Application of MCP-3 inhibitor in preparation of medicine for preventing and treating idiopathic pulmonary fibrosis

Abstract

The invention discloses application of an MCP-3 inhibitor in preparation of a medicine for preventing and treating idiopathic pulmonary fibrosis, and belongs to the technical field of biological medicine. The core effect of MCP-3 secreted by lung fibroblasts in the IPF process is disclosed for the first time, and it is found that MCP-3 activates an MEK-ERK pathway through binding to a receptor, promotes inflammatory aging of lung fibroblasts and transdifferentiation to myofibroblasts, and finally causes collagen deposition. Experiments also prove that the MCP-3 inhibitor Bindarit can be used as a novel therapeutic drug to intervene the IPF process and inhibit lung fibroblast aging and transdifferentiation of lung fibroblast to myofibroblast, a new way is provided for clinical treatment of IPF, and the problems of poor curative effect and large side effect of the current drug for clinically treating IPF are solved.

Description

Technical Field

[0001] The present invention relates to application of an MCP-3 inhibitor in preparing a medicine for preventing and treating idiopathic pulmonary fibrosis, and belongs to the technical field of biomedicine. Background Art

[0002] Senescence-associated pulmonary fibrosis (SAPF) is a common pathological change in aging-related lung diseases that progress to respiratory failure. It is manifested as pulmonary interstitial fibrosis and ultimately progressive worsening of lung dysfunction (Schneider JL, Rowe JH, Garcia-de-Alba C, Kim CF, Sharpe AH and Haigis MC. The aging lung: Physiology, disease, and immunity. Cell. 2021;184:1990-2019). Idiopathic pulmonary fibrosis (IPF), the primary clinical manifestation of SAPF and the most common type of idiopathic interstitial pneumonia, is a chronic, progressive, fibrosing interstitial lung disease of unknown etiology. Median survival after diagnosis is typically less than three years. It is currently believed to be primarily caused by cellular senescence and its associated secretory phenotype (Álvarez D, Cárdenes N, Sellarés J, Bueno M, Corey C, Hanumanthu VS, Peng Y, D'Cunha H, Sembrat J, Nouraie M, Shanker S, Caufield C, Shiva S, Armanios M, Mora AL and Rojas M. IPF lung fibroblasts have asenescent phenotype. American journal of physiology Lung cellular and molecular physiology. 2017;313:L1164-L1173). The incidence of IPF continues to increase with the rapid growth of the world's aging population.

[0003] Currently, the drugs used in clinical treatment of IPF mainly include anti-inflammatory drugs (such as glucocorticoids), immunosuppressants, and anti-fibrotic drugs, which have limited efficacy and are accompanied by side effects. There are currently two FDA-approved anti-fibrotic drugs, pirfenidone and nintedanib. Although these two drugs can slow the rate of deterioration of lung function, they cannot prevent or reverse disease progression, nor can they reduce the mortality rate of IPF patients. In addition, due to the off-target effects of the drugs, patients may experience some adverse reactions, such as nausea, fatigue, and diarrhea, making it difficult for some patients to tolerate the disease in the long term. Therefore, there is an urgent need to find drugs that can effectively reverse the progression of IPF and have fewer side effects.

[0004] Monocyte chemoattractant protein-3 (MCP-3) is an important chemokine. It recruits various immune cells, such as monocytes and neutrophils, to sites of infection or inflammation, thereby participating in immune responses. Previous studies have shown that MCP-3 expression is significantly higher in lung biopsies from patients with SAPF, particularly IPF, than in biopsies from patients with other interstitial lung diseases, and is primarily localized in the lung interstitial regions. This suggests that MCP-3 may play a role in the progression of SAPF, particularly IPF (Choi ES, Jakubzick C, Carpenter KJ, Kunkel SL, Evanoff H, Martinez FJ, Flaherty KR, Toews GB, Colby TV, Kazerooni EA, Gross BH, Travis WD and Hogaboam CM. Enhanced monocyte chemoattractant protein-3 / CC chemokine ligand-7 in usual interstitial pneumonia. American journal of respiratory and critical care medicine. 2004;170:508-15). In addition, there are reports showing that MCP-3 levels in fibroblast cultures of idiopathic interstitial pneumonia (IIP) patients are higher than in non-IIP fibroblasts. Therefore, does MCP-3 primarily act on lung fibroblasts, and what are the effects?

