Application of preparation for blocking Dectin-1 signal transduction in preparation of medicine for treating and / or preventing pulmonary fibrosis

Abstract

The invention relates to application of a preparation for blocking Dectin-1 signal transduction in preparation of a medicine for treating and / or preventing pulmonary fibrosis. The preparation comprises at least one of an antifungal agent, a Dectin-1 antagonist or an RAF1 inhibitor. Belongs to the technical field of biological medicine. At least one of the problems of large side effect, poor treatment effect and the like of the existing medicine for treating pulmonary fibrosis is solved. By deeply understanding the effect of the Dectin-1, important basic support is expected to be provided for a clinical treatment scheme for treating the pulmonary fibrosis by taking the Dectin-1 as a target spot, and a new thought and method are provided for preventing and treating the pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of a preparation for blocking Dectin-1 signal transduction in the preparation of a medicament for treating and / or preventing pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a chronic disease characterized by excessive proliferation and deposition of fibrous tissues in the lung tissue, resulting in a gradual decline in lung function, such as idiopathic pulmonary fibrosis. The immune response plays an important role in the pathogenesis of pulmonary fibrosis, and a variety of immune cells and signaling molecules are involved in the process of fibrosis development. Myeloid cells of innate immunity, especially alveolar macrophages, have been proven to play a key pro-fibrotic role in the process of pulmonary fibrosis, mainly manifested in that alveolar macrophages can promote the fibrotic activities of fibroblasts, myofibroblasts and other matrix cells. Currently, the traditional treatment methods for pulmonary fibrosis include the use of glucocorticoids, immunosuppressants and anti-fibrotic drugs, etc., but these methods generally have limited therapeutic effects, obvious side effects and uncertain long-term survival results, and part of the reason is that the potential mechanisms promoting and regulating fibrosis have not been fully studied.

[0003] Dectin-1 (gene name: CLEC7A) is a type of C-type lectin receptor, mainly present on the surfaces of immune cells such as dendritic cells, macrophages and neutrophils, can recognize β-glucan, and plays an important immunomodulatory role in the process of the body's resistance to fungal invasion. The immune role played by Dectin-1 in pulmonary fibrosis still needs to be further explored. Summary of the Invention

[0004] In view of the above analysis, the present invention aims to provide the use of a preparation for blocking Dectin-1 signal transduction in the preparation of a medicament for treating and / or preventing pulmonary fibrosis, so as to solve at least one of the problems such as large side effects of existing glucocorticoids, immunosuppressants and anti-fibrotic drugs on the medicament for treating pulmonary fibrosis, limited therapeutic effects and uncertain long-term survival results.

[0005] In a first aspect, the present invention provides the use of a preparation for blocking Dectin-1 signal transduction in the preparation of a medicament for treating and / or preventing pulmonary fibrosis, and the preparation includes at least one of an antifungal agent, a Dectin-1 antagonist or a RAF1 inhibitor.

[0006] Further, the antifungal agent includes at least one of an imidazole antifungal agent, a pyrimidine antifungal agent, an allylamine antifungal agent, an echinocandin antifungal agent;

[0007] The described Dectin-1 antagonists include at least one of laminarin, β-glucan with a molecular weight less than 5000, a neutralizing antibody against Dectin-1, or an antagonistic antibody.

[0008] The described 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, Ferroptosis inducer-3, a neutralizing antibody against RAF1, or an antagonistic antibody.

[0009] Furthermore, the described antifungal agent blocks the further exacerbation of pulmonary fibrosis caused by pulmonary fungi by reducing β-glucan on the surface of fungi present in the lungs and blocking Dectin-1 signaling.

[0010] Furthermore, the described fungi are fungi that can express β-glucan and can be recognized by Dectin-1, and the fungi include at least one of Candida, Aspergillus, Penicillium, Eurotium, and Davidiella tassiana.

[0011] Furthermore, the β-glucan expressed on the described fungi activates Dectin-1, further exacerbating pulmonary fibrosis.

[0012] Furthermore, the described Dectin-1 antagonist inhibits the binding of β-glucan to Dectin-1, blocks the downstream signaling pathway of Dectin-1, and reduces transforming growth factor β (TGF-β, gene name Tgfb1), arginase 1 (Arginase-1, gene name Arg1), and matrix metalloproteinase 12 (MMP12, gene name Mmp12) produced by alveolar macrophages (AMs) expressing Dectin-1 to alleviate pulmonary fibrosis.

[0013] Furthermore, the described RAF1 inhibitor blocks Dectin-1 signaling by inhibiting RAF1 and reduces TGF-β, Arginase-1, and MMP12 produced by AMs expressing Dectin-1 to alleviate pulmonary fibrosis.

[0014] In a second aspect, the present invention provides a composition for treating and / or preventing pulmonary fibrosis, including the described preparation.

[0015] Thirdly, the present invention provides a drug for treating and / or preventing pulmonary fibrosis, which comprises an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient comprises the above-mentioned preparation.

[0016] Furthermore, the dosage forms of the drug include one or more of capsules, powders, tablets, granules, drip or intravenous injections, and nasopharyngeal sprays.

[0017] Compared with the prior art, the present invention can at least achieve one of the following beneficial effects:

[0018] (1) The present invention discovers that the fungi existing in the lungs can participate in the occurrence and development process of pulmonary fibrosis by activating the Dectin-1 signaling pathway in the lungs, further aggravating pulmonary fibrosis. The present invention uses antifungal agents to eliminate the fungi in the lungs, thereby blocking the activation of Dectin-1 by β-glucan derived from fungi and inhibiting the further aggravation of pulmonary fibrosis; in addition, the present invention discovers that the RAF1 inhibitor can inhibit RAF1 to block Dectin-1 signal transduction and alleviate pulmonary fibrosis; the present invention also discovers 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.

[0019] (2) By deeply understanding the role of Dectin-1, the present invention is expected to provide important basic support for the clinical treatment plan for treating pulmonary fibrosis targeting Dectin-1, and provide new ideas and methods for the prevention and treatment of pulmonary fibrosis.

[0020] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combined solutions. Other features and advantages of the present invention will be described in the subsequent specification, and some advantages can be made obvious from the specification, or understood by implementing the present invention. The objectives and other advantages of the present invention can be realized and obtained from the content specifically pointed out in the specification and the drawings. Description of the Drawings

[0021] The drawings are only for the purpose of showing specific embodiments, and are not considered as limiting the present invention. Throughout the drawings, the same reference signs represent the same components.

[0022] Figure 1 It is a schematic diagram of the experimental design of fluconazole of the present invention;

[0023] Figure 2 It is to observe the body weight of mice daily after bleomycin (BLM) treatment in the fluconazole experiment of the present invention and compare it with the body weight before treatment;

[0024] Figure 3This is the graph of the change in body weight (in grams) on the last day after BLM treatment in the fluconazole experiment of the present invention;

[0025] Figure 4 This is the display of the fibrotic area by Masson staining of lung tissue at panoramic and 20-fold magnification and the pathological score of the severity of pulmonary fibrosis by the Ashcroft method in the fluconazole experiment of the present invention;

[0026] Figure 5 This is the graph of the relative expression levels of Col1a1 and Col3a1 mRNA in lung tissue measured by qPCR in the fluconazole experiment of the present invention;

[0027] Figure 6 This is the proportion of CD206 + CD11c + cells in immune cells of bronchoalveolar lavage (BAL) of wild-type (WT) mice after treatment with PBS or FCZ detected by flow cytometry in the fluconazole experiment of the present invention;

[0028] Figure 7 This is the graph of the relative expression levels of Arg1 and Spp1 mRNA in BAL cells measured by qPCR in the fluconazole experiment of the present invention;

[0029] Figure 8 This is the schematic diagram of the experimental design of GW5074 in the present invention;

[0030] Figure 9 This is the graph of the daily observation of the body weight of mice after BLM treatment and the comparison with the body weight before treatment, as well as the change in body weight (in grams) on the last day in the GW5074 experiment of the present invention;

