Application of silver forging glycoside in preparation of medicine for preventing and treating pulmonary fibrosis

Abstract

The invention discloses application of silver forging glycoside in preparation of a medicine for preventing and treating pulmonary fibrosis, and belongs to the technical field of biological medicine. The NID1 protein is used as a target spot for treating and / or preventing the fibrosis-type allergic pneumonia and can be specifically combined with a small molecule compound silver forging glycoside, the fibrosis progress of the fibrosis-type allergic pneumonia is effectively inhibited by inhibiting EMT, and the effect of effectively preventing and treating the fibrosis-type allergic pneumonia is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of silver calcinin in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Nidogen-1 (NID1) is a basement membrane protein that plays a crucial role in various physiological and pathological processes. EMT, as a major initiating mechanism of pulmonary fibrosis progression, plays a vital role in this disease. Studies have shown that NID1 promotes epithelial-mesenchymal transition (EMT) by activating the ERK / MAPK signaling pathway. Research has found that carpaine alleviates liver fibrosis associated with non-alcoholic steatohepatitis by regulating the Nid1-JAK2 / STAT3-IL-6 axis. Furthermore, studies have shown significantly increased NID1 expression in an SR-Ag-induced fibrotic allergic alveolitis model, indicating that NID1 plays a multifaceted and crucial role in pulmonary fibrosis and is a potential therapeutic target. However, there is currently a significant gap in the development of small molecule compounds specifically targeting NID1 for the treatment of pulmonary fibrosis. Tiliroside, a glycoside flavonoid, is widely found in plants such as raspberry, rose hips, celandine, and buddleja officinalis in East China. It is a non-competitive inhibitor of α-amylase, inhibiting the digestion of carbohydrates and the absorption of glucose in the gastrointestinal tract. Existing research has primarily reported its anti-diabetic effects. However, no studies have been reported on its application in pulmonary fibrosis.

[0003] In this patent, we have discovered and confirmed for the first time that silver calcinin can specifically target NID1 and inhibit the progression of allergen-induced chronic pulmonary fibrosis by inhibiting the occurrence of EMT. Summary of the Invention

[0004] This invention proposes the application of silagetin in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, by specifically targeting and binding silagetin to NID1 to form a specific drug for the prevention and treatment of pulmonary fibrosis.

[0005] The first objective of this invention is to provide the application of silver calcinin, a small molecule compound that specifically targets NID1, in the preparation of drugs for the prevention and treatment of pulmonary fibrosis.

[0006] Among them, the molecular formula of silage glycoside is C 30 H 26 O 13 Its structural formula is: .

[0007] The pulmonary fibrosis is preferably pulmonary fibrosis caused by allergen-induced chronic allergic pneumonia.

[0008] The silver calcinoside inhibits pulmonary fibrosis by specifically binding to the basement membrane protein NID1 to inhibit EMT.

[0009] The content of the silver calcinin in the preparation of drugs for the prevention and treatment of pulmonary fibrosis is 10-200 μM, more preferably 50-200 μM.

[0010] A second object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, the composition comprising sildenafil and a carrier.

[0011] The carrier is selected from dimethyl sulfoxide, polyethylene glycol 300, polysorbate, 2-hydroxypropyl-β-cyclodextrin, hydrogenated soybean lecithin, and polyethylene glycol distearate phosphatidylethanolamine. Because silver calcinol has low solubility in water, dimethyl sulfoxide can be chosen as the solvent, or any one or more of polyethylene glycol 300, polysorbate, and 2-hydroxypropyl-β-cyclodextrin can be selected to increase the solubility and stability of the drug. Simultaneously, biocompatible natural materials (such as gelatin, albumin, starch, chitosan, etc.) or semi-synthetic materials (such as hydrogenated soybean lecithin and polyethylene glycol distearate phosphatidylethanolamine) are selected as carriers to ensure the safety and efficacy of the drug composition.

[0012] In the pharmaceutical composition of the present invention, silanol can also be mixed with other existing anti-fibrotic active ingredients to form a compound pharmaceutical composition to achieve the purpose of treating pulmonary fibrosis.