[0005] Currently, the potential role and mechanism of MCP-3 in the progression of SAPF diseases, such as IPF, remain unclear. The MCP-3 inhibitor Bindarit (AF-2838) is currently commonly used as a Phase II clinical drug for the treatment of diabetic nephropathy and coronary artery restenosis. However, there are no reports on whether the MCP-3 inhibitor Bindarit (AF-2838) or anti-MCP-3 antibodies can be used as new therapeutic drugs to intervene in the progression of IPF and inhibit the senescence of lung fibroblasts and their transdifferentiation into myofibroblasts. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies of existing drugs for treating idiopathic pulmonary fibrosis (IPF) and to provide a use of an MCP-3 inhibitor in the preparation of drugs for preventing and treating idiopathic pulmonary fibrosis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Use of MCP-3 inhibitors in the preparation of drugs for preventing and treating IPF.

[0009] The inventors have found that MCP-3 is a key regulatory factor in IPF. It activates the MEK-ERK pathway by binding to receptors, promotes inflammatory senescence of lung fibroblasts and their transdifferentiation into myofibroblasts, ultimately leading to collagen deposition. By inhibiting MCP-3, the progression of IPF can be blocked.

[0010] Furthermore, the MCP-3 inhibitor is Bindarit (AF-2838). Bindarit inhibits MCP-3 function and blocks the ERK-MEK signaling pathway, thereby inhibiting the transdifferentiation of lung fibroblasts and alleviating pulmonary fibrosis.

[0011] Furthermore, the drug includes an MCP-3 inhibitor and a pharmaceutically acceptable excipient.

[0012] Furthermore, the dosage form of the drug is granules, capsules, tablets, pills, patches, ointments, nebulizers, aerosols, powder sprays, soft mists, injections, oral solutions or nanoformulations.

[0013] A kit for diagnosing or prognosticating idiopathic pulmonary fibrosis, comprising a reagent for detecting the expression level of MCP-3.

[0014] Beneficial effects of the present invention: This invention reveals for the first time the central role of MCP-3, secreted by lung fibroblasts, in the progression of IPF. It was found that MCP-3 activates the MEK-ERK pathway by binding to its receptor, promoting inflammatory senescence of lung fibroblasts and their transdifferentiation into myofibroblasts, ultimately leading to collagen deposition. The present invention also experimentally demonstrates that the MCP-3 inhibitor Bindarit (AF-2838) or anti-MCP-3 antibodies can be used as novel therapeutic agents to intervene in the progression of IPF, inhibiting the senescence of lung fibroblasts and their transdifferentiation into myofibroblasts, providing a new approach for the clinical treatment of IPF. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the expression level of MCP-3 in the serum of healthy people of different ages;

[0016] Figure 2 This is the GO analysis of differentially expressed genes in the ERK pathway in fibroblasts from healthy controls and IPF patients in the GSE188464 data set;

[0017] Figure 3 Heat map analysis of differentially expressed genes in the ERK pathway in fibroblasts from healthy controls and IPF patients in the GSE188464 data set;

[0018] Figure 4 The expression test results of Collagen I, POSTN, α-SMA, and MCP-3 in lung tissues of IPF patients and controls are shown in Figure 2.

[0019] Figure 5 The results of the detection of MCP-3 protein expression levels in the lung tissues of 2-month-old and 17-month-old mice;

[0020] Figure 6 The results of Transwell crystal violet staining analysis of lung fibroblasts in the control group and MCP-3 treated group;

[0021] Figure 7 These are the results of the scratch test on lung fibroblasts of mice in the control group and the MCP-3 treatment group;

[0022] Figure 8 This is the EdU fluorescence result of lung fibroblasts in the control group and MCP-3 treated group mice;

[0023] Figure 9 The results of gene expression levels of Il6, Il1β, Tnf, Col1a2, Acta2, Postn, p16, p21, p53, and p19 in lung fibroblasts of mice in the control group and MCP-3 treatment group;

[0024] Figure 10The results of the expression of Collagen I, α-SMA, ERK1 / 2, p-ERK1 / 2 (Thr202 / Tyr204), MEK1 / 2, p-MEK1 / 2 (Ser217 / 221), NF-κB-p65, p-p65 (Ser536), POSTN, and p53 proteins in lung fibroblasts of mice in the control group and MCP-3 treatment group are shown;

[0025] Figure 11 H&E staining analysis results of lung tissues of wild-type control mice, mice injected with bleomycin intratracheally, and mice treated with bleomycin + intraperitoneal injection of Bindarit;