[0031] Figure 10 This is the display of the fibrotic area by Masson staining of lung tissue at panoramic and 20-fold magnification and the pathological score of the severity of pulmonary fibrosis by the Ashcroft method in the GW5074 experiment of the present invention;

[0032] Figure 11 This is the relative expression levels of Col1a1, Col3a1 and Acta2 mRNA in lung tissue measured by qPCR in the GW5074 experiment of the present invention;

[0033] Figure 12 This is the proportion of CD206 + CD11c + cells in immune cells of BAL of WT mice after treatment with solvent or GW5074 detected by flow cytometry in the GW5074 experiment of the present invention;

[0034] Figure 13In the GW5074 experiment of the present invention, qPCR was used to determine the relative expression levels of Arg1, Spp1, and Tgfb1 mRNAs in BAL cells;

[0035] Figure 14 In the GW5074 experiment of the present invention, after Dectin-1 gene knockout (Clec7a - / - ) mice were administered GW5074, they were euthanized 14 days after intratracheal injection of BLM and used for experiments (Ctrl n = 5, GW5074 n = 3);

[0036] Figure 15 In the GW5074 experiment of the present invention, qPCR was used to determine the relative expression levels of related genes in BAL cells;

[0037] Figure 16 In the GW5074 experiment of the present invention, qPCR was used to determine the relative expression levels of related genes in BAL lung tissue;

[0038] Figure 17 This is the schematic diagram of the laminarin experiment design of the present invention;

[0039] Figure 18 In the laminarin experiment of the present invention, the body weight of mice was observed daily after BLM treatment and compared with the body weight before treatment, as well as the number of grams of body weight change on the last day;

[0040] Figure 19 In the laminarin experiment of the present invention, Masson staining of lung tissue showed fibrotic areas and the severity of pulmonary fibrosis was scored by the Ashcroft method under panoramic and 20-fold magnification;

[0041] Figure 20 In the laminarin experiment of the present invention, qPCR was used to determine the relative expression levels of Col1a1, Col3a1, Eln, Acta2, and Il11 mRNAs in lung tissue;

[0042] Figure 21 In the laminarin experiment of the present invention, flow cytometry was used to detect the proportion of CD206 + CD11c + cells in BAL immune cells of WT mice after PBS or laminarin treatment;

[0043] Figure 22 In the laminarin experiment of the present invention, flow cytometry was used to detect the proportion of CD206 + CD11c + cells in BAL immune cells of WT mice after PBS or laminarin treatment (PBS n = 5, Laminarin n = 6);

[0044] Figure 23 For the laminarin experiment of the present invention, the relative expression levels of Arg1 and Spp1 mRNAs in BAL cells were determined by qPCR;

[0045] Figure 24 This represents a secondary independent experiment in the laminarin experiment of the present invention. Detailed implementation manners

[0046] The preferred embodiments of the present invention will be specifically described below with reference to the accompanying drawings. The accompanying drawings form a part of the present invention and are used together with the embodiments of the present invention to explain the principles of the present invention, rather than to limit the scope of the present invention.

[0047] A specific embodiment of the present invention discloses the use of a preparation for blocking Dectin-1 signal transduction in the preparation of a drug for treating and / or preventing pulmonary fibrosis. The preparation includes at least one of an antifungal agent, a Dectin-1 antagonist, or a RAF1 inhibitor.

[0048] The present invention discovers that fungi present in the lungs can participate in the occurrence and development process of pulmonary fibrosis by activating the Dectin-1 signaling pathway in the lungs, further aggravating pulmonary fibrosis. The fungi present in the lungs include normal fungi present in the human microbial community. For example, the fungi present in the lungs are some non-pathogenic or opportunistic pathogenic fungi present in healthy lungs. The present invention uses an antifungal agent to eliminate the fungi present in the lungs, thereby blocking the activation of Dectin-1 by fungal-derived β-glucan and inhibiting the further aggravation of pulmonary fibrosis. In addition, the present invention discovers that a RAF1 inhibitor can inhibit RAF1 from blocking Dectin-1 signal transduction and reduce pulmonary fibrosis. The present invention also discovers 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.

[0049] The pulmonary fibrosis of the present invention can be acute pulmonary fibrosis caused by influenza virus infection, or acute and chronic pulmonary fibrosis, idiopathic pulmonary fibrosis, and pulmonary fibrosis caused by other unknown reasons caused by acute pneumonia lung injury caused by virus infection, etc.

[0050] Specifically, the antifungal agent includes at least one of an imidazole antifungal agent, a pyrimidine antifungal agent, an allylamine antifungal agent, and an echinocandin antifungal agent;

[0051] Preferably, the imidazole antifungal agents include at least one of ketoconazole, miconazole, fluconazole, and itraconazole; the pyrimidine antifungal agents include at least one of cytosine, thymine, and uracil; the allylamine antifungal agents include at least one of terbinafine and naftifine; the echinocandin antifungal agents include at least one of caspofungin and micafungin.

[0052] The Dectin-1 antagonists include at least one of laminarin, β-glucan with a molecular weight less than 5000, neutralizing antibodies against Dectin-1, or antagonistic antibodies.

[0053] Preferably, the Dectin-1 antagonists further include all other blocking and neutralizing antibodies against human and murine Dectin-1.

[0054] More preferably, the β-glucan with a molecular weight less than 5000 includes linear β-(1,3)-d-glucan or linear plus branched β-(1,3-1,6)-d-glucan.

[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, Ferroptosis inducer-3, neutralizing antibodies against RAF1, or antagonistic antibodies.

[0056] Specifically, the antifungal agent blocks the further exacerbation of pulmonary fibrosis caused by pulmonary fungi by reducing the β-glucan on the surface of fungi present in the lungs and blocking Dectin-1 signaling.

[0057] Specifically, the fungi are fungi that can express β-glucan and can be recognized by Dectin-1, and the fungi include at least one of Candida, Aspergillus, Penicillium, Eurotium, and Davidiella tassiana.

[0058] Specifically, the β-glucan expressed on the fungi activates Dectin-1, which further exacerbates pulmonary fibrosis.

[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 matrix metalloproteinase 12 (MMP12, gene name Mmp12) by alveolar macrophages (AMs) expressing Dectin-1 to alleviate pulmonary fibrosis.

[0060] Specifically, the RAF1 inhibitor alleviates pulmonary fibrosis by inhibiting RAF1 to block Dectin-1 signaling and reducing the production of TGF-β, Arginase-1, and MMP12 by AMs expressing Dectin-1.

[0061] Another specific embodiment of the present invention discloses a composition for treating and / or preventing pulmonary fibrosis, comprising the said preparation.

[0062] Another specific embodiment of the present invention discloses a drug for treating and / or preventing pulmonary fibrosis, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the said preparation.

[0063] Specifically, the dosage forms of the said drug include one or more of capsules, powders, tablets, granules, drip or intravenous injection agents, and nasopharyngeal sprays.

[0064] By deeply understanding the role of Dectin-1, the present invention is expected to provide important basic support for clinical treatment programs targeting Dectin-1 for treating pulmonary fibrosis, and provide new ideas and methods for the prevention and treatment of pulmonary fibrosis.

[0065] The following further explains and illustrates the technical solutions of the present invention in combination with specific embodiments.

[0066] Example 1

[0067] 1.1 Main instruments and reagents

[0068] The names, models, and manufacturers of the main instruments involved in the present invention are shown in Table 1. The names, models, and manufacturers of the reagents involved in the present invention are shown in Table 2.

[0069] Table 1

[0070]

[0071]

[0072] Table 2

[0073]

[0074]

[0075]

[0076] 1.2 Main experimental methods

[0077] 1.2.1 Human samples

[0078] This invention has been approved by the Clinical Investigation Committee of the First Affiliated Hospital of Sun Yat-sen University (approval number: IIT-2021-654), and the experiments strictly follow ethical regulations and guidelines.

[0079] (1) Extraction of fibroblasts

[0080] ① Lung tissues from patients during surgery were transported to the laboratory biosafety cabinet under sterile and refrigerated conditions.

