Use of formulation for blocking dectin-1 signaling in preparation of drug for treating and / or preventing pulmonary fibrosis
Patent Information
- Application Number
- PCT/CN2025/128848
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-20
- Filing Date
- 2025-10-21
- Publication Date
- 2026-08-27
Smart Images
Figure CN2025128848_27082026_PF_FP_ABST
Abstract
Description
Application of an agent that blocks Dectin-1 signaling in the preparation of drugs for the treatment and / or prevention of pulmonary fibrosis Technical Field
[0001] This application relates to the field of biomedical technology, and in particular to the use of an agent that blocks Dectin-1 signal transduction in the preparation of drugs for the treatment and / or prevention of pulmonary fibrosis. Background Technology
[0002] Pulmonary fibrosis is a chronic disease characterized by excessive proliferation and deposition of fibrous tissue in the lungs, leading to a gradual decline in lung function, such as idiopathic pulmonary fibrosis. Immune responses play a crucial role in the pathogenesis of pulmonary fibrosis, involving multiple immune cells and signaling molecules in its development. Myeloid cells of the innate immune system, particularly alveolar macrophages, have been shown to play a key pro-fibrotic role in pulmonary fibrosis, primarily by promoting the fibrotic activity of fibroblasts, myofibroblasts, and other stromal cells. Current traditional treatments for pulmonary fibrosis include the use of glucocorticoids, immunosuppressants, and anti-fibrotic drugs; however, these methods generally suffer from limited efficacy, significant side effects, and uncertain long-term survival outcomes, partly due to incomplete understanding of the underlying mechanisms promoting and regulating fibrosis.
[0003] Dectin-1 (gene name: CLEC7A) is a type C lectin receptor, mainly found on the surface of immune cells such as dendritic cells, macrophages, and neutrophils. It recognizes β-glucan and plays an important immunomodulatory role in the body's resistance to fungal invasion. The immune role of Dectin-1 in pulmonary fibrosis still needs further investigation. Summary of the Invention
[0004] In view of the above analysis, this application aims to provide an application of a formulation that blocks Dectin-1 signal transduction in the preparation of drugs for the treatment and / or prevention of pulmonary fibrosis, in order to solve at least one of the problems of existing glucocorticoids, immunosuppressants and antifibrotic drugs for the treatment of pulmonary fibrosis, such as large side effects, limited therapeutic effects and uncertain long-term survival outcomes.
[0005] In a first aspect, this application provides the use of an agent that blocks Dectin-1 signal transduction in the preparation of a drug for treating and / or preventing pulmonary fibrosis, said agent comprising at least one of an antifungal agent, a Dectin-1 antagonist, or a RAF1 inhibitor.
[0006] Furthermore, the antifungal agent includes at least one of imidazole antifungal agents, pyrimidine antifungal agents, allylamine antifungal agents, and echinocandins antifungal agents;
[0007] The Dectin-1 antagonist includes at least one of kelp polysaccharide, β-glucan with a molecular weight of less than 5000, neutralizing antibody or antagonistic antibody against Dectin-1;
[0008] The RAF1 inhibitors include at least one of GW5074, Rafinhibitor 1, ZM336372, dabrafenib mesylate, dabrafenib, GSK2118436A, B-Raf inhibitor 1 dihydrochloride, Raf inhibitor 3, C-RAF kinase-IN-1, MEK1 / C-Raf-IN-1, MCP110, AZ628, Kobe2602, Kobe0065, BBO-8520, Bay 43-9006, sorafenib, Ferrottosis inducer-3, and neutralizing or antagonistic antibodies against RAF1.
[0009] Furthermore, the antifungal agent reduces the β-glucan on the surface of fungi present in the lungs, thereby blocking Dectin-1 signaling and further aggravating pulmonary fibrosis caused by fungi in the lungs.
[0010] Furthermore, the fungus is a fungus that can express β-glucan and can be recognized by Dectin-1, including at least one of Candida, Aspergillus, Penicillium, Disyllium, and Diplostomum.
[0011] Furthermore, the fungus exacerbates pulmonary fibrosis by activating Dectin-1 through the expression of β-glucan.
[0012] Furthermore, the Dectin-1 antagonist inhibits the binding of β-glucan to Dectin-1, blocks the downstream signaling pathway of Dectin-1, and reduces the production of transforming growth factor β (TGF-β, gene name Tgfb1), arginase 1 (Arginase-1, gene name Arg1), and metalloproteinase 12 (MMP12, gene name Mmp12) by alveolar macrophages (AM) expressing Dectin-1, thereby alleviating pulmonary fibrosis.
[0013] Furthermore, the RAF1 inhibitors alleviate pulmonary fibrosis by inhibiting RAF1 and blocking Dectin-1 signaling, thereby reducing TGF-β, Arginase-1, and MMP12 produced by AM expressing Dectin-1.
[0014] Secondly, this application provides a composition for treating and / or preventing pulmonary fibrosis, comprising the aforementioned formulation.
[0015] Thirdly, this application provides a medicament for treating and / or preventing pulmonary fibrosis, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the aforementioned formulation.
[0016] Furthermore, the dosage form of the drug includes one or more of the following: capsules, powders, tablets, granules, intravenous drip or injection, and nasopharyngeal spray.
[0017] Compared with the prior art, this application can achieve at least one of the following beneficial effects:
[0018] (1) This application found that fungi present in the lungs can participate in the occurrence and development of pulmonary fibrosis by activating the lung Dectin-1 signaling pathway, which can further aggravate pulmonary fibrosis. This application uses antifungal agents to clear lung fungi, thereby blocking the activation of Dectin-1 by fungal-derived β-glucan and inhibiting the further aggravation of pulmonary fibrosis. In addition, this application found that RAF1 inhibitors can inhibit RAF1 and block Dectin-1 signal transduction, thereby reducing pulmonary fibrosis. This application also found that the Dectin-1 antagonist inhibits the binding of β-glucan to Dectin-1, blocks the downstream signaling pathway of Dectin-1, and reduces pulmonary fibrosis.
[0019] (2) By gaining a deeper understanding of the role of Dectin-1, this application is expected to provide important basic support for clinical treatment plans for pulmonary fibrosis targeting Dectin-1, and provide new ideas and methods for the prevention and treatment of pulmonary fibrosis.
[0020] In this application, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this application will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing this application. The objectives and other advantages of this application can be realized and obtained from the specific points highlighted in the description and accompanying drawings. Attached Figure Description
[0021] The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Throughout the drawings, the same reference numerals denote the same parts.
[0022] Figure 1 is a schematic diagram of the experimental design for fluconazole in this application;
[0023] Figure 2 shows the daily body weight of mice after bleomycin (BLM) treatment in the fluconazole experiment of this application, compared with the body weight before treatment.
[0024] Figure 3 shows the weight change in grams on the last day after BLM treatment in the fluconazole experiment of this application.
[0025] Figure 4 shows the fibrotic areas of lung tissue under panoramic and 20x magnification magnification in the fluconazole test of this application, as well as the severity of pulmonary fibrosis as determined by Ashcroft pathological scoring.
[0026] Figure 5 shows the relative expression levels of Col1a1 and Col3a1 mRNA in lung tissue determined by qPCR in the fluconazole experiment of this application.
[0027] Figure 6 shows the flow cytometry detection of CD206 in bronchoalveolar lavage (BAL) immune cells from wild-type (WT) mice treated with PBS or FCZ in the fluconazole experiment of this application. + CD11c + Cell proportion diagram;
[0028] Figure 7 shows the relative expression levels of Arg1 and Spp1 mRNA in BAL cells determined by qPCR in the fluconazole experiment of this application.
[0029] Figure 8 is a schematic diagram of the experimental design for GW5074 in this application;
[0030] Figure 9 shows the daily weight of mice after BLM treatment in the GW5074 experiment of this application, compared with the weight before treatment, and the weight change in grams on the last day.
[0031] Figure 10 shows the fibrotic areas of lung tissue under panoramic and 20x magnification magnification in the GW5074 experiment of this application, as well as the severity of pulmonary fibrosis as determined by Ashcroft pathological scoring.