[0013] A third object of the present invention is to provide a medicament for treating pulmonary fibrosis, the medicament comprising an active ingredient and a pharmaceutically acceptable carrier, said active ingredient being cypermethrin or a pharmaceutical composition containing cypermethrin.

[0014] In the drugs for treating pulmonary fibrosis, the content of ginsenoside is 10-200 μM per unit. Preferably, it is 50-200 μM per unit.

[0015] The carriers that may be used include sustained-release agents, excipients, or diluents.

[0016] The dosage forms of drugs for treating pulmonary fibrosis include any one or more of the following: oral liquid, injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, powder, spray, and aerosol.

[0017] The beneficial effects of the application of silver glycoside in the preparation of drugs for the prevention and treatment of pulmonary fibrosis are as follows: The present invention uses NID1 protein as a target for the treatment and / or prevention of fibrotic allergic pneumonia, which can specifically bind to the small molecule compound silver glycoside. By inhibiting the occurrence of EMT, it can effectively inhibit the progression of fibrosis in fibrotic allergic pneumonia, thereby achieving the effect of effectively preventing and treating fibrotic allergic pneumonia. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This diagram illustrates the expression of NID1 protein in lung tissue in a mouse model of fibrotic hypersensitivity pneumonitis. A. Hematoxylin and eosin (HE) staining panoramic images of lung tissue sections from both groups of mice (scale bar: 500 μm). B. Bar chart showing the inflammation score of lung tissue in both groups of mice (n=4). C. Representative Masson staining images of lung tissue sections from both groups of mice observed under a microscope (scale bar: 50 μm). D. Bar chart showing the collagen volume fraction in lung tissue from both groups of mice (n=4). E. Immunofluorescence staining of NID1 in paraffin sections from both groups of mice (NID1 is labeled purple, and DAPI staining of cell nuclei is labeled blue) (scale bar: 100 μm). F. Bar chart showing the average fluorescence intensity of NID1 in lung tissue from both groups of mice (n=4).

[0020] Figure 2 This is the conformation of the binding of the compound silver glycoside to the NID1 protein. A. Overall view of the binding position of the compound silver glycoside in the NID1 structure; carbon atoms of the silver glycoside are shown in green, oxygen atoms in red, nitrogen atoms in blue, and the protein in gray. B. 3D partial view of the position of the compound silver glycoside in the NID1 structure; carbon atoms of the silver glycoside are shown in green, oxygen atoms in red, nitrogen atoms in blue, surrounding amino acids are shown in sky blue and labeled in black, red dashed lines represent hydrogen bonds, and yellow represents aromatic ring-hydrogen stacking bonds. C. 2D partial view of the position of the compound silver glycoside in the NID1 structure; green arrows indicate hydrogen bond interactions.

[0021] Figure 3This is a schematic diagram showing the effects of cytosine on epithelial cell viability and EMT. A. A bar chart showing the effect of different concentrations of cytosine on A549 cell viability after 24 hours of treatment. B. Immunoblotting bands showing the expression of FN1, Collagen I, E-cadherin, N-cadherin, β-catenin, Vimentin, α-SMA, and (internal control) α-Tublin proteins in A549 cells from the Ctrl group, NID1 group, cytosine group, and NID1 + cytosine group, n=3.

[0022] Figure 4 The effects of ginsenosides on lung inflammation and fibrosis progression in a mouse model of fibrotic hypersensitivity pneumonia; A. Hematoxylin and eosin (HE) staining panoramic images of different mouse lung tissue pathological sections, scale bar: 1 mm. B. Representative Masson staining images of different mouse lung tissue sections observed under a microscope, scale bar: 50 μm. C. Bar chart showing the collagen volume fraction of different mouse lung tissues, n=5. D. NID1 immunofluorescence staining of different mouse paraffin sections, NID1 labeled purple, DAPI staining of cell nuclei labeled blue, scale bar: 100 μm. E. Bar chart showing the average fluorescence intensity of NID1 in different mouse lung tissues, n=5. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0024] The first objective of this invention is to provide the application of silver calcinin, a small molecule compound that specifically targets NID1, in the preparation of drugs for the prevention and treatment of pulmonary fibrosis.