[0081] ② The lung tissues were rinsed 3 times with sterile pre-cooled PBS to remove the attached blood.

[0082] ③ The lung tissues were minced into small tissue pieces.

[0083] ④ The tissue pieces were resuspended in DMEM medium with 15% FBS. After supplementing the medium to 8 ml, it was transferred to a 10-cm cell culture dish and marked with a cross to evenly distribute it in the dish.

[0084] ⑤ The culture dish containing the tissue pieces was placed in a cell culture incubator at 37°C and 5% CO2.

[0085] ⑥ Two days later, half of the cell supernatant was carefully aspirated and supplemented with DMEM medium with 15% FBS.

[0086] ⑦ On the fifth day, the supernatant was discarded, the cells were rinsed twice with 2 ml PBS, and 1 ml of trypsin was added for digestion for 1 min.

[0087] ⑧ 1 ml of DMEM was added to terminate the digestion. After the cells were resuspended, they were filtered through a 200-mesh filter and collected in a 15-ml centrifuge tube.

[0088] ⑨ After centrifugation at 1500 rmp at 4°C for 5 minutes, the supernatant was discarded. The cells were resuspended in 12 ml of DMEM with 15% FBS and transferred to a 10-cm cell culture dish and marked with a cross to evenly distribute them, and then placed in a cell culture incubator at 37°C and 5% CO2.

[0089] ⑩ The adherent cells obtained were human lung fibroblasts.

[0090] (2) Extraction of cells from bronchoalveolar lavage fluid (BALF)

[0091] ① The BALF from the patient's surgery was transported to the laboratory biosafety cabinet under sterile refrigerated conditions.

[0092] ② The BALF was filtered through a 200-mesh filter and collected in a 15-ml centrifuge tube.

[0093] ③ After centrifugation at 1800 rmp at 4 °C for 5 minutes, the supernatant was discarded. The pellet was resuspended and washed with sterile pre-cooled PBS.

[0094] ④ After centrifugation at 1800 rmp at 4 °C for 5 minutes, the supernatant was discarded. Step ③ was repeated 3 times.

[0095] ⑤ The cell pellet obtained after discarding the supernatant was the BAL cells.

[0096] 1.2.2 Experimental animals

[0097] The animal experiments of the present invention were carried out according to the protocol approved by the Laboratory Animal Management and Use Committee of Sun Yat-sen University (approval number: 2021001262), and the experiments strictly followed ethical regulations and guidelines. All experimental mice were housed in a specific pathogen-free environment (SPF) in an environmentally controlled clean room of the Laboratory Animal Facility of Sun Yat-sen Medical School of Sun Yat-sen University, and sterilized drinking water and food were provided.

[0098] (1) Clec7a - / - , Card9 - / - and Clec4n - / - mice were all of C57BL / 6J background and were introduced from the Animal Disease Model Center of the Institute of Biomedical Sciences, Tokyo University of Science.

[0099] (2) C57BL / 6J wild-type mice were generated by mating heterozygous Clec7a + / - , Card9 + / - and Clec4n + / - littermate Clec7a - / - , Card9 - / - and Clec4n - / - mice and wild-type (WT) mice were used for the experiments.

[0100] (3) C57BL / 6J mice with CD45.1 + were kindly donated by teachers from Sun Yat-sen Medical School of Sun Yat-sen University.

[0101] Only male animals aged 7 - 10 weeks were used in the present invention.

[0102] 1.2.3 Genotyping of mice

[0103] (1) DNA extraction

[0104] ① Cut 2 - 3 mm of the tail of 4-week-old mice about to be weaned.

[0105] ② Add mouse tail and 135 μl of 50 mM NaOH to a 1.5 ml Ep tube.

[0106] ③ Shake and heat in a metal bath at 95 °C for 30 minutes.

[0107] ④ After instantaneous centrifugation, add 15 μl of 1 M Tris HCl and mix well.

[0108] 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.

[0109] ⑥ Take 2 μl of the diluted solution for PCR.

[0110] (2) PCR identification

[0111] ① The PCR system and reaction conditions are shown in Tables 3 and 4:

[0112] Table 3 PCR system

[0113] Primer mixture (F+R) 10 μM dNTPs KODFX Neo KODFx ddH2O DNA 1 μl 4 μl 10 μl 0.2 μl 0.8 μl 4 μl

[0114] Table 4 PCR reaction conditions

[0115]

[0116] ② Clec7a - / - , Card9 - / - and Clec4n - / - The primer sequences for identification are shown in Table 5:

[0117] Table 5 Primer sequences for gene identification

[0118]

[0119] 1.2.4 Construction of bleomycin (BLM)-induced pulmonary fibrosis model

[0120] (1) Preparation of bleomycin

[0121] ① Bleomycin was purchased from Yuanye Bio. Dissolve 10 mg of BLM powder in 1 ml of PBS to prepare a stock solution of 10 mg / ml, and store it at -80 °C after aliquoting.

[0122] ② Dissolve the stock solution before modeling, and dilute it to a working concentration of 0.12 mg / ml with PBS. The drug-dose to body weight ratio is 0.24 mg / kg.

[0123] (2) Preparation and administration strategy of GW5074 (RAF1 inhibitor)

[0124] ① GW5074 was purchased from APExBIO. 5 mg of GW5074 powder was dissolved in 200 μl of PBS to prepare a stock solution at a concentration of 25 mg / ml. After aliquoting, it was stored at -80 °C.

[0125] ② The stock solution was dissolved before use and diluted with PBS to a working concentration of 0.25 mg / ml.

[0126] ③ The drug was intraperitoneally injected into the mice once a day for 3 consecutive days, 3 times in total, at a dose - to - body - weight ratio of 2 mg / kg, 3 days before establishing the mouse model of pulmonary fibrosis.

[0127] ④ After establishing the pulmonary fibrosis model, GW5074 was administered once every other day, 4 times in total.

[0128] ⑤ GW5074 was administered 7 times in total.

[0129] (3) Preparation and administration strategy of Laminarin

[0130] ① Laminarin was purchased from InvivoGen. 100 mg of Laminarin powder was dissolved in 5 ml of PBS to prepare a stock solution at a concentration of 20 mg / ml. After aliquoting, it was stored at -80 °C.

[0131] ② The stock solution was dissolved before use and diluted with PBS to a working concentration of 1 mg / ml.

[0132] ③ 50 μl of the working solution was instilled into the nasal cavity of the mice once a day for 3 consecutive days, 3 times in total, 3 days before establishing the mouse model of pulmonary fibrosis.

[0133] ④ After establishing the pulmonary fibrosis model, it was instilled into the nasal cavity once every other day, 4 times in total.

[0134] ⑤ Laminarin was administered 7 times in total.

[0135] (4) Preparation and administration strategy of fluconazole

[0136] ① Fluconazole was purchased from Sigma - Aldrich. 1 g of fluconazole powder was dissolved in 2 L of PBS to prepare a working solution at a concentration of 0.5 mg / ml. After aliquoting, it was stored at -80 °C.

[0137] ② The working solution was dissolved before use.

[0138] ③ 50 μl of the working solution was instilled into the nasal cavity of the mice once every other day for 7 times, 14 days before establishing the mouse model of pulmonary fibrosis.

[0139] ④ After establishing the pulmonary fibrosis model, it was instilled into the nasal cavity once every other day, 4 times in total.

[0140] ⑤ Fluconazole was administered 11 times in total.

[0141] (5) Intratracheal injection in mice

[0142] ① Anesthetize the mice by intraperitoneal injection of 1% sodium pentobarbital at 30 mg / kg.

[0143] ② After the mice are completely anesthetized, weigh them and record the weight. Shave the hair on the neck to expose the neck skin.

[0144] ③ According to the weight of the mice, aspirate the corresponding volume of BLM working solution and then inhale it into a 1-ml syringe with a needle.

[0145] ④ Place the mice on their backs and fix their limbs with medical tape. Disinfect the neck skin with iodophor.

[0146] ⑤ Use ophthalmic scissors to make a longitudinal incision about 0.5 cm in the neck, and use ophthalmic forceps to bluntly separate the muscles to expose the trachea.

