Use of colistin in the preparation of a medicament for the treatment and prevention of acute respiratory distress syndrome

Abstract

The application relates to application of colistin in preparation of a drug for treating and preventing acute respiratory distress syndrome, and relates to the technical field of medicines. Colistin effectively inhibits intracellular flow of calcium ions by inhibiting a mechanical sensitivity channel of Piezo1 protein, thereby preventing and reducing pathological changes such as lung bleeding, neutrophil infiltration, lung capillary hyperemia and septal thickening in ARDS, and has an important role in preventing or treating acute respiratory distress syndrome, and has great application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of Colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome. Background Technology

[0002] Acute respiratory distress syndrome (ARDS) is one of the leading causes of death in intensive care patients. ARDS is an acute, diffuse inflammatory lung injury that leads to increased pulmonary vascular permeability and lung weight, and a reduction in air-containing lung tissue. Its etiology is complex, and its pathogenesis is not fully understood. Major causes include sepsis, massive blood transfusions, severe pneumonia, chest trauma, pulmonary embolism, mechanical ventilation, pure oxygen inhalation, and acute pancreatitis. Numerous studies have shown that pulmonary inflammation is the most important cause of ARDS. Activation of innate and adaptive immunity leads to a series of inflammatory responses, and the release of large amounts of cytokines and inflammatory mediators exacerbates lung damage. The ultimate result of this uncontrolled pulmonary inflammatory response is damage to alveolar epithelial cells and capillary endothelial cells, leading to alveolar hemorrhage, pulmonary edema, and hyaline membrane formation.

[0003] Treatment for ARDS includes mechanical ventilation and non-mechanical ventilation. Mechanical ventilation involves endotracheal intubation using a ventilator and includes lung-protective ventilation strategies, low tidal volume ventilation strategies, and inverse ratio ventilation. Non-mechanical ventilation mainly includes lung tissue clearance and fluid management, alveolar surfactant supplementation, the use of beta-agonists and statins, blood purification, and nutritional intervention.

[0004] ARDS has a complex pathogenesis involving multiple injury mechanisms, and it is a highly heterogeneous syndrome with varying degrees of severity among patients. Currently, drugs in clinical trials for ARDS include glucocorticoids, ulinastatin, vitamin C, mesenchymal stromal cells (MSCs), anti-TNFR1, epithelial / endothelial cell targets, and alveolar epithelial sodium channel activators (AP-301), but their reliable efficacy has not yet been established. To date, there are no reports in the literature regarding the use of colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to provide the use of Colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome.

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

[0007] The purpose of this invention is to provide the use of Colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome.

[0008] The beneficial effects of adopting the present invention are as follows: There are currently no reports on the use of Colistin in the treatment of acute respiratory distress syndrome. The present invention demonstrates that Colistin interacts with Piezo1 protein, thereby playing a role in the treatment of ARDS. Furthermore, Colistin has no potential toxicity to human health and survival, and thus provides the application of Colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome.

[0009] Furthermore, Colistin is a polypeptide antibiotic produced by Polymyxin Bacillus.

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

[0011] The further beneficial effects of adopting the above are: Colistin is an FDA-approved and marketed drug with well-established safety profile.

[0012] Furthermore, Colistin treats acute respiratory distress syndrome by inhibiting neutrophil infiltration.

[0013] Furthermore, Colistin inhibits neutrophil infiltration by suppressing the mechanosensitive channels of the Piezo1 protein.

[0014] Specifically, Colistin prevents and reduces the infiltration and number of neutrophils in ARDS by inhibiting the mechanosensitive channel of Piezo1 protein in neutrophils in vivo. This significantly reduces alveolar rupture, vascular congestion, and interstitial edema in the lungs, thus significantly alleviating lung pathological phenomena. Colistin plays an important role in the prevention and treatment of acute respiratory distress syndrome and has great application potential.

