Use of elsigluar for the preparation of a medicament for the treatment of pulmonary fibrosis

Abstract

This invention discloses the application of ilismo in the preparation of anti-pulmonary fibrosis drugs, belonging to the field of biomedical technology. The research and verification of this invention demonstrates that ilismo can selectively inhibit the activity of activated lung myofibroblasts and reduce the expression of fibrosis-related markers, significantly improving the degree of pulmonary fibrosis in animal models. This invention provides a new candidate drug for the treatment of pulmonary fibrosis and has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the application of irismo in the preparation of anti-pulmonary fibrosis drugs. Background Technology

[0002] Pulmonary fibrosis is a progressive and fatal disease characterized by chronic lung tissue damage, abnormal repair, and excessive deposition of extracellular matrix, with a poor clinical prognosis. Currently, commonly used drugs can only slow disease progression, and there are no effective treatments to reverse fibrosis.

[0003] Recent studies suggest that copper ion homeostasis disorder may be involved in the fibrosis process, but related research is mostly still in the stage of mechanism exploration and no clear drug strategy that can be used to treat pulmonary fibrosis has yet been formed.

[0004] Therefore, it is of great significance to develop a new drug that can target key pathogenic cells and exert an anti-fibrotic effect. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and explore the application of irismo in the preparation of drugs for treating pulmonary fibrosis.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides the use of irismo in the preparation of a drug for treating pulmonary fibrosis.

[0007] In one possible design, ilismo is able to reduce α-SMA and / or collagen expression levels.

[0008] In one possible design, ilismo could be used to inhibit the activation of lung myofibroblasts.

[0009] The present invention also provides a composition for treating pulmonary fibrosis, comprising irismo as an active substance.

[0010] The present invention also provides a medicament for treating pulmonary fibrosis, comprising the composition of claim 4, and further comprising a pharmaceutically acceptable carrier or excipient.

[0011] In one possible design, the dosage form of the drug includes any one of tablets, capsules, granules, oral liquids, and injections.

[0012] In one possible design, bleomycin was used to construct a mouse model of pulmonary fibrosis, with illismoxine administered at a dose of 20 mg / kg via intraperitoneal injection.

[0013] This invention also provides the use of irismo in the preparation of drugs that reduce the expression levels of α-SMA and / or collagen.

[0014] The present invention also provides the use of irismo in the preparation of a drug that inhibits the activation of pulmonary myofibroblasts.

[0015] The present invention also provides a composition for inhibiting the activation of pulmonary myofibroblasts, comprising ilimismo as an active substance.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. First public disclosure of a new use: The first discovery and verification that Elesclomol can be used to treat pulmonary fibrosis.

[0017] 2. Cell state-selective action: It can selectively act on activated lung myofibroblasts, while having less effect on resting cells.

[0018] 3. High degree of unpredictability: The above-mentioned selective effect has not been reported in existing copper ion carriers and is a technical effect that cannot be expected by those skilled in the art.

[0019] 4. Effective both in vivo and in vitro: It exhibits stable anti-fibrotic effects in both cell and animal models.

[0020] 5. Possesses clear translational potential: Provides new drug candidates for the treatment of pulmonary fibrosis. Elesclomol, as a copper ion carrier, has been used in anti-tumor research. However, due to the significant differences in cell selectivity and biological effects among different copper carriers, their effects cannot be directly deduced by existing technologies. To date, there are no reports of the application of Elesclomol in the treatment of pulmonary fibrosis. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0022] Figure 1 To construct a bleomycin-induced mouse pulmonary fibrosis and ES intervention model, pathological examination was performed. In this study, A represents HE staining of lung tissue from different groups of mice, B represents Sirius red staining of lung tissue from different groups of mice, and C represents Masson staining of lung tissue from different groups of mice. Figure 2To detect the different groups of mouse primary lung fibroblasts, the following data were extracted: A represents cell copper content, B represents cell DLAT protein oligomerization, C represents the levels and quantification of different intracellular proteins, D represents cell immunofluorescence (green fluorescence for DLAT, red fluorescence for mitochondria, and blue fluorescence for DAPI), E represents cell immunofluorescence (green fluorescence for Ki67 and blue fluorescence for DAPI), F represents cell glutathione content, G represents cell viability, H represents cell FN mRNA expression level, and I represents cell α-SMA mRNA expression level. Figure 3 Different concentrations of ES were used to treat TGFβ-induced activated fibroblasts in vitro. A represents cell viability, B represents cell copper content, C represents cell glutathione content, D represents cell FN and α-SMA mRNA expression levels, and E represents cell protein oligomerization. Figure 4 The effect of ES on copper death induced by fibroblasts and myofibroblasts in vitro is shown in Figure A, where A represents different protein levels in cells, B represents cell immunofluorescence, green fluorescence represents DLAT, red fluorescence represents mitochondria, and blue fluorescence represents DAPI. Figure 5 The effect of ES on copper-induced death in primary fibroblasts and myofibroblasts is shown in the figures. A represents cell viability, B represents immunoblotting and different protein levels in cells, C represents the expression levels of FN, ATP7A, and FDX1 mRNA in cells, D represents cell protein oligomerization, E represents cell glutathione content, F represents cell copper content, and G represents cell immunofluorescence. Green fluorescence represents DLAT, red fluorescence represents mitochondria, and blue fluorescence represents DAPI. Figure 6 The effect of ES on primary ATII cells and macrophages is shown in Figure 1. A and D represent the same cell content, B and E represent the cellular glutathione content, and C represents different cellular protein levels and DLAT protein oligomerization. Detailed Implementation

