Application of Colistin in preparation of medicine for treating and preventing acute respiratory distress syndrome

Abstract

The invention relates to application of Colistin in preparation of a medicine for treating and preventing acute respiratory distress syndrome, and relates to the technical field of medicines. The Colistin effectively inhibits calcium ion cell influx by inhibiting a mechanical sensitive channel of Piezo1 protein, further prevents and reduces pathological changes such as pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary hyperemia and interval thickening in the ARDS disease, plays an important role in preventing or treating the acute respiratory distress syndrome, and has a huge application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of Colistin in preparing medicines for treating and preventing acute respiratory distress syndrome. Background Art

[0002] Acute Respiratory Distress Syndrome (ARDS) is one of the leading causes of death in patients admitted to intensive care units. ARDS is an acute, diffuse inflammatory lung injury that leads to increased pulmonary vascular permeability and lung weight, and a decrease in air-filled lung tissue. Its etiology is complex, and its pathogenesis remains incompletely elucidated. Major causes include sepsis, massive blood transfusion, severe pneumonia, chest trauma, pulmonary embolism, mechanical ventilation, pure oxygen inhalation, and acute pancreatitis. Numerous studies have demonstrated that pulmonary inflammation is the most important cause of ARDS. Activation of innate and adaptive immunity leads to a series of inflammatory reactions, with the release of large amounts of cytokines and inflammatory mediators exacerbating lung damage. This uncontrolled pulmonary inflammation ultimately results in damage to alveolar epithelial cells and capillary endothelial cells, leading to alveolar hemorrhage, pulmonary edema, and the development of hyaline membranes.

[0003] Treatment options for ARDS include mechanical ventilation and non-mechanical ventilation. Mechanical ventilation involves the use of a ventilator after endotracheal intubation and includes strategies such as lung-protective ventilation, low tidal volume ventilation, and inverse ratio ventilation. Non-mechanical ventilation, on the other hand, primarily involves lung tissue clearance and fluid management, surfactant supplementation, the use of beta-agonists and statins, blood purification, and nutritional intervention.

[0004] The pathogenesis of ARDS is complex, involving multiple mechanisms of injury. Furthermore, ARDS is a highly heterogeneous syndrome, with varying degrees of severity across patients. Currently, clinical trials for ARDS include glucocorticoids, ulinastatin, vitamin C, mesenchymal stromal cells (MSCs), anti-TNFR1 (anti-TNFR1), epithelial / endothelial cell targets, and the alveolar epithelial sodium channel activator (AP-301). However, the efficacy of these agents has yet to be established. To date, there have been no reports of the use of colistin in the development of drugs for the treatment and prevention of acute respiratory distress syndrome. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide the use of Colistin in the preparation of drugs for treating and preventing acute respiratory distress syndrome.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The purpose of the present invention is to provide an application of Colistin in preparing a medicine for treating and preventing acute respiratory distress syndrome.

[0008] The beneficial effects of the scheme of the present invention are: there are currently no reports on the use of colistin in the treatment of acute respiratory distress syndrome. The present invention proves that colistin and Piezo1 protein interact, thereby playing a role in the treatment of ARDS, and colistin has no potential toxicity to human health and survival, and thus provides the use of colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome.

[0009] Furthermore, the colistin is a polypeptide antibiotic produced by Bacillus polymyxa.

[0010] Furthermore, the CAS number of the colistin is No: 1264-72-8.

[0011] The above-mentioned further beneficial effects are: Colistin is an FDA-approved drug that has been marketed and its safety has been fully verified.

[0012] Furthermore, the Colistin treats acute respiratory distress syndrome by inhibiting the infiltration of neutrophils.

[0013] Furthermore, Colistin inhibits the infiltration of neutrophils by inhibiting the mechanical sensitivity channel of Piezo1 protein.

[0014] Specifically, Colistin inhibits the mechanical sensitivity channel of the Piezo1 protein in neutrophils in the body, thereby preventing and reducing the infiltration and number of neutrophils in ARDS. It significantly alleviates phenomena such as alveolar structure rupture, vascular congestion, and interstitial edema in the lungs, and significantly reduces lung pathology. It plays an important role in the prevention and treatment of acute respiratory distress syndrome and has great application prospects.

[0015] Furthermore, the active ingredient of the drug is Colistin.

[0016] Furthermore, the active ingredient of the drug is a pharmaceutically acceptable salt of Colistin.

[0017] Furthermore, the medicine also includes pharmaceutically acceptable carriers and excipients.

[0018] Furthermore, the dosage form of the drug is any medically approved dosage form.