[0025] Among them, the molecular formula of silage glycoside is C 30 H 26 O 13 Its structural formula is: .

[0026] The content of ginsenoside in the preparation of drugs for the prevention and treatment of pulmonary fibrosis is 10-200 μM per unit, and more preferably 50-200 μM per unit.

[0027] A second objective of this invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, comprising silanol and a carrier. The carrier is selected from dimethyl sulfoxide, polyethylene glycol 300, polysorbate, 2-hydroxypropyl-β-cyclodextrin, hydrogenated soybean lecithin, and polyethylene glycol distearate phosphatidylethanolamine. Since silanol has low solubility in water, dimethyl sulfoxide can be chosen as the solvent, or any one or more of polyethylene glycol 300, polysorbate, and 2-hydroxypropyl-β-cyclodextrin can be selected to increase the solubility and stability of the drug. Simultaneously, biocompatible natural materials (such as gelatin, albumin, starch, chitosan, etc.) or semi-synthetic materials (such as hydrogenated soybean lecithin and polyethylene glycol distearate phosphatidylethanolamine) are selected as the carrier to ensure the safety and efficacy of the pharmaceutical composition.

[0028] In the pharmaceutical composition of the present invention, silanol can also be mixed with other existing anti-fibrotic active ingredients to form a compound pharmaceutical composition to achieve the purpose of treating pulmonary fibrosis.

[0029] A third object of the present invention is to provide a medicament for treating pulmonary fibrosis, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is silagetinib or a pharmaceutical composition containing silagetinib. In the medicament for treating pulmonary fibrosis, the content of silagetinib is 10-200 μM per unit dose, preferably 50-200 μM per unit dose.

[0030] The carriers used may include sustained-release agents, excipients, or diluents. Dosage forms of drugs for treating pulmonary fibrosis include any one or more of the following: oral liquids, injections, suspensions, emulsions, solutions, syrups, tablets, capsules, granules, powders, sprays, and aerosols.

[0031] Example 1

[0032] Validation of the therapeutic target of NID1 protein in fibrotic hypersensitivity pneumonitis Experimental materials: 6-8 week old male C57BL / 6J mice; all animal experiments were conducted in accordance with the guidelines of relevant animal protection associations; *Polyspora linearis* ( Saccharopolyspora rectivirgula The following reagents were commercially available: SR, SR-Ag (a type of sclerotium spp. antigen extract), hematoxylin-eosin (H&E) staining kit, Masson's trichrome staining kit, and NID1 rabbit polyclonal antibody.

[0033] Test method: (1) Construction of fibrotic allergic pneumonia After one week of acclimatization, C57BJ / 6J mice were randomly divided into two groups: a control group treated with PBS (PBS group) and a model group treated with *Streptococcus linearis* antigen extract (SR-Ag) (SR-Ag group). Mice were rapidly anesthetized with 200 μL of isoflurane. Mice in the SR-Ag group received SR-Ag (20 μg / mouse) via airway inhalation into their lungs for three consecutive days per week for six weeks. Mice in the PBS group received the same treatment with an equal volume of PBS. Finally, the mice were euthanized by cervical dislocation under CO2 anesthesia, and lung tissue was collected for pathological sections.

[0034] (2) Pathological staining The mouse lung tissue obtained in step (1) was soaked in 4% paraformaldehyde for 48 hours, then eluted with ethanol solutions of progressively increasing concentrations of 70%, 80%, 90%, 95% and 100%, cleared with xylene, embedded in paraffin, and cut into 5µm thick sections for staining. HE staining procedure: Sections were dewaxed in xylene for 10-40 minutes, then hydrated in a gradient (100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water) → hematoxylin staining for 5 minutes, followed by washing away excess hematoxylin with deionized water → differentiation with 1% hydrochloric acid ethanol for 5-10 seconds → eosin staining for 30 seconds, followed by washing away excess eosin with deionized water → dehydration and clearing (sections were sequentially immersed in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes for dehydration and clearing) → sections were removed and air-dried, mounted with neutral resin, and then observed and scanned using a whole-section scanning system (VS200, Olympus), and the inflammatory cell infiltration of lung tissue was scored. The results are as follows. Figure 1 A and Figure 1 As shown in B.