[0023] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0024] In at least one embodiment, the present invention provides the use of irismo in the preparation of a medicament for treating pulmonary fibrosis.

[0025] Irismoxol can reduce the expression levels of α-SMA and / or collagen.

[0026] In at least one embodiment, irismo can be used to inhibit the activation of lung myofibroblasts.

[0027] In at least one embodiment, the present invention also provides a composition for treating pulmonary fibrosis, comprising ilimismo as an active substance.

[0028] In at least one embodiment, the present invention also provides a medicament for treating pulmonary fibrosis, comprising the composition of claim 4, and further comprising a pharmaceutically acceptable carrier or excipient.

[0029] The dosage form of the drug includes any one of tablets, capsules, granules, oral liquids, and injections.

[0030] In at least one embodiment, a mouse model of pulmonary fibrosis was constructed using bleomycin, and ilimismo was administered at a dose of 20 mg / kg via intraperitoneal injection.

[0031] In at least one embodiment, the present invention also provides the use of ilisimo in the preparation of a medicament that reduces the expression levels of α-SMA and / or collagen.

[0032] In at least one embodiment, the present invention also provides the use of irismo in the preparation of a medicament for inhibiting the activation of pulmonary myofibroblasts.

[0033] In at least one embodiment, the present invention also provides a composition for inhibiting the activation of pulmonary myofibroblasts, comprising ilimismo as an active substance.

[0034] Compared with the prior art, the present invention has the following beneficial effects: 1. First public disclosure of a novel use: The discovery that Elesclomol can be used to treat pulmonary fibrosis is novel.

[0035] 2. Cell state-selective action: It can selectively act on activated lung myofibroblasts, while having less effect on resting cells.

[0036] 3. High degree of unpredictability: The above-mentioned selective effect has not been reported in existing copper ion carriers and is a technical effect that cannot be expected by those skilled in the art.

[0037] 4. Effective both in vivo and in vitro: It exhibits stable anti-fibrotic effects in both cell and animal models.

[0038] 5. Possesses clear translational potential: Provides new drug candidates for the treatment of pulmonary fibrosis.

[0039] Specifically, the preparation method and verification method of the present invention are as follows: Step 1: Constructing a bleomycin-induced mouse model of pulmonary fibrosis and ilismo intervention. Specifically, the bleomycin (BLOM) tracheal perfusion model: Mice were anesthetized with sodium pentobarbital and fixed supine on a worktable. The neck fur was moistened with alcohol, and the skin was cut along the midline of the neck. The trachea was exposed by separating the peritracheal tissue with forceps. 50 μL of BLM solution (5 mg / kg) was instilled into the mice. After the mice recovered from anesthesia, they were fed and ate normally for 24 hours. Starting 14 days after BLM instillation, mice were given intraperitoneal injections of elixromol (ES) at a concentration of 20 mg / kg (20 mg / kg of body weight) every three days. Mice were sacrificed on day 28 after BLM modeling, and bronchoalveolar lavage fluid and lung tissue were collected.

[0040] Preparation of lung pathology slides: Mouse lung tissue was fixed in 4% paraformaldehyde solution for one week. The tissue was then removed, trimmed, and residual paraformaldehyde was washed away. Gradual dehydration was then performed (treatment with 85% ethanol solution for 2 hours, 95% ethanol solution for 1 hour, followed by another 1 hour of treatment with 95% ethanol solution, and then anhydrous ethanol solution for 0.5 hours). After dehydration, the tissue was treated with xylene for 30 minutes, followed by another 10 minutes of treatment with new xylene to achieve transparency. After transparency, the tissue was then subjected to paraffin embedding (paraffin wax was changed every hour for a total of 3 hours). Subsequently, the paraffin-embedded tissue, along with the molten paraffin, was poured into a container and immersed in cold water to immediately solidify into a wax block. The tissue was then embedded, sectioned, mounted on glass slides, and finally dried in a 60°C oven for 5 hours before use.