[0032] Figure 11 shows the relative expression levels of Col1a1, Col3a1, and Acta2 mRNA in lung tissue determined by qPCR in the GW5074 experiment of this application;
[0033] Figure 12 shows the CD206 cells in WT mouse BAL immune cells after flow cytometry detection of the solvent or GW5074 after treatment with GW5074 in the GW5074 experiment of this application. + CD11c + Cell ratio;
[0034] Figure 13 shows the relative expression levels of Arg1, Spp1, and Tgfb1 mRNA in BAL cells determined by qPCR in the GW5074 experiment of this application;
[0035] Figure 14 shows the Decin-1 gene knockout (Clec7a) in the GW5074 experiment of this application. - / - Mice were administered GW5074 and then injected with BLM via the airway. They were euthanized 14 days later and used in experiments (Ctrl n=5, GW5074 n=3).
[0036] Figure 15 shows the relative expression levels of BAL cell-related genes determined by qPCR in the GW5074 experiment of this application;
[0037] Figure 16 shows the relative expression levels of related genes in BAL lung tissue determined by qPCR in the GW5074 experiment of this application;
[0038] Figure 17 is a schematic diagram of the experimental design of laminarin in this application;
[0039] Figure 18 shows the daily weight of mice after BLM treatment in the laminarin experiment of this application, compared with the weight before treatment, and the weight change in grams on the last day.
[0040] Figure 19 shows the fibrotic areas of lung tissue under panoramic and 20x magnification magnification in the laminarin experiment of this application, as well as the severity of pulmonary fibrosis as determined by the Ashcroft method pathological score.
[0041] Figure 20 shows the relative expression levels of Col1a1, Col3a1, Eln, Acta2, and Il11 mRNA in lung tissue determined by qPCR in the laminarin experiment of this application;
[0042] Figure 21 shows the flow cytometry detection of CD206 in WT mouse BAL immune cells after PBS or laminarin treatment in the laminarin experiment of this application. + CD11c + Cell ratio;
[0043] Figure 22 shows the flow cytometry detection of CD206 in WT mouse BAL immune cells after PBS or laminarin treatment in the laminarin experiment of this application. + CD11c + Absolute cell count (PBS n=5, Laminarin n=6);
[0044] Figure 23 shows the relative expression levels of Arg1 and Spp1 mRNA in BAL cells determined by qPCR in the laminarin experiment of this application;
[0045] Figure 24 shows two independent experiments in the laminarin experiment of this application. Detailed Implementation
[0046] The preferred embodiments of this application are described in detail below with reference to the accompanying drawings, which constitute a part of this application and are used together with the embodiments of this application to illustrate the principles of this application, but are not intended to limit the scope of this application.
[0047] One specific embodiment of this application discloses the use of an agent that blocks Dectin-1 signal transduction in the preparation of drugs for the treatment and / or prevention of pulmonary fibrosis, wherein the agent comprises at least one of an antifungal agent, a Dectin-1 antagonist, or a RAF1 inhibitor.
[0048] This application discovers that fungi present in the lungs can participate in the occurrence and development of pulmonary fibrosis by activating the pulmonary Dectin-1 signaling pathway, further aggravating pulmonary fibrosis. The fungi present in the lungs include normal fungi present in the human microbiome, such as non-pathogenic or opportunistically pathogenic fungi present in healthy lungs. This application uses antifungal agents to eliminate fungi present in the lungs, thereby blocking the activation of Dectin-1 by fungal-derived β-glucan and inhibiting further aggravation of pulmonary fibrosis. Furthermore, this application finds that RAF1 inhibitors can inhibit RAF1, blocking Dectin-1 signal transduction and alleviating pulmonary fibrosis. This application also finds that the aforementioned Dectin-1 antagonists inhibit the binding of β-glucan to Dectin-1, blocking downstream signaling pathways of Dectin-1 and alleviating pulmonary fibrosis.
[0049] The pulmonary fibrosis in this application can be acute pulmonary fibrosis caused by influenza virus infection, or acute or chronic pulmonary fibrosis caused by acute pneumonia lung injury caused by viral infection, idiopathic pulmonary fibrosis, or pulmonary fibrosis caused by other unknown causes.
[0050] Specifically, the antifungal agents include at least one of imidazole antifungal agents, pyrimidine antifungal agents, allylamine antifungal agents, and echinocandins antifungal agents;
[0051] Preferably, the imidazole antifungal agent includes at least one of ketoconazole, miconazole, fluconazole, and itraconazole; the pyrimidine antifungal agent includes at least one of cytosine, thymine, and uracil; the allylamine antifungal agent includes at least one of terbinafine and naftifine; and the echinocandin antifungal agent includes at least one of caspofungin and micafungin.
[0052] The Dectin-1 antagonist includes at least one of kelp polysaccharide, β-glucan with a molecular weight of less than 5000, neutralizing antibody or antagonistic antibody against Dectin-1;
[0053] Preferably, the Dectin-1 antagonist also includes all other blocking and neutralizing antibodies against human and mouse Dectin-1.
[0054] More preferably, β-glucans with a molecular weight of less than 5000 include linear β-(1,3)-d-glucan or linear β-(1,3-1,6)-d-glucan with side chains.
[0055] The RAF1 inhibitors include at least one of GW5074, Rafinhibitor 1, ZM336372, dabrafenib mesylate, dabrafenib, GSK2118436A, B-Raf inhibitor 1 dihydrochloride, Raf inhibitor 3, C-RAF kinase-IN-1, MEK1 / C-Raf-IN-1, MCP110, AZ628, Kobe2602, Kobe0065, BBO-8520, Bay 43-9006, sorafenib, Ferrottosis inducer-3, and neutralizing or antagonistic antibodies against RAF1.
[0056] Specifically, the antifungal agent reduces the β-glucan on the surface of fungi present in the lungs, thereby blocking Dectin-1 signaling and further aggravating pulmonary fibrosis caused by fungi in the lungs.
[0057] Specifically, the fungus is a fungus that can express β-glucan and be recognized by Dectin-1, including at least one of Candida, Aspergillus, Penicillium, Disyllium, and Diplostomum.
[0058] Specifically, the fungus further exacerbates pulmonary fibrosis by activating Dectin-1 through the expression of β-glucan.
[0059] Specifically, the Dectin-1 antagonist inhibits the binding of β-glucan to Dectin-1, blocks the downstream signaling pathway of Dectin-1, and reduces the production of transforming growth factor β (TGF-β, gene name Tgfb1), arginase 1 (Arginase-1, gene name Arg1), and metalloproteinase 12 (MMP12, gene name Mmp12) by alveolar macrophages (AM) expressing Dectin-1, thereby alleviating pulmonary fibrosis.
[0060] Specifically, the RAF1 inhibitors reduce pulmonary fibrosis by inhibiting RAF1 and blocking Dectin-1 signaling, thereby reducing TGF-β, Arginase-1, and MMP12 produced by AM expressing Dectin-1.
[0061] Another specific embodiment of this application discloses a composition for treating and / or preventing pulmonary fibrosis, comprising the aforementioned formulation.
[0062] Another specific embodiment of this application discloses a medicament for treating and / or preventing pulmonary fibrosis, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the aforementioned formulation.
[0063] Specifically, the dosage form of the drug includes one or more of the following: capsules, powders, tablets, granules, intravenous drip or injection, and nasopharyngeal spray.
[0064] This application, through a deeper understanding of the role of Dectin-1, is expected to provide important basic support for clinical treatment plans targeting Dectin-1 in the treatment of pulmonary fibrosis, and to offer new ideas and methods for the prevention and treatment of pulmonary fibrosis.
[0065] The technical solution of this application will be further explained below with reference to specific embodiments.
[0066] Example 1
[0067] 1.1 Main Instruments and Reagents
[0068] The names, models, and manufacturers of the main instruments involved in this application are shown in Table 1. The names, models, and manufacturers of the reagents involved in this application are shown in Table 2.
[0069] Table 1
[0070] Table 2
[0071] 1.2 Main Experimental Methods
[0072] 1.2.1 Human Samples
[0073] This application has been approved by the Clinical Investigation Committee of the First Affiliated Hospital of Sun Yat-sen University (Approval No.: IIT-2021-654), and the experiment strictly follows ethical regulations and guidelines.