[0019] Furthermore, the dosage of the drug is any therapeutically acceptable dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of establishing and validating the ARDS model of Example 1 of the present invention are presented; (A) is an H&E staining image of mouse lung tissue; (B) is a graph showing the expression levels of inflammatory factors in mouse blood; (C) is a graph showing the number of neutrophils in mouse blood; (D) is the expression level of MPO in mouse lung tissue; and (E) is a gene difference analysis of transcriptome sequencing results.

[0021] Figure 2 The results of the discovery of mouse Piezo1 protein inhibitors and the inhibition of Piezo1 protein function by HY-A0089 in Example 2 of the present invention are presented; (A) is a diagram of the virtual screening process based on the structure of Piezo1 protein; (B) is a diagram of the interaction between the 9 compounds screened virtually and the Piezo1 protein; (C) is the experimental result of immunofluorescence detection of HY-0089 inhibiting Piezo1-mediated calcium influx; (D) is the flow cytometry detection of intracellular Ca 2+ Content; (E) Effect of HY-A0089 on cell survival;

[0022] Figure 3 The results of affinity testing between HY-A0089 and Piezo1 protein in Example 3 of the present invention are presented; wherein (A) is the molecular formula of HY-A0089 and the molecular docking results of HY-A0089 and the CTD domain of Piezo1 protein; (B) ITC detection of the affinity between the CTD domain and HY-A0089; (C) is the SDS-PAGE results of CTD domain protein expression; (D) is the ion exchange chromatography of CTD protein; (E) is the gel filtration chromatography of CTD protein; (F) is the SDS-PAGE results of gel filtration chromatography;

[0023] Figure 4 The results of the therapeutic effect of HY-A0089 on AIDS in Example 4 of the present invention are presented; (A) is a schematic diagram of the mouse model construction and experimental process of Example 4; (B) is the lung injury assessment; (C) is an H&E staining image of the lung tissue of each group of mice; (D) Immunofluorescence detection of neutrophil infiltration in lung tissue; (E) Flow cytometry detection of neutrophil count in lung tissue;

[0024] Figure 5The results of the preventive effect of HY-A0089 on AIDS in Example 5 of the present invention are presented; wherein (A) is a schematic diagram of the construction of the mouse model and the experimental process of Example 5; (B) is an analysis diagram of the expression level of the inflammatory cytokine IL-6 in the mouse blood; (C) is an analysis diagram of the expression level of the inflammatory cytokine TNF-α in the mouse blood; (D) is an analysis diagram of the expression level of the inflammatory cytokine IL-1β in the mouse blood; (E) is an analysis diagram of the activity of MPO in the mouse lung tissue; (F) H&E staining of the lung tissue of each group of mice; (G) Immunofluorescence detection of neutrophil infiltration in the lung tissue; (H) lung injury score; (I) comparison of the wet-to-dry ratio of the lung tissue of each group of mice; (J) flow cytometry detection of the number of neutrophils in the lung tissue of each group;

[0025] Figure 6 The results of the in vivo safety assessment of HY-A0089 in Example 6 of the present invention are presented, including: (A) detection of alanine aminotransferase content in mouse blood; (B) detection of albumin content in mouse blood; (C) detection of aspartate aminotransferase content in mouse blood; (D) detection of alkaline phosphatase content in mouse blood; (E) detection of total protein content in mouse blood; (F) detection of creatinine content in mouse blood; and (G) H&E staining results of the heart, liver, spleen, and kidney of mice. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0027] Colistin (colistin sulfate) used in the specific embodiment of the present invention has a CAS number of 1264-72-8 and a product number of HY-A0089. It was purchased from MedChemExpress in the United States. Colistin is produced by Polymyxa bacteria and has a strong antibacterial effect on Gram-negative bacteria. It is used to treat intestinal diseases caused by Gram-negative bacteria and is used as a feed additive. It has a significant growth-promoting effect and is better when used in combination with sulfadiazine.

[0028] Example 1: Establishment and validation of ARDS mouse model

[0029] (1) Establishment of mouse ARDS model

[0030] Twenty-four SPF-grade BALB / c mice, 19.7±2.2 g, male, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd. During the feeding period, the mice had free access to water, a normal diet, and were fed ordinary maintenance feed. They were randomly divided into a control group (CTL) and three ARDS groups. The control group did not receive any treatment. ARDS group mice were injected with 25 μg, 50 μg, and 100 μg LPS (Shanghai Andy Biotechnology Co., Ltd.) into the airways of the three ARDS group mice through a nebulizer (AP-1 Air Pump; Beijing Yuansen Kaide) to establish ARDS mouse models (25 μg), ARDS mouse models (50 μg), and ARDS mouse models (100 μg). After modeling, the lung tissues of each group of mice were isolated.