[0041] Hematoxylin-Eosin (H&E) staining: Dewaxing and hydrating mouse lung paraffin sections: First, soak in xylene for 10 minutes, then remove and soak again in fresh xylene for 10 minutes; then soak in anhydrous ethanol for 4 minutes, then soak again in fresh anhydrous ethanol for 4 minutes; then soak in 95% ethanol for 4 minutes, then soak again in 80% ethanol for 4 minutes; finally rinse with running water for 5 minutes, air dry, and await staining. After dehydration, the sections soaked in distilled water are stained in hematoxylin solution for 5 minutes, then rinsed with tap water and the remaining water is blotted dry; the sections are then differentiated in acidic water and ammonia water for a few seconds; rinsed with running water for 2 hours, then placed in distilled water for a short time; then dehydrated in 70% and 90% ethanol for 10 minutes each; then stained with alcohol-eosin staining solution for 2-3 minutes. A stepwise dehydration process is performed, soaking in 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 5 minutes each. Finally, the sections are soaked in xylene for 5 minutes to obtain neutral resin mountings.

[0042] HE staining results are as follows Figure 1 As shown in Figure A, ES significantly alleviated BLM-induced lung injury in mice, manifested as reduced alveolar wall thickening, decreased inflammatory nodules, and restoration of alveolar structure.

[0043] Sirius Red staining: Paraffin-embedded mouse tissue sections were dewaxed sequentially in xylene I and II for 10 minutes each, then rehydrated by treatment with a gradient of ethanol (100%, 95%, 80%, and 70% ethanol) for 2 minutes each. After rinsing twice with distilled water, the sections were stained at room temperature for 1 hour in Chondrex's Sirius Red staining solution. The staining solution was discarded, and the sections were quickly rinsed twice with 0.1% acetic acid solution to remove unbound dye. Subsequently, the sections were dehydrated sequentially in anhydrous ethanol I, II, and III for 2 minutes each, then cleared with xylene I and II for 5 minutes each, and finally mounted with neutral resin. Under a regular optical microscope, the collagen fibers appeared red against a green background.

[0044] Sirius red staining results are as follows: Figure 1 As shown in Figure B, ES significantly alleviated BLM-induced collagen fibrosis deposition in mouse lung tissue, as evidenced by a significant reduction in red collagen fibers.

[0045] Masson's trichrome staining: Paraffin-embedded mouse tissue sections were sequentially dewaxed to water by immersing them in environmentally friendly dewaxing and clearing solution I for 15 min, environmentally friendly dewaxing and clearing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, followed by rinsing with water. The sections were then immersed in Masson A solution and placed in a 65°C oven for 30 min, after which they were rinsed with running water. Simultaneously, Masson D and Masson F solutions were preheated in a 55°C water bath. The sections were then stained for 1 min in a mixture of equal parts Masson B and Masson C solutions, rinsed with tap water, differentiated with differentiation solution for a few seconds, and then rinsed with tap water. Next, the sections were stained in preheated Masson D solution for 6 min and rinsed with tap water. Finally, they were stained in Masson E solution for 1 min, slightly drained, and then directly immersed in preheated Masson A solution. Stain in solution F for 2-30 seconds; after removal, rinse with 1% acetic acid for differentiation, then immerse in anhydrous ethanol I and II for dehydration, then soak in anhydrous ethanol III for 5 minutes, clear with xylene for 5 minutes, and finally mount with neutral resin; under a microscope, collagen fibers appear blue, muscle fibers and cytoplasm appear red, and cell nuclei appear blue-black.

[0046] Masson staining results are as follows: Figure 1 As shown in Figure C, BLM induces collagen deposition in mouse lung tissue, manifested as a significant increase in blue collagen, while ES intervention significantly reduces collagen fibrosis deposition in mouse lung tissue.

[0047] Immunohistochemistry (IHC) staining: Routine dewaxing and hydration of paraffin sections: First, soak the tissue sections in xylene for 10 minutes, then remove and soak again in fresh xylene for 10 minutes. Next, soak in anhydrous ethanol for 4 minutes, then replace with fresh anhydrous ethanol and soak for another 4 minutes. Then soak in 95% ethanol for 4 minutes, followed by 80% ethanol for 4 minutes. Finally, rinse with running water for 5 minutes. Place the sections on a slide holder and put them in a container containing 1× citrate buffer. Bring to a boil on an induction cooker, then boil for 8 minutes. Turn off the cooker and cool for 5 minutes. Repeat this process (antigen retrieval) 3 times. After antigen retrieval, cool the sections on ice for 20 minutes. After cooling, wash with water for 5 minutes. Place the sections in a staining jar containing 1× PBS solution and wash on a shaker for 5 minutes, repeating this process three times. Wipe the sections dry. Add 3% H2O2 / methanol solution (volume not specified, just enough to completely cover the tissue) to the tissue and allow it to act for 15 minutes (in a humidified chamber) to eliminate endogenous peroxidase activity. After washing with water for 5 minutes, place the sections in 1×PBS solution and wash on a shaker for 5 minutes. Add mouse α-SMA antibody and incubate overnight at 4°C, then wash three times with PBS for 5 minutes each time. Add the secondary antibody provided in the kit (Kangwei Century, SP Rabbit & Mouse HRP Kit (DAB), Rabbit / Mouse Universal Streptavidin-HRP Kit (DAB)), incubate at 37°C for 20 minutes, and wash three times with PBS for 5 minutes each time. Add DAB staining solution for 2 minutes to achieve staining effect, then wash with water; counterstain with hematoxylin for 1 minute, and wash with water for 5 minutes. Dehydrate with a gradient of ethanol (95% v / v, 95% v / v, 100% v / v, 100% v / v each for 4 minutes), wash three times with xylene for 10 minutes each time, and dry in an oven. Finally, mount with neutral resin and observe the results under a microscope.