[0074] (1) Extraction of fibroblasts
[0075] ① Lung tissue from the patient's surgery was transported to the laboratory biosafety cabinet under sterile refrigeration conditions.
[0076] ② Rinse the lung tissue three times with sterile, pre-cooled PBS to remove any attached blood.
[0077] ③ Cut the lung tissue into small tissue fragments.
[0078] ④ Resuspend the tissue block in DMEM medium with 15% FBS, add medium to 8ml, transfer to a 10cm cell culture dish and draw a cross to distribute it evenly in the culture dish.
[0079] ⑤ Place the culture dish containing tissue fragments in a cell culture incubator at 37℃ and 5% CO2.
[0080] ⑥ Two days later, carefully aspirate half of the cell supernatant and replenish with DMEM medium containing 15% FBS.
[0081] ⑦ On the fifth day, discard the supernatant, rinse the cells twice with 2ml PBS, and add 1ml trypsin to digest for 1min.
[0082] ⑧Terminate digestion with 1ml DMEM, resuspend cells and filter through a 200-mesh filter, then collect in a 15ml centrifuge tube.
[0083] ⑨ Centrifuge at 1500 rpm for 5 minutes at 4℃ and discard the supernatant. Resuspend the cells in 12 ml of 15% FBS DMEM and transfer them to a 10 cm cell culture dish. Draw a cross on the dish to distribute the cells evenly and place it in a cell culture incubator at 37℃ and 5% CO2.
[0084] ⑩ The adherent cells obtained are human lung fibroblasts.
[0085] (2) Cell extraction from bronchoalveolar lavage fluid (BALF)
[0086] ① The BALF from the patient's surgery was transported to the laboratory biosafety cabinet under sterile refrigeration conditions.
[0087] ②The BALF was filtered through a 200-mesh filter and collected in a 15ml centrifuge tube.
[0088] ③ Centrifuge at 1800 rpm for 5 minutes at 4℃ and discard the supernatant. Resuspend and wash with sterile, pre-cooled PBS.
[0089] ④ Centrifuge at 1800 rpm for 5 minutes at 4℃ and discard the supernatant. Repeat step ③ 3 times.
[0090] ⑤ The cell pellet obtained after discarding the supernatant is BAL cells.
[0091] 1.2.2 Laboratory Animals
[0092] The animal experiments in this application were conducted in accordance with the protocol approved by the Laboratory Animal Management and Use Committee of Sun Yat-sen University (Approval No.: 2021001262), and the experiments strictly followed ethical regulations and guidelines. All experimental mice were housed in a specific pathogen-free (SPF) environment in the environmentally controlled cleanroom of the Laboratory Animal Facility of Sun Yat-sen University School of Medicine, and were provided with sterilized drinking water and food.
[0093] (1)Clec7a - / - Card9 - / - With Clec4n - / - All mice were C57BL / 6J mice, introduced from the Animal Disease Model Center of the Institute of Biomedical Science, Tokyo University of Science.
[0094] (2) C57BL / 6J wild-type mice via heterozygote Clec7a + / - Card9 + / - and Clec4n + / - Produced by mating. Clec7a from the same litter. - / - Card9 - / - and Clec4n - / - Mice and wild-type (WT) mice were used in the experiments.
[0095] (3)CD45.1 + The C57BL / 6J mice were a gift from a professor at Sun Yat-sen University School of Medicine.
[0096] This application uses only male animals aged 7-10 weeks.
[0097] 1.2.3 Mouse Genotype Identification
[0098] (1) DNA extraction
[0099] ① Cut off 2-3 mm of the tail of a 4-week-old mouse that is about to be weaned.
[0100] ② Add rat tail and 135 μl of 50 mM NaOH to a 1.5 ml Ep tube.
[0101] ③ Heat in a metal bath at 95℃ for 30 minutes with vibration.
[0102] ④ After a brief centrifugation, add 15 μl of 1M Tris HCl and mix well.
[0103] ⑤ After centrifuging at 5000 rpm for 2 minutes, take 2 μl of the supernatant and dilute it 20 times with 38 μl of ultrapure water.
[0104] ⑥ Take 2 μl of the dilution solution for PCR.
[0105] (2) PCR identification
[0106] ①The PCR system and reaction conditions are shown in Tables 3 and 4:
[0107] Table 3 PCR System
[0108] Table 4 PCR reaction conditions
[0109] ②Clec7a - / - Card9 - / - With Clec4n - / - The primer sequences for identification are shown in Table 5:
[0110] Table 5 Primer sequences for gene identification
[0111] 1.2.4 Construction of a bleomycin (BLM)-induced pulmonary fibrosis model
[0112] (1) Preparation of bleomycin
[0113] ① Bleomycin was purchased from Yuanye Biotechnology. 10 mg of BLM powder was dissolved in 1 ml of PBS to make a 10 mg / ml stock solution, which was then aliquoted and stored at -80°C.
[0114] ② Dissolve the storage solution before modeling and dilute with PBS to a working concentration of 0.12 mg / ml, with a drug-to-body weight ratio of 0.24 mg / kg.
[0115] (2) Formulation and administration strategy of GW5074 (RAF1 inhibitor)
[0116] ①GW5074 was purchased from APExBIO. 5 mg of GW5074 powder was dissolved in 200 μl of PBS to prepare a 25 mg / ml stock solution, which was then aliquoted and stored at -80°C.
[0117] ② Dissolve the storage solution before use and dilute with PBS to a working concentration of 0.25 mg / ml.
[0118] ③ Three days before establishing a mouse pulmonary fibrosis model, the drug was injected intraperitoneally three times a day, with a dosage of 2 mg / kg of body weight.
[0119] ④ After pulmonary fibrosis modeling, GW5074 was administered once every other day for a total of 4 times.
[0120] ⑤GW5074 was administered a total of 7 times.
[0121] (3) Preparation and administration strategy of laminarin (kelp polysaccharide)
[0122] ①Laminarin was purchased from InvivoGen. 100mg of Laminarin powder was dissolved in 5ml of PBS to prepare a 20mg / ml stock solution, which was then aliquoted and stored at -80℃.
[0123] ② Dissolve the storage solution before use and dilute with PBS to a working concentration of 1 mg / ml.
[0124] ③ Three days before establishing a mouse pulmonary fibrosis model, administer 50 μl of the working solution via intranasal drip three times daily.
[0125] ④ After pulmonary fibrosis modeling, administer nasal drops once every other day for a total of 4 times.
[0126] ⑤Laminarin was administered a total of 7 times.
[0127] (4) Fluconazole preparation and administration strategy
[0128] ① Fluconazole was purchased from Sigma-Aldrich. 1g of fluconazole powder was dissolved in 2L of PBS to make a working solution of 0.5mg / ml. After aliquoting, the solution was stored at -80℃.
[0129] ② Dissolve the working solution before use.
[0130] ③ 50 μl of working solution was administered intranasally every other day for 7 days, 14 days before the establishment of the mouse pulmonary fibrosis model.
[0131] ④ After pulmonary fibrosis modeling, administer nasal drops once every other day for a total of 4 times.
[0132] ⑤ Fluconazole was administered a total of 11 times.
[0133] (5) Intratracheal injection in mice
[0134] ①Anesthetize mice by intraperitoneal injection of 1% sodium pentobarbital at 30 mg / kg.
[0135] ②After the mouse is completely anesthetized, weigh it and record its weight, shave off the hair on its neck, and expose the skin on its neck.
[0136] ③ Draw the corresponding volume of BLM working solution according to the mouse's weight, and then aspirate it into a 1ml syringe with a needle.
[0137] ④ Place the mouse belly up and fix its limbs with medical tape, then disinfect the skin on its neck with iodine.
[0138] ⑤ Use ophthalmic scissors to make a longitudinal incision of about 0.5 cm in the neck, and use ophthalmic forceps to bluntly separate the muscles to expose the trachea.
[0139] ⑥ Insert a syringe containing BLM liquid into the trachea and quickly expel the liquid. When the mouse is quickly upright and rotated left and right, the liquid is evenly distributed in the lungs.