[0031] (2) H&E staining: H&E staining was used to determine the damage of mouse lung tissue. 8 h after LPS inhalation, lung tissues from the control group and the three ARDS groups were placed in 10% neutral buffered formalin solution, fixed at 4°C for 48 h, then embedded in paraffin and cut into 5 μm thick sections. The sections were stained with hematoxylin-eosin (H&E) for pathological evaluation and analyzed using a microscope (Olympus CKX53) and CaseViewer Native Windows application software. The results are shown in Figure 2. Figure 1 As shown in A.

[0032] (3) Detection of inflammatory factors by enzyme-linked immunosorbent assay:

[0033] ① The ARDS mouse model was re-established according to the method (1) in this example, with only the LPS dose being different, which was 4 mg / kg. The other reagents and doses used were the same.

[0034] ② Blood was collected from mice through the retinal venous plexus, and the expression levels of inflammatory cytokines TNF-α (88-7324-22), IL-1β (88-7013-22), and IL-6 (BMS603-2) in the blood of mice in the CTL group and ARDS group were detected using ELISA kits (eBioscience Co, Invitrogen, San Diego, CA). 96-well plates (Corning Costar 9018) were coated with 100 μL / well of capture antibody and incubated at 4°C overnight. 50 uL of sample was added to each well and incubated at 18-25°C for 3 hours. Then, 40 μL of horseradish peroxidase (HRP)-conjugated antibody was added to each well. The absorbance was read at 450 nm to analyze the expression levels of inflammatory cytokines. The results are shown in Figure 2. Figure 1 As shown in B.

[0035] (4) Detection of neutrophil count using a blood cell counter

[0036] Blood was collected from the CTL group mice and ARDS group mice in Example (2) through the orbital venous plexus, and the collected blood was added to a blood collection tube containing EDTA (ethylenediaminetetraacetic acid) anticoagulant. Neutrophil counts were performed using a Beckman DxH 900 fully automatic blood cell analyzer. The results are shown in Figure 2. Figure 1 As shown in C.

[0037] (5) Detection of myeloperoxidase (MPO) in mouse lung tissue: Myeloperoxidase is a heme protein that is mainly present in neutrophils. LPS stimulation can aggregate and activate neutrophils to release MPO. The number of neutrophils in lung tissue can be determined by detecting the expression of MPO. Detection method: 100 mg of lung tissue from the CTL group mice and ARDS group mice in Example (2) were taken respectively, 1 mL of reaction solution (50 mM hexadecyltrimethylammonium bromide, 50 mM KH2PO4 solution at pH 6.0, 0.5 mM EDTA solution) was added, the lung tissue was homogenized, and the supernatant was removed after centrifugation at 12000 r / min and 4°C for 15 min. 100 μL of buffer solution (0.167 mg / mL O-dianisidine solution, 50 mM KH2PO4 solution at pH 6.0, 0.0005% mM H2O2 solution) was added, and the OD value was measured at 460 nm to analyze the expression of MPO in the mouse lung tissue. The results are as follows: Figure 1 As shown in D.

[0038] (6) Transcriptome sequencing: 200 mg of lung tissue was collected from ARDS model mice, placed in tissue preservation solution (Miltenyi Biopharm), and sent to a sequencing company (Beijing Xunyin Biotechnology Co., Ltd.) for transcriptome sequencing and gene difference analysis. The results are as follows Figure 1 As shown in E.

[0039] Result analysis:

[0040] (1) By Figure 1 A shows that LPS-induced ARDS mice had significant lung damage, and the gradual increase in LPS dose was positively correlated with the severity of alveolar structural destruction, neutrophil infiltration, and perivascular and interstitial edema. When the ARDS mouse model was subsequently established by LPS induction, an LPS dose of 4 mg / kg was selected.

[0041] To elucidate the pathological progression of ARDS and select an appropriate sampling protocol, the temporal expression profiles of inflammatory factors, including TNF-α, IL-1β, and IL-6, in the blood of mice were evaluated. Figure 1 As shown in B, the inflammatory factors in the ARDS group mice were significantly increased, and their expression levels reached a peak within 8 to 12 hours after LPS inhalation, which was about 3 times that before LPS inhalation.

[0042] (3) Figure 1 As shown in C, the number of neutrophils (PMN) in the blood of the ARDS group increased significantly compared with the control group, reaching a peak at about 12 hours, indicating that the number of neutrophils in the blood increased significantly when ARDS occurred. Figure 1 As shown in Figure D, the level of peroxidase (MPO) in the lung tissue of mice in the ARDS group was significantly higher than that in the control group, indicating a large number of neutrophils infiltrating the lung tissue, reaching a peak at 12 hours. This result is consistent with the increase in the number of neutrophils in the blood. Therefore, in subsequent experiments, blood samples were collected 12 hours after LPS inhalation so that the samples obtained could more accurately and objectively reflect the changes in PMN cell numbers.