[0048] IHC staining results showed that BLM-induced α-SMA levels in mouse lung tissue were significantly increased, manifested as an increase in brown granules, while ES significantly inhibited α-SMA levels in mouse lung tissue.

[0049] Step 2: Extract primary fibroblasts / myofibroblasts from mice in different treatment groups and detect their activity. Isolation and processing of primary fibroblasts: Lung tissue was collected from mice in the designated treatment group. Under aseptic conditions, the surrounding connective tissue and trachea were dissected, and the tissue was rinsed 2-3 times in pre-cooled PBS containing 1% penicillin / streptomycin to remove as much blood as possible. The rinsed lung tissue was transferred to a sterile culture dish and finely minced to approximately 1 mm using ophthalmic scissors. 3 Cut the tissue into small, pasty pieces. Add an appropriate amount of 0.25% trypsin to the chopped tissue and digest at 37°C with shaking for 15 minutes, intermittently agitating to ensure complete digestion. Once the tissue pieces are loose and flocculent, add DMEM / F12 medium containing 10% fetal bovine serum to stop digestion. Filter the digested cell suspension through a 70μm cell sieve to remove incompletely digested tissue residue. Collect the filtrate and centrifuge at 1000 rpm (approximately 200 g) for 5 minutes at 4°C. Discard the supernatant and resuspend the cell pellet in DMEM / F12 complete medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, and 1% glutamine. Seed the pellet into culture flasks and incubate statically at 37°C with 5% CO2. After 24-48 hours of culture, take advantage of the rapid adhesion of fibroblasts by changing the medium to remove non-adherent suspended cells (such as blood cells and epithelial cells), achieving preliminary purification. The culture medium was changed every 2-3 days thereafter. When the cells reached 80-90% confluence, they were digested and passaged with 0.25% trypsin (v / v). After 2-3 passages, high-purity fibroblasts were obtained for testing. For the in vivo copper death assessment experiment of activated fibroblasts, fibroblasts were isolated from ES-treated mice and then treated with 20 μM TTM.

[0050] Copper content in fibroblasts: Fibroblasts isolated and purified from lung tissue of BLM and BLM+ES mice were used to detect intracellular copper ions. The specific method is as follows: The culture medium was aspirated from the culture dish, and the cells were gently washed 1-2 times with pre-chilled PBS. Lysis buffer was added to each well of a 6-well plate at a ratio of 150-200 μL. The cells were pipetted several times to ensure sufficient contact between the lysis buffer and the cells, and then placed on ice for 5-10 minutes for lysis. The lysed cell suspension was collected into centrifuge tubes and centrifuged at 14000g for 3-5 minutes at 4°C. The supernatant was carefully collected and used. An appropriate amount of supernatant was taken and the procedure was performed according to the instructions of the Nanjing Jiancheng Bioengineering Institute Copper Assay Kit (catalog number: E010-1-1). The principle of this kit is based on the determination of Cu under acidic conditions. 2+ It dissociates from ceruloplasmin and albumin, and ascorbic acid reduces it to Cu. + Cu +The reagent reacts with the complexing agent to form a blue complex, and the absorbance is measured at 600 nm. Following the kit instructions, add the appropriate reagents and sample, mix well, and measure the absorbance of each tube at 600 nm. Calculate the copper ion content in the sample based on the standard curve.

[0051] Copper content results as follows Figure 2 As shown in Figure A, ES treatment significantly increased the copper content in mouse lung fibroblasts.