[0140] ⑦ Suture the neck incision and disinfect the surface skin.
[0141] ⑧ After surgery, the mice were placed on a 37°C constant temperature heating pad until they recovered, and then transferred to an IVC cage.
[0142] 1.2.5 RNA Extraction and Real-Time Quantitative PCR
[0143] (1) RNA extraction
[0144] ①The Mammalian Total RNAMiniprep kit is used for RNA extraction.
[0145] ② For tissue samples, use a grinding bead homogenizer to homogenize the tissue before proceeding with subsequent operations; for cell samples, use lysis buffer to mix.
[0146] ③ Take 200 μl of the lysis buffer containing the sample into the filter column and centrifuge at 12,000 rpm for 2 min.
[0147] ④ Discard the upper column, add an equal amount of 70% ethanol to the lower column and mix well. Transfer the mixture to the nucleic acid purification column and centrifuge at 12,000 rpm for 20 seconds.
[0148] ⑤ Discard the waste liquid at the bottom of the column, add 330 μl Wash SolutionⅠ to the top of the column, and centrifuge at 12000 rpm for 20 s.
[0149] ⑥ Discard the waste liquid at the bottom of the column, add 300 μl of Wash Solution II to the top of the column, and centrifuge at 12000 rpm for 20 s.
[0150] ⑦ Discard the waste liquid at the bottom of the column, add 330 μl of Wash Solution II to the top of the column, and centrifuge at 12000 rpm for 1 min.
[0151] ⑧ Discard the waste liquid from the lower column, centrifuge at 12000 rpm for 20 seconds, and then replace the lower column with a collection tube.
[0152] ⑨ Add 15-40 μl of RNase-free H2O to the center of the column and centrifuge at 12000 rpm for 1 min.
[0153] ⑩Nano Drop assay to determine RNA concentration.
[0154] (2) RNA is reverse transcribed into cDNA
[0155] ① The reverse transcription reagent Evo M-MLVRTMaster Mix was purchased from Aikerui Biotechnology. The RNA mass used for reverse transcription was 50-500 ng.
[0156] ②The reverse transcription system is shown in Table 6:
[0157] Table 6 RNA reverse transcription system
[0158] ③ The reverse transcription procedure is shown in Table 7:
[0159] Table 7 Reverse Transcription Reaction Conditions
[0160] ④ The cDNA was diluted 10-20 times with ultrapure water before being used for qPCR.
[0161] (3) Real-time quantitative PCR (qPCR)
[0162] ① The Green Pro Taq HS Premix qPCR Kit is used for qPCR.
[0163] ②Reverse transcription procedure:
[0164] Table 8 qPCR system
[0165] ③ qPCR program:
[0166] Table 9 qPCR reaction conditions
[0167] ④The primer list is shown in Table 10.
[0168] Table 10
[0169] ⑤ Gapdh or GADPH is used for the standardization of relative mRNA expression levels.
[0170] 1.2.6 Cell Extraction
[0171] (1) The cells were extracted from mice after euthanasia.
[0172] (2) Extraction of BAL cells
[0173] ① Place the mouse on its abdomen with its limbs fixed to the operating table.
[0174] ② Cut open the abdominal skin to expose the abdominal cavity, and cut open the diaphragm to allow the lung tissue to communicate with atmospheric pressure.
[0175] ③ Cut open the skin of the neck and separate the muscle tissue around the trachea to fully expose it.
[0176] ④ Make a small incision in the trachea and insert a 22G soft syringe.
[0177] ⑤ Carefully and slowly inject 0.5 ml of pre-cooled HBSS into the syringe through the tubing. After holding for 20 seconds, aspirate the liquid and transfer it to a 15 ml centrifuge tube. The liquid recovery rate is approximately 80%. Repeat this operation 4 times.
[0178] ⑥ The collected BALF was centrifuged at 1800 rpm and 4℃ for 5 min.
[0179] ⑦ Discard the supernatant, resuspend in 2ml of erythrocyte lysis buffer (ACK) and let stand for 5 minutes, then add an equal volume of HBSS to stop hemolysis.
[0180] ⑧ Centrifuge at 1800 rpm and 4℃ for 5 min, discard the supernatant, and wash twice with 5 ml of HBSS.
[0181] ⑨ Centrifuge at 1800 rpm and 4℃ for 5 min, discard the supernatant, and the resulting cell pellet is BAL cells.
[0182] (3) Extraction of single cells from lung tissue
[0183] ① Place the mouse on its abdomen with its limbs fixed to the operating table.
[0184] ② Cut open the abdominal skin to expose the chest cavity, remove the ribs, and expose the lung tissue and heart.
[0185] ③ Cut open the inferior vena cava, insert the needle into the right ventricle, and push in PBS to perfuse the lungs and flush out the blood.
[0186] ④ Cut off the lung tissue and mince it into pieces about 1mm in size. 3 Fragments.
[0187] ⑤ Resuspend the fragments in 5 ml of culture medium containing 200 U / ml type IV collagenase and 5 U / ml DNase I, and incubate at 37°C for 2 h.
[0188] ⑥ After the water bath, shake the suspension for 10 seconds using Vortex, then filter it through a 200-mesh filter to remove large pieces.
[0189] ⑦ Centrifuge at 1800 rpm and 4℃ for 5 min, discard the supernatant, gently resuspend the cells in 2 ml of red blood cell lysis buffer, let stand for 5 min, and add an equal amount of HBSS to stop hemolysis.
[0190] ⑧ Centrifuge at 1800 rpm and 4℃ for 5 min, discard the supernatant, and wash twice with 5 ml of HBSS.
[0191] ⑨ Centrifuge at 1800 rpm and 4℃ for 5 min, discard the supernatant, and the resulting cell pellet is a single cell of lung tissue.
[0192] 1.2.7 Purification and Culture of Lung Fibroblasts
[0193] (1) The cells were extracted from mice after euthanasia.
[0194] (2) Extraction of lung fibroblasts
[0195] ① Place the mouse on its abdomen with its limbs fixed to the operating table.
[0196] ② Cut open the abdominal skin to expose the chest cavity, remove the ribs, and expose the lung tissue and heart.
[0197] ③ Cut open the inferior vena cava, insert the needle into the right ventricle, and push in PBS to perfuse the lungs and flush out the blood.
[0198] ④ Cut off the lung tissue and mince it into pieces about 1mm in size. 3 Fragments.
[0199] ⑤ Resuspend the fragments in 8 ml of DMEM containing 15% FBS and 1% penicillin, transfer to a 10 cm cell culture dish, and culture in a cell culture incubator at 37°C and 5% CO2.
[0200] ⑥ Change the medium halfway after 2 days and continue culturing.
[0201] ⑦ Five days later, long, spindle-shaped, densely packed adherent cells, i.e., pulmonary fibrotic cells, can be seen under a microscope.
[0202] (3) Purification of lung fibroblasts
[0203] ① Discard the fibroblast supernatant, rinse the cells twice with 2ml PBS, and digest with 1ml trypsin for 1min.
[0204] ②Terminate digestion with 1ml DMEM, mix the cells by pipetting, filter through a 70μm cell filter, and collect the cells in a 15ml centrifuge tube.
[0205] ③ Centrifuge at 1500 rpm for 5 minutes at 4℃ and discard the supernatant. Resuspend the cells in 100 μl of basal culture medium containing 1 μl of anti-mouse CD45-biotin, 1 μl of anti-mouse CD31-biotin, and 1 μl of anti-mouse CD326-biotin, and incubate at 4℃ for 20 minutes.
[0206] ④ Centrifuge at 1500 rpm and 4℃ for 5 min, discard the supernatant, and wash twice with 2 ml of basal culture medium.
[0207] ⑤ Discard the supernatant, resuspend the cells in 100 μl of basal culture medium containing 10 μl of avidin microparticle magnetic beads, and incubate at 4°C for 20 min.
[0208] ⑥ Centrifuge at 1500 rpm for 5 min at 4℃, discard the supernatant, and wash twice with 2 ml of basal culture medium.