[0026] Figure 12 The results of Masson trichrome staining and analysis of lung tissues of wild-type control mice, mice injected with bleomycin intratracheally, and mice treated with bleomycin + intraperitoneal injection of Bindarit;

[0027] Figure 13 The results of protein expression tests of Collagen I, POSTN, α-SMA, p53, and p16 in lung tissues of wild-type control mice, mice injected with intratracheal bleomycin, and mice treated with bleomycin plus intraperitoneal injection of Bindarit;

[0028] Figure 14 The results of H&E staining analysis of liver and kidney tissues of wild-type control mice and mice treated with intraperitoneal injection of Bindarit;

[0029] Figure 15 The results of Masson trichrome staining analysis of liver and kidney tissues of wild-type control mice and mice treated with intraperitoneal injection of Bindarit;

[0030] Figure 16 The results of serum ELISA analysis of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen levels in wild-type control mice and mice treated with intraperitoneal injection of Bindarit. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1

[0033] MCP-3 may be an important factor inducing SAPF disease IPF:

[0034] 1. The inventors used a public database developed by Stanford University (https: / / twc-stanford.shinyapps.io / aging_plasma_proteome / ) to analyze MCP-3 changes in the human plasma proteome to investigate the role of MCP-3 in aging. They also analyzed signaling pathways in IPF patients and controls from a public high-throughput RNA sequencing dataset.

[0035] Figure 1 is the expression level of MCP-3 in the serum of healthy people of different ages. It can be seen that the expression of MCP-3 is positively correlated with age.

[0036] Figure 2 This is the GO analysis diagram of differentially expressed genes in the ERK pathway of fibroblasts between healthy controls and IPF patients in the GSE188464 data. Figure 3 This is a heat map analysis of differentially expressed genes in the ERK pathway in fibroblasts from healthy controls and IPF patients in the GSE188464 data. It can be seen that compared with the control group, the ERK signaling pathway is enriched in lung fibroblasts from IPF patients, and among the differentially expressed genes in this pathway, CCR1 expression is increased. It is generally considered to be one of the receptors for MCP-3, suggesting that MCP-3 may be elevated in the IPF population.

[0037] 2. The expression of MCP-3 in lung tissues from IPF patients and controls was detected.

[0038] Lung tissue specimens from 30 IPF patients undergoing lung transplantation who were diagnosed with pulmonary fibrosis by histopathological examination were collected from surgically removed specimens. For comparative analysis, 30 patients undergoing surgery for pulmonary nodules were also selected and normal lung tissue specimens adjacent to the nodules were collected from surgically removed specimens as controls.

[0039] Note: All these samples were obtained from the Department of Critical Care Medicine, Wuxi People's Hospital Affiliated to Nanjing Medical University, and the above clinical sample collection implementation plan has been approved by the Scientific Research Ethics Committee of Wuxi People's Hospital (approval number: KY25009).

[0040] (1) Protein extraction:

[0041] 1) Take 20 mg of tissue sample, add 300 μL of lysis buffer and homogenize;

[0042] 2) Lyse the sample on ice for 30 minutes, shaking once every 5 minutes;

[0043] 3) Centrifuge at 13,000 rpm / min for 15 minutes at 4°C, collect the supernatant, and record the volume;

[0044] 4) Add 5× SDS Loading Buffer to each sample based on the measured protein concentration. Mix thoroughly and heat in a 100°C metal bath for 10 minutes to denature the protein. Denatured samples can be used immediately for subsequent experiments or aliquoted and stored in a -80°C freezer for long-term storage.

[0045] (2) Western blot protein detection:

[0046] electrophoresis

[0047] 1) Clean and assemble the gel plates, and add the prepared electrophoresis solution;

[0048] 2) Load 20-30 μg protein per well;

[0049] 3) Run the electrophoresis at a constant voltage of 80 V until the loading buffer reaches the bottom of the separation gel.

[0050] Transfer

[0051] 1) Cut the NC membrane and soak it in pre-cooled transfer buffer;

[0052] 2) Remove the gel, make a "sandwich" structure, and place it in the transfer tank;

[0053] 3) Pour in the transfer solution and transfer the membrane at a constant current of 280mA. The transfer time depends on the molecular weight.

[0054] Antibody incubation and exposure

[0055] 1) After transfer, block the NC membrane in 5% skim milk at room temperature for 2 hours;

[0056] 2) Incubate with primary antibody at 4°C overnight;

[0057] 3) The next day, wash the NC membrane with PBST for 7 minutes each time, repeat three times;

[0058] 4) Incubate with the prepared secondary antibody of the corresponding species at room temperature for 2 hours;

[0059] 5) Wash three times with PBS, 7 min each time;

[0060] 6) Prepare exposure solution, expose and take photos.