[0052] Western blotting: Prepare the gel according to the reagent instructions. Place the prepared SDS-PAGE gel into the electrophoresis apparatus, slowly pour in the electrophoresis buffer, remove the comb from the gel, and add the sample and protein marker to each well (10 µL). Set the voltage to 80 V and run the gel at a constant voltage. When protein marker bands appear, increase the voltage to 180 V. Stop electrophoresis when the sample reaches near the bottom of the gel. Turn off the power, remove the gel plate, and rinse off excess gel buffer with water. Activate the PVDF membrane with methanol for 15 seconds beforehand, and place it in the transfer buffer along with the filter paper and sponge required for transfer. To prepare for transfer, place the gel in the transfer buffer, then layer the thick sponge, thick filter paper, gel, PVDF membrane, thin filter paper, and thin sponge in that order. Set the voltage to 75 V and transfer for 2 hours. The transferred membrane was then removed and activated sequentially with methanol, rinsed with triple-distilled water, and stained with Ponceau S for 5 minutes to visualize proteins. Raw data were recorded by photographing, and a small corner was cut off from the upper right corner of the membrane according to the sample loading direction for marking. Primary antibody was incubated overnight at 4°C, followed by washing four times with PBST for 5 minutes each time. Secondary antibody was then incubated for 1 hour, followed by washing four times with PBST for 5 minutes each time. Finally, protein bands on the membrane were observed using developing solution.

[0053] Immunoblotting results as follows Figure 2 As shown in B and C, ES treatment significantly increased the oligomerization level of DLAT protein. DLAT oligomers are one of the gold standards for verifying copper death, and the high molecular weight DLAT oligomer bands were significantly enhanced. Simultaneously, ES treatment significantly increased the levels of ETFDH, SDHB, and HSP proteins (copper death-related proteins), suggesting that ES can induce copper death in fibroblasts. Consistently, we examined FN and α-SMA levels, and the results showed that ES treatment significantly reduced both levels, suggesting that ES treatment can inhibit fibrosis.

[0054] Cellular immunofluorescence assay: Transfer a small amount of cells from each tube to a 1.5 mL EP tube, centrifuge at 4°C, and resuspend in an equal volume of 4% paraformaldehyde at room temperature for 45 minutes. Take 5 µL of each resuspended cell and place it on an adhesive slide. Spread the resuspended cell in an area approximately the size of an 8 mm coverslip using a pipette tip. After drying at room temperature (until slightly whitish, but not completely dry), draw circles around the cells with a histochemical pen, and wash with PBS. Wipe away excess liquid, block with sheep serum working solution for 2 hours, wash with PBS for 5 minutes three times, wipe away excess liquid, add primary antibody and incubate overnight at 4°C, wash with PBS for 5 minutes three times, add secondary antibody (1:500, diluted with 5% BSA), and incubate at room temperature or 37°C for 1 hour, wash with PBS for 5 minutes three times, mount with Hoechst mounting medium, and observe under a fluorescence microscope.

[0055] Immunofluorescence results as follows Figure 2 In fibroblasts isolated from the lung tissue of mice in groups D and ES, DLAT (green) was significantly increased in mitochondria (red portion), appearing as yellow granules, indicating copper death in fibroblasts. However, after combined TTM treatment, the yellow granules decreased, suggesting an alleviation of copper death in fibroblasts.

[0056] Immunofluorescence results (2E) showed that Ki67 (cell proliferation protein) was significantly reduced in fibroblasts isolated from lung tissue of mice in the ES group, indicating insufficient fibroblast activity. However, after combined treatment with TTM, Ki67 significantly increased, indicating increased fibroblast activity.

[0057] Glutathione (GSH) content in fibroblasts: The level of GSH in cells was detected using a commercial kit (A006-2-1, Nanjing Jiancheng Biotechnology Institute, China). Fibroblasts isolated and purified from lung tissue of BLM and BLM+ES mice were used to detect intracellular GSH. The specific method was as follows: Cells were lysed by sonication on ice. 0.1 ml of the sonicated cell suspension was mixed with 0.1 ml of reagent one, centrifuged at 3500 rpm for 10 minutes, and 0.1 ml of the supernatant was mixed with 0.1 ml of reagent two and 25 μl of reagent three. After standing for 5 minutes, the absorbance was measured at 405 nm using a microplate reader. The cellular GSH content was calculated based on the standard curve.

[0058] GSH content results are as follows Figure 2 As shown in Figure F, ES treatment significantly increased copper content in mouse lung fibroblasts. Cell viability assay: Fibroblasts in the logarithmic growth phase were digested with trypsin and resuspended in DMEM / F12 medium containing 10% fetal bovine serum, and the cell density was adjusted to 5 × 10⁶ cells / year. 3Cells were seeded per well in 96-well plates at a volume of 200 μL per well and incubated overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, different concentrations of the drug were added according to the experimental design, with 3-5 replicates per group. Blank wells (culture medium only, no cells) and control wells (cells without drug treatment) were also included, and cells were incubated for the corresponding time. After incubation, 20 μL of MTT solution (2 mg / mL, prepared with PBS, sterilized by 0.22 μm filter) was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. The supernatant was carefully aspirated, taking care not to remove the formazan crystals at the bottom. 100 μL of isopropanol / hydrochloric acid mixture (isopropanol diluted with concentrated hydrochloric acid to a final concentration of 0.04 M) was added to each well, and the plates were shaken on a shaker at low speed for 10 minutes to dissolve the crystals completely. The absorbance (OD value) of each well was measured at a wavelength of 570 nm using a Synergy 2 multi-microplate reader (Biotek, USA).