[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 drug also includes pharmaceutically acceptable carriers and excipients.

[0018] Furthermore, the dosage form of the drug can be any medically recognized dosage form.

[0019] Furthermore, the dosage of the drug is any pharmacologically acceptable dosage. Attached Figure Description

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

[0021] Figure 2 This document presents the discovery of mouse Piezo1 protein inhibitors and the results of HY-A0089 inhibiting Piezo1 protein function in Example 2 of this invention. (A) is a diagram of the virtual screening process based on the Piezo1 protein structure; (B) is a diagram showing the interaction between the nine virtually screened compounds and the Piezo1 protein; (C) is the experimental results of immunofluorescence detection of HY-0089 inhibiting Piezo1-mediated calcium ion influx; and (D) is the flow cytometry detection of intracellular calcium ions. 2+ Content; Effect of (E)HY-A0089 on cell survival;

[0022] Figure 3 This document presents the results of affinity testing between HY-A0089 and Piezo1 proteins in Example 3 of this invention. (A) shows the molecular structure of HY-A0089 and the docking results between HY-A0089 and the CTD domain of Piezo1 protein; (B) shows the affinity between the CTD domain and HY-A0089 detected by ITC; (C) shows the SDS-PAGE results of CTD domain protein expression; (D) shows the CTD protein ion exchange chromatography; (E) shows the CTD protein gel filtration chromatography; and (F) shows the SDS-PAGE results of the gel filtration chromatography.

[0023] Figure 4 This document presents the results of the therapeutic effect of HY-A0089 on AIDS in Example 4 of the present invention; wherein (A) is a schematic diagram of the mouse model construction and experimental process of Example 4; (B) is the evaluation of lung injury; (C) is the H&E staining image of lung tissue of mice in each group; (D) immunofluorescence detection of neutrophil infiltration in lung tissue; (E) flow cytometry detection of neutrophil count in lung tissue;

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

[0025] Figure 6 The following is a demonstration of the in vivo safety assessment results of HY-A0089 in Example 6 of this invention; wherein (A) the detection of alanine aminotransferase content in mouse blood; (B) the detection of albumin content in mouse blood; (C) the detection of aspartate aminotransferase content in mouse blood; (D) the detection of alkaline phosphatase content in mouse blood; (E) the detection of total protein content in mouse blood; (F) the detection of creatinine content in mouse blood; and (G) the H&E staining results of the heart, liver, spleen, and kidney of mice. Detailed Implementation

[0026] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0027] The Colistin (colistin sulfate) used in the specific embodiments of this invention has CAS No. 1264-72-8 and product number HY-A0089. It was purchased from MedChemExpress in the United States. Colistin is produced by Polymyxin Bacillus and has a strong antibacterial effect against Gram-negative bacteria. It is used to treat intestinal diseases caused by Gram-negative bacteria, and is used as a feed additive. It also has a significant growth-promoting effect and works well 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 male SPF-grade BALB / c mice, weighing 19.7±2.2g and aged 6-8 weeks, were purchased from Beijing Huafukang Biotechnology Co., Ltd. During the rearing period, the mice had free access to water and a normal diet, fed a standard maintenance diet. They were randomly divided into a control group (CTL) and three ARDS groups. The control group received no treatment. In the ARDS groups, 25μg, 50μg, and 100μg of LPS (Shanghai Andy Biotechnology Co., Ltd.) were injected into the airways of the three ARDS groups via an AP-1 Air Pump (Beijing Yuansen Kaide) to establish ARDS mouse models (25μg, 50μg, and 100μg, respectively). After modeling, lung tissue was isolated from each group of mice.

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

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

[0033] ①The ARDS mouse model was re-established according to the method in (1) of this embodiment, except that the LPS dose was different. The LPS dose was 4 mg / kg, and the other reagents and doses were the same.