[0043] (4) Figure 1 As shown in Figure E, transcriptome sequencing results showed that Piezo1 expression was significantly upregulated in the lung tissues of mice in the ARDS group compared to the control group, indicating that Piezo1 plays a key role in the development and progression of ARDS. Therefore, using Piezo1 as a target, a virtual screening approach was used to identify inhibitors of the Piezo1 protein, thereby potentially treating ARDS.

[0044] Example 2: Screening and experimental verification of mouse Piezo1 protein inhibitors

[0045] (1) Virtual screening of mouse Piezo1 protein inhibitors

[0046] The cryo-electron microscopy structure of mouse Piezo1 protein (PDB: 7WLT) was obtained from the RCSB (Protein Structure Database). Protein structure preparation was performed using the Protein Preparation Wizard tool in Maestro 13.1 software, which included steps such as optimizing the hydrogen bond network, adding missing atoms, and correcting incorrect residues.

[0047] 3200 FDA-approved compounds were used as a small molecule library for virtual screening, and the LigPrep tool in the maestro software was used to prepare small molecules. The SiteMap tool of the maestro software was used to discover possible small molecule binding pockets of proteins. The top three pockets among the discovered binding pockets were used for virtual screening of molecules. The virtual screening simulation was performed using the Glide tool of the maestro software. In Glide, the "Receptor Grid Generation" module was selected to define a grid for docking. The scale factor of the van der Waals radius was set to 1.0 and the partial charge cutoff was set to 0.25 during molecular docking. All compounds in the small molecule library were docked into the three binding pockets using the high-precision XP mode. The three compounds with the highest docking scores in the three binding pockets, a total of 9 compounds, were selected for subsequent experiments. The virtual screening process of Piezo1 protein inhibitors is as follows. Figure 2 As shown in A, the binding of the 9 screened compounds to Piezo1 protein is as follows Figure 2 As shown in B.

[0048] (2) MTT assay to determine the effect of HY-A0089 on cell survival

[0049] HEK293 cells in logarithmic phase were adjusted to 1x10 5 100 μL / mL cell suspension was added with HY-A0089 solution at a final concentration of 50 μM, and incubated at 5% CO2 and 37°C for 72 h. 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) was added to each well and cultured for another 4 h. 150 μL of dimethyl sulfoxide was then added to each well, and the cells were shaken at low speed on a shaker for 10 min to fully dissolve the crystals. The absorbance at OD 490 nm was measured. The results were as follows: Figure 2 As shown in E.

[0050] (3) Experimental verification of Piezo1 protein target inhibitors

[0051] Piezo1 is a mechanosensitive ion channel that belongs to the Piezo protein family. Piezo1 is a transmembrane protein that can form pores on the cell membrane. When the cell membrane is mechanically stimulated, Piezo1 undergoes conformational changes, causing the pores to open, allowing specific ions (such as calcium ions Ca 2+ ) flows into or out of cells, thereby changing the intracellular ion concentration and electrical potential, triggering a series of biological reactions. When cells are exposed to a high concentration of calcium ions, extracellular calcium ions flow into the cells. Adding an inhibitor affects the influx of calcium ions. Therefore, by examining the influx of calcium ions, we can reflect the inhibitor's effect on Piezo1 protein function.

[0052] (a) Intracellular Ca2+ Content determination

[0053] HEK293 cells were plated overnight in calcium-containing DMEM medium (thermo scientific) and incubated for 8 hours with 10 μM of each of the inhibitors purchased from MCE: HY-A0089, HY-P0205B, HY-17405, HY-14667, HY-16974, HY-17003, HY-17571A, HY-100373, and HY-A0089. HEK293 cells were treated with 4 μM Fluo-4-AM (MCE, USA) for 30 minutes and photographed using a fluorescence microscope (Leica M205 FCA). The results are shown in Figure 2. Figure 2 As shown in C.

[0054] (b) Flow cytometry detection of intracellular Ca 2+ content

[0055] HEK293 cells were incubated with calcium-containing DMEM medium (thermo scientific) and 100 μM HY-0089 inhibitor for 12 hours. 4 μM Fluo-4-AM working solution was added and incubated for 30 minutes. The cells were washed once with PBS buffer and the fluorescence intensity was measured using a BD FACSCantoII flow cytometer. The results are shown in Figure 2. Figure 2 As shown in D.

[0056] Result Analysis

[0057] The nine small molecules screened out by virtual screening are HY-A0089, HY-P0205B, HY-17405, HY-14667, HY-16974, HY-17003, HY-17571A, HY-100373, and HY-A0089. Their binding to Piezo1 protein is shown in Figure 2. Figure 2 As shown in B. Through intracellular Ca 2+ Content determination found that HY-A0089 can effectively inhibit the influx of calcium ions, such as Figure 2 C. Flow cytometry detection of intracellular Ca 2+ The results also showed that the small molecule HY-A0089 could significantly inhibit the influx of calcium ions. Figure 2 D. In addition, we also tested the effect of HY-A0089 on cell survival. The results showed that the HY-A0089 solution with a final concentration of 50 μM had no effect on cell survival. Figure 2 E. The above results indicate that HY-A0089 can effectively inhibit the calcium influx function of Piezo1 protein, and higher concentrations of HY-A0089 have no effect on cell survival.