[0059] It should be noted that the concentration unit M in this article is an abbreviation of mol / L. M = mole / liter, i.e., mol / L; mM = millimole / liter, i.e., mmol / L; μM = micromole / liter, i.e., μmol / L; nM = nanomole / liter, i.e., nmol / L.

[0060] MTT results are as follows Figure 2 As shown in G, after ES treatment, the activity of fibroblasts in the BLM group mice was significantly reduced, while after treatment with the combined copper ion chelator (TTM), the activity of fibroblasts inhibited by ES was significantly restored.

[0061] qRT-PCR experiment: Total RNA was extracted from treated fibroblasts using the Trizol method. RNA concentration and purity were measured (A260 / A280 ratio between 1.8 and 2.0). Following the reverse transcription kit instructions, the RNA was reverse transcribed into cDNA. The cDNA obtained from reverse transcription was used as a template to prepare a real-time quantitative PCR reaction system on ice: 10 μL SYBR Green premix, 0.4 μL each of forward and reverse primers (10 μM), 2 μL cDNA template, and nuclease-free water to a final volume of 20 μL. Each sample was tested in triplicate, and a template-free control was included. The PCR plate was briefly centrifuged to remove air bubbles and then placed in a real-time PCR instrument for amplification. The reaction program was: 94℃ pre-denaturation for 2 minutes; 94℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 40 cycles. Melting curve analysis was performed after the reaction to verify the specificity of the amplified products. Using β-actin as an internal reference gene, the relative expression level of the target gene was calculated using the 2^-ΔΔCt method.

[0062] qRT-PCR results are as follows Figure 2 As shown in H and I, after ES treatment, the mRNA expression of α-SMA and FN in fibroblasts of mice in the BLM group was significantly reduced, while after treatment with the copper ion chelator (TTM), the mRNA expression of α-SMA and FN inhibited by ES was significantly restored.

[0063] Step 3: Verification of ES-induced copper death in myofibroblasts in vitro Cell culture and processing: Primary fibroblasts were extracted from mouse lung tissue and cultured (see step 2 for the method). They were then induced to become activated fibroblasts (i.e., myofibroblasts) by TGFβ. Subsequently, the cells were treated with different concentrations of ES, namely 0 nM (CTRL), 6.25 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM.

[0064] Cell viability assay: Cell viability was assessed in different groups, as described in step 2. The cell viability results are as follows: Figure 3 As shown in Figure A, ES treatment in vitro significantly inhibited cell activity in a dose-dependent manner; the higher the ES concentration, the lower the cell activity.

[0065] Cellular copper content detection: The copper content of cells from different groups was measured, as described in step 2. The results of the cell copper content measurement are as follows: Figure 3 As shown in B, ES treatment in vitro significantly increased intracellular copper content in a dose-dependent manner; the higher the ES concentration, the higher the cellular copper content.

[0066] Cellular glutathione content detection: Glutathione levels were measured in different groups of cells, as described in step 2. The results of the cellular glutathione content detection are as follows: Figure 3 As shown in C, ES treatment in vitro can significantly inhibit the intracellular glutathione content, and this effect is dose-dependent; the higher the ES concentration, the lower the cellular glutathione content.

[0067] qRT-PCR experiment: See step 2 for the method. The qRT-PCR results are as follows: Figure 3 As shown in Figure D, ES treatment significantly reduced the mRNA expression of α-SMA and FN, and the higher the ES concentration, the lower the mRNA expression of α-SMA and FN.

[0068] Immunoblotting assay: The method is described in step 2. The immunoblotting results are as follows: Figure 3As shown in Figure E, ES treatment significantly increased the oligomerization level of DLAT protein (DLAT oligomers are one of the gold indicators for verifying copper death), and the high molecular weight DLAT oligomer bands were significantly enhanced.

[0069] Step 4: ES induces copper death in myofibroblasts, but has no effect on fibroblasts. Cell culture and processing: Primary fibroblasts were extracted from mouse lung tissue and cultured (see step 2 for the method). The cells were induced to activate into myofibroblasts by TGFβ treatment or without treatment, followed by ES treatment (50 nM) or without treatment. The cells were divided into four groups: CTRL group, ES group, TGFβ group, and ES+TGFβ group. Immunoblotting and immunofluorescence detection were performed on each group, as detailed in step 2.

[0070] Immunoblotting results as follows Figure 4 A showed that TGFβ1 treatment activated fibroblasts into myofibroblasts, characterized by a significant increase in FN and α-SMA protein levels. ES treatment significantly inhibited TGFβ1-induced FN and α-SMA protein levels. Furthermore, ES significantly inhibited the iron-sulfur protein clusters (SDHB and ETFDH, copper death markers) in the TGFβ1-treated group, while having no significant effect on the iron-sulfur protein clusters in the untreated group, suggesting that ES only affects activated fibroblasts.