[0035] Masson staining procedure: Sections are dewaxed in xylene for 10-40 minutes, then hydrated in a gradient (100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water). Next, they are stained sequentially with Weigert's iron hematoxylin for 5 minutes, then with Ponceau S-acid fuchsin for 5-10 minutes, washed with deionized water, treated with 1% phosphomolybdic acid for 3-5 minutes, treated with aniline blue for 5 minutes, differentiated with 1% acetic acid for 1 minute, and then dehydrated and mounted (sections are sequentially immersed in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes for clearing). After removing the sections from the xylene and allowing them to air dry slightly, they are mounted with neutral resin. The sections are then observed under a microscope, and the collagen volume fraction is calculated. The results are as follows: Figure 1 C and Figure 1 As shown in D.

[0036] Immunofluorescence staining procedure: Sections were dewaxed with xylene for 10-40 minutes → hydrated in a gradient (100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water), followed by antigen retrieval (sodium citrate, 95℃, 15 minutes) → blocked with 5% goat serum for 30 minutes → incubated overnight at 4℃ with rabbit NID1 antibody (1:200 dilution) → bound to primary antibody with goat anti-rabbit Alexa Fluor™ 647 antibody (1:1000 dilution) at room temperature for 2 hours → stained nuclei with DAPI for 10 minutes → mounted with anti-fluorescence quencher, observed using a whole-section scanning system, and fluorescence intensity was recorded. Results are as follows: Figure 1 E and Figure 1 As shown in F.

[0037] In this embodiment, HE staining, Masson staining, and immunofluorescence staining are all routine procedures performed by those skilled in the art.

[0038] Results of HE staining of lung tissue ( Figure 1 A) showed that the SR-Ag group mice exhibited extensive inflammatory cell infiltration around the trachea and blood vessels in the lung tissue, and the alveolar walls were significantly thickened. No such changes were observed in the PBS group; the inflammation score in the SR-Ag group was significantly higher than that in the PBS group, and the difference was statistically significant. P <0.0001) ( Figure 1 B). Masson staining results ( Figure 1 (C) showed that the SR-Ag group had significantly more local collagen deposition and alveolar fibrosis in the lung tissue compared with the PBS group; the collagen volume fraction in the SR-Ag group was significantly higher than that in the PBS group, and the difference was statistically significant. P=0.029 ) ( Figure 1 D). Immunofluorescence results of lung tissue are as follows: Figure 1 E and such Figure 1 F showed that, compared with the PBS group, the SR-Ag group had increased NID1 expression in lung tissue; compared with the PBS group, the SR-Ag group had significantly increased fluorescence (F). P =0.0002). The above results indicate that NID1 protein expression was significantly increased in the lung tissue of mice with SR-Ag-induced fibrotic hypersensitivity pneumonia, suggesting that NID1 protein has the potential to serve as a target for the treatment and / or prevention of fibrotic hypersensitivity pneumonia.

[0039] Example 2

[0040] Verification of specific targeting and binding of silver calcinate to NID1 Verification method: (1) Preparation of protein structure and binding site The predicted alphafold structure of NID1 was optimized at the bond level using the ProteinPreparation Wizard module in the Schrodinger software. Irrelevant ligands and redundant parts were removed, hydrogen atoms were added, and an energy minimization optimization step was performed. The MOE (Minimum Orientation of Energy) was used to predict the protein-binding compound pockets, revealing four sites. These four pockets were selected for further screening, and the optimal binding pocket was chosen based on affinity comparisons for the next experimental step.

[0041] (2) Preparation of small molecule compound library The L1000 (marketed drug library) and L6810 (traditional Chinese medicine single-molecule library) compound libraries from Taoshu Biotechnology Co., Ltd. were selected. The compound libraries were preprocessed using the LigPrep module in Schrodinger software (LigPrep, Schrodinger, LLC, New York, NY, 2021), including adding hydrogen atoms, removing salt ions, and adjusting charge distribution. Energy minimization calculations were performed using molecular force fields to generate small molecule three-dimensional structures suitable for molecular docking analysis.