[0058] Example 3: Affinity detection of HY-A0089 and Piezo1 protein

[0059] In order to verify that HY-A0089 indeed acts on Piezo1 protein, the affinity between HY-A0089 and Piezo1 protein was tested.

[0060] (1) Expression of Piezo1 CTD domain protein:

[0061] The CTD domain gene of the Piezo1 protein was synthesized by Sangon Biotech (Shanghai) and cloned into the pET28a plasmid. It was expressed using the BL21 (DE3) strain of TransGen Biotech (China) and then cultured in LB medium (Sigma-Aldrich) containing 50 mg / mL kanamycin to an OD 600 value of 0.6-0.8. Subsequently, 0.5 mM IPTG (Sigma-Aldrich) was added to induce expression at 16°C overnight. The cells were collected by centrifugation at 4°C and 4000g for 10 minutes, resuspended in lysis buffer containing 25 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 8.0, and lysed using a JNBIO high-pressure homogenizer at 6°C. After centrifugation of the lysis buffer at 4°C and 26000g for 40 minutes, the supernatant was taken and purified by Ni-NTA affinity chromatography column (GE Healthcare, USA), and the purity and size of the protein were verified by SDS-PAGE ( Figure 3 C).

[0062] The obtained protein was further purified by anion exchange chromatography. Figure 3 D. Finally, gel filtration chromatography was performed using a GE Healthcare Superdex 200 10 / 300GL column ( Figure 3 E), the obtained protein was verified by SDS-PAGE (e.g. Figure 3 F). The final protein was stored in 25 mM Tris, 200 mM NaCl, 5% glycerol, pH 8.0 buffer and used immediately for isothermal titration calorimetry (ITC) experiments or stored at -80°C until use.

[0063] (2) Determination of affinity between HY-A0089 and Piezo1 protein

[0064] In this experiment, isothermal titration calorimetry (ITC) was used to investigate the affinity between the small molecule Colistin and the protein Piezo1. The experiment used a NANO ITC instrument produced by TA Corporation in the United States. The results are shown in Figure 2. Figure 3 As shown in B.

[0065] A 50 μL solution of 0.1 mM HY-A0089 was drawn into a syringe, and 200 μL of a 10 μM Piezo1 solution was added to the sample cell. At 25°C, 25 drops of HY-A0089 were gradually added to the Piezo1 solution, with 180 seconds between each drop. Stirring was performed at 350 rpm during the titration to ensure uniform mixing. A control group was also established. Only the Piezo1 solution in the experimental group was replaced with the solvent buffer, while all other conditions remained unchanged. This allowed for accurate evaluation of the binding interaction between HY-A0089 and Piezo1. Comparing the thermal changes in the experimental and control groups allowed for more accurate characterization of the thermal effects of the binding process.

[0066] Result analysis:

[0067] (1) Virtual screening results showed that HY-A0089 mainly binds to the C-terminal extracellular domain (CTD) of the Piezo1 protein. The CTD consists of amino acids 2214-2457, and the crystal structure of the CTD has been solved (PDB: 4RAX). The molecular docking of HY-0089A and CTD was re-performed, and the binding free energy was 34.62 kcal / mol. The main amino acids involved in the binding of HY-0089A and the interaction forces formed were analyzed ( Figure 3 A). The binding of HY-0089A to CTD is mainly achieved through salt bridges formed by GLU2334, GLU2236, and ASP2225, hydrogen bonds with ARG2318, ASN2332, and ASN2337, and cation-π interactions (Pi-cation) with TYR 2335.

[0068] (2) The obtained protein was purified by anion exchange chromatography to obtain a relatively uniformly charged protein ( Figure 3 D) In ​​order to obtain a more uniform protein, gel filtration chromatography was performed on a GE Healthcare Superdex200 10 / 300GL column ( Figure 3 E), the obtained protein was verified by SDS-PAGE, and the protein purity was greater than 95% ( Figure 3 F).

[0069] (3) The affinity of CTD protein expressed in vitro was determined by Figure 3 B. The affinity results show that the affinity between HY-A0089 and the CTD domain is 6.19 mM, indicating that HY-A0089 and Piezo1 protein have strong binding affinity. Therefore, HY-A0089 can act on the CTD domain of Piezo1 protein.