[0209] ⑦ Discard the supernatant, resuspend the cells in 500 μl of basal culture medium, and transfer to a flow cytometer.
[0210] ⑧ Place the flow cytometry tube on a magnetic rack. After 5 minutes, the positive cells will be adsorbed onto the tube wall. Carefully aspirate the liquid and add 2 ml of basal culture medium to the aspirated liquid to resuspend the cells.
[0211] ⑨ Centrifuge at 1500 rpm and 4℃ for 5 min, discard the supernatant, and the obtained cells are the purified lung fibroblasts.
[0212] 1.2.8 Induction of bone marrow-derived macrophages and type II macrophages
[0213] (1) The cells were extracted from mice after euthanasia.
[0214] (2) Extraction of bone marrow cells
[0215] ① Place the mouse on its belly, cut open the skin of the lower limbs, separate the femur, tibia and humerus and remove the attached muscles.
[0216] ② Draw 1ml of basal RPMI 1640 culture medium into a syringe, flush out the bone marrow and collect it in a 15ml centrifuge tube.
[0217] ③ Centrifuge at 1500 rpm and 4℃ for 5 min, discard the supernatant, gently resuspend the cells in 5 ml of red blood cell lysis buffer, let stand for 5 min, and add an equal volume of RPMI 1640 medium containing 10% FBS to stop hemolysis.
[0218] ④ Centrifuge at 1500 rpm for 5 min at 4℃, discard the supernatant, and wash twice with 5 ml of culture medium.
[0219] ⑤ Centrifuge at 1800 rpm and 4℃ for 5 min, discard the supernatant, and the resulting cell pellet is bone marrow cells.
[0220] (3) Induction of bone marrow macrophages (BMDM) and type II macrophages (M2)
[0221] ① The cell pellet from the previous step was resuspended in 8 ml of RPMI 1640 whole culture containing 30 ng / ml mouse M-CSF and transferred to a 10 cm cell culture dish, and cultured in a cell culture incubator at 37℃ and 5% CO2.
[0222] ② On day 3, use RPMI1640 full culture half replacement medium containing 30 ng / ml mouse M-CSF.
[0223] ③On day 7, the adherent cells obtained are BMDM.
[0224] ④ Discard the supernatant, add 8 ml of RPMI 1640 whole culture containing 25 ng / ml mouse IL-4 and 25 ng / ml mouse IL-13, and continue culturing for 24 h.
[0225] ⑤After 24 hours, the cells obtained are M2 macrophages.
[0226] 1.2.9 Masson staining and immunohistochemical staining of pathological sections
[0227] (1) Tissue fixation, paraffin embedding and sectioning
[0228] ① Mouse lung tissue or human lung tissue blocks after pulmonary circulation perfusion were fixed by immersion in 10% formalin.
[0229] ②The tissue was dehydrated overnight in anhydrous ethanol and then embedded in paraffin.
[0230] ③ The paraffin block was sectioned at a thickness of 5 μm.
[0231] (2) Dewaxing
[0232] ① Immerse the baked paraffin sections on the staining rack in the oven in xylene I for 20 minutes.
[0233] ②Immerse the sample in xylene II for 20 minutes.
[0234] ③ After the wax on the sample dissolves, transfer the sample to anhydrous ethanol I and soak for 5 minutes.
[0235] ④ Immerse the sample in anhydrous ethanol II for 5 minutes.
[0236] ⑤ Rinse the sample with anhydrous ethanol for 20 seconds, then transfer it to a basin of water and rinse it off with running tap water.
[0237] (3) Masson staining
[0238] ①Incubate the slices overnight in Masson's stain 1 in a microwave repair box.
[0239] ② Remove the slice and rinse it quickly with tap water in a staining cup until it is colorless.
[0240] ③ Place the slices into Masson's stain 2 in the microwave repair box (preheat at 65℃ for 30 minutes before staining), then put them back into the oven for staining for 3-5 minutes. After that, take the staining cup and wash it with tap water 2-3 times.
[0241] ④ Stain the sections in Masson's stain solution 3 in the microwave repair box for 30 seconds to 1 minute.
[0242] ⑤ Remove the section, drain it slightly, and place it in Masson's stain 4 in the microwave repair box (preheat at 65℃ for 30 minutes before staining, and return to the oven to preheat after use) for 5-20 seconds.
[0243] ⑥ Immerse the slices in 1% glacial acetic acid (you need to prepare your own) for a few seconds to differentiate. Repeat this process in three batches, differentiating for 5-10 seconds in each batch.
[0244] ⑦ Place the slices into three cylinders of anhydrous ethanol in the microwave repair box, and microwave for 5-10 seconds in each cylinder.
[0245] ⑧ Transfer the slice into n-butanol in the microwave repair box for 10-20 seconds.
[0246] ⑨ Place the slices into a staining jar containing xylene I for 5 minutes.
[0247] ⑩ Transfer the xylene II into the staining tank for 5 minutes, quickly dry the sections through the vent, and then mount them with neutral resin.
[0248] (4) Immunohistochemical staining
[0249] ① Antigen retrieval: Immerse the slides in citric acid (pH 6.0) overnight at 60°C.
[0250] ② Endogenous enzyme blockade: The slices were placed in 3% H2O2 solution and incubated at room temperature for 20 min. They were then washed three times with PBS for 5 min each time.
[0251] ③ Serum blocking: Draw a circle around the tissue with a histochemical pen, then add serum to the tissue and incubate at 37°C for 30 minutes.
[0252] ④ Primary antibody incubation: Dilute the antibody (anti-human-Dectin-1, 1 μg / ml) with antibody diluent, remove the serum from the slide, add the antibody working solution to the tissue, and incubate overnight at 4°C.
[0253] ⑤ Secondary antibody incubation: Prepare HRP-labeled secondary antibody with PBST, add an appropriate amount of secondary antibody to the tissue, incubate at 37℃ for 1 hour, and wash three times with PBST for 5 minutes each time.
[0254] ⑥ DAB staining: Add DAB working solution to the tissue and observe it under a microscope. When a specific brown color appears, rinse the tissue with water to remove the DAB staining solution, and then soak the slide in water.
[0255] ⑦ Counterstaining with hematoxylin: Place the slide in hematoxylin staining solution and stain for 3-5 minutes. Wash away excess hematoxylin staining solution with water. After the cell nuclei turn blue, immerse the slide in 0.5% hydrochloric acid-alcohol differentiation solution and differentiate for 1-2 seconds. Immediately rinse with water. Then immerse the slide in reverse blue solution for 3-5 seconds, followed by rinsing with water.
[0256] ⑧ Place the slides sequentially into containers containing anhydrous ethanol 1, anhydrous ethanol 2, anhydrous ethanol 3, n-butanol 1, n-butanol 2, xylene 1, and xylene 2, soaking for 5 minutes each time. After air drying, drop an appropriate amount of neutral resin onto the tissue, cover the tissue with a coverslip, and let it air dry.
[0257] 1.2.10 Immunofluorescence staining
[0258] (1) Human pulmonary fibrosis tissue sections were prepared according to the method described above.
[0259] (2) Immunofluorescence staining
[0260] ① Serum blocking: After antigen retrieval, draw a circle around the tissue with a histochemical pen, add 1% goat serum to the tissue, and incubate at 37°C for 1 hour.
[0261] ② Primary antibody incubation: Dilute the antibody (anti-human-Dectin-1, 10 μg / ml; anti-human-α-SMA, 1:250) with 1% goat serum, discard the serum on the slide, add the antibody working solution to the tissue, and incubate overnight at 4°C.
[0262] ③ Wash with PBS 3 times, 5 minutes each time.
[0263] ④ Secondary antibody incubation: Add an appropriate amount of secondary antibody (goat-anti-rabbit-Alexa594, 1:500) to the tissue, incubate at room temperature for 1 hour, and wash three times with PBS for 5 minutes each time.
[0264] ⑤ Apply DAPI to the tissue and cover with a coverslip.
[0265] 1.2.11 Flow cytometry
[0266] (1) Perform cell extraction according to the steps in “1.2.6”. After the extracted cells are washed twice with 500 μl FACS buffer (HBSS containing 2% FBS), they can be used for flow cytometry staining.