[0061] Figure 4 The expression levels of Collagen I, POSTN, α-SMA, and MCP-3 in lung tissues of IPF patients and the control group are tested. Figure 4 A is the Western Blot test result, Figure 4B is a statistical graph, which shows that compared with the control group, the expression of Collagen I, POSTN, and α-SMA in the lung tissue of IPF patients was increased, and the protein expression of MCP-3 was significantly increased.

[0062] 3. Six wild-type mice of C57BL / 6J background, aged 2 months and 17 months, were obtained (purchased from the Medical Laboratory Animal Center of Nanjing Medical University). Mice were anesthetized with 3% sodium pentobarbital and sacrificed by cervical dislocation. The chest cavity was exposed and the lung tissue was cut and immersed in PBS solution. Tissue protein was extracted from the right lung. Protein extraction method: (1) 20 mg of tissue sample was taken and homogenized with 300 μL lysis buffer; (2) the sample was lysed on ice for 30 minutes, with shaking once every 5 minutes; (3) the sample was centrifuged at 13,000 rpm / min at 4°C for 15 minutes, the supernatant was collected and the volume was recorded; (4) 5× SDS loading buffer was added to each sample according to the measured protein concentration, mixed thoroughly, and then heated in a 100°C metal bath for 10 minutes to denature the protein. The denatured sample can be used immediately for subsequent experiments or aliquoted and stored in a -80°C refrigerator for long-term storage. The expression level of MCP-3 protein was detected by Western blot. The Western blot protein detection method is as follows:

[0063] Electrophoresis:

[0064] 1) Clean and assemble the gel plates, and add the prepared electrophoresis solution;

[0065] 2) Load 20-30 μg protein per well;

[0066] 3) Run the electrophoresis at a constant voltage of 80 V until the loading buffer reaches the bottom of the separation gel.

[0067] Transfer:

[0068] 1) Cut the NC membrane and soak it in pre-cooled transfer buffer;

[0069] 2) Remove the gel, make a "sandwich" structure, and place it in the transfer tank;

[0070] 3) Pour in the transfer solution and transfer the membrane at a constant current of 280mA. The transfer time depends on the molecular weight.

[0071] Antibody incubation and exposure:

[0072] 1) After transfer, block the NC membrane in 5% skim milk at room temperature for 2 hours;

[0073] 2) Add primary antibody and incubate overnight at 4°C;

[0074] 3) The next day, wash the NC membrane with PBST for 7 minutes each time, repeat three times;

[0075] 4) Incubate with the prepared secondary antibody of the corresponding species at room temperature for 2 hours;

[0076] 5) Wash three times with PBS, 7 min each time;

[0077] 6) Prepare exposure solution, expose and take photos.

[0078] The expression level of MCP-3 protein and its relative expression level were detected by Western blot, and t-test was used with β-actin as internal reference. The inter-group quantitative data were expressed as Mean ± SEM. * P < 0.05 compared with 2-month-old mice.

[0079] Figure 5 The results of the detection of MCP-3 protein expression levels in the lung tissues of 2-month-old and 17-month-old mice are shown in Figure 2. Figure 5 A is the Western blot test result, Figure 5 B is a statistical graph, which shows that the expression level of MCP-3 in the lung tissue of aged mice (4 months old) is higher than that of young mice (2 months old).

[0080] These results suggest that MCP-3 may promote the disease progression of IPF.

[0081] Example 2

[0082] MCP3 activates the ERK-MEK pathway through positive feedback, promoting senescence of lung fibroblasts and their transdifferentiation into myofibroblasts:

[0083] To clarify how MCP-3 promotes the development of IPF, we treated mouse lung fibroblasts with recombinant MCP-3 protein (HY-P7243, Med Chem Express, USA) and observed changes in their biological properties. We isolated and cultured primary lung fibroblasts from WT mice using the following extraction method:

[0084] Mice were anesthetized with 3% sodium pentobarbital (intraperitoneal injection, 50 mg / kg) and sacrificed by cervical dislocation. The mice were then disinfected by immersion in 70% alcohol for 5 minutes. Within the cleanroom, the mice were removed with forceps, and the chest was opened with sterile instruments to expose the lungs. The lung tissue was transferred to a sterile 4mL EP tube and washed thoroughly with sterile PBS containing 200 U / mL penicillin and 200 μg / mL streptomycin in double-antibody-containing buffer. 3mL of Collagenase D (1mg / mL) was then added, and the cells were digested on a shaker at 175 rpm in a 37°C incubator for 45-60 minutes. After digestion, the cells were centrifuged at 1500 rpm for 5 minutes, the collagenase was discarded, and the cells were washed three times with PBS containing double-antibody-containing buffer. During digestion, 7mL of complete culture medium was added to a large dish. Afterward, resuspend the tissue fragments in 3 mL of complete culture medium (α-MEM medium + 10% FBS + 1% double-antibody), drip into a large dish, mix gently, and incubate in a 37°C incubator for 3 days. After 3 days, most cells will have attached, and the medium should be replaced halfway. After one week, when cells are essentially fully attached, the medium should be replaced as usual and culture should continue for subsequent experiments.

[0085] The cells were passed down to the third generation for use in the experiment. After induction with 100 ng / mL MCP-3 for 24 hours, a Transwell assay was performed. The specific method was as follows: (1) Cell suspensions of different groups were obtained using serum-free culture medium and counted, and the cell concentration was adjusted to 2*10^5 cells per milliliter of cell suspension; (2) 600 μL of culture medium (10% FBS) and drugs were added to each well of a 24-well plate, and a Transwell chamber was placed inside, with 200 μL of cell suspension added to each chamber; (3) The Transwell chamber was placed in an incubator at 37°C and 5% CO2 for 24 hours; (4) After the incubation period, the Transwell chamber was removed and the upper and lower chambers were washed with PBS; (5) The cells were fixed with 4% paraformaldehyde for 20 minutes and washed three times with PBS; (6) The cells that did not pass through the chamber were gently wiped off with cotton and washed three times with PBS; (7) The cells were stained with crystal violet stain for 10-15 minutes and then washed with PBS to remove unbound dye. (8) Observe and count the cells on the lower chamber side using a microscope. Select 5 random fields of view to count and calculate the average value.

[0086] In order to detect the migration ability of cells, a scratch test was performed. The specific method is as follows: (1) cells of different groups were seeded into 6-well plates; (2) after the cell fusion rate reached 100%, scratches of equal distance were made vertically with a yellow pipette tip; (3) the cells were washed three times with PBS solution; (4) photos were taken and observed after 20 hours.

[0087] To detect cell proliferation, after 48 hours of MCP-3 treatment, EdU fluorescent staining was used to mark proliferating cells. The specific method is as follows: (1) EdU solution (component A) was diluted to an appropriate concentration with complete cell culture medium and then added to the cells and mixed well; a negative control group without EdU treatment was set up. (2) Incubated in a cell culture incubator for 2 hours. (3) After incubation, the culture medium was removed. The cells were washed twice with 1× PBS for 5 minutes each time to remove the EdU residue that was not incorporated into DNA. Cells that were not firmly attached to the wall can reduce the washing intensity. 50 μL of 4% paraformaldehyde fixative was added, incubated at room temperature for 20 minutes, and then the fixative was removed. (4) 50 μL of 2 mg / mL glycine solution was added to each well and incubated at room temperature for 5 minutes to neutralize the residual fixative. (5) The cells were washed twice with 100 μL of 3% BSA per well. (6) The washing solution was removed and 100 μL of 0.5% Triton X-100 was added and incubated at room temperature for 10 minutes. (7) Remove the permeation enhancer and wash the cells twice with 100 μL of 3% BSA wash buffer per well. (8) Add 100 μL of Click-iT working solution to each well to evenly cover the cells. (9) Incubate at room temperature in the dark for 30 min. (10) Remove the Click-iT working solution and wash the cells twice with 100 μL of 3% BSA. Counterstain the cell nuclei with DAPI and count the number of EdU-positive cells.

[0088] In addition, the expression levels of Il6, Il1β, Tnf, Col1a2, Acta2, Postn, p16, p21, p53, and p19 genes were detected by RT-qPCR, with β-actin as an internal reference. Cell proteins were extracted after 48 hours of MCP-3 treatment, and the expression levels of Collagen I, α-SMA, ERK1 / 2, p-ERK1 / 2 (Thr202 / Tyr204), MEK1 / 2, p-MEK1 / 2 (Ser217 / 221), NF-κB-p65, p-p65 (Ser536), POSTN, and p53 proteins were detected by Western blot, with β-actin as an internal reference ( Figure 10 The above cell experiments were repeated three times. The data were statistically analyzed using the t-test.