[0070] Example 4: The therapeutic effect of HY-A0089 on AIDS

[0071] (1) Model establishment

[0072] Eighteen 6-8 week old male Balb / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., Beijing, China) were divided into three groups (6 mice in each group), namely control group (CTL), ARDS group and HY-A0089 group.

[0073] The mice in the control group (CTL) had free access to water, a normal diet, and were fed with ordinary maintenance feed without any special treatment.

[0074] Mice in the ARDS group were placed on a fixed rack and the drug was administered quantitatively through the airway of the mice. 4 mg / kg LPS was aerosolized and injected into the airways of Balb / c mice using a nebulizer (AP-1AirPump; Beijing Yuansen Kaide; HY-LWH02).

[0075] In the HY-A0089 group, 4 mg / kg LPS was aerosolized into the airways of Balb / c mice using a nebulizer (AP-1 Air Pump; Beijing Yuansen Kaide; HY-LWH02). HY-A0089 was administered intravenously at a dose of 5 mg / kg 1 and 6 hours after inhalation of 4 mg / kg LPS. Twelve hours after modeling, lung tissue was isolated from each group, and blood was collected from the orbital venous plexus for pharmacodynamic evaluation and mechanism studies.

[0076] (2) H&E staining and evaluation

[0077] Lung tissues from mice in each group were placed in 10% neutral buffered formalin solution, fixed at 4°C for 48 h, then embedded in paraffin and cut into 5-μm-thick sections. The sections were stained with hematoxylin-eosin (H&E) for pathological evaluation;

[0078] The severity of lung injury was scored as follows: based on four independent indicators of pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening, the severity of lung injury was divided into grades 0 to 4; grade 0: normal; grade 1: mild injury (<25% injury); grade 2: moderate injury (25% to 50% injury); grade 3: severe injury (50% to 75% injury); grade 4: extremely severe injury (>75% injury). The score of each mouse was calculated as the average of five randomly selected areas. H&E staining and lung injury evaluation were as follows: Figure 4 B and 4C.

[0079] (3) Flow cytometry to detect the number of neutrophils in lung tissue

[0080] By using flow cytometry technology, combined with specific monoclonal antibody labeling, the neutrophils in mouse lung tissue were accurately counted and analyzed. Specifically, the lung tissues of mice in the control group, ARDS group, and HY-A0089 group were dissected and placed on a filter containing phosphate buffered saline (PBS) for grinding to prepare a cell suspension. In order to specifically label and count neutrophils, this experiment used three monoclonal antibodies for staining: anti-CD45-FITC (Thermo Scientific), anti-CD11b-APC (Thermo Scientific), and anti-Ly6G-BV421 (Thermo Scientific). CD45 is a common surface marker of leukocytes, while CD11b and Ly6G are specific markers of neutrophils. The combined use of these markers can ensure the accurate identification and counting of neutrophils.

[0081] Mouse lung tissue was placed on a 70mm cell strainer, which was then placed in a 6-well plate. 2mL of PBS was added to the sample and filtered to obtain a grinding solution. The solution was then placed in a flow cytometer and centrifuged at 6000 rpm for 2 minutes. The supernatant was discarded and the cell pellet was collected. 2mL of erythrocyte lysis buffer (source) was added to the cell pellet, mixed thoroughly, and lysed for 10 minutes. The cell pellet was then centrifuged at 6000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was collected and centrifuged at 6000 rpm for 5 minutes. The cell pellet was washed once with 2mL of PBS and centrifuged again to obtain the cell pellet. After resuspending in 200µL of PBS, 1µL each of anti-CD45-FITC, anti-CD11b-APC, and anti-Ly6G-BV421 at a concentration of 0.2ug / µL was added to each well. The cells were vortexed to mix thoroughly, incubated at 4°C in the dark for 45 minutes, washed once with 2mL of PBS, and resuspended in 150µL of PBS.

[0082] Subsequently, the results were analyzed by BD FACSCanto II flow cytometry. Figure 4 As shown in E.

[0083] (4) Immunofluorescence detection of neutrophil infiltration in lung tissue