[0147] ⑥ Insert the syringe containing BLM liquid into the trachea and quickly push out the liquid. When the mice are quickly held upright and rotated left and right, the liquid is evenly distributed in the lungs.

[0148] ⑦ Suture the neck incision and disinfect the surface skin.

[0149] ⑧ Place the postoperative mice on a 37°C constant temperature heating pad until they recover and then transfer them to an IVC cage.

[0150] 1.2.5 RNA Extraction and Real-Time Fluorescent Quantitative PCR

[0151] (1) RNA Extraction

[0152] ① The Mammalian Total RNA Miniprep kit is used for RNA extraction.

[0153] ② For tissue samples, use a grinding bead homogenizer to homogenize the tissue and then perform subsequent operations; for cell samples, mix them with lysis buffer.

[0154] ③ Take 200 μl of lysis buffer containing the sample and place it in the filter column, and centrifuge at 12,000 rpm for 2 min.

[0155] ④ Discard the supernatant in the column, add an equal volume of 70% ethanol to the column and mix well. Transfer the mixture to the nucleic acid purification column and centrifuge at 12,000 rpm for 20 s.

[0156] ⑤ Discard the waste liquid in the lower column, add 330 μl of Wash Solution Ⅰ to the upper column, and centrifuge at 12,000 rpm for 20 s.

[0157] ⑥ Discard the waste liquid in the lower column, add 300 μl of Wash Solution Ⅱ to the upper column, and centrifuge at 12,000 rpm for 20 s.

[0158] ⑦Discard the waste liquid from the lower column, add 330 μl of Wash Solution Ⅱ to the upper column, and centrifuge at 12,000 rpm for 1 min.

[0159] ⑧Discard the waste liquid from the lower column, centrifuge at 12,000 rpm for 20 s, and then replace the lower column with a collection tube.

[0160] ⑨Add 15 - 40 μl of RNase free H2O to the center of the upper column and centrifuge at 12,000 rpm for 1 min.

[0161] ⑩Measure the RNA concentration using Nano Drop.

[0162] (2) Reverse transcription of RNA into cDNA

[0163] ①The reverse transcription reagent Evo M - MLV RT Master Mix was purchased from Aikrui Biotechnology, and the RNA quality used for reverse transcription was 50 - 500 ng

[0164] ②The reverse transcription system is shown in Table 6:

[0165] Table 6 RNA reverse transcription system

[0166] RNA <![CDATA[RNasefreeH2O]]> 5×RT MIX Total amount RNA quality / RNA concentration 8 μl - RNA quality / RNA concentration 2 μl 10 μl

[0167] ③The reverse transcription program is shown in Table 7:

[0168] Table 7 Reverse transcription reaction conditions

[0169]

[0170] ④The cDNA was diluted 10 - 20 times with ultrapure water and used for qPCR.

[0171] (3) Real - time fluorescence quantitative PCR (qPCR)

[0172] ① Green Pro Taq HS Premix qPCR Kit was used for qPCR. ②Reverse transcription program:

[0173] Table 8 qPCR system

[0174] SYBR green (Rox plus) cDNA Primer <![CDATA[ddH2O]]> Total volume 2.7 μl 2 μl 1 μl 4.3 μl 10 μl

[0175] ③qPCR program:

[0176] Table 9 qPCR reaction conditions

[0177]

[0178] ④The primer list is shown in Table 10.

[0179] Table 10

[0180]

[0181]

[0182]

[0183] ⑤Gapdh or GADPH is used for the normalization of the relative mRNA expression level.

[0184] 1.2.6 Cell extraction

[0185] (1) After the mice are euthanized, they are used for cell extraction.

[0186] (2) Extraction of BAL cells

[0187] ① Place the mouse on its back and fix its four limbs on the operating table.

[0188] ② Cut open the abdominal skin to expose the abdominal cavity, and cut open the diaphragm to make the lung tissue communicate with the atmospheric pressure.

[0189] ③ Cut open the neck skin and separate the muscle tissue around the trachea to fully expose it.

[0190] ④ Make a small cut in the trachea and insert a 22G soft syringe needle.

[0191] ⑤ Carefully and slowly push the pre-cooled 0.5 ml HBSS into the syringe through the hose, wait for 20 s and then aspirate the liquid, transfer it to a 15 ml centrifuge tube, and the liquid recovery rate is about 80%. Repeat this operation 4 times.

[0192] ⑥ Centrifuge the collected BALF at 1800 rpm at 4 °C for 5 min.

[0193] ⑦ Discard the supernatant, resuspend the cells in 2 ml of red blood cell lysis buffer (ACK) and let it stand for 5 min, then add an equal volume of HBSS to terminate hemolysis.

[0194] ⑧ Centrifuge at 1800 rpm at 4 °C for 5 min, discard the supernatant, and wash the cells twice with 5 ml of HBSS.

[0195] ⑨ Centrifuge at 1800 rpm at 4 °C for 5 min, discard the supernatant, and the obtained cell pellet is the BAL cells.

[0196] (3) Extraction of single cells from lung tissue

[0197] ① Place the mouse on its back and fix its four limbs on the operating table.

[0198] ② Cut open the abdominal skin to expose the thoracic cavity, remove the ribs, and expose the lung tissue and the heart.

[0199] ③ Cut open the inferior vena cava, insert a needle into the right ventricle, and push PBS to perform pulmonary circulation perfusion to flush out the blood in the lungs.

[0200] ④ Cut the lung tissue and cut it into pieces about 1 mm 3 in size.

[0201] ⑤ Resuspend the pieces in 5 ml of medium containing 200 U / ml type IV collagenase and 5 U / ml DNase I, and incubate in a water bath at 37 °C for 2 h.

[0202] ⑥ After the water bath, vortex for 10 s, filter the suspension through a 200-mesh filter, and remove large pieces.

[0203] ⑦ Centrifuge at 1800 rpm at 4 °C for 5 min, discard the supernatant, gently resuspend the cells with 2 ml of red blood cell lysate, let stand for 5 min, and add an equal volume of HBSS to terminate hemolysis.

[0204] ⑧ Centrifuge at 1800 rpm at 4 °C for 5 min, discard the supernatant, and wash twice with 5 ml of HBSS.

[0205] ⑨ Centrifuge at 1800 rpm at 4 °C for 5 min, discard the supernatant, and the resulting cell pellet is the single cells of the lung tissue.

[0206] 1.2.7 Purification and culture of lung fibroblasts

[0207] (1) After the mice are euthanized, they are used for cell extraction.

[0208] (2) Extraction of lung fibroblasts

[0209] ① Place the mouse on its back with its limbs fixed to the operating table.

[0210] ② Cut open the abdominal skin, expose the chest cavity, remove the ribs, and expose the lung tissue and heart.

[0211] ③ Cut open the inferior vena cava, insert a needle into the right ventricle, and inject PBS to perfuse the pulmonary circulation to flush out the blood in the lungs.

[0212] ④ Cut the lung tissue and cut it into pieces about 1 mm 3 in size.

[0213] ⑤ Resuspend the pieces in 8 ml of DMEM containing 15% FBS and 1% double antibody, transfer to a 10-cm cell culture dish, and culture in a cell culture incubator at 37 °C and 5% CO2.

[0214] ⑥ After 2 days, change half of the medium and continue culturing.

[0215] ⑦ After 5 days, long spindle-shaped and densely arranged adherent cells can be seen under the microscope, which are lung fibrotic cells.

[0216] (3) Purification of lung fibroblasts

[0217] ① Discard the fibroblast supernatant, wash the cells twice with 2 ml of PBS, and add 1 ml of trypsin to digest for 1 minute.

[0218] ② Add 1 ml of DMEM to terminate the digestion, pipette the cells to mix well, filter them through a 70-μm cell strainer, and collect the cells in a 15-ml centrifuge tube.

[0219] ③ After centrifuging at 1500 rmp at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 100 μl of basal 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°C for 20 minutes.

[0220] ④ Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and wash twice with 2 ml of basal medium.