[0267] (2) Staining with Dectin-1
[0268] ① Anti-mouse-Dectin-1 was purchased from InvivoGen. 100 μg of antibody powder was dissolved in 1 ml of double-distilled water to prepare a 0.1 mg / ml stock solution, which was then aliquoted and stored at -80℃.
[0269] ① Prepare a 2.4G2 blocking antibody secondary antibody (anti-rabbit-IgG-FITC) dilution buffer using FACS buffer at a dilution ratio of 1:250.
[0270] ② Resuspend the cells in 500 μl of FACS buffer in a 1.5 ml EP tube and centrifuge at 5000 rpm for 2 min at 4 °C.
[0271] ③ Discard the supernatant, aspirate 30 μl of 2.4G2 blocking antibody to resuspend the cell pellet, and incubate at 4°C for 30 min.
[0272] ④ Wash twice with 500 μl FACS buffer, then centrifuge at 5000 rpm and 4℃ for 2 min.
[0273] ⑤ Discard the supernatant, resuspend the cells in 30 μl of FACS buffer and 2 μl of anti-mouse-Dectin-1 stock solution, and incubate at 4°C for 30 min.
[0274] ⑥ Wash twice with 500 μl FACS buffer, then centrifuge at 5000 rpm and 4℃ for 2 min.
[0275] ⑦ Discard the supernatant, resuspend the cell pellet in 30 μl of secondary antibody dilution buffer, and incubate at 4°C for 30 min.
[0276] ⑧ Wash twice with 500 μl FACS buffer, then centrifuge at 5000 rpm and 4℃ for 2 min.
[0277] 9. Discard the supernatant, and you can proceed with staining for other antibodies.
[0278] (3) Cell surface staining
[0279] ① Prepare antibody dilution buffer and 2,4G2 blocking antibody using FACS buffer at a dilution ratio of 1:250.
[0280] ② Resuspend the cells in 500 μl of FACS buffer in a 1.5 ml EP tube and centrifuge at 5000 rpm for 2 min at 4 °C.
[0281] ③ Discard the supernatant, aspirate 30 μl of 2.4G2 blocking antibody to resuspend the cell pellet, and incubate at 4°C for 30 min.
[0282] ④ Wash twice with 500 μl FACS buffer, then centrifuge at 5000 rpm and 4℃ for 2 min.
[0283] ⑤ Discard the supernatant, take 30 μl of antibody dilution buffer to resuspend the cell pellet, and incubate at 4°C for 30 min.
[0284] ⑥ Wash twice with 500 μl FACS buffer, then centrifuge at 5000 rpm and 4℃ for 2 min.
[0285] ⑦ Discard the supernatant, resuspend in 400 μl of FACS buffer, filter through a 200-mesh filter, and then perform flow cytometry detection, or further perform intracellular antibody staining.
[0286] (4) Intracellular staining
[0287] ① Anti-mouse-CD206 and anti-mouse-Arg-1 require intracellular staining.
[0288] ② Resuspend the cell pellet in 120 μl of Cytofix / Cytoperm and incubate at 4°C for 20 min or overnight at 4°C.
[0289] ③ Prepare antibody dilution solution using BD Perm / Wash Buffer at a dilution ratio of 1:250.
[0290] ④ Wash twice with 500μl BD Perm / Wash Buffer, then centrifuge at 5000rpm at 4℃ for 2min.
[0291] ⑤ Discard the supernatant, take 30 μl of antibody dilution buffer to resuspend the cell pellet, and incubate at 4°C for 30 min.
[0292] ⑥ Wash twice with 500 μl BD Perm / Wash Buffer, then centrifuge at 5000 rpm at 4℃ for 2 min.
[0293] ⑦ Discard the supernatant, resuspend in 400 μl of FACS buffer, filter through a 200-mesh filter, and then perform flow cytometry analysis.
[0294] (5) autoMACS fully automatic magnetic bead sorting
[0295] ①BAL cells are used for magnetic bead sorting.
[0296] ② Resuspend cells in 100 μl of basal medium containing 1 μl of anti-mouse CD11c-biotin and incubate at 4°C for 20 min.
[0297] ③ Centrifuge at 1500 rpm and 4℃ for 5 min, discard the supernatant, and wash twice with 2 ml of basal culture medium.
[0298] ④ Discard the supernatant, resuspend the cells in 100 μl of basal medium containing 10 μl of anti-biotin microbeads, and incubate at 4°C for 20 min.
[0299] ⑤ Centrifuge at 1500 rpm for 5 min at 4℃, discard the supernatant, and wash twice with 2 ml of basal culture medium.
[0300] ⑦ Discard the supernatant, resuspend the cells in 500 μl of basal culture medium, and transfer to a flow cytometer.
[0301] ⑧ AutoMAC upper-level sorting, select fine positive selection mode.
[0302] ⑨ The cells obtained by positive selection were resuspended in 2 ml of basal culture medium and centrifuged at 1500 rpm at 4℃ for 5 min.
[0303] ⑩ Discard the supernatant, and obtain cells and CD11c in BALF. + Cells, and alveolar macrophages.
[0304] 1.2.12 Construction of bone marrow chimera
[0305] (1) Preparation of recipient mice
[0306] ① One week before irradiation, add 250 mg / L gentamicin, 320 mg / L erythromycin and 10% sucrose to the drinking water of 6-week-old WT (CD45) animals.
[0307] ② One week later, the recipient mice were irradiated with 6.5 Gy of X-rays.
[0308] (2) Donor cell preparation
[0309] ①WT(CD45.1) and Clec7a - / - (CD45) mice were used to extract bone marrow cells.
[0310] ② Extract bone marrow cells according to the steps in “1.2.8(2)”.
[0311] ③ Combine WT-derived cells with Clec7a - / - The source cells were mixed 1:1 and the cell concentration was adjusted to 1×10⁻⁶. 8 / ml
[0312] (3) Bone marrow transplantation
[0313] ①The recipient mice underwent bone marrow transplantation on the same day they were irradiated.
[0314] ② Inject via tail vein: 100 μl / animal 1×10 7 cells / each.
[0315] ③The bone marrow reconstruction is completed one month later, and the experiment can then be performed.
[0316] (4) Flow cytometry sorting of WT sources after bone marrow reconstitution and Clec7a - / - Cells of origin
[0317] ① Perform cell extraction according to the steps in “1.2.6”. After extraction, wash the cells twice with 500 μl FACS buffer (HBSS containing 2% FBS) before using them for flow cytometry staining.
[0318] ② Prepare antibody (FITC-anti-mouse-CD45.1, Pacific Blue-anti-mouse-CD45) dilution buffer at a dilution ratio of 1:250 using FACS buffer.
[0319] ③ Perform flow cytometry staining according to the steps in “1.2.11(3)”.
[0320] ④ Flow sorting strategy: CD45 + CD45.1 + That is, cells derived from WT, CD45 + CD45.1 is equivalent to Clec7a - / - The source of the cells.
[0321] 1.2.13 Macrophage clearance and transplantation
[0322] (1) Clearance of alveolar macrophages: 60 μl of chlorophosphate liposomes were administered nasally 3 days before BLM-induced pulmonary fibrosis to clear alveolar macrophages.
[0323] (2) Donor cell preparation: Obtain M2 according to the steps in “1.2.8(3)”.
[0324] (3) Macrophage transplantation
[0325] ① Three days after BLM-induced pulmonary fibrosis, 50 μl 4×10 5Cells were transplanted into the lungs via nasal drops.
[0326] ② Fourteen days after cell transplantation, mice were euthanized to assess the severity of pulmonary fibrosis.
[0327] 1.2.14 In vitro cell stimulation and co-culture
[0328] (1) Co-culture of alveolar macrophages and lung fibroblasts
[0329] ① One day in advance, lung fibroblasts were seeded into 48-well plates at a density of 1×10⁻⁶ cells / well. 5 / well.
[0330] ② One day later, discard the lung fibroblast culture medium and magnetically sort and purify 2×10⁶ alveolar macrophages. 5 / well is added to the pores of lung fibroblasts.