[0034] ② Blood was collected from mice via the fundus 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 CTL and ARDS mice were detected using an ELISA kit (eBioscience Co., Invitrogen, San Diego, CA). 96-well plates (Corning Costar 9018) were coated with 100 μL / well of capture antibody and incubated overnight at 4°C. 50 μL of sample was added to each well, and the plates were incubated at 18-25°C for 3 h. Then, 40 μL of horseradish peroxidase (HRP)-conjugated antibody was added to each well, and absorbance was read at 450 nm to analyze the expression levels of inflammatory cytokines. Results are as follows: Figure 1 As shown in B.

[0035] (4) Detect the number of neutrophils using a blood cell counter.

[0036] In this embodiment (2), blood was collected from the orbital venous plexus of CTL group mice and ARDS group mice. The collected blood was added to blood collection tubes containing EDTA (ethylenediaminetetraacetic acid) anticoagulant, and neutrophil counts were performed using a Beckman DxH 900 fully automated hematology analyzer. The results are as follows: Figure 1 As shown in C.

[0037] (5) Detection of myeloperoxidase (MPO) in mouse lung tissue: Myeloperoxidase is a heme protein mainly found 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 level of MPO. Detection method: 100 mg of lung tissue from CTL group mice and ARDS group mice in this example (2) was taken respectively, and 1 mL of reaction solution (50 mM hexadecyltrimethylammonium bromide, 50 mM pH 6.0 KH2PO4 solution, 0.5 mM EDTA solution) was added. The lung tissue was homogenized and centrifuged at 12000 r / min and 4℃ for 15 min. The supernatant was removed, and 100 μL of buffer (0.167 mg / mL O-dianisidine solution, pH 6.0 50 mM KH2PO4 solution, 0.0005% mM H2O2 solution) was added. The OD value was measured at 460 nm to analyze the expression level of MPO in mouse lung tissue. The results are as follows. Figure 1 As shown in D.

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

[0039] Results analysis:

[0040] (1) By Figure 1 As shown in A, LPS-induced ARDS mice showed significant lung damage. The increasing LPS dose was positively correlated with the severity of alveolar structure destruction, neutrophil infiltration, and perivascular and interstitial edema. Therefore, when establishing ARDS mouse models in the future, the LPS dose was selected as 4 mg / kg.

[0041] To elucidate the pathological progression of ARDS and select an appropriate sampling protocol, the temporal expression profiles of inflammatory factors in mouse blood, including TNF-α, IL-1β, and IL-6, were assessed. Figure 1 B shows that inflammatory factors in ARDS mice were significantly increased. Their expression levels peaked 8 to 12 hours after LPS inhalation, approximately three times higher than before LPS inhalation.

[0042] (3) Figure 1 As shown in Figure C, the number of neutrophils (PMNs) in the blood was significantly increased in the ARDS group compared to the control group, reaching a peak at approximately 12 hours, indicating a significant increase in neutrophils in the blood during ARDS. Figure 1 As shown in Figure D, the level of lung peroxidase (MPO) in the ARDS group mice was significantly higher than that in the control group, indicating a large number of neutrophil infiltrations in the lung tissue, which peaked 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 at 12 hours after LPS inhalation to ensure that the obtained samples could more accurately and objectively reflect the changes in PMN cell count.

[0043] (4) Figure 1 As shown in Figure E, transcriptome sequencing results indicated that Piezo1 expression was significantly upregulated in the lung tissue of ARDS-affected mice compared to the control group, suggesting that Piezo1 plays a crucial role in the development and progression of ARDS. Therefore, using Piezo1 as a target, a virtual screening method was employed to identify inhibitors of Piezo1 protein, thereby enabling the treatment of ARDS.

[0044] Example 2: Screening and experimental validation 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, including steps such as optimizing the hydrogen bond network, adding missing atoms, and correcting incorrect residues.

