Use of a small peptide in the preparation of a drug for preventing and treating klebsiella pneumoniae infection and its related lung injury

Abstract

The application discloses an application of a small peptide in the preparation of a drug for preventing and treating Klebsiella pneumoniae infection and lung injury related thereto, and belongs to the technical field of new application of bioactive peptides. Research proves that the small peptide can regulate host immune response related to Klebsiella pneumoniae infection in vivo and in vitro. It is further confirmed that the small peptide mainly plays a role by inducing and regulating pyroptosis of alveolar macrophages. Mechanism research proves that the small peptide can promote activation of inflammasome-related signals, enhance Caspase-1 activation and GSDMD lysis, promote maturation and release of proinflammatory factors such as IL-1β, thereby regulating inflammatory response under the condition of infection and improving lung tissue injury. The above results show that the small peptide inhibits the pathological process related to Klebsiella pneumoniae infection through a pyroptosis-related immune regulation mechanism, and provides an experimental basis for the small peptide to be used for preparing a drug for preventing and treating diseases related to Klebsiella pneumoniae infection.

Description

Technical Field

[0001] This invention belongs to the field of new applications of bioactive peptides, specifically relating to the application of a small peptide in the preparation of drugs for the prevention and treatment of Klebsiella pneumoniae infection and related lung injuries. Background Technology

[0002] Klebsiella pneumoniae is an important opportunistic pathogen and one of the leading causes of hospital-acquired infections and severe community-acquired infections. In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) and highly virulent Klebsiella pneumoniae (hvKP) have continued to circulate clinically, leading to a significant increase in anti-infective treatment failure rates and mortality. Current treatments primarily rely on antibiotics to control the pathogen load; however, in the context of widespread drug-resistant strains, their efficacy is severely limited, and they struggle to effectively control infection-induced excessive inflammatory responses and tissue damage.

[0003] During lung infections, alveolar macrophages, as the first line of defense in the lung's innate immunity, play a crucial role in pathogen recognition, phagocytosis and clearance, and inflammation regulation. However, under severe bacterial infection conditions, alveolar macrophage function becomes imbalanced, often leading to amplified inflammatory responses, abnormal cell death, and exacerbated tissue damage. Therefore, exploring new intervention strategies to alleviate infection-related damage by regulating host immune cell function has become an important direction in current anti-infection research.

[0004] Recent studies have discovered that non-coding RNAs contain translatable small open reading frames that encode biologically active short peptide molecules. The roles of these encoded small peptides in immune regulation and disease development are increasingly attracting attention, but their function and application in bacterial infection-related lung injury still require further investigation. Summary of the Invention

[0005] Technical problem solved: To address the above-mentioned technical problems, this invention provides an application of encoded small peptides in the preparation of drugs for the prevention and treatment of Klebsiella pneumoniae infection and related lung injuries, breaking through the traditional anti-infective treatment route that mainly relies on antibiotics, and providing a new biological therapy strategy for the prevention and treatment of infections in the context of drug-resistant strains.

[0006] Technical solution: The application of a small peptide in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injury, wherein the amino acid sequence of the small peptide is shown in SEQ NO: 1.

[0007] Preferably, the encoded small peptide is used to promote the activation of inflammasome-related signals, induce changes in cell membrane permeability and release of inflammatory factors under infection conditions, thereby regulating infection-related immune responses.

[0008] Furthermore, the inflammatory factors include IL-1β, IL-6, TNF-α, and IL-18.

[0009] Preferably, the drug exerts its immunomodulatory effect by inducing or regulating pyroptosis in alveolar macrophages, wherein the pyroptosis is a programmed cell death induced in alveolar macrophages by a small peptide encoded under Klebsiella pneumoniae infection conditions.

[0010] Furthermore, the target proteins of the encoded small peptide in alveolar macrophages are STK24 and Samm50.

[0011] Furthermore, the prerequisite for pyroptosis is the activation of the inflammasome signaling pathway.

[0012] Furthermore, the activation of the inflammasome signaling pathway includes the assembly and activation of the NLRP3 inflammasome, activation of Caspase-1, cleavage of GSDMD and its N-terminal fragment-mediated cell membrane pore formation.