[0084] In cases of acute lung injury (ALI) or acute respiratory syndrome (ARDS), neutrophil infiltration is a key pathological feature. Immunofluorescence analysis of neutrophil counts in lung tissue is used to assess disease severity. The antibody used in this experiment was a neutrophil-specific Ly-6G antibody (Thermo Scientific). First, mouse lung tissue was removed and frozen sections were made. The slides were then washed three times with PBS for 3 minutes each. The slides were then fixed with 4% paraformaldehyde in PBS buffer for 15 minutes and then washed three times with PBS for 3 minutes each. Next, 300 μL of 0.5% Triton X-100 (Shanghai Bioengineering) in PBS buffer was added to each well and permeabilized at room temperature for 20 minutes. The slides were then washed again with PBS buffer three times for 3 minutes each. The PBS was then blotted dry with absorbent paper. Normal goat serum or 5% BSA (ThermoFisher Scientific) was then added to the slides and blocked at room temperature for 30 minutes. Subsequently, the blocking solution was removed with absorbent paper. Without washing, a sufficient amount of diluted primary antibody (1:200) was added to each slide for soaking, and the slides were placed in an immunohistochemical humidified box (1% BSA with primary antibody) and incubated overnight at 4°C. The next day, the slides were washed with PBST (1L PBS with 1mL Tween) three times for 3 minutes each time. After the excess liquid on the slides was dried with absorbent paper, the diluted fluorescent secondary antibody (1% BSA with 1:200) was added and incubated in a humidified box at 20-37°C (room temperature) for 1 hour. The sections were then washed with PBST three times for 3 minutes each time, and then observed and collected under a fluorescence microscope (Leica M205 FCA). The results are shown in the figure. Figure 2 As shown in D.

[0085] Result analysis:

[0086] After intravenous injection of HY-A0089 into mice, H&E staining results showed that pathological manifestations such as pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening in the HY-A0089 group were significantly alleviated compared with the ARDS group ( Figure 4 B and 4C). Figure 4 As shown in D, immunofluorescence results showed that the neutrophil infiltration in the lung tissue of mice in the HY-A0089 group was significantly reduced compared with the ARDS group, approaching the level of the control group (4D). Similarly, flow cytometry analysis of the number of neutrophils in the lung tissue showed that the neutrophil infiltration in the lung of the HY-A0089 group was reduced by more than 50% compared with the ARDS group ( Figure 4 E), HY-A0089 has a significant inhibitory effect on neutrophil infiltration in lung tissue caused by ARDS. In summary, HY-A0089 has a good therapeutic effect on ARDS.

[0087] Example 5: Preventive Effect of HY-A0089 on ARDS

[0088] (1) Experimental grouping and animal modeling

[0089] Eighteen 6-8 week old male Balb / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., Beijing, China) were divided into three groups (6 mice in each group), namely control group (CTL), ARDS group and HY-A0089 group.

[0090] The mice in the control group (CTL) had free access to water and a normal diet, and were fed with ordinary maintenance feed without any special treatment.

[0091] In the ARDS group, 4 mg / kg LPS was injected into the airways of Balb / c mice using a nebulizer (AP-1 Air Pump; Beijing Yuansen Kaide; HY-LWH02).

[0092] In the HY-A0089 group, 4 mg / kg LPS was aerosolized into the airways of Balb / c mice using a nebulizer (AP-1 Air Pump; Beijing Yuansen Kaide; HY-LWH02). HY-A0089 was then administered intravenously three times at a dose of 5 mg / kg, 12 hours before, 6 hours after, and 6 hours after LPS injection.

[0093] 12 hours after modeling, lung tissues of mice in each group were isolated and blood was collected through the retinal venous plexus for subsequent analysis.

[0094] (2) Detection of inflammatory factors IL-6, TNF-α and IL-1β: The method is the same as the enzyme-linked immunosorbent assay in part (3) of Example 1 to detect inflammatory factors. The results are as follows Figure 5 As shown in B, 5C and 5D.

[0095] (3) Detection of MPO activity in mouse lung tissue: The method is the same as the detection of myeloperoxidase in mouse lung tissue in part (5) of Example 1. The results are as follows. Figure 5 As shown in E.

[0096] (4) Lung tissue H&E staining and lung injury evaluation: The method is the same as that of H&E staining test in part (2) of Example 4. The results are as follows. Figure 5 F and 5H.

[0097] (5) Pulmonary edema measurement: Lung tissues isolated from mice in the control group, ARDS group, and HY-A0089 group were taken and weighed for wet weight (W). The lung tissues were dried in an oven at 60°C for 72 h. The samples were then reweighed to obtain the dry weight (D) of the lung tissues. The wet-to-dry ratio (W / D) was calculated. The results are shown in Table 1. Figure 5 As shown in I.

[0098] (6) Immunofluorescence detection of neutrophil infiltration in lung tissue: The method was the same as that in part (4) of Example 4, except that the experimental subjects were mice in the control group, ARDS group, and HY-A0089 group of this example. The number of neutrophils was detected by blood cell counting. The results are shown in Figure 4. Figure 5 As shown in G;

[0099] (7) Flow cytometry detection of neutrophil count in lung tissue: The method was the same as that of part (3) of Example 4, immunofluorescence detection of neutrophil infiltration in lung tissue, except that the experimental subjects were mice in the control group, ARDS group, and HY-A0089 group. The results were as follows: Figure 5 As shown in J.

