isopropanol (Sinopharm Group Co., Ltd.) at -20 °C for 2 h. After centrifugation (17,500 × g, 25 min, 4°C), the precipitate was washed once with 0.9 mL of 75% ethanol, centrifuged (17,500 × g, 3 min, 4°C), briefly blown dry (3–5 min), and resuspended with 20–200 μL of RNase-free water (Biyuntian Biotech) depending on the amount of precipitate for subsequent analysis.

[0098] This example conducted a validation test of differentially expressed genes.

[0099] To explore the changes in inflammatory cytokines after MAC4 treatment, RT-PCR analysis was performed on specific genes related to inflammation (IL-10, IL-6, IL-1β, and TNF-α) in the model group and the medium-dose MAC4 treatment group. The primer sequences used for this analysis (Shanghai Sangon Biotechnology Co., Ltd.) are listed in the sequence table, where F and R represent the forward primer and reverse primer of the corresponding gene, respectively. The GAPDH gene was used as a reference gene. RNA was extracted using the Trizol kit (Ambion, USA), followed by reverse transcription using the PrimeScript RT Master Mix kit (Takara) to obtain cDNA. RT-PCR was performed using SYBR Green Master Mix (Yisheng Biotechnology (Shanghai) Co., Ltd.) and the Applied Biosystems QuantStudio 6Flex real-time fluorescence quantitative PCR system (Applied Biosystems, CA, USA).

[0100] like Figure 3 As shown, this example identified 1150 differentially expressed genes (DEGs), of which 490 were up-regulated and 660 were down-regulated (e.g. Figure 3 B). GO enrichment analysis of down-regulated genes revealed that they were focused on membrane-related components, especially the integral components of the membrane (GO: 0005886), cell membrane (GO: 0005886) and outer membrane (GO: 0019867) (as shown in Figure 3 C). This result suggests that MAC4 specifically disrupts membrane integrity and function. In addition, the downregulated DEGs showed enrichment in DNA binding and recombination activities, indicating that MAC4 impairs genetic processes that are critical for bacterial survival and adaptation.

[0101] KEGG pathway analysis showed that there were significant enrichments in pathways such as glycerophospholipid metabolism (ko00564), ABC transporters (ko02010), DNA replication (ko03030), lipopolysaccharide biosynthesis (ko00540), and bacterial invasion of epithelial cells (ko05100). Figure 3 D. In particular, the ABC transporter pathway, which is essential for nutrient uptake and toxin excretion, emerged as a major target for downregulation, along with pathways involved in energy metabolism and biofilm formation. Figure 3 The results showed that the bactericidal mechanism of MAC4 covers a broad spectrum of interference with key bacterial functions, especially from membrane integrity to energy metabolism and genetic processes.

[0102] Furthermore, in order to confirm the accuracy of the research results of differentially expressed genes, this example performed RT-qPCR verification on some randomly selected genes (frdC, yniA, yqhD, dhaL, cspI, yeeJ, rbsA, tdcR, evgS, fumD), using rrsG (16S rRNA gene) as a standardized control. The primer sequences used are shown in the sequence table, where F and R represent the forward primer and reverse primer of the corresponding gene, respectively. Figure 3 As shown in E, the high correlation coefficient (R 2 =0.77) verified the reliability of the DEGs identified in this example. The results of this example illustrate the complex antibacterial mechanism of MAC4, indicating that it not only plays a role in inhibiting biofilm formation and affecting bacterial adhesion, but also plays a dual role in disrupting bacterial morphology and cellular processes. The transcriptomic changes observed in this example further reveal the inhibitory ability of MAC4 in key pathways that bacterial survival and communication depend on.

[0103] This example also provides protein extraction and Western blot analysis content, the specific steps are as follows:

[0104] A portion of lung tissue was cut into small pieces. Subsequently, 150-250 μL RIPA lysis buffer (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.) was added to each 20 mg of tissue, and a protease inhibitor (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., GRF101) and a phosphatase inhibitor (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., GRF102) were added to the RIPA lysis buffer at a ratio of 1:100. The tissue was thoroughly homogenized using a glass homogenizer until it was completely lysed. After the sample was completely lysed, it was centrifuged at 12000xg for 30 min at 4°C. The supernatant containing the protein sample was transferred to a new centrifuge tube, and 80 μL of the supernatant and 20 μL of 5x non-deformable protein sampling buffer (Solabo, China) were added to it and treated at 100°C for 10 min for subsequent experimental analysis. In this example, IL-10 (ab225820, Abcam, UK), β-actin (ab8226, Abcam, UK), IL-6 (D5H4V, CST, USA), IL-1β (D6D6T, CST, USA) and TNF-α (AF7014, Affinity Biosciences, USA) were subjected to Western blotting analysis. Anti-rabbit IgG-HRP secondary antibody (BL003A, Biosharp) and anti-mouse IgG-HRP secondary antibody (BL001A, Biosharp) were also used in this example. β-actin was used as an internal reference for quantitative analysis. Three-color pre-stained protein (Shanghai Yamei Biopharmaceutical Technology Co., Ltd., WJ103) was used to track the molecular weight of the protein. PVDF membrane (Merck Millipore, Germany, IPVH00010) was used for membrane transfer. Protein immunoblotting bands were detected in the Bio-Rad GelDoc XR+ imaging system, and semi-quantitative analysis was performed using ImageJ software.

[0105] Figure 4 C~ Figure 4 D The results show that after MAC4 treatment, the expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α) is significantly reduced. The expression of anti-inflammatory cytokine IL-10 increases. This result is supported by RT-PCR and Western blot analysis, indicating that MAC4 not only has antibacterial effects, but also can regulate host immune response and reduce inflammatory response. The data of this example show that MAC4 has potential antibacterial and immunomodulatory effects in the treatment of bacterial pneumonia, which is of great significance for the treatment or relief of various bacterial lung infections and related inflammations.

[0106] This example provides research content on the antibacterial mechanism of MAC4 against Escherichia coli in vitro and in vivo. The steps of library construction and transcriptome sequencing are as follows:

[0107] A total of eight E. coli samples were extracted and treated, four of which were treated with MAC4 at 0.1 times the minimum inhibitory concentration (MIC), and the other four served as controls. Eight E. coli samples were used for in vitro analysis. Total RNA was extracted from these samples using the TRIzol kit (Qiagen Life Sciences). The quality of RNA was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2200 TapeStation (Agilent Technologies). Ribosomal RNA was removed from total RNA using the QIAseq FastSelect-5S / 16S / 23S kit (Qiagen Life Sciences). The remaining RNA was fragmented and first-strand cDNA was synthesized by reverse transcription using random primers and amitramycin D. The synthesized double-stranded cDNA was then purified, followed by end repair, dA tailing, and adapter ligation. DNA fragments of approximately 400 bp in length were isolated by magnetic bead-based size selection. Each sample was PCR amplified using specific P5 and P7 primers and then purified again using a magnetic bead-based method. The quality and concentration of the library were verified using a Qsep100 bio-fragment analyzer (Bioptic, Taiwan, China) and a Qubit 3.0 spectrophotometer (Invitrogen Life Sciences, Inc., USA). Finally, sequencing was performed using the Illumina Novaseq 6000 platform, using a 2×150 bp paired-end sequencing mode.

[0108] The in vivo analysis steps provided in this embodiment are as follows:

[0109] For six lung tissue samples (three samples per group), samples were collected before and after MAC4 treatment. Total RNA samples that passed the quality check were digested with DNase I. Oligo(dT) magnetic beads were used to enrich mRNA; after mRNA was fragmented, it was combined with random primers to synthesize the first strand of cDNA. dUTP was used instead of dTTP for the synthesis of the second strand of cDNA. After the double-strand synthesis of cDNA was completed, end repair, "A" tail addition, and adapter ligation were performed. After the PCR reaction, the adapter ligation product was treated with UDG enzyme to digest the second strand template labeled with U. PCR amplification was then performed and the PCR product was recovered. After the quality assessment of the library, the PCR product was single-stranded circularized and the uncircularized linear DNA molecules were removed to obtain the final library. Finally, through rolling circle replication, the single-stranded circular DNA molecules formed DNA nanoballs (DNBs) for subsequent sequencing.

[0110] Data processing and enrichment analysis

[0111] 1. Raw data filtering

[0112] For in vitro samples, Cutadapt (v1.9.1) was used to trim reads to remove low-quality parts and adapter sequences.

[0113] For in vivo samples, SOAPnuke (v1.5.2) was used to complete similar tasks.

[0114] 2. Alignment with reference genome

[0115] Reads from in vitro samples were aligned to the reference sequence of Escherichia coli ATCC 25922 (ASM74325v1).

[0116] Reads from in vivo samples were aligned to the reference genome of mouse (Mus musculus) (GCF_000001635.27_GRCm39).

