Application of Compound 428 in the preparation of drugs for the prevention, control or treatment of idiopathic pulmonary fibrosis or GPX4 stabilizers

Abstract

This invention provides the application of compound 428 in the preparation of drugs or GPX4 stabilizers for the prevention, control, or treatment of idiopathic pulmonary fibrosis, belonging to the field of biomedical technology. This invention uses a bleomycin (BLM)-induced pulmonary fibrosis mouse model to verify the in vivo effects of 428. The results showed that, compared with the control group, 428 could delay the progression of BLM-induced pulmonary fibrosis, significantly prolong the survival time of mice, and reduce the level of lipid peroxidation MDA. It significantly alleviated the massive inflammatory cell infiltration and alveolar structural destruction in mouse lung tissue caused by BLM, and significantly reduced BLM-induced pulmonary fibrosis. This invention also provides the application of compound 428 in the preparation of GPX4 stabilizers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of compound 428 in the preparation of drugs for the prevention, control or treatment of idiopathic pulmonary fibrosis or GPX4 stabilizers. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease that seriously threatens human health. It commonly affects middle-aged and elderly men (median age at diagnosis is 65 years), causing changes in lung compliance, impaired alveolar gas exchange, and ultimately leading to dyspnea and even death. The incidence and mortality rates of IPF are increasing annually, with a median survival of only 2.5–3.5 years after diagnosis. Two antifibrotic drugs (nintedanib and pirfenidone) are approved for clinical treatment of IPF; however, these drugs only delay the decline in lung function and do not stop the progression of the disease. Currently, lung transplantation is a last resort for treating IPF; however, factors such as donor matching, high surgical costs, and the use of anti-rejection drugs result in a very low rate of lung transplantation in China. Therefore, developing new drugs for the treatment of IPF is of great value. Summary of the Invention

[0003] The purpose of this invention is to provide the use of compound 428 in the preparation of a medicament for the prevention, control or treatment of idiopathic pulmonary fibrosis or a GPX4 stabilizer, wherein compound 428 can be used for the prevention, control or treatment of idiopathic pulmonary fibrosis.

[0004] This invention provides the use of compound 428 in the preparation of medicaments for the prevention, control, or treatment of idiopathic pulmonary fibrosis, wherein the chemical structural formula of compound 428 is shown in Formula 1: Formula 1.

[0005] Preferably, the dosage form of the drug includes an injection.

[0006] Preferably, the drug is administered in unit doses; the drug contains compound 428 which is effective in preventing, controlling or treating idiopathic pulmonary fibrosis, in the range of 30-60 mg / kg of compound 428 per day.

[0007] Preferably, the idiopathic pulmonary fibrosis includes bleomycin-induced idiopathic pulmonary fibrosis.

[0008] The present invention also provides the application of compound 428 in the preparation of GPX4 stabilizer, wherein the chemical structure of compound 428 is shown in Formula 1.

[0009] Preferably, compound 428 can bind to GPX4 and improve the stability of GPX4.

[0010] Preferably, improving the stability of GPX4 includes improving the thermal stability of GPX4.

[0011] Preferably, compound 428 can also inhibit the covalent binding of RSL3 and GPX4 and / or inhibit ferroptosis.

[0012] The present invention also provides the use of compound 428 in the preparation of products with antioxidant, cardiovascular protection or anti-aging properties, the chemical structure of which is shown in Formula 1.

[0013] Preferably, the product includes a drug.

[0014] Beneficial effects: This invention provides the use of compound 428 in the preparation of medicaments for the prevention, control, or treatment of idiopathic pulmonary fibrosis, wherein the chemical structural formula of compound 428 is shown in Formula 1: Formula 1.