[0042] (3) Molecular docking and screening After the small molecule compound library and protein were prepared, Schrödinger software was used to perform compound docking on each compound in the library, selecting compounds that bind to proteins and have an affinity value below -8 kcal / mol. MMGBSA binding free energy was calculated, selecting compounds that bind to proteins and have an MMGBSA value below -50. PLIF analysis was performed to select compounds that interact with the protein structure, then clustered based on 70% structural similarity, followed by binding mode analysis, finally identifying the compound with the best binding potential, silver calcinyl glycoside.

[0043] (4) SPR verification The affinity of ginsenoside for interaction with NID1 protein was determined using Biacore. The assay was performed using a CM5 chip-coupled protein assay, with a signal value RU > 20.

[0044] (5) Visualization of protein-small molecule compound binding The structure file of the NID1 protein-silver glycoside complex was imported into PyMOL software for visualization analysis, and the results are as follows: Figure 2 As shown.

[0045] Using Schrodinger software, a high-throughput screening strategy was implemented on the marketed compound library and traditional Chinese medicine monomer library provided by Taoshu Biotechnology Co., Ltd., successfully identifying several compounds that can effectively bind to the NID1 protein. All of these compounds formed stable three-dimensional bindings to the target protein. The affinity between the compounds and the NID1 protein was determined using Biacore, validating the most promising small molecule identified through high-throughput screening – silver glycoside. Table 1 lists partial information of the top 50 candidate compounds with high CADD binding energy screened by SPR. Figure 2 A shows the structure of the NID1 protein. The binding mode and energy stability of the candidate compound, cypermethrin, were analyzed using Schrodinger software. Cypermethrin and NID1 have a binding energy of -13.07 kcal / mol, while the binding free energy of MMGBSA is -60.88 kcal / mol, indicating a very stable binding. Figure 1 B shows that the hydroxyl groups of silver calcinate can form hydrogen bonds with D1072, F985, V1028, D11177, and S1158, respectively, and T1157 can form hydrogen bonds with the carbonyl group of the compound. The benzene ring of silver calcinate can form an aromatic ring-hydrogen stacking conjugation with I1069.

[0046] Table 1. Partial information on the top 8 small molecule compounds after high-throughput screening and SPR validation. name CAS Molecular formula CADD binding energy Silver calcin 20316-62-5 <![CDATA[C 30 H 26 O 13 ]]> -13.0732 Diquaphosphoryltetrasodium 211427-08-6 <![CDATA[C 18 H 22 N4Na4O 23 P4]]> -12.8555 Tibrutinib Hydrochloride 1439901-97-9 <![CDATA[C 25 H 23 ClN6O3]]> -10.9485 Nebivolol hydrochloride 152520-56-4 <![CDATA[C 22 H 25 F2NO4·HCl]]> -11.00115 Salinomycin 32780-64-6 <![CDATA[C 19 H 25 ClN2O3]]> -11.32637 TA-0910 acetate 1549593-23-8 <![CDATA[C 19 H 27 N7O7]]> -11.2579 Cefotetanil hydrochloride 95789-30-3 <![CDATA[C 27 H 37 N9O7S3.2ClH]]> -11.5076 Dabigatran mesylate 872728-81-9 <![CDATA[C 34 H 41 N7O5.CH4O3S]]> -11.5666 Example 3 Inhibitory effect of silver calcinate on NID1-induced epithelial cell EMT Experimental materials: Commercially available silver calcinin (T5S1172, TargetMol, USA, 50mg), recombinant human NID1 protein (Ag4739, 50ug) and antibodies, including: FN1 (15613-1-AP), Collagen-I (14695-1-AP), E-cadherin (20874-1-AP), N-cadherin (66219-1-Ig), Vimentin (10366-1-AP), and α-Tublin (14555-1-AP).

[0047] Specific experimental steps: (a) Cell Culture Human alveolar type II epithelial cells A549 were cultured in high-glucose DMEM basal medium containing 10% FBS and 1% streptomycin or penicillin antibiotics, and were stably passaged and grown in a 5% CO2, 37°C constant temperature and humidity cell culture incubator.