[0117] The comparison tool is Bowtie2 (v2.2.6).

[0118] 3. Expression level calculation

[0119] HTSeq (v0.6.1) and RSEM (v1.2.8) were used to quantify gene expression levels.

[0120] 4. Differential Expression Analysis

[0121] Differential expression analysis was performed using the DESeq2 package to compare the differences between the control and treatment groups to identify differentially expressed genes (DEGs).

[0122] For in vitro samples, the criteria for DEGs were log2(fold change) of absolute value ≥1 and adjusted P value (Padj) <0.05.

[0123] For in vivo samples, the criteria were log2 of absolute value (fold change) ≥ 1 and P value < 0.05.

[0124] 5. Functional enrichment analysis

[0125] For in vitro samples, GOSeq (1.34.1) and KOBAS (v2.0) software were used to perform enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). For in vivo samples, a similar enrichment analysis was performed using the DAVID database. This embodiment validated the selected differentially expressed genes (DEGs) from in vitro and in vivo studies by RT-PCR analysis. To ensure optimal amplification efficiency, gene-specific primers (Shanghai Bioengineering) were designed in this embodiment for gene amplification. In in vitro experiments, the rrsG gene was selected as an internal reference gene. For in vivo experiments, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was selected as an internal reference gene. RNA extraction and RT-PCR procedures followed the previously described protocol.

[0126] like Figure 5 As shown in A, RNA-Seq analysis identified 535 differentially expressed genes (DEGs), of which 310 were upregulated and 225 were downregulated after MAC4 treatment. Figure 5 As shown in F, the selected DEGs were validated by RT-qPCR in this example, confirming the strong positive correlation with RNA-Seq data (R 2 =0.85), thus confirming the reliability of the transcriptomics results. The primer sequences used are shown in the sequence table, where F and R represent the forward primer and reverse primer of the corresponding gene, respectively. In particular, MAC4 treatment upregulated genes involved in immune response and neutrophil production, such as Csf3 and Alox15, indicating that the host defense mechanism was enhanced. At the same time, the downregulation of genes such as Ccl7 and Adtrp indicated that the immune response was strategically regulated, which may reduce excessive inflammation and tissue damage.

[0127] Figure 5 Gene enrichment analysis of B showed that the upregulated genes were mainly involved in immune response process (GO:0002376 and GO:0045087), transcriptional regulation (GO:0006357 and GO:0000122), and metabolic adjustment (GO:0006629), which are crucial for infection control. Figure 5C showed that the downregulated genes were related to cell adhesion (GO: 0007155) and apoptosis processes (GO: 0006915), suggesting the existence of mechanisms to limit pathogen spread and cell death. The reduction in expression in inflammatory response (GO: 0006954) can reduce the inflammatory response after infection. In addition, changes in lipid metabolism (GO: 0006629) can affect the integrity and function of the cell membrane, which is essential for resisting pathogen invasion. KEGG pathway analysis found upregulation of immune-related pathways, including cytokine-cytokine receptor interaction (mmu04060) and Toll-like receptor signaling pathway (mmu04620), indicating an enhanced immune response, such as Figure 5 D. Downregulation of the P13K-Akt signaling pathway (mmu04151) indicated a reduction in the activation and survival of inflammatory cells, leading to reduced inflammation, as Figure 5 E. Therefore, the data in this example show that MAC4 is able to inhibit the proliferation of pathogens during lung infection and modulate the host immune response, which highlights its therapeutic potential against bacterial pathogens.

Claims

1. A pharmaceutical composition for treating bacterial infection, characterized in that: The pharmaceutical composition comprises malic acid, fumaric acid, glycine and hippuric acid.

2. The pharmaceutical composition according to claim 1, characterized in that The mass percentage of malic acid is 40 to 80%.

3. The pharmaceutical composition according to claim 1, characterized in that The mass percentage of fumaric acid is 5-10%.

4. The pharmaceutical composition according to claim 1, characterized in that The mass percentage of glycine is 5-10%.

5. The pharmaceutical composition according to claim 1, characterized in that The mass percentage of hippuric acid is 10-20%.

6. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of malic acid, fumaric acid, glycine and hippuric acid is 12:1:1:

3.

7. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a substance for inhibiting the formation of bacterial biofilm.

8. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a medicament for treating infections caused by microorganisms.

9. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of daily chemical products for reducing oral pathogenic bacteria colonization.

10. The use according to claim 9, characterized in that: The daily chemical products include toothpaste, mouthwash or oral spray.

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