[0015] This invention uses a bleomycin (BLM)-induced pulmonary fibrosis mouse model to verify the in vivo effects of 428. The results showed that, compared with the control group, 428 could delay the progression of BLM-induced pulmonary fibrosis, specifically manifested in a significant prolongation of the survival time of mice in the treatment group. HE staining of mouse lung tissue showed that BLM caused extensive inflammatory infiltration and alveolar structure destruction in mouse lung tissue, which was significantly alleviated in the 428 group. Furthermore, Masson staining of mouse lung tissue indicated that 428 could significantly reduce BLM-induced pulmonary fibrosis. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0017] Figure 1Compound 428 can inhibit ferroptosis induced by various ferroptosis inducers. A shows the chemical structure of 428; B shows HFL1 cells pretreated with different concentrations of 428 for 6 h, followed by overnight treatment with corresponding concentrations of RSL3, FIN56, or Erastin, with cell viability assessed by CCK8; C shows HFL1 cells treated with 200 nM RSL3 and 10 μM 428 for 8 h, with microscopic results showing that 428 inhibits RSL3-induced cell death; D shows BEAS-2B cells pretreated with 10 µM 428 for 6 h, followed by overnight treatment with 2 µM RSL3, with cell viability assessed by CCK8; E shows BEAS-2B cells pretreated with 10 µM 428 for 6 h, followed by overnight treatment with 5 µM FIN56, with cell viability assessed by CCK8; F shows HBE cells pretreated with 10 µM 428 for 6 h, followed by overnight treatment with 2.5 µM... RSL3 was treated overnight, and cell viability was detected by CCK8. G showed that 293T cells were pretreated with 5 µM 428 for 6 h, then treated with 1 µM RSL3 overnight, and cell viability was detected by CCK8. Figure 2 To investigate the effects of 428 on downregulating RSL3-induced lipid peroxidation and stabilizing intracellular GSH levels, the study included: A showing the results of flow cytometry analysis after pretreatment of HFL1 cells with different concentrations of 428 overnight, followed by treatment with RSL3 for 2 h, and incubation with the C11-bodipy 581 / 591 lipid peroxidation fluorescent probe; B showing the statistical results of the proportion of C11-Bodipy-positive cells in HFL1 cells; C showing the results of flow cytometry analysis after pretreatment of BEAS-2B cells with different concentrations of 428 for 4 h, followed by treatment with RSL3 for 2 h, and incubation with the C11-bodipy 581 / 591 lipid peroxidation fluorescent probe; D showing the statistical results of the proportion of C11-Bodipy-positive cells in BEAS-2B cells; and E showing the results of flow cytometry analysis after pretreatment of HBE cells with different concentrations of 428 overnight, followed by treatment with RSL3 for 4 h, and incubation with the C11-bodipy 581 / 591 lipid peroxidation fluorescent probe; Flow cytometry was performed after incubation with the 581 / 591 lipid peroxidation fluorescent probe; F shows the statistical results of the proportion of C11-Bodipy positive cells in HBE cells; G shows the changes in GSH levels in HFL1 cells after pretreatment with 10 μM 428 for 6–8 h followed by overnight treatment with 0.1 μM RSL3; H shows the changes in GSH levels in BEAS-2B cells after pretreatment with 10 μM 428 for 6–8 h followed by overnight treatment with 2 μM RSL3. vs. Con, & vs. RSL3 Figure 3To delay the progression of pulmonary fibrosis in vivo, A shows the survival curves of mice in the BLM+Vehicle and BLM+428 groups, which are mouse models of pulmonary fibrosis induced by BLM (BLM). B shows the MDA level in the lung tissue of each group of mice. C shows HE staining (top) and Masson staining (bottom) in the lung tissue of each group of mice. D shows the statistical results of the proportion of fibrotic area in the lung tissue of each group of mice.