[0331] ③ After 24 hours, cells were collected for RNA extraction and qPCR detection.
[0332] (2) Curdlan in vitro stimulation
[0333] ① Following the steps in “1.2.8”, the obtained BMDMs were used for subsequent experiments.
[0334] ②BMDMs are laid in a 24-well plate at 250 μl / well, 1×10 6 / well.
[0335] ③ Add 25 ng / ml mouse IL-4, 25 ng / ml mouse IL-13, and 0, 10, 50, and 100 μg / ml curdlan to RPMI 1640 complete culture medium.
[0336] ④ Add curdlan medium containing 0, 10, 50, and 100 μg / ml to the wells of BMDMs respectively.
[0337] ⑤ After 24 hours, cells were collected for RNA extraction and qPCR detection.
[0338] (3) In vitro stimulation with Curdlan and Laminarin
[0339] ① Following the steps in “1.2.8”, the obtained BMDMs were used for subsequent experiments.
[0340] ②BMDMs are laid in a 24-well plate at 250 μl / well, 1×10 6 / well.
[0341] ③ Three hours before curdlan treatment, treat BMDMs with RPMI 1640 complete medium containing 1 mg / ml laminarin.
[0342] ④ Add 25 ng / ml mouse IL-4, 25 ng / ml mouse IL-13, and 100 μg / ml curdlan to the BMDMs culture medium.
[0343] ⑤ After 24 hours, cells were collected for RNA extraction and qPCR detection.
[0344] (4) In vitro stimulation with Curdlan and GW5074
[0345] ① Following the steps in “1.2.8”, the obtained BMDMs were used for subsequent experiments.
[0346] ②BMDMs are laid in a 24-well plate at 250 μl / well, 1×10 6 / well.
[0347] ③ Add 25 ng / ml mouse IL-4, 25 ng / ml mouse IL-13, and 0, 0.1, 0.5, and 5 μM GW5074 to RPMI 1640 complete medium.
[0348] ④ Add GW5074 medium containing 0, 0.1, 0.5, and 5 μM respectively to the wells of BMDMs.
[0349] ⑤ Add 100 μg / ml of curdlan to the BMDMs culture medium.
[0350] ⑥ After 24 hours, cells were collected for RNA extraction and qPCR detection.
[0351] 1.2.15 Bulk RNA Sequencing and Analysis
[0352] (1) Bulk RNA sequencing: WT and Clec7a – / – Lung tissue from mice with BLM-induced pulmonary fibrosis was used for RNA extraction and subsequent RNA sequencing. An Agilent Bioanalyzer 2100 system was used to detect RNA integrity (RIN); samples with RIN > 8 were used for further RNA sequencing. The NEBNext Ultra™ RNA Library Prep Kit for Illumina was used for library construction. NovaSeq6000 was used for sequencing and generating paired-end reads.
[0353] (2) Sequencing Data Analysis: Reads containing adapters, poly-N sequences, and low-quality reads were removed from the raw data to obtain clean reads. The Q20, Q30, GC content, and sequence repetition level of the clean reads were calculated. The data were aligned to the reference genome GRCm38 (mm10) using the Hisat2 software tool. The data were normalized using total count correction and then converted to log2(TPM+1) for further analysis. Genes with log2(fold change) > 1 or < -1 were considered upregulated or downregulated, respectively. KEGG pathway analysis was performed on genes with log2(fold change) > 1 or < -1 using the Benjamini and Hochberg methods. P < 0.05 and q < 0.1 were considered significant. Heatmaps were generated using Tbtools software, and the z-score was calculated using transcripts per million (TPM) as (TPM - average of TPM) / standard deviation. Other graphics were generated by imageGP (www.bic.ac.cn) and Gene Denovo (www.omicshare.com), online bioinformatics graphics tools.
[0354] 1.2.16 Data Analysis
[0355] Statistical analysis between two groups typically employs a two-tailed unpaired t-test. For experiments involving more than two correlated groups, one-way ANOVA combined with Tukey's multiple comparison test is used. The log-rank test is used to compare survival curves. Prism v8.0 and v9.0 (GraphPad Software) were used for statistical analysis. The Pearson correlation test for analyzing correlations was performed using R (version 4.1.3, R Foundation). Statistical significance was defined as a p-value less than 0.05.
[0356] 1.3 Experimental Conclusions
[0357] (1) This application found through experiments that clearing lung fungi with fluconazole can alleviate the BLM-induced pulmonary fibrosis phenotype. The specific experiments and conclusions are as follows:
[0358] To elucidate the role of pulmonary fungi in the regulation of Dectin-1-mediated pulmonary fibrosis, mice were administered fluconazole (FCZ), a potent antifungal agent, via intranasal administration before and during BLM treatment to eliminate normally present pulmonary fungi, thereby blocking the activation of Dectin-1 by fungal-derived β-glucan (Figure 1). WT and Clec7a – / –Mice were treated with PBS or FCZ, then induced with BLM pulmonary fibrosis, and euthanized after 14 days for experimental use (n = 4 in WT-PBS, WT-FCZ, and Clec7a). – / – -FCZ groups, n = 3 in Clec7a – / – -PBS group). After pulmonary fungal clearance with fluconazole, the weight loss induced by airway-administered BLM in WT mice was significantly reduced and approached the level seen in Clec7a mice treated with BLM. – / – Levels of weight change in mice (Figures 2 and 3).
[0359] Analysis of Masson staining results on lung tissue sections revealed that in WT mice with fibrosis modeling after clearance of pulmonary fungi, the degree of collagen deposition and the area of fibrosis were reduced. However, for Clec7a... – / – In mice, regardless of whether they received FCZ intranasal treatment, the area of fibrosis in the lungs caused by BLM was reduced (Figure 4).
[0360] Compared with the control group WT mice that did not receive FCZ in the nose, the expression levels of collagen-encoding genes (Col1a1 and Col3a1) in the lung tissue of WT mice treated with FCZ after BLM-induced modeling were also suppressed (Figure 5).
[0361] After flow cytometry analysis of bronchoalveolar lavage fluid cells following fibrosis modeling, no difference was observed in the proportion of alveolar macrophages (AM) (Figure 6), but the expression levels of Arg1 and Spp1 genes in BAL cells of FCZ-treated WT mice were significantly reduced (Figure 7).
[0362] The data (Figures 2-7) represent two independent experiments. Data in Figure 2 are presented as mean ± standard deviation. Data (Figures 2-5 and 7) were analyzed using one-way ANOVA combined with Tukey's multiple comparison test, and data (Figure 6) were analyzed using a two-tailed unpaired t-test. (ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
[0363] Since BLM causes alveolar epithelial cells to gradually form fibrosis during the damage and repair process by destroying the DNA of alveolar epithelial cells, this application uses an antifungal agent to clear normally present fungi in the lungs, and then observes that the fibrosis is improved in wild-type mice after clearance.
[0364] In summary, this indicates that when Dectin-1 is in a state of activation without fungal-derived β-glucan, the ability of AM to promote pulmonary fibrosis is reduced. Notably, clearing Clec7a... – / – Fungi in the mouse lungs did not affect the phenotype of reduced pulmonary fibrosis. These results suggest that lung fungi participate in the occurrence and development of pulmonary fibrosis by activating the Dectin-1 signaling pathway in the lungs (Figures 1–7).
[0365] Symbiotic fungi, found on most mucosal surfaces and skin, are considered another important component of symbiotic microorganisms besides bacteria. The interaction between symbiotic fungi and the immune system is crucial for host defense and immune system regulation, influencing not only intestinal diseases but also systemic diseases. Although some specific species of non-gut symbiotic fungi have been reported to play an important role in suppressing Dectin-1-mediated allergic airway inflammation by promoting the expansion of intestinal regulatory T cells.
[0366] However, the current study in this application shows that the intranasal antifungal drug fluconazole can significantly inhibit the progression of pulmonary fibrosis, and this phenomenon was only observed in WT mice, while no similar phenomenon was observed in Dectin-1 knockout mice. This suggests that pulmonary fungi play an important role in promoting Dectin-1 signaling-mediated pulmonary fibrosis.