[0047] A library of 3200 FDA-approved compounds was used as a virtual screening small molecule library, and the LigPrep tool in Maestro software was used for small molecule preparation. The SiteMap tool in Maestro software was used to identify potential small molecule binding pockets for proteins. The top three binding pockets were then used for virtual screening. The virtual screening simulation was performed using the Glide tool in Maestro software. In Glide, the “Receptor Grid Generation” module was selected to define a grid for docking, with the van der Waals radius scaling factor set to 1.0 and the partial charge cutoff set to 0.25 for 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 from the three binding pockets, a total of nine compounds, were selected for subsequent experiments. The virtual screening process for Piezo1 protein inhibitors is as follows. Figure 2 As shown in Figure A, the binding of the nine screened compounds to the Piezo1 protein is as follows: Figure 2 As shown in B.

[0048] (2) Effect of HY-A0089 on cell viability determined by MTT assay

[0049] The logarithmic growth phase HEK293 cells were adjusted to 1x10⁻¹¹. 5 Cell suspension of cells / mL was added to a final concentration of 50 μM HY-A0089 solution and incubated at 37°C for 72 h at 5% CO2. Then, 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) was added to each well, and the mixture was incubated for another 4 h. Subsequently, 150 μL of dimethyl sulfoxide was added to each well, and the mixture was shaken at low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance at OD 490 nm was measured, and the results are as follows: Figure 2 As shown in E.

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

[0051] Piezo1 is a mechanosensitive ion channel belonging to the Piezo protein family. It is a transmembrane protein that forms pores in the cell membrane. When the cell membrane is mechanically stimulated, Piezo1 undergoes a conformational change, causing the pores to open and allowing specific ions (such as calcium ions) to pass through. 2+ Calcium ions flow into or out of cells, altering intracellular ion concentration and potential, triggering a series of biological reactions. When cells are in a high-concentration calcium ion environment, extracellular calcium ions flow into the cell. Adding inhibitors affects this influx of calcium ions; therefore, examining the influx of calcium ions can reflect the effect of inhibitors on the function of Piezo1 protein.

[0052] (a) Intracellular Ca2+ Content determination

[0053] HEK293 cells were seeded overnight in calcium-containing DMEM medium (Thermo Scientific), and then 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 then treated with 4 μM Fluo-4-AM (MCE, USA) for 30 min, and photographs were taken using a fluorescence microscope (Leica M205 FCA). Results are as follows: Figure 2 As shown in C.

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

[0055] HEK293 cells were co-incubated for 12 h with calcium-containing DMEM medium (Thermo Scientific) and 100 μM HY-0089 inhibitor. After incubation for 30 min with 4 μM Fluo-4-AM working solution, cells were washed once with PBS buffer, and fluorescence intensity was detected using a BD FACSCantoII flow cytometer. Results are as follows: Figure 2 As shown in D.

[0056] Results Analysis

[0057] The nine small molecules selected through virtual screening were HY-A0089, HY-P0205B, HY-17405, HY-14667, HY-16974, HY-17003, HY-17571A, HY-100373, and HY-A0089. Their binding to the Piezo1 protein was as follows... Figure 2 As shown in B. Through intracellular Ca... 2+ Content analysis revealed that HY-A0089 can effectively inhibit the influx of calcium ions, such as... Figure 2 As shown in Figure C. Flow cytometry detection of intracellular Ca. 2+ The content results also showed that the small molecule HY-A0089 could significantly inhibit the influx of calcium ions, such as Figure 2 As shown in D. Furthermore, we examined the effect of HY-A0089 on cell survival. The results showed that a final concentration of 50 μM HY-A0089 solution had no effect on cell survival, as indicated by [example data]. Figure 2 As shown in Figure E. The above results indicate that HY-A0089 can effectively inhibit the calcium ion 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 proteins

[0059] To verify that HY-A0089 does indeed act on the Piezo1 protein, the affinity between HY-A0089 and the Piezo1 protein was tested.