[0018] Figure 4 The study investigated the effects of 428 on GPX4 protein levels without affecting its transcriptional levels. Specifically, A showed the changes in GPX4 protein levels (Western Blot) and GPX4 mRNA levels in HFL1 cells treated with different concentrations of 428 for corresponding time periods; B showed the changes in GPX4 protein levels (Western Blot) and GPX4 mRNA levels (Real-Time PCR) in BEAS-2B cells treated with different concentrations of 428 for corresponding time periods; C showed the changes in GPX4 protein levels (Western Blot) and GPX4 mRNA levels (Real-Time PCR) in HBE cells treated with different concentrations of 428 for corresponding time periods; and D showed the changes in GPX4 protein levels (Western Blot) and GPX4 mRNA levels (Real-Time PCR) in 293T cells treated with different concentrations of 428 for corresponding time periods. Changes in mRNA levels; E shows changes in GPX4 protein levels in HFL1 cells treated with a fixed concentration of 428 at corresponding time points, as detected by Western blotting; F shows changes in GPX4 protein levels in BEAS-2B cells treated with a fixed concentration of 428 at corresponding time points, as detected by Western blotting. Figure 5 The results showed that in animal experiments of BLM-induced pulmonary fibrosis, lung tissues of mice in the control group (PBS), BLM and BLM+428 groups were taken, and the changes in GPX4 protein levels were detected by Western blotting. Figure 6 The study aimed to prolong the half-life of GPX4 protein using 428. Specifically, A showed HFL1 cells treated with 20 μg / mL actinomycete ketone (CHX) with or without 10 μM 428, followed by protein collection at different time points and Western blotting (WB) analysis of GPX4 protein changes; B showed HBE cells treated with 20 μg / mL actinomycete ketone (CHX) with or without 10 μM 428, followed by protein collection at different time points and WB analysis of GPX4 protein changes; C showed BEAS-2B cells treated with 20 μg / mL actinomycete ketone (CHX) with or without 10 μM 428, followed by protein collection at different time points and WB analysis of GPX4 protein changes. Figure 7To improve the thermostability of GPX4 protein, the following methods were employed: A) HBE cell lysate was incubated with DMSO or 50 μM 428 at 37°C for 30 min, then aliquoted into 8-tube strips and heated at different temperatures. After high-speed centrifugation, the supernatant was collected for Western blotting (WB) to detect GPX4 protein levels. B) 293T cell lysate was incubated with DMSO or 50 μM 428 at 37°C for 30 min, then aliquoted into 8-tube strips and heated at different temperatures. After high-speed centrifugation, the supernatant was collected for WB to detect GPX4 protein levels. C) HBE cell lysate was incubated with different concentrations of 428 for 30 min, then heated in a PCR instrument at 52°C, and after high-speed centrifugation, the supernatant was collected for WB to detect GPX4 protein levels. D) 293T cell lysate was incubated with different concentrations of 428 for 30 min, then heated in a PCR instrument at 52°C, and after high-speed centrifugation, the supernatant was collected for WB to detect GPX4 protein levels. Figure 8 428 may compete with RSL3 for binding to GPX4 protein. In study A, recombinant GPX4 protein was first incubated with 50 µM 428 at 37°C for 30 min, then 50 µM RSL3 was added, and incubation continued at 37°C for another 30 min. The mixture was then divided into 8-tube strips, heated at different temperatures, centrifuged at high speed, and the supernatant was collected for Western blotting to detect GPX4 protein levels. Study B showed that BEAS-2B cells were treated with different concentrations of RSL3, followed by the addition of 428 at corresponding time points, and CCK8 assays were performed overnight. Study C showed that BEAS-2B cells were treated with different concentrations of FIN56, followed by the addition of 428 at corresponding time points, and CCK8 assays were performed overnight. Figure 9 Different doses of 428 were injected into the tail vein of mice, and changes in mouse body weight were recorded. Detailed Implementation

[0019] This invention provides the use of compound 428 in the preparation of medicaments for the prevention, control, or treatment of idiopathic pulmonary fibrosis, wherein the chemical structural formula of compound 428 is shown in Formula 1: Formula 1.

[0020] Compound 428 of this invention is a small molecule compound with the molecular formula C4. 30 H 37 NO7Se has a molecular weight of 603.17; the preferred preparation method of compound 428 is described in CN 111909162 B; compound 428 is derived from the natural product noscapine, has high safety and strong pharmacological effects, and shows good potential for medicinal use.

[0021] In this invention, the dosage form of the drug preferably includes an injection.

[0022] In this invention, the drug is administered in unit dose form; the drug contains compound 428 which can effectively prevent, control or treat idiopathic pulmonary fibrosis, preferably in the range of 30 to 60 mg / kg of compound 428 per day.

[0023] In this invention, the idiopathic pulmonary fibrosis preferably includes bleomycin-induced pulmonary fibrosis.

[0024] The present invention also provides the application of compound 428 in the preparation of GPX4 stabilizer, wherein the chemical structure of compound 428 is shown in Formula 1.

[0025] In this invention, compound 428 can bind to GPX4 and improve the stability of GPX4, thereby improving the level of cell anti-ferroptosis. This invention opens up new application areas for compound 428.

[0026] In this invention, improving the stability of GPX4 preferably includes improving the thermal stability of GPX4. The fact that 428 improves the thermal stability of GPX4 suggests that 428 may bind to the GPX4 protein, and the two may interact.

[0027] In this invention, compound 428 can also inhibit the covalent binding of RSL3 and GPX4 and / or inhibit ferroptosis.

[0028] The present invention also provides the use of compound 428 in the preparation of products with antioxidant, cardiovascular protection or anti-aging properties, the chemical structure of which is shown in Formula 1.

[0029] In this invention, the product preferably comprises a drug. In this invention, compound 428 is capable of scavenging free radicals in the body.

[0030] Compound 428 of the present invention has the characteristics of easy absorption and good stability, and is suitable for drug development.