[0221] ⑤ Discard the supernatant, resuspend the cells in 100 μl of basal medium containing 10 μl of anti-biotin microparticle magnetic beads, and incubate at 4°C for 20 minutes.

[0222] ⑥ Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and wash twice with 2 ml of basal medium.

[0223] ⑦ Discard the supernatant, resuspend the cells in 500 μl of basal medium, and transfer them to a flow cytometry tube.

[0224] ⑧ Place the flow cytometry tube on a magnetic stand. After 5 minutes, the positive cells will adhere to the tube wall. Carefully aspirate the liquid, and resuspend the aspirated liquid in 2 ml of basal medium.

[0225] ⑨ Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and the obtained cells are the purified lung fibroblasts.

[0226] 1.2.8 Induction of bone marrow-derived macrophages and M2 macrophages

[0227] (1) After euthanizing the mice, they are used for cell extraction.

[0228] (2) Extraction of bone marrow cells

[0229] ① Place the mouse on its back, cut open the skin of the lower limbs, separate the femur, tibia, and humerus, and remove the attached muscles.

[0230] ② Use a 1-ml syringe to aspirate basal RPMI1640 medium, flush out the bone marrow, and collect it in a 15-ml centrifuge tube.

[0231] ③ Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells gently by pipetting with 5 ml of red blood cell lysate, let it stand for 5 minutes, and then add an equal volume of RPMI1640 medium containing 10% FBS to terminate hemolysis.

[0232] ④ Centrifuge at 1500 rpm at 4 °C for 5 min, discard the supernatant, and wash twice with 5 ml of medium.

[0233] ⑤ Centrifuge at 1800 rpm at 4 °C for 5 min, discard the supernatant, and the resulting cell pellet is the bone marrow cells.

[0234] (3) Induction of bone marrow macrophages (BMDM) and type II macrophages (M2)

[0235] ① Resuspend the cell pellet from the previous step in 8 ml of RPMI 1640 complete medium containing 30 ng / ml murine M-CSF and transfer it to a 10-cm cell culture dish. Culture in a cell culture incubator at 37 °C with 5% CO2.

[0236] ② On the 3rd day, replace half of the medium with RPMI 1640 complete medium containing 30 ng / ml murine M-CSF.

[0237] ③ On the 7th day, the adherent cells obtained are BMDM.

[0238] ④ Discard the supernatant, add 8 ml of RPMI 1640 complete medium containing 25 ng / ml murine IL-4 and 25 ng / ml murine IL-13, and continue culturing for 24 h.

[0239] ⑤ After 24 h, the resulting cells are M2 macrophages.

[0240] 1.2.9 Masson staining and immunohistochemical staining of pathological sections

[0241] (1) Tissue fixation, paraffin embedding, and sectioning

[0242] ① Immerse the mouse lung tissue after pulmonary circulation perfusion or human postoperative lung tissue block in 10% formalin for fixation.

[0243] ② Dehydrate the tissue overnight in absolute ethanol and then perform paraffin embedding.

[0244] ③ Cut the tissue paraffin block into sections with a thickness of 5 μm.

[0245] (2) Deparaffinization

[0246] ① Place the paraffin section sample baked on the staining rack in the oven into xylene I and soak for 20 min.

[0247] ② Transfer the sample to xylene II and soak for 20 min.

[0248] ③ After the wax on the sample dissolves, transfer the sample to absolute ethanol I and soak for 5 min.

[0249] ④ Transfer the sample to absolute ethanol II and soak for 5 min.

[0250] ⑤ Scrub with absolute ethanol for 20 s, transfer to a washbasin, and rinse the alcohol on the sample thoroughly with running tap water.

[0251] (3) Masson staining

[0252] ① Place the sections in the Masson staining solution 1 in a microwave repair box and incubate overnight.

[0253] ② Take out the sections, use a staining cup to quickly wash them with tap water until colorless.

[0254] ③ Place the sections in the Masson staining solution 2 (preheat at 65 °C for 30 min before staining) in a microwave repair box, put them back in the oven for staining for 3 - 5 min, then use a staining cup to wash them 2 - 3 times with tap water.

[0255] ④ Place the sections in the Masson staining solution 3 in a microwave repair box and stain for 30 s - 1 min.

[0256] ⑤ Take out the sections, drain them slightly, place them in the Masson staining solution 4 (preheat at 65 °C for 30 min before staining and put it back in the oven for preheating after use) in a microwave repair box and stain for 5 - 20 s.

[0257] ⑥ Place the sections in 1% glacial acetic acid (to be prepared by yourself) for differentiation for several seconds, in three tanks, with 5 - 10 s of differentiation in each tank.

[0258] ⑦ Place the sections in absolute ethanol in three tanks in a microwave repair box, with 5 - 10 s in each tank.

[0259] ⑧ Transfer the sections to n - butanol in a microwave repair box for 10 - 20 s.

[0260] ⑨ Place the sections in xylene Ⅰ in a staining cylinder for 5 min

[0261] ⑩ Transfer to xylene Ⅱ in a staining cylinder for 5 min, quickly dry the sections at the ventilation opening, and then mount the sections with neutral balsam.

[0262] (4) Immunohistochemical staining

[0263] ① Antigen retrieval: Immerse the sections in citric acid (pH 6.0) and incubate overnight at 60 °C.

[0264] ② Block endogenous enzymes: Place the sections in 3% H2O2 solution and incubate at room temperature for 20 min. Wash with PBS 3 times, 5 min each time.

[0265] ③ Serum blocking: Use a histochemical pen to draw a circle around the tissue, then drop serum on the tissue and incubate at 37 °C for 30 min.

[0266] ④ Incubate with primary antibody: Dilute the antibody (anti - human - Dectin - 1, 1 μg / ml) with an antibody diluent, discard the serum on the slides, drop the antibody working solution on the tissue, and incubate overnight at 4 °C.

[0267] ⑤ Secondary antibody incubation: Prepare the secondary antibody labeled with HRP enzyme using PBST. Add an appropriate amount of the secondary antibody dropwise onto the tissue and incubate at 37°C for 1 h. Wash with PBST three times, 5 min each time.

[0268] ⑥ DAB color development: Drop the DAB working solution onto the tissue and observe under a microscope. Wait until specific brown color appears, then rinse the DAB color development solution on the tissue with water, and subsequently soak the slide in water.

[0269] ⑦ Counterstaining with hematoxylin: Place the slide in the hematoxylin staining solution and stain for 3 - 5 min. Wash off the excess hematoxylin staining solution with water. After the cell nuclei turn blue, immerse the slide into the 0.5% hydrochloric acid alcohol differentiation solution for 1 - 2 s, and immediately rinse with water. Then place the slide in the blueing solution and soak for 3 - 5 s, and subsequently rinse with water.

[0270] ⑧ Place the slide successively into containers containing absolute ethanol 1, absolute ethanol 2, absolute ethanol 3, n-butanol 1, n-butanol 2, xylene 1, and xylene 2, soak for 5 min each time. After air drying, drop an appropriate amount of neutral gum onto the tissue, cover the tissue with a coverslip, and let it dry.

[0271] 1.2.10 Immunofluorescence staining

[0272] (1) Sections of human lung fibrosis tissue are prepared according to the method described above.

[0273] (2) Immunofluorescence staining

[0274] ① Serum blocking: After antigen retrieval, use a histochemical pen to draw a circle around the tissue on the section, and drop 1% goat serum onto the tissue and incubate at 37°C for 1 h.

[0275] ② Primary antibody incubation: Dilute the antibodies (anti-human - Dectin-1, 10 μg / ml; anti-human - α-SMA, 1:250) using 1% goat serum. Discard the serum on the slide, and drop the antibody working solution onto the tissue and incubate overnight at 4°C.

[0276] ③ Wash with PBS three times, 5 min each time.

[0277] ④ Secondary antibody incubation: Drop an appropriate amount of the secondary antibody (goat-anti-rabbit-Alexa594, 1:500) onto the tissue and incubate at room temperature for 1 h. Wash with PBS three times, 5 min each time.