[0048] (II) Cell proliferation and toxicity detection To investigate the toxicity and proliferation effects of the compound silanol on A549 cells, the effects of different concentrations (10 μM, 50 μM, 150 μM, and 200 μM) of silanol on A549 cell viability were examined using a CCK8 assay; the procedure was as follows: (1) The cells were divided into a blank group, a drug group, and a control group. A549 cells were digested with trypsin to prepare a cell suspension, seeded into 96-well plates, and cultured for 24 hours. The drug group was added with different concentrations of silver glycoside (10 μM, 50 μM, 150 μM and 200 μM) and cultured for another 24 hours. The control group was not given any drug. The blank group was not seeded with cells and was given the same volume of DMEM high glucose medium.

[0049] (2) Remove the supernatant and add CCK 8 reagent to each well at a mass ratio of (CCK 8 reagent: serum-free DMEM = 1:10); (3) After incubating in a 37℃ incubator for 0.5 hours and 1 hour, the culture plate was removed and the absorbance (OD value) at 450nm was measured using an ELISA reader to calculate cell viability.

[0050] .

[0051] The results obtained are as follows Figure 3 As shown in Figure A, after 24 hours of treatment with the compound silagetin, the viability of A549 cells decreased with increasing compound concentration. At a concentration of 10 μM, silagetin had no significant effect on A549 cell viability, indicating no significant cytotoxicity. However, at concentrations of 50 μM, 150 μM, and 200 μM, silagetin significantly reduced A549 cell viability.

[0052] (III) Immunoblotting detection (1) Experimental grouping: 5 × 10⁻⁶ per well 4 A549 cells were evenly seeded in 6-well plates, with four groups: control group (Ctrl group), NID1 group, silver glycoside group, and NID1 + silver glycoside group. The Ctrl group received no silver glycoside or recombinant protein. The NID1 group was treated with NID1 (2 μg / mL) for 24 hours. The silver glycoside group was treated with a concentration of silver glycoside (50 μM) for 24 hours. The NID1 + silver glycoside group was treated with both NID1 (2 μg / mL) and silver glycoside (50 μM) for 24 hours. Cell proteins were extracted after cell collection.

[0053] (2) Protein extraction: Cells were lysed on ice for 30 minutes with RIPA lysis buffer (containing protein / phosphatase inhibitor), centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatant was collected.

[0054] (3) The protein in the supernatant is quantified and denatured by heating to obtain the protein sample. The protein sample and the protein molecular weight standard (marker) are added to the sample well of the polyacrylamide gel respectively, and SDS-PAGE electrophoresis is performed at 120V.

[0055] (3) The gel after electrophoresis is combined with a PVDF membrane to form a sandwich structure. The membrane is transferred at a constant voltage of 100V for 2 hours to transfer the protein from the gel to the PVDF membrane. The transferred PVDF membrane is placed in 5% skim milk powder and incubated at room temperature for 2 hours to block the non-specific binding sites on the PVDF membrane.

[0056] (4) Add the test antibodies (FN1, Collagen I, E-cadherin, N-cadherin, β-catenin, Vimentin, α-SMA and (internal control) α-Tublin) to the blocked PVDF membrane and incubate overnight at 4°C to allow the antibodies to specifically bind to the target protein.

[0057] (5) After washing the membrane to remove unbound antibodies, add a secondary antibody that matches the antibody (horseradish peroxidase-labeled anti-mouse secondary antibody or anti-rabbit secondary antibody) and incubate at room temperature for 2 hours.

[0058] (6) After washing the membrane thoroughly, use a chemiluminescence imaging system (eBLOT) to detect and observe the target protein band.