[0031] To further illustrate the present invention, the application of compound 428 provided by the present invention in the preparation of medicaments for the prevention, control or treatment of idiopathic pulmonary fibrosis or GPX4 stabilizers is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Experimental Example 1 (a) Laboratory mice: Male C57 / BL6 mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and housed in a specific pathogen-free (SPF) environment at the Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine.

[0033] (II) Reagents: Fetal bovine serum (Hyclone, Logan, UT); RPMI-1640 medium, DMEM medium, DMEM-F12 medium (Sigma-Aldrich, St Louis, MO); dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO); Trizol reagent kit (Invitrogen, Paisley, Scotland, UK); reverse transcription kit (Promega, Madison, WI); PCR kit (TaKara, Dalian, China); cDNA synthesis kit (Applied Biosystems, Foster City, CA); SYBR Green PCR Core reagent (PE Biosystems, Warrington, UK); anti-GPX4, SLC7A11, HRP-mouse anti-human, rabbit anti-human secondary antibodies; anti-β-actin, tubulin, vinculin antibodies (Santa Cruz, Cell Signaling, Abclonal, Proteintech); C11-Bodipy 581 / 591 lipid peroxidation fluorescent probe (Abclonal), GSH / GSSG detection kit (Beyotime), Bleomycin (Sigma); (III) Cells and Cell Culture The HBE cell line used in this invention was cultured in RPMI-1640 medium containing 10% fetal bovine serum; the 293T cell line and the BEAS-2B cell line were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum; and the HFL1 cell line was cultured in DMEM-F12 medium containing 10% fetal bovine serum at 37°C in an incubator containing 5% CO2 to 95% O2.

[0034] (iv) Detection of intracellular lipid peroxides Different human lung cell lines were collected and divided into control group, RSL3 group, and 428+RSL3 group. Cells were treated with RSL3 ferroptosis inhibitor and 428 was added in a concentration gradient (0, 1, 2, 5, 10, 20 μM). Then, 1 µM C11-Bodipy 581 / 591 lipid peroxidation fluorescent probe was added and incubated in a cell culture incubator for 45 min. After digestion with trypsin, the cells were washed twice with PBS and resuspended with an appropriate volume of PBS. The intracellular C11-Bodipy positive cell population was detected by flow cytometry. (v) GSH / GSSG testing Sample preparation: HFL1 cells or BEAS-2B cells were treated with the corresponding concentrations of RSL3 and 428 for 16-24 hours, and the cell pellets were collected. The changes in intracellular GSH levels were detected according to the instructions of the GSH / GSSH detection kit.

[0035] (vi) Western Blot Cells were collected and washed twice with pre-cooled PBS, discarding as much residual PBS as possible. Cells were resuspended in an appropriate volume of PBS, and an equal volume of 2X SDS was added. The cells were boiled in a 99°C metal bath for 10 min, then on ice for 5 min, repeated three times. Samples with the same total protein content from different treatments were subjected to 12% SDS-polyacrylamide gel electrophoresis, and the proteins were then transferred to an NC membrane. The transferred NC membrane was first blocked with 5% skim milk at room temperature for 1 h. Then, the primary antibody was prepared in 5% skim milk and incubated overnight at 4°C. Next, the membrane was incubated with a horseradish peroxidase (HRP)-labeled secondary antibody. Finally, protein expression changes were detected using a chemiluminescence phototope-HRP kit. After membrane removal, the membrane was incubated with internal control antibodies such as β-actin to ensure consistency in the total protein load.