[0013] A drug for the prevention and treatment of Klebsiella pneumoniae infection and related lung injury, comprising a small peptide encoded with an amino acid sequence as shown in SEQ NO: 1 and pharmaceutically acceptable excipients.

[0014] Preferably, the drug is a tablet, capsule, powder, granule, solution, suspension, injection, inhaler, or spray.

[0015] Preferably, the excipients include one or more of the following: fillers, stabilizers, diluents, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, or lubricants. Beneficial effects

[0016] New applications of anti-infectives are expanding: This invention uses encoded small peptides to prepare drugs for the prevention and treatment of Klebsiella pneumoniae infection and related lung injury, breaking through the traditional anti-infective treatment route that mainly relies on antibiotics, and providing a new biotherapy strategy for the prevention and treatment of infection in the context of drug-resistant strains. Clear immune regulation mechanisms: This invention reveals that the encoded small peptide exerts its anti-infective effect by inducing and regulating pyroptosis of alveolar macrophages, which is different from the direct pathogen-killing mechanism of traditional antibiotics. This mechanism promotes the release of inflammatory factors and enhances the host's immune response by activating the inflammasome-Caspase-1-GSDMD signaling axis, thereby improving the pathogen clearance efficiency. Protective effect against infection-related lung injury: In vitro and in vivo experimental results show that the encoded small peptide can effectively regulate the function of alveolar macrophages, improve the infection-induced inflammatory microenvironment, reduce lung tissue inflammation and tissue damage, and has no obvious toxicity to normal tissues, showing good potential for application safety. Promising application prospects: The encoded small peptide provided by this invention is a novel immunomodulatory active molecule that can be used to develop new anti-infective biological agents or immunotherapeutic drugs, and has broad application prospects in the prevention and treatment of Klebsiella pneumoniae infection and related lung injury. Attached Figure Description

[0017] Figure 1 Figure showing the survival and cell status of alveolar macrophages after treatment with encoded small peptides; Figure 2 Figure showing the protein and RNA levels of pro-inflammatory factors in alveolar macrophages after treatment with small peptides; Figure 3 The image shows the detection results of pro-inflammatory factors in the supernatant of cultured cells after treatment with small peptides. Figure 4 The image shows the results of Western blot analysis of pyroptosis-related proteins after treatment with small peptides. Figure 5 To screen and identify target proteins encoding small peptides in macrophages; Figure 6 This diagram illustrates the mechanism of action of the small peptides. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0019] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are conventional methods; unless otherwise specified, the materials and reagents used in the embodiments are commercially available products.

[0020] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0021] Example 1: Application of encoded small peptides in the preparation of drugs for preventing and treating lung injury associated with Klebsiella pneumoniae infection. This embodiment provides the application of a small peptide (Ptges-AS1-52aa) derived from the antisense strand of the PTGES locus in the preparation of drugs for the prevention and treatment of Klebsiella pneumoniae infection and related lung injury. The peptide is derived from a non-coding transcript of the antisense strand of the PTGES locus, which consists of multiple exons and is approximately 1772 bases in length. Following lncRNA nomenclature rules, this transcript is named Ptges-AS1. This transcript contains a translatable small open reading frame (ORF), which encodes a small peptide of approximately 52 amino acids. The amino acid sequence of the peptide is shown in SEQ NO: 1. MPHIPATQLRKSLSRRLSLPRAQEERKLQAGLAMSFLQLEANWKMQGSLFKG.

[0022] The encoded small peptide exerts an immunomodulatory effect by regulating pyroptosis of alveolar macrophages.

[0023] I. Experimental Materials and Setup: Cell culture: Macrophage cell lines were all purchased from the American Type Culture Collection (ATCC). In accordance with the ATCC guidelines, all cells used in this application were cultured in DMEM / 1640 medium containing 10% FBS and 1% antibiotics under humidified conditions of 37°C and 5% carbon dioxide.

[0024] II. Experimental Methods: 1. Cell viability assay: 3000 cells were seeded into 96-well plates with 6 replicates. Cell viability was assessed using a CCK-8 assay kit.