[0100] Result analysis:

[0101] The results showed that the serum levels of IL-6, TNF-α and IL-1β in mice in the HY-A0089 group were significantly reduced, approaching the levels in the control group ( Figure 5 B, 5C, and 5D). This indicates that HY-A0089 can effectively reduce the levels of inflammatory factors in the blood during ARDS. H&E staining of lung tissue showed that the HY-A0089 group had significantly reduced lung pathological changes, characterized by granulocytopenia, improved alveolar structural integrity, reduced alveolar wall thickening, and reduced pulmonary capillary congestion ( Figure 5 F), lung damage was significantly reduced ( Figure 5 H). The wet-dry ratio of lung tissue in the HY-A0089 group was significantly lower than that in the ARDS group, approaching that in the control group, indicating that pulmonary edema in the HY-A0089 group was significantly alleviated ( Figure 5 I). The superoxide MPO activity in the HY-A0089 group was significantly lower than that in the ARDS group ( Figure 5 E), reflecting that the neutrophil infiltration in the lung tissue of mice in the HY-A0089 group was significantly reduced compared with the ARDS group. Similarly, immunofluorescence results showed that the neutrophil infiltration in the lung tissue of mice in the HY-A0089 group was significantly reduced and had dropped to normal levels ( Figure 5 G). Flow cytometry results showed that the percentage of neutrophils in the lung tissue of mice in the HY-A0089 group was significantly lower than that in the ARDS group ( Figure 5 H), which is consistent with the results of lung tissue immunofluorescence staining. In summary, HY-A0089 has a good preventive effect on ARDS.

[0102] Example 6: In vivo safety evaluation of HY-A0089

[0103] (1) Experimental groups

[0104] BALB / C mice (6-8 weeks old) were randomly divided into two groups, each consisting of six mice. The control group (CTL) received free access to water, a normal diet, and a standard maintenance diet without any special treatment. The HY-A0089 group received an intravenous injection of 110 μg of HY-A0089. Twenty-four hours after injection, blood was collected from the control and HY-A0089 groups via the orbital venous plexus. Organs including the heart, spleen, liver, and kidney were also collected for histochemical analysis.

[0105] (2) Mouse blood biochemical test

[0106] Siemens automatic biochemical analyzer (ADVIA 2400) to detect the expression levels of alanine aminotransferase (ALT), albumin (ALB), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP) and creatinine (CREA) in the blood of mice in each group. Figure 6 A. Figure 6 B. Figure 6 C. Figure 6 D. Figure 6 E and Figure 6 As shown in F.

[0107] (3) H&E staining of mouse organs

[0108] In this example, the steps and reagents used in the H&E staining method are the same as those in Example 1(2), except that the organs to be stained are different, namely the mouse heart, spleen, liver and kidney.

[0109] The results are as follows Figure 6 As shown in G.

[0110] Result analysis:

[0111] ALT (a marker of liver and gallbladder damage in the blood) Figure 6 A), ALB( Figure 6 B)AST( Figure 6 C) ALP( Figure 6 D), TP( Figure 6 E) and creatinine ( Figure 6 F), the results showed that there was no significant difference between the HY-A0089 group and the control group. In addition, H&E staining results showed that there was no inflammation and tissue damage in the heart, spleen, liver and kidney of mice in the HY-A0089 group, and there was no significant difference compared with the control group ( Figure 6 G) HY-A0089 had no damaging effect on the important organs of mice, including the heart, spleen, liver, and kidney.

[0112] In summary, the piezo1-specific compound inhibitor Colistin has good clinical application prospects in the treatment and prevention of ARDS.

[0113] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of Colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome.

2. The use of Colistin according to claim 1 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that The colistin is a polypeptide antibiotic produced by Bacillus polymyxa.

3. The use of Colistin according to claim 1 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that The CAS number of the colistin is 1264-72-8.

4. The use of Colistin according to claim 3 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that The colistin treats acute respiratory distress syndrome by inhibiting the infiltration of neutrophils.

5. The use of Colistin according to claim 4 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that Colistin inhibits the infiltration of neutrophils by inhibiting the mechanical sensitivity channel of Piezo1 protein.

6. Use of Colistin according to claim 1 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that: The active ingredient of the medicine is Colistin.

7. The use of Colistin according to claim 1 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that The active ingredient of the medicine is a pharmaceutically acceptable salt of Colistin.

8. Use of the colistin according to any one of claims 6 to 7 in the preparation of a drug for treating and preventing acute respiratory distress syndrome, characterized in that: The medicine also includes pharmaceutically acceptable carriers and excipients.

9. Use of Colistin according to claim 8 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that: The dosage form of the medicine is any dosage form recognized medically.

10. Use of Colistin according to claim 9 in preparing a drug for treating and preventing acute respiratory distress syndrome, characterized in that: The dosage of the drug is any therapeutically acceptable dose.

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