[0036] (vii) RNA extraction and Real-time PCR Extraction of total RNA from cells Total RNA was extracted from cells according to the method provided by the Invitrogen TRIzol kit. The specific experimental steps are as follows: 1) Collect 5~10×10 6 Cells were resuspended in 1 ml TRIzol and repeatedly pipetted to achieve complete lysis; 2) Incubate at room temperature for 5 minutes to allow for complete lysis of the nucleoprotein complex; 3) Add 0.2 ml of chloroform to every 1 mL of TRIzol, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes; 4) Centrifuge at 12000 g for 15 min at 4℃; 5) Carefully transfer the upper aqueous phase to another centrifuge tube, add 0.5 mL of isopropanol to each 1 mL of TRIzol, mix the solution thoroughly and slowly, incubate at room temperature for 10 min to precipitate RNA, and centrifuge at 12000 g at 4℃ for 10 min. 6) Discard the supernatant, add 1 mL of 75% ethanol to every 1 mL of TRIzol, and wash the RNA precipitate. Vortex briefly for a few seconds, then centrifuge at 7500 g at 4℃ for 5 min; dry the RNA precipitate, dissolve it in an appropriate amount of DEPC water, and store it at -80℃ for later use. Real-time PCR Total RNA from cells was reverse transcribed into cDNA using an RT-PCR kit (TaKaRa, Dalian, China). Real-time quantitative PCR was performed on an ABI PRISM 7900 real-time quantitative PCR instrument (Perkin-Elmer, Torrance, CA). The reaction mixture was as follows: 5 μl of 2×SYBR Green PCR Master Mixture (Applied Biosystems, Warrington, UK), 0.05 μl each of forward and reverse primers for β-actin or the target gene product, 1 μl of cDNA, and 3.8 μl of ddH2O. The reaction procedure was as follows: 50℃ for 2 min (UNG incubation), 95℃ for 10 min (hot-start PCR), repeated 40 times with the following cycle: 95℃ for 15 s, 59℃ for 15 s, 72℃ for 15 s, and finally a dissociation curve (or melting curve) was established at 65℃ to 95℃. The primer sequences for β-actin were as follows: β-actin-F: 5'-CACCATTGGCAATGAGCGGTTC-3' (SEQ ID NO.1) and β-actin-R: 5'-AGGTCTTTGCGGATGTCCACGT-3' (SEQ ID NO.2); The primer sequences for GPX4 were as follows: GPX4-F: 5'-GGGACGACTGGCGCTG-3' (SEQ ID NO.3); GPX4-R: 5'-CGCAAACCACACTCAGCGTATC-3' (SEQ ID NO.4). All reactions and assays were performed on MicroAmp optical 384-well plates covered with a transparent sealing film. Triple samples were used for all PCR reactions, and the standard deviation of the reaction experiment error was calculated. All data were analyzed using ABI PRISM SDS 2.0 software. The threshold cycle (Ct) calculated by this instrument's software represents the number of cycles required to reach a certain fluorescence intensity. The "ΔCt method" was applied, with β-actin used to correct for the amount of RNA used in different reactions.

[0037] (viii) Cellular Thermal Shift Assay (CETSA) 1. Cell collection: Collect 10 million cells, wash twice with 1×PBS, aspirate any residual liquid, and store at -80℃.

[0038] 2. Add 500 μl of 1×PBS (with a 1:100 dose of proteasome inhibitor cocktail), freeze and thaw three times in liquid nitrogen, centrifuge at 20,000 g for 20 min at 4°C, discard the precipitate, and transfer the supernatant to another EP tube.

[0039] 3. Divide the supernatant into two equal portions. Add the corresponding concentration of compound to one portion and add an equal volume of DMSO to the other portion. Incubate at 37°C for 30 min to 1 h.

[0040] 4. Aliquot 50 μL of the incubated mixture into 8-tube PCR apparatus, set an appropriate temperature gradient, heat in PCR instrument for 3 min, then at room temperature for 3 min, and maintain at 4℃.

[0041] 5. Centrifuge the heated sample at 12,000 rpm and 4°C for 20 min. Collect the supernatant from each tube, add the same volume of 2×SDS to lyse the protein, heat at 98°C for 10 min, and perform Western blotting.

[0042] (ix) Establishment of a pulmonary fibrosis model On day 0, after anesthetizing the mice, each mouse was injected intrabronchially with 1.75 mg / kg Bleomycin (dissolved in PBS, total volume 50 µL). Administration method of 428: Dissolution and preparation of 428: The purity of 428 used in this experiment was measured to be 99%, the solvent was DMSO, and the storage concentration was 100 mg / mL. The 428 solution formulation was 10% DMSO + 18% hydroxypropyl-β-cyclodextrin + 72% H2O.

[0043] Starting from day 1 after model establishment, each mouse in the experimental group was administered 50 mg / kg of 428 (100 μl volume) daily via tail vein injection for 5 consecutive days, followed by a 2-day break, and this treatment was repeated for two cycles. Each mouse in the control group was administered 100 µL LDMSO / hydroxypropyl-β-cyclodextrin (the ratio was the same as in the treatment group) via tail vein injection. Each group consisted of 15 mice. The survival time of the mice was observed and recorded. (x) Statistical Analysis Differences between the two groups were analyzed using Student's t-test. Homogeneity of variance was analyzed using one-way ANOVA before performing two-sided paired or unpaired Student's t-tests. A p-value < 0.05 was considered statistically significant. p <0.033, p <0.002, p <0.001; ns, not significant Experimental results 1. 428 inhibited cell death induced by different ferroptosis inducers in vitro.