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

[0061] The CTD domain gene of the Piezo1 protein was synthesized by Sangon Biotech (Shanghai) and cloned into the pET28a plasmid. Expression was performed using the BL21(DE3) strain from TransGen Biotech (China). Cells were cultured in LB medium (Sigma-Aldrich) containing 50 mg / mL kanamycin until an OD600 of 0.6–0.8 was reached, followed by overnight induction with 0.5 mM IPTG (Sigma-Aldrich) at 16°C. Cells were collected by centrifugation at 4°C, 4000 g for 10 min, resuspended in lysis buffer (pH 8.0) containing 25 mM Tris, 500 mM NaCl, and 10 mM imidazole, and lysed using a JNBIO high-pressure homogenizer at 6°C. After centrifugation at 4°C, 26000 g for 40 min, the supernatant was purified using a Ni-NTA affinity column (GE Healthcare, USA). Protein purity and size were then verified by SDS-PAGE. Figure 3 C).

[0062] The obtained protein was further purified using anion exchange chromatography, and the results are as follows: Figure 3 As shown in 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 immediately used for isothermal titration calorimetry (ITC) experiments or stored at -80°C for later use.

[0063] (2) Determination of the 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 was conducted using a NANO ITC instrument manufactured by TA Instruments (USA). The results are as follows: Figure 3 As shown in B.

[0065] 50 μL of 0.1 mM HY-A0089 solution was drawn into a syringe, and 200 μL of 10 μM Piezo1 protein solution was added to the sample cell. At 25°C, 25 drops of HY-A0089 solution were gradually added to the Piezo1 protein solution at 2 μL intervals, with 180-second intervals between each drop. The solution was stirred at 350 rpm during the titration to ensure homogeneous mixing. A control group was also set up, in which the Piezo1 protein solution was replaced with a solvent buffer, while all other conditions remained unchanged, to accurately assess the binding interaction between HY-A0089 and Piezo1. Comparing the thermal changes between the experimental and control groups allowed for a more accurate differentiation of the thermal effects during the binding process.

[0066] Results 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 from positions 2214 to 2457, and its crystal structure has been resolved (PDB: 4RAX). Molecular docking of HY-0089A and the CTD was re-performed, with a binding free energy of 34.62 kcal / mol. The main amino acids involved in HY-0089A binding and their interactions 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 formed with ARG2318, ASN2332 and ASN2337, and cation-π interaction (Pi-cation) formed with TYR 2335.

[0068] (2) The obtained protein was purified by anion exchange chromatography to obtain a protein with a relatively uniform charge. Figure 3 D). To obtain a more homogeneous protein, 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, and the protein purity was greater than 95% (e.g., Figure 3 F).

[0069] (3) Affinity was determined by in vitro expression of the CTD protein. Figure 3 Affinity results (B) showed that the affinity between HY-A0089 and the CTD domain was 6.19 mM, indicating a strong binding force between HY-A0089 and the Piezo1 protein. Therefore, HY-A0089 can act on the CTD domain of the Piezo1 protein.

[0070] Example 4: 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 (n=6 per group): control group (CTL), ARDS group, and HY-A0089 group.

[0073] Control group (CTL) mice had free access to water, a normal diet, and were fed a standard maintenance diet without any special treatment.

[0074] ARDS mice were placed on a fixed frame and administered a quantitative dose of LPS via their airways. 4 mg / kg LPS was nebulized into the airways of Balb / c mice using an AP-1 Air Pump (Beijing Yuansen Kaide; HY-LWH02).

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

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

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

[0078] The severity of lung injury was scored as follows: based on four independent indicators—pulmonary hemorrhage, neutrophil infiltration, pulmonary capillary congestion, and septal thickening—the severity of lung injury was graded from 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 for each mouse was calculated as the average of five randomly selected regions. H&E staining and lung injury assessment are as follows. Figure 4 As shown in B and 4C.