[0278] ⑤ Drop DAPI onto the tissue and cover with a coverslip.

[0279] 1.2.11 Flow cytometry

[0280] (1) Extract cells according to the steps of "1.2.6". After the extracted cells are washed twice with 500 μl of FACS buffer (HBSS containing 2% FBS), they can be used for flow cytometry staining.

[0281] (2) Staining of Dectin-1

[0282] 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 stock solution of 0.1 mg / ml, and after aliquoting, it was stored at -80 °C.

[0283] ① Prepare a secondary antibody (anti-rabbit-IgG-FITC) dilution solution and 2.4G2 blocking antibody at a dilution ratio of 1:250 with FACS buffer.

[0284] ② Resuspend the cells in 500 μl of FACS buffer in a 1.5 ml EP tube and centrifuge at 5000 rpm at 4 °C for 2 min.

[0285] 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.

[0286] ④ Wash twice with 500 μl of FACS buffer and centrifuge at 5000 rpm at 4 °C for 2 min.

[0287] ⑤ Discard the supernatant, resuspend and mix the cells with 30 μl of FACS buffer and 2 μl of anti-mouse-Dectin-1 stock solution, and incubate at 4 °C for 30 min.

[0288] ⑥ Wash twice with 500 μl of FACS buffer and centrifuge at 5000 rpm at 4 °C for 2 min.

[0289] ⑦ Discard the supernatant, aspirate 30 μl of the secondary antibody dilution solution to resuspend the cell pellet, and incubate at 4 °C for 30 min.

[0290] ⑧ Wash twice with 500 μl of FACS buffer and centrifuge at 5000 rpm at 4 °C for 2 min.

[0291] ⑨ Discard the supernatant, and then proceed with the staining of other antibodies.

[0292] (3) Cell surface staining

[0293] ① Prepare an antibody dilution solution and 2.4G2 blocking antibody at a dilution ratio of 1:250 with FACS buffer.

[0294] ② Resuspend the cells in 500 μl of FACS buffer in a 1.5 ml EP tube and centrifuge at 5000 rpm at 4 °C for 2 min.

[0295] ③Discard the supernatant, aspirate 30 μl of 2.4G2 blocking antibody to resuspend the cell pellet, and incubate at 4°C for 30 min.

[0296] ④Wash twice with 500 μl of FACS buffer, and centrifuge at 5000 rpm at 4°C for 2 min.

[0297] ⑤Discard the supernatant, aspirate 30 μl of antibody diluent to resuspend the cell pellet, and incubate at 4°C for 30 min.

[0298] ⑥Wash twice with 500 μl of FACS buffer, and centrifuge at 5000 rpm at 4°C for 2 min.

[0299] ⑦Discard the supernatant, resuspend with 400 μl of FACS buffer, filter through a 200-mesh filter, and then perform flow cytometry analysis, or further perform intracellular antibody staining.

[0300] (4) Intracellular staining

[0301] ①Anti-mouse-CD206 and anti-mouse-Arg-1 require intracellular staining.

[0302] ②Resuspend the cell pellet with 120 μl of Cytofix / Cytoperm, and incubate at 4°C for 20 min or overnight at 4°C.

[0303] ③Prepare the antibody diluent with BD Perm / Wash Buffer at a dilution ratio of 1:250.

[0304] ④Wash twice with 500 μl of BD Perm / Wash Buffer, and centrifuge at 5000 rpm at 4°C for 2 min.

[0305] ⑤Discard the supernatant, aspirate 30 μl of antibody diluent to resuspend the cell pellet, and incubate at 4°C for 30 min.

[0306] ⑥Wash twice with 500 μl of BD Perm / Wash Buffer, and centrifuge at 5000 rpm at 4°C for 2 min.

[0307] ⑦Discard the supernatant, resuspend with 400 μl of FACS buffer, filter through a 200-mesh filter, and then perform flow cytometry analysis.

[0308] (5) autoMACS automatic magnetic bead sorting

[0309] ①BAL cells are used for magnetic bead sorting.

[0310] ②Resuspend the cells with 100 μl of basal medium containing 1 μl of anti-mouse CD11c-biotin, and incubate at 4°C for 20 min.

[0311] ③ Centrifuge at 1500 rpm at 4°C for 5 min, discard the supernatant, and wash twice with 2 ml of basal medium.

[0312] 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.

[0313] ⑤ Centrifuge at 1500 rpm at 4°C for 5 min, discard the supernatant, and wash twice with 2 ml of basal medium.

[0314] ⑦ Discard the supernatant, resuspend the cells in 500 μl of basal medium, and transfer to a flow cytometry tube.

[0315] ⑧ Perform autoMAC upper sorting and select the fine positive selection mode.

[0316] ⑨ Resuspend the positively selected cells in 2 ml of basal medium and centrifuge at 1500 rpm at 4°C for 5 min.

[0317] ⑩ Discard the supernatant to obtain the cells with CD11c in the obtained cells and BALF + cells, and alveolar macrophages.

[0318] 1.2.12 Construction of bone marrow chimeras

[0319] (1) Preparation of recipient mice

[0320] ① Add 250 mg / l gentamicin, 320 mg / l erythromycin, and 10% sucrose to the drinking water of 6-week-old WT (CD45) mice one week before irradiation.

[0321] ② Irradiate the recipient mice one week later with 6.5 Gy of X-rays.

[0322] (2) Preparation of donor cells

[0323] ① WT (CD45.1) and Clec7a - / - (CD45) mice are used to extract bone marrow cells.

[0324] ② Extract bone marrow cells according to the steps of "1.2.8(2)".

[0325] ③ Mix the cells from WT and Clec7a - / - sources at a ratio of 1:1 and adjust the cell concentration to 1×10 8 / ml

[0326] (3) Bone marrow transplantation

[0327] ① Perform bone marrow transplantation on the recipient mice on the day of irradiation.

[0328] ②Perform tail vein injection: 100 μl / mouse, 1×10 7 cells / mouse.

[0329] ③After one month, bone marrow reconstitution is completed and the experiment can be carried out.

[0330] (4) Flow cytometry sorting of cells from WT and Clec7a - / - sources after bone marrow reconstitution

[0331] ①Extract cells according to the steps in "1.2.6". The extracted cells can be used for flow cytometry staining after being washed twice with 500 μl of FACS buffer (HBSS containing 2% FBS).

[0332] ②Prepare the antibody (FITC - anti - mouse - CD45.1, PacificBlue - anti - mouse - CD45) dilution with FACS buffer at a dilution ratio of 1:250.

[0333] ③Perform flow cytometry staining according to the steps in "1.2.11(3)".

[0334] ④Flow cytometry sorting strategy: CD45 + CD45.1 + are the cells from WT source, CD45 + CD45.1 - are the cells from Clec7a - / - source.

[0335] 1.2.13 Clearance and transplantation of macrophages

[0336] (1) Clearance of alveolar macrophages: Three days before BLM - induced pulmonary fibrosis, instill 60 μl of clodronate liposomes into the nasal cavity to clear alveolar macrophages.

[0337] (2) Preparation of donor cells: Obtain M2 according to the steps in "1.2.8(3)".

[0338] (3) Macrophage transplantation

[0339] ①Three days after BLM - induced pulmonary fibrosis, instill 50 μl of 4×10 5 cells into the lungs by nasal drip for transplantation.

[0340] ②Fourteen days after cell transplantation, euthanize the mice and evaluate the severity of pulmonary fibrosis.

[0341] 1.2.14 In vitro stimulation and co - culture of cells

[0342] (1) Co - culture of alveolar macrophages and lung fibroblasts

[0343] ① Seed lung fibroblasts in a 48-well plate one day in advance at a density of 1×10 5 / well.

[0344] ② One day later, discard the culture medium of lung fibroblasts, and add 2×10 5 / well of purified alveolar macrophages sorted by magnetic force to the wells containing lung fibroblasts.

[0345] ③ Harvest the cells after 24 h for RNA extraction and qPCR detection.

[0346] (2) In vitro stimulation with curdlan

[0347] ① According to the steps of "1.2.8", the obtained BMDMs are used for subsequent experiments.