[0059] like Figure 3 As shown in Figure B, this embodiment used a 50 μM concentration for immunoblotting. The results showed that after treating A549 cells with 2 μg / mL NID1 for 24 hours, the expression of collagen deposition-related proteins (FN1 and Collagen I) was significantly increased compared to the Ctrl group, and the expression of EMT-related proteins (N-cadherin, β-catenin, Vimentin, and α-SMA) was also increased, while the expression of E-cadherin was decreased. In the NID1 + silver cadherin group, the expression of both collagen deposition-related proteins and EMT-related proteins was significantly decreased compared to the NID1 group, while the expression of E-cadherin was increased. Meanwhile, compared to the Ctrl group, silver cadherin treatment alone had no significant effect on the expression of collagen deposition-related proteins and EMT-related proteins in A549 cells. These results indicate that under the condition of treating cells with 50 μM silver cadherin for 24 hours, this compound is non-toxic to A549 cells and can significantly inhibit NID1-induced epithelial cell EMT.

[0060] Example 4 The preventive and therapeutic effects of cytosolic acid on fibrotic allergic pneumonia. Experimental materials: Tribuloside, recombinant human NID1 protein (Ag4739, 50ug) and antibodies, including FN1 (15613-1-AP), Collagen-I (14695-1-AP), E-cadherin (20874-1-AP), N-cadherin (66219-1-Ig), Vimentin (10366-1-AP), and α-Tublin (14555-1-AP), all of which were commercially available.

[0061] Animal model: 24 C57BJ / 6J mice (average weight of about 20g) were randomly divided into 4 groups: solvent-treated control group (control group), SR-Ag-treated model group (SR-Ag group), silver calcinate-treated control group (silver calcinate group), and SR-Ag + silver calcinate-treated experimental group (SR-Ag + silver calcinate group). Mice in the SR-Ag group and the SR-Ag+silver glycoside group were rapidly anesthetized with 200 μL of isoflurane and then inhaled SR-Ag (20 μg / mouse) into their lungs via the airway for 3 consecutive days each week for 6 weeks. Mice in the SR-Ag+silver glycoside group and the SR-Ag+silver glycoside group were intraperitoneally injected with 10 mg / kg of silver glycoside one hour before each airway inhalation. Mice in the control group were treated in the same way with an equal volume of drug dissolving solvent (5% dimethyl sulfoxide + 40% polyethylene glycol 300 + 5% polysorbate + 50% physiological saline). Finally, the mice were anesthetized with CO2 and euthanized by cervical dislocation, and lung tissue was collected for pathological sections. Pathological staining The obtained mouse lung tissue was soaked in 4% paraformaldehyde for 48 hours, then eluted with ethanol solutions of progressively increasing concentrations of 70%, 80%, 90%, 95% and 100%, cleared with xylene, embedded in paraffin, and cut into 5µm thick sections for staining. HE staining procedure: Sections were dewaxed in xylene for 10-40 minutes, then hydrated in a gradient (100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water) → hematoxylin staining for 5 minutes, followed by washing away excess hematoxylin with deionized water → differentiation with 1% hydrochloric acid ethanol for 5-10 seconds → eosin staining for 30 seconds, followed by washing away excess eosin with deionized water → dehydration and clearing (sections were sequentially immersed in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes for dehydration and clearing) → sections were removed and air-dried, mounted with neutral resin, and then observed and scanned using a whole-section scanning system (VS200, Olympus), and the inflammatory cell infiltration of lung tissue was scored. The results are as follows. Figure 4 As shown in Figure A.

[0062] Masson staining procedure: Sections are dewaxed in xylene for 10-40 minutes, then hydrated in a gradient (100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water). Next, they are stained sequentially with Weigert's iron hematoxylin for 5 minutes, then with Ponceau S-acid fuchsin for 5-10 minutes, washed with deionized water, treated with 1% phosphomolybdic acid for 3-5 minutes, treated with aniline blue for 5 minutes, differentiated with 1% acetic acid for 1 minute, and then dehydrated and mounted (sections are sequentially immersed in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes for clearing). After removing the sections from the xylene and allowing them to air dry slightly, they are mounted with neutral resin. The sections are then observed under a microscope, and the collagen volume fraction is calculated. The results are as follows: Figure 4 As shown in B.