[0089] Figure 6 The results of Transwell crystal violet staining of mouse lung fibroblasts in the control group and MCP-3 treatment group were used to detect cell migration ability. Figure 6 A is a representative microscopic image of Transwell crystal violet staining. Figure 6B is a statistical diagram of the number of migrated cells in a single field of view. It can be seen that compared with the control group, MCP-3 treatment can increase the migration ability of lung fibroblasts.

[0090] Figure 7 The results of the scratch test of mouse lung fibroblasts in the control group and the MCP-3 treatment group were used to detect cell migration ability. Figure 7 A is a representative microscopic image of scratching at 0 hours and 20 hours. Figure 7 B is the quantitative statistical analysis of scratch wound healing, which shows that MCP-3 treatment can increase the migration ability of lung fibroblasts compared with the control group.

[0091] Figure 8 EdU fluorescence results of lung fibroblasts in the control group and MCP-3 treated group were used to detect cell proliferation ability. Figure 8 A is a representative microscopic image of proliferating cells marked by EdU fluorescent staining and cell nuclei counterstained with DAPI. Figure 8 B is a statistical graph of the proportion of EdU-positive cells, which shows that compared with the control group, MCP-3 treatment can reduce the proliferation ability of lung fibroblasts.

[0092] Figure 9 These are the results of gene expression level detection of Il6, Il1β, Tnf, Col1a2, Acta2, Postn, p16, p21, p53, and p19 in the lung fibroblasts of mice in the control group and the MCP-3-treated group. It can be seen that after MCP-3 treatment, the mRNA expression of inflammatory genes, pro-fibrotic genes, and aging-related genes in the lung fibroblasts of mice increased.

[0093] Figure 10 The results of protein expression of Collagen I, α-SMA, ERK1 / 2, p-ERK1 / 2 (Thr202 / Tyr204), MEK1 / 2, p-MEK1 / 2 (Ser217 / 221), NF-κB-p65, p-p65 (Ser536), POSTN, and p53 in lung fibroblasts of mice in the control group and MCP-3 treatment group were shown. Figure 10 A is the Western Blot test result, Figure 10 B is a statistical graph, which shows that MCP-3 treatment promotes the expression of Collagen I and α-SMA in mouse lung fibroblasts, and activates ERK-MEK pathway proteins and NF-κB-p65 signals.

[0094] The above results indicate that MCP-3 can positively feedback activate the ERK-MEK pathway, promote the senescence of lung fibroblasts and their transdifferentiation into myofibroblasts.

[0095] Example 3

[0096] The MCP-3 inhibitor Bindarit alleviates bleomycin-induced IPF in mice:

[0097] To determine whether administration of an MCP-3 inhibitor in mice could alleviate bleomycin-induced pulmonary fibrosis, we induced pulmonary fibrosis in mice by intratracheal injection of bleomycin. Twelve 9-week-old wild-type C57BL / 6J mice (purchased from the Medical Laboratory Animal Center of Nanjing Medical University) were divided into three treatment groups (n=4): a control group, a bleomycin-induced group, and a bleomycin-induced group with concurrent intraperitoneal administration of Bindarit. Both the bleomycin-induced and bleomycin-induced groups were treated with intraperitoneal administration of bleomycin (HY-17565, Med ChemExpress, USA) at 5 mg / kg on day 0 to induce pulmonary fibrosis. In the bleomycin-induced, concurrent intraperitoneal Bindarit treatment group, mice were intraperitoneally injected with the MCP-3 inhibitor Bindarit (HY-B0498, Med Chem Express, USA) at 25 mg / kg on day 3 and day 5 after bleomycin injection. On day 21, mice were anesthetized with 3% sodium pentobarbital and sacrificed by cervical dislocation. The thoracic cavity was exposed, lung tissue was excised, and immersed in PBS. The left lung was immersed in 4% paraformaldehyde, ensuring complete immersion, and then transferred to a 4°C refrigerator for 24 hours to preserve tissue morphology and prevent autolysis. Following fixation, the lung tissue was dehydrated in a series of ethanol solutions (70%, 80%, 90%, 95%, and 100%). The dehydration time for each step was adjusted based on tissue size, typically 1-2 hours. After dehydration, the tissue was transferred to paraffin for embedding. The embedded tissue block was cut into 5 μm thick slices using a microtome. The slices were attached to slides and stained with H&E and Masson.