[0348] ② Seed BMDMs in a 24-well plate at a density of 1×10 6 / well in 250 μl / well.

[0349] ③ Add 25 ng / ml murine IL-4, 25 ng / ml murine IL-13, and 0, 10, 50, 100 μg / ml of curdlan to the RPMI 1640 complete medium.

[0350] ④ Add the curdlan-containing medium at 0, 10, 50, 100 μg / ml to the wells of BMDMs respectively.

[0351] ⑤ Harvest the cells after 24 h for RNA extraction and qPCR detection.

[0352] (3) In vitro stimulation with curdlan and laminarin

[0353] ① According to the steps of "1.2.8", the obtained BMDMs are used for subsequent experiments.

[0354] ② Seed BMDMs in a 24-well plate at a density of 1×10 6 / well in 250 μl / well.

[0355] ③ Three hours before curdlan treatment, treat BMDMs with the RPMI 1640 complete medium containing 1 mg / ml laminarin

[0356] ④ Add 25 ng / ml murine IL-4, 25 ng / ml murine IL-13, and 100 μg / ml of curdlan to the culture medium of BMDMs.

[0357] ⑤ Harvest the cells after 24 h for RNA extraction and qPCR detection.

[0358] (4) In vitro stimulation of curdlan and GW5074

[0359] ① According to the steps in "1.2.8", the obtained BMDMs were used for subsequent experiments.

[0360] ② The BMDMs were seeded in 24-well plates at 250 μl / well, 1×10 6 / well.

[0361] ③ 25 ng / ml of murine IL-4, 25 ng / ml of murine IL-13, and 0, 0.1, 0.5, 5 μM of GW5074 were added to the RPMI1640 complete medium.

[0362] ④ The media containing 0, 0.1, 0.5, 5 μM of GW5074 were added to the BMDMs wells respectively.

[0363] ⑤ Then 100 μg / ml of curdlan was added to the BMDMs culture medium.

[0364] ⑥ After 24 h, the cells were harvested for RNA extraction and qPCR detection.

[0365] 1.2.15 Bulk RNA sequencing and analysis

[0366] (1) Bulk RNA sequencing: Lung tissues of WT and Clec7a – / – mice after BLM-induced pulmonary fibrosis were used for RNA extraction and subsequent RNA sequencing. The Agilent Bioanalyzer 2100 system was used to detect RNA integrity (RIN), and samples with RIN > 8 were used for further RNA sequencing. The NEBNext UltraTM RNA Library Prep Kit for Illumina was used for library construction. The NovaSeq6000 was used for sequencing and generating paired-end reads.

[0367] (2) Sequencing data analysis: Remove reads containing adapters, poly-N, and low-quality reads from the raw data to obtain clean reads. Calculate the Q20, Q30, GC content, and sequence duplication level of the clean reads. Use the Hisat2 tool software to align the data with the reference genome GRCm38 (mm10). The data is normalized by total count number correction and then transformed into log2(TPM + 1) for further analysis. Genes with log2(fold change) > 1 or < -1 are considered upregulated or downregulated genes. The KEGG pathway analysis is performed on genes with log2(fold change) > 1 or < -1 using the Benjamini and Hochberg method. P < 0.05 and q < 0.1 are considered significant. The Heatmap is drawn using the Tbtools software, and the z-score is calculated by transcripts per million (TPM) with the formula (TPM - average of TPM) / standard deviation. Other figures are generated by the online bioinformatics graphic tools imageGP (www.bic.ac.cn) and Gene Denovo (www.omicshare.com).

[0368] 1.2.16 Data analysis

[0369] The statistical analysis between two groups usually adopts the two-tailed unpaired t-test. For experiments involving more than two related 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) are used for statistical analysis. The Pearson correlation test for analyzing correlations is performed through the R language (version 4.1.3, R Foundation). Statistical significance is defined as a P-value less than 0.05.

[0370] 1.3 Experimental conclusions

[0371] (1) Through experiments, it was found that the clearance of pulmonary fungi by fluconazole can alleviate the BLM-induced pulmonary fibrosis phenotype. The specific experiments and conclusions are as follows:

[0372] To clarify the influence of fungi present in the lungs on Dectin-1-mediated pulmonary fibrosis regulation, before and during BLM treatment, mice were administered fluconazole (FCZ), a highly effective antifungal drug, by intranasal (i.n.) instillation to clear the fungi normally present in the lungs, thereby blocking the activation of Dectin-1 by fungal-derived β-glucan ( Figure 1 ). WT and Clec7a– / – Mice were treated with PBS or FCZ and then subjected to BLM-induced pulmonary fibrosis modeling and euthanized after 14 days for experiments (n = 4 in WT-PBS, WT-FCZ, and Clec7a – / – -FCZ groups, n = 3 in Clec7a – / – -PBS group). After clearing pulmonary fungi with fluconazole, the weight loss caused by intratracheal injection of BLM in WT mice was significantly reduced and was close to the weight change level of Clec7a – / – mice after BLM treatment ( Figure 2 and 3 ).

[0373] Analysis of the Masson staining results of lung tissue sections revealed that in the lungs of WT mice after fibrosis modeling following fungal clearance, the degree of pulmonary collagen deposition and the fibrotic area decreased. For Clec7a – / – mice, regardless of whether they were treated with intranasal FCZ or not, the fibrotic area in the lungs caused by BLM was in a decreasing state ( Figure 4 ).

[0374] Compared with the control WT mice without intranasal FCZ, the expression levels of collagen-encoding genes (Col1a1 and Col3a1) in the lung tissues of WT mice treated with FCZ after BLM-induced modeling were also inhibited ( Figure 5 ).

[0375] After flow cytometry of bronchoalveolar lavage fluid cells after fibrosis modeling, although no differences were observed in the proportion of alveolar macrophages (AM) ( Figure 6 ), the expression levels of Arg1 and Spp1 genes in BAL cells of WT mice treated with FCZ were significantly reduced ( Figure 7 ).

[0376] Among them, the data ( Figure 2 - 7 ) represent secondary independent experiments. The data ( Figure 2 ) are shown as mean ± standard deviation. The data ( Figure 2 - 5 and 7) were analyzed by one-way ANOVA combined with Tukey's multiple comparison test and the data ( Figure 6 ) were analyzed by two-tailed unpaired t-test. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0377] Since BLM causes fibrosis by damaging the DNA of alveolar epithelial cells and leading to their gradual formation of fibrosis during injury and repair, the present invention uses an antifungal agent to eliminate the fungi normally present in the lungs, and then observes that fibrosis is improved in wild-type mice after elimination.

[0378] In summary, it is shown that when Dectin-1 is in an unactivated state by fungal-derived β-glucan, the ability of AM to promote pulmonary fibrosis is weakened. Notably, eliminating Clec7a – / – fungi in the lungs of mice does not affect the phenotype of alleviated pulmonary fibrosis. These results indicate that pulmonary fungi are involved in the occurrence and development of pulmonary fibrosis by activating the Dectin-1 signaling pathway in the lungs ( Figure 1 - 7 ).

[0379] Commensal fungi, which exist on most mucosal surfaces and the skin, are considered to be another important component of commensal microorganisms in addition to bacteria. The interaction between commensal fungi and the immune system is crucial for host defense and immune system regulation, and can affect not only intestinal diseases but also systemic diseases. Although it has been reported that certain specific strains of intestinal commensal fungi do not play an important role in suppressing Dectin-1-mediated allergic airway inflammation by promoting the expansion of intestinal regulatory T cells.

[0380] However, the current research of the present invention shows that intranasal administration of the antifungal drug fluconazole can significantly inhibit the progression of pulmonary fibrosis, and this phenomenon is only found in WT mice, and a similar phenomenon is not observed in Dectin-1 knockout mice. This indicates that pulmonary fungi play an important role in promoting Dectin-1 signaling-mediated pulmonary fibrosis.