[0071] Immunofluorescence results as follows Figure 4 B showed that only in TGFβ1-treated fibroblasts did DLAT protein significantly increase after ES treatment.

[0072] Step 5: In vivo validation of ES-specific induction of copper death in myofibroblasts To further determine the cell specificity of ES, we validated it in a mouse pulmonary fibrosis model. Primary fibroblasts (i.e., fibroblasts and myofibroblasts) were extracted from mice in the CTRL and BLM groups and treated with ES (50 nM), with or without copper ion chelating agent intervention. Cell extraction and culture are described in step 2. Cell viability, Western blotting, qRT-PCR, copper content, glutathione content, and immunofluorescence were detected in each group, as described in step 2.

[0073] In the cell viability assay, copper ions were added to the cell culture medium because the cells were deprived of the in vivo environment (where copper ions were absent). The cell viability assay results showed that activated fibroblasts (fibroblasts extracted from the BLM group) exhibited significantly higher ES-induced cytotoxicity than normal fibroblasts (fibroblasts extracted from the CTRL group). Higher ES concentrations resulted in lower activity of the activated fibroblasts.

[0074] Immunoblotting results showed the effects of different ES treatment times (24 hours and 48 hours) on activated fibroblasts and fibroblasts. Results are as follows... Figure 5 As shown in Figure B, BLM-related fibroblast activation was manifested by increased FN protein levels; ES significantly inhibited FN and ATP7A (a copper ion transporter responsible for transporting monovalent copper ions out of cells) in the BLM group, while significantly increasing FDX1 (responsible for converting divalent copper ions into monovalent toxic copper ions). Further immunoblotting results of DLAT were also provided. Figure 5 D indicates that ES can significantly induce DLAT oligomerization, and the DLAT-induced DLAT oligomerization was significantly inhibited after treatment with the copper ion chelator TTM.

[0075] qRT-PCR results showed the effects of different ES treatment times (24 h and 48 h) on activated fibroblasts and fibroblasts. Results are as follows... Figure 5 As shown in C, BLM-component fibroblast activation is manifested by increased FN mRNA expression; ES can significantly inhibit FN and ATP7A in the BLM group, while significantly increasing FDX1 mRNA.

[0076] Glutathione results as follows Figure 5 E showed that ES significantly inhibited glutathione levels in the BLM group but had no significant effect on the CTRL group, suggesting that copper death occurred in the BLM group after ES administration.

[0077] Copper content results as follows Figure 5 As shown in F, ES significantly induced the copper content of BLM-component fibroblasts, but had no significant effect on the copper content of the CTRL group, suggesting that activated fibroblasts are more sensitive to ES.

[0078] Immunofluorescence results as follows Figure 5 G and ES significantly induced DLAT aggregation in BLM-composing fibroblasts, but had no significant effect on CTRL-composing cells. Further treatment with a copper ion chelating agent weakened DLAT aggregation, suggesting that ES induced copper death in activated fibroblasts.

[0079] Step 6: ES has no effect on macrophages or type II alveolar epithelial cells. To further verify the specific targeting and activation of fibroblasts by ES, we extracted macrophages and alveolar type II epithelial cells (ATII) from mice in the ES-treated group and the control group.

[0080] Primary ATII cell extraction: Mice in different treatment groups were euthanized, and immediately perfused with PBS via the right ventricle to remove blood from the lungs. The trachea was exposed and ligated with a cannula. Dispase enzyme solution (Roche Diagnostics) was slowly injected into the trachea, followed immediately by low-melting-point agarose solution to fully inflate the lungs. The lung tissue was incubated on ice to solidify the agarose, and the lung tissue was carefully separated and transferred to Dispase enzyme solution containing DNase I (Roche Diagnostics). Digestion continued for 45 minutes at room temperature. The digested lung tissue was gently minced and filtered sequentially through 70 μm, 40 μm, and 25 μm cell sieves to obtain single-cell suspensions. ATII cell purification was performed using a two-step immunomagnetic bead sorting (MACS) method: First, negative selection was performed to remove lineage cells. A crude lung cell suspension was incubated with a mixture of biotinylated anti-CD45 and anti-CD16 / CD32 antibodies (Miltenyi Biotec). Then, streptavidin beads (Miltenyi Biotec) were added for labeling. The cell suspension was then passed through an MS sorting column (Miltenyi Biotec) placed in a magnetic field to remove labeled hematopoietic lineage cells (CD45+ and CD16 / 32+ cells). After collecting the flow-through, positive selection was performed to enrich epithelial cells. These cells were then incubated with FITC-labeled anti-mouse SPC antibody (Santa Cruz), followed by the addition of anti-FITC magnetic beads (Miltenyi Biotec). SPC-positive cells obtained by passing the cells through an MS sorting column were considered purified ATII cells. The purity of the isolated cells was assessed by immunofluorescence staining and flow cytometry. The isolated cells were used for subsequent copper and GSH content detection and Western blotting.