[0025] 2. Cell phagocytosis assay: Macrophages were co-incubated with KP (MOI=10) for 30 min, washed with PBS, and treated with Gentamicin (100 μg / mL) for 30 min. After washing with PBS, cells were lysed with 0.1% Triton X-100 for 5 min. The lysates were collected, serially diluted 10-fold, and plated on LB agar plates. After incubation at 37°C overnight, colony-forming units (CFU) were counted to evaluate the intracellular phagocytic / endocytic viable bacterial load.

[0026] 3. Protein Immunoblot Assay: Total protein was extracted from cultured macrophages, washed twice with 1×PBS, and then lysed thoroughly with RIPA lysis buffer on ice. The lysis buffer was centrifuged at 12,000×g for 10 min (4℃), and the supernatant was collected. Protein concentration was determined using the BCA method. Equal volumes of protein samples were separated by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose (NC) membrane. The membrane was blocked in 5% skim milk at room temperature for 1 h, and then incubated overnight at 4℃ with the corresponding primary antibody. Primary antibodies included pyroptosis and inflammation-related antibodies: NLRP3 (CST, 15101S, 1:1000), ASC (Pycard) (CST, 67824S, 1:1000), Caspase-1 (CST, 3866S, 1:1000), Cleaved Caspase-1 (p20) (AdipoGen, AG-20B-0042, 1:1000), GSDMD (CST, 96458S, 1:1000), Cleaved GSDMD (GSDMD-N) (Abcam, ab215203, 1:1000), IL-1β (CST, 12703S, 1:1000), and IL-18 (Abcam, ab191860, 1:1000); inflammatory signaling proteins NF-κB p65 and p-p65 (CST, 8242S / ) were also detected. 3033S (1:1000) and Actin (HUABIO, ET1601-4, 1:10000) were used as internal controls. Subsequently, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 h, and then developed and images were acquired using a chemiluminescent reagent.

[0027] 4. ELISA detection of pro-inflammatory cytokine secretion: Cells were processed according to experimental groups, and culture supernatants were collected at set time points. Cell debris was removed by centrifugation at 12,000×g, 4℃ for 10 min, and the supernatants were used. The levels of pro-inflammatory cytokines such as IL-1β, IL-18, TNF-α, and IL-6 in the supernatant were detected using a commercially available ELISA kit according to the manufacturer's instructions. Two to three technical replicates were set up for each sample. Cytokine concentrations were calculated using a standard curve, and results are expressed as pg / mL.

[0028] 5. RT-qPCR assay for detecting inflammatory factor transcription: After treatment, the culture medium was discarded, cells were washed once with PBS, and total RNA was extracted using the TRIzol method. RNA concentration and purity were then measured. Following the kit instructions, an equal volume of RNA was reverse transcribed into cDNA. Real-time quantitative PCR was performed using the SYBR Green system to detect the mRNA expression levels of inflammation-related genes such as Il1b, Il18, Tnf-α, Il-6, and Nlrp3. Gapdh was used as an internal control gene, and relative expression levels were calculated using the 2^-ΔΔCt method. At least three technical replicates and at least three biological replicates were performed for each sample.

[0029] 6. LDH Release Detection: Cells were seeded in 96-well plates and treated according to experimental groups. Culture supernatants were collected at set time points, and the LDH release was detected according to the LDH detection kit instructions. Cells treated with lysis buffer served as the maximum release control, and cell-free wells served as blank controls. After the reaction, absorbance values ​​were read at the specified wavelength using a microplate reader, and the LDH release rate was calculated using the formula to assess the degree of cell membrane integrity disruption.

[0030] 7. Statistical Data: The statistical analyses used in this application include t-tests and Pearson correlation coefficients applied to the publicly available data. Data are expressed as mean ± standard deviation of mean (SD), * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0031] III. Experimental Results 1. Encoding small peptides improves the survival rate of KP-infected cells. like Figure 1 As shown, under Klebsiella pneumoniae (KP) infection conditions, the cell viability of the group treated with the encoded small peptide was significantly improved. Compared with the KP group, the treatment with the encoded small peptide reduced the degree of infection-induced cell damage and increased the cell survival rate, suggesting that the encoded small peptide has a certain protective effect on host cells during the infection response.