[0098] The specific experimental method of HE staining is as follows: (1) dewax the paraffin sections as usual; (2) add hematoxylin stain solution, stain for 3 to 5 minutes, and rinse in running water for 3 to 5 minutes. Then, lift the sections in 1% hydrochloric acid alcohol twice and rinse in running water for 10 minutes. When the cell nuclei are observed to be clear blue under an optical microscope, the rinsing can be stopped; (3) add eosin solution, stain for 30 seconds, dehydrate with gradient alcohol for 3 minutes each time, and finally immerse in xylene for 3 minutes each time to make it transparent, and seal the sections with neutral gum.

[0099] The specific experimental method of Masson staining is as follows: (1) First, immerse the paraffin sections in gradient concentrations of xylene (e.g., 100%, 95%, 90%) in sequence, soaking for 5-10 minutes at each step to completely remove the paraffin on the surface of the sections. Subsequently, transfer the sections to gradient concentrations of alcohol (e.g., 100%, 95%, 80%, 70%) for hydration, soaking for 2-3 minutes at each step, and finally rinse with distilled water for 1 minute. Next, immerse the slices in hematoxylin stain for 3-5 minutes to fully stain the cell nuclei; (2) After staining, add acidic ethanol differentiation solution to the slices and let it stand for 2-10 seconds to remove excess hematoxylin stain, then rinse slowly with running water for 1 minute until the slices appear light blue; (3) Immerse the slices in Masson blueing solution and perform blueing treatment until the cell nuclei appear clear blue under a microscope, then rinse with distilled water for 1 minute to remove the residual blueing solution; (4) Add ponceau fuchsin solution to the slice sample and let it stand for 3-5 minutes to fully stain the cytoplasm and muscle fibers; (5) Gently rinse the slices with weak acid working solution to remove excess fuchsin stain until the background is clear; (6) Add phosphomolybdic acid solution to the slices and let it stand for 1-2 minutes to enhance the staining effect, then add weak acid working solution again and let it stand for 1 minute to further remove unbound dye; (7) Immerse the sections in aniline blue staining solution for 1 minute to make the collagen fibers appear blue, then rinse twice with weak acid working solution for 1 minute each time to remove excess staining solution; (8) Finally, immerse the sections in gradient concentrations of alcohol (e.g., 70%, 80%, 95%, 100%) for dehydration, soaking for 2-3 minutes each step. After dehydration, transfer the sections to gradient concentrations of xylene (e.g., 50%, 75%, 100%) for transparent treatment, soaking for 5 minutes each step. After transparentization, seal the sections with neutral gum and cover with a coverslip. After the gum solidifies, the sections can be used for microscopic observation and analysis.

[0100] Figure 11 The results of H&E staining analysis of lung tissues of wild-type control mice, mice injected with intratracheal bleomycin, and mice treated with bleomycin + intraperitoneal injection of Bindarit. It can be seen that bleomycin can induce inflammatory infiltration of lung tissue, while Bindarit can partially alleviate the inflammatory infiltration induced by bleomycin.

[0101] Figure 12 The results of Masson trichrome staining and analysis of lung tissues of wild-type control mice, mice injected with bleomycin intratracheally, and mice treated with bleomycin + intraperitoneal injection of Bindarit. Figure 12 A is a representative photomicrograph of Masson staining of alveoli and bronchi in lung tissue. Figure 12B is a statistical diagram of the area of ​​Masson-positive collagen fibers in and around the alveoli and bronchi. It can be seen that bleomycin can induce pulmonary fibrosis and collagen deposition, while Bindarit can partially alleviate bleomycin-induced pulmonary fibrosis and collagen deposition.

[0102] Figure 13 Results of the detection of Collagen I, POSTN, α-SMA, p53, and p16 protein expressions in lung tissues of wild-type control mice, mice injected with bleomycin intratracheally, and mice treated with bleomycin plus intraperitoneal injection of Bindarit. Figure 13 A is the Western blot test result, Figure 13 B is a statistical graph, which shows that bleomycin can aggravate fibrosis and collagen deposition in the lung tissue of mice and promote their aging, while Bindarit can partially alleviate fibrosis and aging.

[0103] Masson, H&E and Western blot results showed that bleomycin could induce pulmonary fibrosis and inflammatory senescence, while Bindarit could partially alleviate bleomycin-induced pulmonary fibrosis and inflammatory senescence.

[0104] These results indicate that in vivo administration of the MCP-3 inhibitor Bindarit can alleviate bleomycin-induced IPF in mice.