[0081] Method for extracting primary lung macrophages from mice (bronchoalveolar lavage): Mice were euthanized by cervical dislocation and fixed in a supine position. The skin of the chest and abdomen was disinfected with 75% ethanol. The skin and peritoneum were incised along the midline of the abdomen, and the diaphragm was incised upwards to expose the thoracic cavity. The peritracheal tissues were carefully dissected to fully expose the trachea. A small V-shaped incision was made on the anterior wall of the trachea below the larynx using ophthalmic scissors. A 22G blunt-tipped cannula or intravenous catheter was inserted into the trachea through the tracheal incision about 0.5-1 cm and secured with sutures to prevent slippage. Pre-cooled lavage fluid (containing 2% fetal bovine serum and ethanol) was drawn up with a 1 mL syringe. Slowly inject penicillin-streptomycin in PBS (pH 7.4) into the lungs. The lung lobes will gradually fill and turn white. Gently massage the pleural cavity for 5-10 seconds, then slowly aspirate to collect the lavage fluid into a 15 mL centrifuge tube placed on ice. Inject 0.8-1 mL each time, repeating 8-10 times, for a total lavage fluid volume of approximately 8-10 mL. Avoid excessive force during the procedure to prevent lung tissue damage and bleeding. Filter the collected bronchoalveolar lavage fluid through a 70 μm cell sieve into a new centrifuge tube to remove mucus and cell clumps. Centrifuge at 250 g for 10 minutes at 4°C, carefully discarding the supernatant. Observe the cell pellet. If there are many red blood cells (the pellet is red), add 2 mL of red blood cell lysis buffer (such as ACK lysis buffer) to resuspend the cells. Incubate on ice or at room temperature for 2-3 minutes, add an equal volume of PBS to stop lysis, and centrifuge again at 250 g for 10 minutes at 4°C, discarding the supernatant. The cell pellet was resuspended in complete culture medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) for cell counting and trypan blue rejection assay to assess cell viability. The cell density was adjusted to 1×10^6 cells / mL. The cell suspension was seeded into culture plates or dishes and incubated at 37°C, 5% CO2 for 40-60 minutes. Taking advantage of the rapid adhesion of macrophages, the culture medium was gently aspirated, and the cells were gently washed 1-2 times with pre-warmed PBS to remove non-adherent cells (mainly lymphocytes). The adherent cells were then purified lung macrophages. The isolated cells were used for subsequent copper and GSH content detection and immunoblotting.

[0082] Copper content results as follows Figure 6 As shown in A and D, ES had no significant effect on the copper content of ATII cells or Macrophages, suggesting that ES does not induce copper death in ATII cells and Macrophages.

[0083] Glutathione results as follows Figure 6 As shown in B and E, ES had no significant effect on glutathione levels in ATII cells or Macrophages, suggesting that ES does not induce copper death in ATII cells and Macrophages.

[0084] Immunoblotting results as follows Figure 6 As shown in C and F, the ATII cells and Macrophages iron-sulfur protein clusters (ETFDH1 and SDHB) and DLAT oligomerization in the ES-treated group of mice were not significantly different from those in the BLM group, suggesting that ES does not induce copper death in ATII cells and Macrophages.

[0085] In this invention, ilismo can exert its anti-fibrotic effect in a low-dose range.

[0086] In some embodiments, the concentration used is, for example, 0.001 to 1 μmol / L, but is not limited to this range.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any indirect modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Application of ilismo in the preparation of drugs for the treatment of pulmonary fibrosis.

2. The application according to claim 1, characterized in that, Irismoxol can reduce the expression levels of α-SMA and / or collagen.

3. The application according to claim 1, characterized in that, Ilisimor can be used to inhibit the activation of lung myofibroblasts.

4. A composition for treating pulmonary fibrosis, characterized in that, Including ilismo as an active ingredient.

5. A drug for treating pulmonary fibrosis, characterized in that, The composition includes the composition of claim 4, and further includes a pharmaceutically acceptable carrier or excipient.

6. The drug according to claim 5, characterized in that, The dosage form of the drug includes any one of tablets, capsules, granules, oral liquids, and injections.

7. The drug according to claim 5, characterized in that, Bleomycin was used to establish a mouse model of pulmonary fibrosis. Ilisimor was administered at a dose of 20 mg / kg via intraperitoneal injection.

8. Application of ilisimor in the preparation of drugs that reduce the expression levels of α-SMA and / or collagen.

9. Application of ilismo in the preparation of drugs that inhibit the activation of pulmonary myofibroblasts.

10. A composition for inhibiting the activation of lung myofibroblasts, characterized in that, Including ilismo as an active ingredient.

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