[0063] Immunofluorescence staining procedure: Sections were dewaxed with xylene for 10-40 minutes → hydrated in a gradient (100% ethanol → 95% ethanol → 80% ethanol → 70% ethanol → distilled water), followed by antigen retrieval (sodium citrate, 95℃, 15 minutes) → blocked with 5% goat serum for 30 minutes → incubated overnight at 4℃ with rabbit NID1 antibody (1:200 dilution) → bound to primary antibody with goat anti-rabbit Alexa Fluor™ 647 antibody (1:1000 dilution) at room temperature for 2 hours → stained nuclei with DAPI for 10 minutes → mounted with anti-fluorescence quencher, observed using a whole-section scanning system, and fluorescence intensity was recorded. Results are as follows: Figure 4 D and Figure 4 As shown in E.

[0064] Results of HE staining of lung tissue ( Figure 4 A) shows that the SR-Ag group mice exhibited extensive inflammatory cell infiltration around the trachea and blood vessels in the lung tissue, and significant alveolar wall thickening. Compared with the SR-Ag group, the SR-Ag + silver glycoside group showed significantly reduced inflammatory cell infiltration around the airways and blood vessels, and reduced alveolar wall thickening. There were no significant changes in inflammatory cell infiltration or changes in the lung septa between the silver glycoside group and the control group.

[0065] Masson staining results of lung tissue ( Figure 4 (B) showed that the SR-Ag group had significantly more local collagen deposition and alveolar fibrosis in the lung tissue compared to the control group; compared with the SR-Ag group, the SR-Ag + ginseng group had significantly less local collagen deposition and alveolar fibrosis in the lung tissue. The collagen volume fraction in the SR-Ag group was significantly higher than that in the control group, and the difference was statistically significant. P=0.002 The collagen volume fraction in the SR-Ag + silver calcinate group was significantly lower than that in the SR-Ag group, and the difference was statistically significant. P=0.018 ) ( Figure 4 C).

[0066] Immunofluorescence results of lung tissue as follows Figure 4 D and Figure 4 E showed that, compared with the control group, the SR-Ag group had increased NID1 expression in lung tissue ( P <0.0001); compared with the SR-Ag group, the fluorescence of the SR-Ag + silver calcinate group was significantly reduced ( P<0.0001 ).

[0067] The above results indicate that ginsenosides can reduce inflammatory response and collagen deposition in the lung tissue of mice with SR-Ag-induced fibrotic hypersensitivity pneumonia, while also inhibiting the expression of NID1 protein and slowing the progression of pulmonary fibrosis.

[0068] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The application of small molecule compounds specifically targeting NID1 in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, characterized in that: The specific small molecule compound targeting NID1 is silicoside.

2. The application of the NID1-specific small molecule compound according to claim 1 in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, characterized in that: The pulmonary fibrosis mentioned refers to interstitial fibrosis of fibrotic hypersensitivity pneumonitis.

3. The application of the NID1-specific small molecule compound according to claim 1 in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, characterized in that: The silver calcinoside inhibits pulmonary fibrosis by specifically binding to basement membrane proteins and inhibiting EMT.

4. The application of the NID1-specific small molecule compound according to claim 3 in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, characterized in that: The basement membrane protein is NID1 protein.

5. The application of the NID1-specific small molecule compound according to claim 3 in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The content of the silver calcinin in the drug is 10-200 μM.

6. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that: The pharmaceutical composition includes sildenafil and a carrier.

7. The pharmaceutical composition for treating pulmonary fibrosis according to claim 6, characterized in that: The carrier is selected from dimethyl sulfoxide, polyethylene glycol 300, polysorbate, 2-hydroxypropyl-β-cyclodextrin, polyethylene glycol 300, polysorbate, 2-hydroxypropyl-β-cyclodextrin, hydrogenated soybean lecithin, and polyethylene glycol distearate phosphatidylethanolamine.

8. A drug for treating pulmonary fibrosis, characterized in that: It includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is cypermethrin or the pharmaceutical composition of claim 7.

9. The medicament for treating pulmonary fibrosis according to claim 8, characterized in that: The content of the silver calcinin in the drug is 10-200 μM.

10. The medicament for treating pulmonary fibrosis according to claim 8, characterized in that: The dosage forms of the drug include any one or more of the following: oral liquid, injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, powder, spray, and aerosol.

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