[0105] Example 4

[0106] Bindarit treatment had no adverse effects on liver and kidney function or histology in mice:

[0107] To determine whether administration of an MCP-3 inhibitor causes hepatotoxicity in mice, we administered Bindarit intraperitoneally to wild-type mice. Eight 9-week-old wild-type mice with a C57BL / 6J background (purchased from the Medical Laboratory Animal Center of Nanjing Medical University) were divided into two treatment groups: a control group and a Bindarit-treated group. Each group consisted of four mice. The Bindarit-treated group received 25 mg / kg of the MCP-3 inhibitor Bindarit (HY-B0498, Med Chem Express, USA) intraperitoneally on days 1 and 3. On day 10, mice were anesthetized with 3% sodium pentobarbital. Blood was collected by eye removal and allowed to stand at room temperature for approximately 20 minutes. The blood was then centrifuged at 13,000 rpm for 15 minutes at 4°C to collect serum. Mice were then sacrificed by cervical dislocation, and the peritoneal cavity was exposed. Liver and kidney tissues were excised and then immersed in PBS. Serum was tested for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN). The following steps were followed: After equilibration at room temperature for 20 minutes, the desired strips were removed from the aluminum foil pouch. The remaining strips were sealed in a ziplock bag and returned to 4°C. Standard and sample wells were set up, and 50 μL of a standard of varying concentrations was added to each standard well. 10 μL of the sample to be tested was added to the sample wells, followed by 40 μL of sample diluent. 100 μL of horseradish peroxidase (HRP)-conjugated detection antibody was added to each standard and sample well, except for the blank well. The wells were sealed with sealing film and incubated at 37°C in a waterbath or incubator for 60 minutes. The liquid was discarded and the plate was patted dry on absorbent paper. Washing solution was then added to each well, allowing it to stand for 1 minute. The wash solution was then discarded and the plate was patted dry on absorbent paper. This process was repeated five times (a plate washer can also be used). Add 50 μL each of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 minutes. The left lobe of the liver and left kidney (cut longitudinally) were immersed in 4% paraformaldehyde solution, ensuring complete immersion of the tissue. The tissues were then transferred to a 4°C refrigerator for fixation for 24 hours to fully preserve tissue morphology and prevent autolysis. After fixation, the liver and kidney tissues were dehydrated in a series of ethanol solutions (70%, 80%, 90%, 95%, and 100%). The dehydration time for each step was adjusted according to tissue size, typically 1-2 hours. After dehydration, the tissues were transferred to paraffin and embedded. The embedded tissue blocks were sliced ​​into 5 μm sections using a microtome, mounted on slides, and stained with H&E and Masson staining.

[0108] Figure 14The results of H&E staining analysis of lung tissues of wild-type control mice and mice treated with intraperitoneal injection of Bindarit show that intraperitoneal injection of Bindarit has no effect on the liver and kidney tissues of mice.

[0109] Figure 15 The results of Masson trichrome staining and analysis of lung tissues of wild-type control mice and mice treated with intraperitoneal injection of Bindarit are shown in Figure 2. Figure 15 A is a representative micrograph of liver and kidney tissues stained by Masson staining. Figure 15 B is a statistical diagram of the Masson-positive collagen fiber area in liver and kidney tissues. It can be seen that intraperitoneal injection of Bindarit does not lead to fibrosis and collagen deposition in liver and kidney tissues.

[0110] Figure 16 The results of serum ELISA analysis of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen levels in wild-type control mice and mice treated with intraperitoneal injection of Bindarit show that intraperitoneal injection of Bindarit has no effect on the liver and kidney functions of mice.

[0111] These results indicate that the MCP-3 inhibitor Bindarit exhibits good liver and kidney safety in mice.

[0112] The above technical solution only reflects the preferred technical solution of the technical solution of the present invention and does not impose any formal limitations on the present invention. Any simple modification, equivalent replacement, improvement, etc. made by technical personnel in this field to the above technical implementation method based on the technical essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of MCP-3 inhibitors in the preparation of drugs for preventing and treating idiopathic pulmonary fibrosis.

2. The use according to claim 1, characterized in that The MCP-3 inhibitor is Bindarit.

3. The use according to claim 1, characterized in that The drug comprises an MCP-3 inhibitor and a pharmaceutically acceptable excipient.

4. The use according to claim 1, wherein The dosage form of the medicine is granules, capsules, tablets, pills, patches, ointments, sprays, aerosols, powder sprays, soft sprays, injections, oral solutions or nano preparations.

5. A kit for diagnosing or prognosticating idiopathic pulmonary fibrosis, characterized in that: The kit comprises a reagent for detecting the expression level of MCP-3.

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