[0381] (2) The present invention discovers through experiments that the RAF1 inhibitor GW5074 inhibits the RAF1 pathway to block Dectin-1 signal transduction and alleviate pulmonary fibrosis. The specific experiments and conclusions are as follows:

[0382] By intraperitoneally injecting the WT mice with GW5074, a highly efficient and specific RAF1 inhibitor ( Figure 8 ). After treating the WT mice with GW5074, the degree of weight loss caused by BLM is reduced ( Figure 9 ), the degree of pulmonary collagen deposition and the fibrotic area are reduced ( Figure 10 ), and the expression level of fibrosis-related genes in the lung tissue is downregulated ( Figure 11 ).

[0383] These results indicate that the development of pulmonary fibrosis can be inhibited by blocking the RAF1 pathway. After injecting GW5074, although the proportion of AMs in the GW5074-treated group among the BAL cells of mice with fibrotic models did not change significantly compared with the control group ( Figure 12 ), the gene expression level of Arg1, which characterizes the function of type II (M2) AMs, decreased, and the gene expression level of the profibrotic factor Tgfb1 also decreased ( Figure 13 ). This indicates that the profibrotic ability of AMs is inhibited after blocking the RAF1 pathway. In addition, treating Clec7a – / – mice with GW5074 had no effect on the phenotype of alleviated pulmonary fibrosis ( Figure 14 - 16 ), that is, it was confirmed that Dectin-1 is the main upstream of the RAF1 signaling pathway in the case of BLM-induced pulmonary fibrosis. These results indicate that in the BLM-induced pulmonary fibrosis model, Dectin-1 on AMs affects the phenotype of pulmonary fibrosis through RAF1.

[0384] Data ( Figure 14 - 17 ) represent secondary independent experiments. Data (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).

[0385] In summary, RAF1 is a downstream molecule of Dectin-1 signal transduction. By using the RAF1 inhibitor GW5074 to block RAF1 signal transduction, the present invention observed a significantly alleviated pulmonary fibrosis phenotype in WT mice. Treating Dectin-1 knockout mice with GW5074 had no obvious effect on their pulmonary fibrosis phenotype. That is, Dectin-1 can promote the development of pulmonary fibrosis through a CARD9-independent and RAF1-dependent pathway.

[0386] (3) The present invention found through experiments that the Dectin-1 antagonist laminarin alleviates 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:

[0387] To explore the effect of blocking the Dectin-1 signaling pathway by laminarin on pulmonary fibrosis, the present invention performed laminarin nasal drops treatment on WT mice before and during the induction of pulmonary fibrosis by BLM airway injection ( Figure 17) WT mice were treated with PBS or laminarin and then subjected to BLM-induced pulmonary fibrosis modeling and euthanized after 14 days for experiments. Treatment of WT animals with laminarin significantly alleviated the weight loss caused by BLM ( Figure 18 ) and significantly inhibited the expansion of pulmonary parenchymal and fibrotic regions caused by fibrosis modeling ( Figure 19 ). After treatment with laminarin, the expression of genes related to BLM-induced pulmonary tissue fibrosis was also significantly reduced ( Figure 20 ). Although after treatment with laminarin, the proportion and cell number of AMs in the bronchoalveolar lavage fluid after BLM-induced pulmonary fibrosis did not change compared with the group without laminarin treatment ( Figure 21 、 Figure 22 ), the mRNA expression levels of Arg1 and Spp1 in the lungs were significantly reduced, indicating that the profibrotic characteristics of AMs were inhibited by blocking Dectin-1 ( Figure 23 ). On the other hand, when bone marrow-derived M2 macrophages were treated with Dectin-1 ligands, it was found that the expression of Arg1 and Tgfb1 was upregulated after treatment with the Dectin-1 agonist curdlan, while treatment with the antagonist laminarin or the RAF1 inhibitor GW5074 inhibited this upregulation caused by Dectin-1 stimulation ( Figure 24 ). These results indicate that Dectin-1 signaling can directly induce or promote the profibrotic characteristics of M2 macrophages.

[0388] Among them, mouse M2 macrophages cultured from bone marrow cells were pretreated with laminarin (1 mg / ml) or GW5074 (1 μM) for 3 hours and then stimulated with curdlan (100 μg / ml) for 20 hours, and the relative expression levels of Arg1 and Tgfb1 mRNAs were measured by qPCR (using Gapdh as a standard, n = 3 wells / group). The data ( Figure 18 - 23 ) represent three independent experiments, and the data ( Figure 24 ) represent two independent experiments. The data ( Figure 18 - 20 、 Figure 22 - 24 ) are shown as mean ± standard deviation. The data ( Figure 18 - 23 ) were analyzed by two-tailed unpaired t-test and the data ( Figure 24 ) were analyzed by 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).

[0389] In summary, intranasal administration of the Dectin-1 antagonist laminarin can also significantly inhibit pulmonary fibrosis, indicating that Dectin-1 activating ligands in the lungs play a promoting role in pulmonary fibrosis.

[0390] By deeply understanding the role of Dectin-1, the present invention is expected to provide important basic support for the development of new drugs targeting Dectin-1 for the treatment of pulmonary fibrosis and clinical treatment plans, and provide new ideas and methods for the prevention and treatment of pulmonary fibrosis.

[0391] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. Use of a preparation for blocking Dectin-1 signal transduction in the preparation of a drug for treating and / or preventing pulmonary fibrosis, characterized in that: The formulation comprises at least one of an antifungal agent, a Dectin-1 antagonist or a RAF1 inhibitor.

2. The use according to claim 1, characterized in that: The antifungal agent comprises at least one of an imidazole antifungal agent, a pyrimidine antifungal agent, an acrylamine antifungal agent, and an echinocandin antifungal agent; The Dectin-1 antagonist comprises at least one of laminarin, β-glucan with a molecular weight less than 5000 Da, and neutralizing antibodies or antagonistic antibodies against Dectin-1; The RAF1 inhibitor includes at least one of GW5074, Rafinhibitor 1, ZM336372, dabrafenib mesylate, dabrafenib, GSK2118436A, B-Raf inhibitor 1dihydrochloride, Raf inhibitor 3, C-RAF kinase-IN-1, MEK1 / C-Raf-IN-1, MCP110, AZ628, Kobe2602, Kobe0065, BBO-8520, Bay 43-9006, sorafenib, Ferroptosis inducer-3, and anti-RAF1 neutralizing antibody or antagonistic antibody.

3. The use according to claim 1 or 2, characterized in that: The antifungal agent reduces the β-glucan on the surface of fungi in the lungs and blocks the Dectin-1 signal transduction, thereby further aggravating the pulmonary fibrosis caused by pulmonary fungi.

4. The use according to claim 3, characterized in that: The fungus is a fungus that can express beta-glucan and be recognized by Dectin-1, and the fungus includes at least one of Candida, Aspergillus, Penicillium, Eurotium, and Dioscorea.

5. The use according to claim 3, characterized in that: The fungus further aggravates pulmonary fibrosis by activating Dectin-1 through the expression of β-glucan.

6. The use according to claim 1 or 2, characterized in that: The Dectin-1 antagonist inhibits the binding of β-glucan to Dectin-1, blocks the Dectin-1 downstream signaling pathway, and alleviates pulmonary fibrosis.

7. The use according to claim 1 or 2, characterized in that: The RAF1 inhibitor blocks Dectin-1 signal transduction by inhibiting RAF1, thereby alleviating pulmonary fibrosis.

8. A composition for treating and / or preventing pulmonary fibrosis, characterized in that: The invention comprises the preparation for use in preparing a drug for treating and / or preventing pulmonary fibrosis as described in claim 1.

9. A drug for treating and / or preventing pulmonary fibrosis, characterized in that: It comprises an effective ingredient and a pharmaceutically acceptable carrier, wherein the effective ingredient comprises the preparation for use in preparing a drug for treating and / or preventing pulmonary fibrosis as described in claim 1.

10. A drug for treating and / or preventing pulmonary fibrosis according to claim 8 or 9, characterized in that: The dosage form of the medicine includes one or more of capsules, powders, tablets, granules, drip or intravenous injections, and nasopharyngeal sprays.

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