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

[0080] Accurate counting and analysis of neutrophils in mouse lung tissue was achieved using flow cytometry combined with specific monoclonal antibody labeling. Specifically, lung tissue from mice in the control, ARDS, and HY-A0089 groups was dissected and ground on a filter containing phosphate-buffered saline (PBS) to prepare cell suspensions. To specifically label and count neutrophils, three monoclonal antibodies were used 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 ensured accurate identification and counting of neutrophils.

[0081] Mouse lung tissue was placed on a 70 mm cell filter and then in a 6-well plate. 2 mL of PBS was added, and the tissue was ground and filtered to obtain a homogenate. The homogenate was then placed in a flow cytometry tube and centrifuged at 6000 rpm for 2 min. The supernatant was discarded, and the cell pellet was collected. 2 mL of erythrocyte lysis buffer (source) was added to the cell pellet, mixed, and lysed for 10 min. The mixture was then centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the cell pellet was collected. The cell pellet was centrifuged again at 6000 rpm for 5 min, washed once with 2 mL of PBS, and centrifuged again. The cell pellet was resuspended in 200 μL of PBS. 1 μL each of anti-CD45-FITC, anti-CD11b-APC, and anti-Ly6G-BV421 (at a concentration of 0.2 μg / μL) was added to each well, vortexed, and incubated at 4°C in the dark for 45 min. The mixture was washed once with 2 mL of PBS and resuspended in 150 μL of PBS.

[0082] Subsequently, analysis was performed using a BD FACSCanto II flow cytometer, and the results are as follows: 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 characteristic of the pathological changes. Immunofluorescence assays are used to detect the number of neutrophils in lung tissue to assess disease severity. The antibody used in this experiment was a neutrophil-specific Ly-6G antibody (Thermo Scientific). First, mouse lung tissue was collected and frozen sections were prepared. Then, the sections were washed three times with PBS for 3 minutes each time, followed by fixation with 4% paraformaldehyde in PBS buffer for 15 minutes. The sections were then washed three times with PBS for 3 minutes each time. Next, 300 μL of 0.5% Triton X-100 (Shanghai Sangon Biotech) in PBS buffer was added to each well, and the slides were permeated at room temperature for 20 minutes. The slides were then washed three times again with PBS buffer for 3 minutes each time. The PBS was blotted dry with absorbent paper, and normal goat serum or 5% BSA (Thermo Fisher Scientific) was added to the slides. The slides were then blocked at room temperature for 30 minutes. Subsequently, the blocking solution was absorbed with absorbent paper. Without washing, a sufficient amount of diluted primary antibody (1:200) was added to each slide for immersion, and the slides were placed in an immunohistochemistry humidifier (primary antibody prepared with 1% BSA) and incubated overnight at 4°C. The next day, the slides were washed three times with PBST (1 mL Tween added to 1 L PBS), 3 minutes each time. After absorbing excess liquid with absorbent paper, diluted fluorescent secondary antibody (1:200 prepared with 1% BSA) was added, and the slides were incubated in a humidifier at 20-37°C (room temperature) for 1 hour. The slides were then washed three times with PBST, 3 minutes each time. Images were then observed under a fluorescence microscope (Leica M205 FCA). The results are as follows: Figure 2 As shown in D.

[0085] Results analysis:

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

[0087] Example 5: The 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 (n=6 per group): control group (CTL), ARDS group, and HY-A0089 group.

[0090] Control group (CTL) mice had free access to water, a normal diet, and were fed a standard maintenance diet without any special treatment.

[0091] ARDS mice received 4 mg / kg LPS via nebulizer (AP-1 Air Pump; Beijing Yuansen Kaide; HY-LWH02) into the airways of Balb / c mice.

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

[0093] Twelve hours after modeling, lung tissue from each group of mice was isolated and blood was collected from the fundus venous plexus for subsequent analysis.