[0032] 2. Encoding small peptides reduces the expression of inflammatory factors. like Figure 2 As shown, in the KP infection model, treatment with the encoded small peptide significantly affected the expression of inflammation-related factors at both the transcriptional and protein levels. Compared with the KP group, the expression levels of multiple inflammation-related genes / proteins in the peptide-treated group changed significantly, showing an overall downregulation trend, suggesting that the encoded small peptide can participate in and regulate the infection-induced inflammatory response.

[0033] 3. Encoding small peptides promote the release of inflammatory factors under infectious conditions. like Figure 3As shown, the secretion levels of IL-1β, TNF-α, IL-6 and IL-18 in the cell culture supernatant of the small peptide-encoded group were significantly higher than those in the control group, indicating that the small peptide-encoded group can enhance the release of inflammatory factors under infectious conditions, thereby affecting the transmission of inflammatory signals and the intensity of the immune response.

[0034] 4. The role of pyroptosis inhibition in reversibly encoding small peptides like Figure 4 As shown in the PI staining results, the proportion of pyroptosis / membrane integrity-impaired cells in the pretreatment group encoding the small peptide was significantly lower than that in the KP group. Simultaneously, Western blot results indicated that treatment with the small peptide significantly inhibited KP-induced Caspase-1 activation and GSDMD cleavage. These results suggest that the effect of the small peptide on infection-related cell damage is closely related to the pyroptosis pathway; inhibition of pyroptosis signaling alters the effects of the small peptide, further supporting the view that the small peptide functions through the pyroptosis pathway.

[0035] 5. STK24 and Samm50 are involved in encoding small peptides that regulate pyroptosis in alveolar macrophages. like Figure 5 As shown, interaction protein screening analysis revealed STK24 and Samm50 as potential binding proteins encoding small peptides. Further experimental results confirmed that both STK24 and Samm50 can specifically bind to the encoded small peptides.

Claims

1. The application of a small peptide encoding a drug in the preparation of drugs for the prevention and treatment of Klebsiella pneumoniae infection and related lung injury, characterized in that, The amino acid sequence encoding the small peptide is shown in SEQ NO:

1.

2. The application of the encoded small peptide according to claim 1 in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injuries, characterized in that, The encoded small peptide is used to promote the activation of inflammasome-related signals, induce changes in cell membrane permeability and release of inflammatory factors under infection conditions, thereby regulating infection-related immune responses.

3. The application of the encoded small peptide according to claim 2 in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injuries, characterized in that, The inflammatory factors include IL-1β, IL-6, TNF-α, and IL-18.

4. The application of the encoded small peptide according to claim 1 in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injuries, characterized in that, The drug exerts its immunomodulatory effect by inducing or regulating pyroptosis in alveolar macrophages, wherein the pyroptosis is a programmed cell death induced in alveolar macrophages by a small peptide encoded under Klebsiella pneumoniae infection conditions.

5. The application of the encoded small peptide according to claim 4 in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injuries, characterized in that, The target proteins of the encoded small peptide in alveolar macrophages are STK24 and Samm50.

6. The application of the encoded small peptide according to claim 4 in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injuries, characterized in that, The prerequisite for pyroptosis is the activation of the inflammasome signaling pathway.

7. The application of the encoded small peptide according to claim 6 in the preparation of drugs for preventing and treating Klebsiella pneumoniae infection and related lung injuries, characterized in that, The activation of the inflammasome signaling pathway includes the assembly and activation of the NLRP3 inflammasome, activation of Caspase-1, cleavage of GSDMD and its N-terminal fragment-mediated cell membrane pore formation.

8. A drug for preventing and treating Klebsiella pneumoniae infection and related lung injury, characterized in that, Includes small peptides encoded with amino acid sequences as shown in SEQ NO: 1, and pharmaceutically acceptable excipients.

9. A drug for preventing and treating Klebsiella pneumoniae infection and related lung injury according to claim 8, characterized in that, The drug is in the form of tablets, capsules, powders, granules, solutions, suspensions, injections, inhalers, or sprays.

10. A drug for preventing and treating Klebsiella pneumoniae infection and related lung injury according to claim 8, characterized in that, The excipients include one or more of the following: fillers, stabilizers, diluents, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, or lubricants.

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