[0367] (2) This application found through experiments that the RAF1 inhibitor GW5074 inhibits the RAF1 pathway, blocks Decin-1 signaling, and reduces pulmonary fibrosis. The specific experiments and conclusions are as follows:
[0368] WT mice were intraperitoneally injected with GW5074, a highly effective and specific RAF1 inhibitor (Figure 8). After GW5074 treatment, the degree of weight loss caused by BLM was reduced (Figure 9), the degree of collagen deposition and fibrosis area in the lungs was reduced (Figure 10), and the expression level of fibrosis-related genes in lung tissue was downregulated (Figure 11).
[0369] These results demonstrate that blocking the RAF1 pathway can inhibit the development of pulmonary fibrosis. After injection of GW5074, although the proportion of AM in the GW5074-treated group of fibrotic mouse BAL cells did not change significantly compared to the control group (Figure 12), the gene expression level of Arg1, which characterizes type II (M2) AM function, was decreased, and the gene expression level of the pro-fibrotic factor Tgfb1 was also downregulated (Figure 13). This indicates that the pro-fibrotic capacity of AM is inhibited after blocking the RAF1 pathway. Furthermore, for Clec7a... – / –Treatment of mice with GW5074 did not affect their phenotype of reduced pulmonary fibrosis (Figures 14-16), confirming that Dectin-1 is a major upstream pathway of RAF1 in BLM-induced pulmonary fibrosis. These results indicate that in the BLM-induced pulmonary fibrosis model, Dectin-1 on AM influences the pulmonary fibrosis phenotype through RAF1.
[0370] Data (Figures 14-17) represent two independent experiments. Data in (14-17) are shown as mean ± standard deviation. Data (14-16) were analyzed using a two-tailed unpaired t-test. (ns: not significant, *p<0.05, **p<0.01).
[0371] In summary, RAF1 is a downstream molecule of Dectin-1 signaling. By blocking RAF1 signaling using the RAF1 inhibitor GW5074, this application observed a significantly reduced pulmonary fibrosis phenotype in WT mice. However, treatment of Dectin-1 knockout mice with GW5074 had no significant effect on their pulmonary fibrosis phenotype. This indicates that Dectin-1 promotes the development of pulmonary fibrosis through a non-CARD9-dependent but RAF1-dependent pathway.
[0372] (3) This application found through experiments that the Dectin-1 antagonist laminarin reduces pulmonary fibrosis by inhibiting the binding of β-glucan to Dectin-1 and blocking the downstream signaling pathway of Dectin-1. The specific experiments and conclusions are as follows:
[0373] To investigate the effect of blocking the Dectin-1 signaling pathway with laminarin on pulmonary fibrosis, WT mice were treated with laminarin via nasal drops before and during BLM-induced pulmonary fibrosis (Figure 17). WT mice were treated with PBS or laminarin before BLM-induced pulmonary fibrosis modeling and euthanized 14 days later for experimental use. Laminarin treatment significantly reduced the weight loss induced by BLM in WT animals (Figure 18) and significantly inhibited the expansion of lung consolidation and fibrotic areas caused by fibrosis modeling (Figure 19). The expression of BLM-induced pulmonary fibrosis-related genes was also significantly reduced after laminarin treatment (Figure 20). Although the proportion and number of AM cells in the bronchoalveolar lavage fluid after BLM-induced pulmonary fibrosis were unchanged compared to the untreated group after laminarin treatment (Figures 21 and 22), the mRNA expression levels of Arg1 and Spp1 in the lungs were significantly reduced, indicating that blocking Dectin-1 inhibited the pro-fibrotic characteristics of AM (Figure 23). On the other hand, when bone marrow-derived M2 macrophages were treated with Dectin-1 ligands, treatment with the Dectin-1 agonist curdlan upregulated the expression of Arg1 and Tgfb1, while treatment with the antagonist laminarin or the RAF1 inhibitor GW5074 inhibited this upregulation induced by Dectin-1 stimulation (Figure 24). These results suggest that Dectin-1 signaling can directly induce or promote the profibrotic characteristics of M2 macrophages.
[0374] In this study, mouse M2 macrophages obtained from bone marrow cell culture were pretreated with laminarin (1 mg / ml) or GW5074 (1 μM) for 3 hours, followed by stimulation with curdlan (100 μg / ml) for 20 hours. The relative expression levels of Arg1 and Tgfb1 mRNA were determined by qPCR (using Gapdh as the standard, n = 3 wells / group). Data (Figures 18-23) represent three independent experiments, and data (Figure 24) represent two independent experiments. Data in Figures 18-20 and 22-24 are shown as mean ± standard deviation. Data in Figures 18-23 were analyzed using a two-tailed unpaired t-test, and data in Figure 24 were analyzed using one-way ANOVA combined with Tukey's multiple comparison test. (ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
[0375] In summary, laminarin, an antagonist of Dectin-1, administered via nasal drops, can also significantly inhibit pulmonary fibrosis, indicating that Dectin-1 activating ligands in the lungs play a promoting role in pulmonary fibrosis.
[0376] This application, through a deeper understanding of the role of Dectin-1, is expected to provide important basic support for the development of novel drugs and clinical treatment plans targeting Dectin-1 for the treatment of pulmonary fibrosis, and to provide new ideas and methods for the prevention and treatment of pulmonary fibrosis.
[0377] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.
Claims
1. Use of an agent that blocks Dectin-1 signaling in the manufacture of a medicament for the treatment and / or prevention of pulmonary fibrosis, characterized in that, The formulation includes at least one of an antifungal agent, a Dectin-1 antagonist, or a RAF1 inhibitor.
2. Use according to claim 1, characterized in that, The antifungal agents mentioned include at least one of imidazole antifungal agents, pyrimidine antifungal agents, allylamine antifungal agents, and echinocandins antifungal agents; The Dectin-1 antagonist includes at least one of kelp polysaccharide, β-glucan with a molecular weight of less than 5000 Da, neutralizing antibody or antagonistic antibody against Dectin-1; The RAF1 inhibitors include at least one of GW5074, Rafinhibitor 1, ZM336372, dabrafenib mesylate, dabrafenib, GSK2118436A, B-Raf inhibitor 1 dihydrochloride, Raf inhibitor 3, C-RAF kinase-IN-1, MEK1 / C-Raf-IN-1, MCP110, AZ628, Kobe2602, Kobe0065, BBO-8520, Bay 43-9006, sorafenib, Ferrottosis inducer-3, and neutralizing or antagonistic antibodies against RAF1.
3. Use according to claim 1 or 2, characterized in that, The antifungal agent reduces the β-glucan on the surface of fungi present in the lungs, thereby blocking Dectin-1 signaling and further aggravating pulmonary fibrosis caused by fungi in the lungs.
4. Use according to claim 3, characterized in that, The fungus is a fungus that can express β-glucan and can be recognized by Dectin-1, including at least one of Candida, Aspergillus, Penicillium, Disyllium, and Diplostomum.
5. Use according to claim 3, characterized in that, The fungus activates Dectin-1 through the expression of β-glucan, which further aggravates pulmonary fibrosis.
6. The application according to claim 1 or 2, characterized in that, The Dectin-1 antagonist inhibits the binding of β-glucan to Dectin-1, blocks the downstream signaling pathway of Dectin-1, and alleviates pulmonary fibrosis.
7. The application according to claim 1 or 2, characterized in that, The RAF1 inhibitors mentioned above alleviate pulmonary fibrosis by inhibiting RAF1 and blocking Dectin-1 signaling.
8. A composition for treating and / or preventing pulmonary fibrosis, characterized in that, The formulations included in claim 1 for use in the preparation of medicaments for the treatment and / or prevention of pulmonary fibrosis.
9. A medicament for treating and / or preventing pulmonary fibrosis, characterized in that, It includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient includes the formulation of claim 1 for use in the preparation of a medicament for the treatment and / or prevention of pulmonary fibrosis.
10. A medicament for treating and / or preventing pulmonary fibrosis according to claim 8 or 9, characterized in that, The dosage forms of the drug include one or more of the following: capsules, powders, tablets, granules, intravenous drip or injection, and nasopharyngeal spray.