[0094] (2) Detection of inflammatory factors IL-6, TNF-α and IL-1β: The method was the same as the enzyme-linked immunosorbent assay (ELISA) for detecting inflammatory factors in Part (3) of Example 1. 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 was 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) H&E staining of lung tissue and evaluation of lung injury: The method was the same as the H&E staining detection in Part (2) of Example 4. The results are as follows: Figure 5 As shown in F and 5H.

[0097] (5) Measurement of pulmonary edema: Lung tissues from mice isolated from the control group, ARDS group, and HY-A0089 were collected and weighed for wet weight (W). The lung tissues were then dried in a 60℃ oven for 72 hours. The samples were then weighed again to obtain the dry weight (D) of the lung tissue, and the wet-to-dry weight ratio (W / D) was calculated. The results are shown below. Figure 5 As shown in Figure I.

[0098] (6) Immunofluorescence detection of neutrophil infiltration in lung tissue: Except for the control group, ARDS group, and HY-A0089 group mice in this embodiment, the method was the same as in part (4) of embodiment 4, using a blood cell counter to detect the number of neutrophils. The results are as follows: Figure 5 As shown in G;

[0099] (7) Flow cytometry detection of neutrophil count in lung tissue: Except for the control group, ARDS group, and HY-A0089 group mice in this embodiment, the method was the same as in part (3) of Example 4, which describes the detection of neutrophil infiltration in lung tissue by immunofluorescence. The results are as follows: Figure 5 As shown in J.

[0100] Results analysis:

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

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

[0103] (1) Experimental Grouping

[0104] BALB / c mice (6-8 weeks old) were randomly divided into two groups of six each. The control group (CTL) mice had free access to water and were fed a normal maintenance diet without any special treatment. Mice in the HY-A0089 group were intravenously injected with 110 μg of HY-A0089. Twenty-four hours after injection, blood was collected from both the control and HY-A0089 groups via the orbital venous plexus, and organs such as the heart, spleen, liver, and kidneys were collected for histochemical analysis.

[0105] (2) Blood biochemical tests in mice

[0106] Using the Siemens fully automated biochemical analyzer (ADVIA) 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 were detected (2400). The results are as follows: 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 embodiment, except for the different organs stained (mouse heart, spleen, liver and kidney), the steps and reagents used for H&E staining are the same as those in Example 1(2) H&E staining.

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

[0110] Results analysis:

[0111] ALT (a blood indicator reflecting liver and gallbladder damage) Figure 6 A) ALB Figure 6 B), AST Figure 6 C), ALP Figure 6 D), TP Figure 6 E) and creatinine, which reflects kidney damage ( Figure 6 F), the results showed no significant difference between the HY-A0089 group and the control group. Furthermore, H&E staining results showed no inflammation or tissue damage in the heart, spleen, liver, and kidneys of the HY-A0089 group mice, with no significant difference compared to the control group. Figure 6 G). This indicates that HY-A0089 has no damaging effect on the heart, spleen, liver, and kidneys, which are vital organs in mice.

[0112] In conclusion, Colistin, a piezo1-specific compound inhibitor, shows promising clinical application potential in the treatment and prevention of ARDS.

[0113] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of Colistin in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome, characterized in that, Colistin treats and prevents acute respiratory distress syndrome by inhibiting the mechanosensitive channel of the Piezo1 protein in neutrophils in vivo, thereby preventing and reducing the infiltration and number of neutrophils. The active ingredient of the drug is Colistin or a pharmaceutically acceptable salt thereof; The drug also includes pharmaceutically acceptable carriers and excipients; The dosage form of the drug can be any medically approved dosage form; The dosage of the drug is any pharmacologically acceptable dosage.

2. The use of Colistin according to claim 1 in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome, characterized in that, Colistin is a polypeptide antibiotic produced by Polymyxin Bacillus.

3. The use of Colistin according to claim 1 in the preparation of drugs for the treatment and prevention of acute respiratory distress syndrome, characterized in that, The CAS number for Colistin is No. 1264-72-8.

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