[0044] This invention screened a series of noscapine derivatives for a small molecule compound, 428, which can inhibit RSL3-induced ferroptosis using a CCK8 cell proliferation assay. The results showed that noscapine derivative 428 (… Figure 1 A) exhibits a significant protective effect against ferroptosis. RSL3 is a classic ferroptosis inducer, its mechanism of action primarily involving direct inhibition of GPX4 protein activity and degradation of GPX4 protein. This invention found that pretreatment with 428 in the human embryonic lung fibroblast (HFL1) cell line dose-dependently inhibited the ferroptosis effect of RSL3. Furthermore, 428 also dose-dependently inhibited FIN56- and Erastin-induced ferroptosis. Importantly, within the effective concentration range, 428 itself did not exhibit significant cytotoxicity to HFL1 cells. Figure 1 B in Figure 1 (C in the middle).

[0045] This invention also examined the protective effect of 428 against ferroptosis in human bronchial epithelial cells (HBE) and human lung epithelial cells (BEAS-2B and 293T). The results showed that 428 could also inhibit the ferroptosis induced by RSL3 and FIN56, and 428 itself was not cytotoxic. Figure 1 D~ Figure 1 (G in the middle).

[0046] 2.428 inhibits ferroptosis and lipid peroxidation.

[0047] Lipid peroxidation is a marker of ferroptosis. This invention used the C11-bodipy 581 / 591 fluorescent probe for lipid peroxidation detection and found that RSL3 treatment significantly upregulated lipid peroxidation levels in HFL1, BEAS-2B, and HBE cells; while 428 dose-dependently downregulated the proportion of C11-bodipy-positive cells in the cells. Figure 2 A~ Figure 2 In addition, this invention also detected changes in intracellular GSH levels in HFL1 cells. RSL3 treatment significantly reduced intracellular GSH levels in HFL1 cells, indicating GSH depletion, while 428 supplementation maintained intracellular GSH homeostasis. Figure 2 In BEAS-2B cells, similar experimental results were obtained (G in the text). Figure 2 (H in the text). All the above results suggest that 428 has a protective effect against ferroptosis, and this effect is dose-dependent.

[0048] 3.428 can inhibit the progression of pulmonary fibrosis in vivo.

[0049] This invention uses a bleomycin (BLM)-induced pulmonary fibrosis mouse model to verify the in vivo effects of 428. The results showed that, compared with the control group, 428 could delay the progression of BLM-induced pulmonary fibrosis, and the survival time of mice in the treatment group was significantly prolonged. Figure 3 A). MDA can reflect the level of cellular lipid peroxidation, and 428 reduces the increase in MDA level in mouse lung tissue induced by BLM ( Figure 3 (B in the text); In addition, HE staining results of mouse lung tissue showed that BLM could cause extensive inflammatory infiltration and alveolar structure destruction in mouse lung tissue, while the 428 group showed significant relief; furthermore, Masson staining results of mouse lung tissue showed that 428 could significantly reduce BLM-induced pulmonary fibrosis. Figure 3 C in Figure 3 D in the middle 4.428 can upregulate the key protein GPX4, which is responsible for ferroptosis.

[0050] GPX4 is a key protein affecting ferroptosis. This invention found that 428 can upregulate GPX4 protein levels in different cells in a dose- and time-dependent manner without affecting its transcriptional level. Figure 4 More importantly, in animal experiments with BLM-induced pulmonary fibrosis, this invention confirmed that 428 can also increase GPX4 protein levels in vivo. Figure 5 ) 5. 428 inhibits the degradation of GPX4.

[0051] 428 can upregulate the protein level of GPX4 without affecting its transcription, suggesting that 428 may affect the translation or stability of GPX4. To clarify whether 428 can affect the stability of GPX4, this invention conducted an actinomycin (CHX) experiment. Western blotting results showed that 428 can prolong the half-life of the GPX4 protein ( Figure 6 The information suggests that 428 can stabilize the GPX4 protein.

[0052] 6.428 can bind GPX4 inside cells.

[0053] To investigate whether there is an interaction between 428 and GPX4, this invention conducted a CETSA (Cellular Thermal Shift Assay), which revealed that 428 can improve the thermal stability of the GPX4 protein. Figure 7 A in Figure 7 (B in the text), and it is dose-dependent ( Figure 7 C in Figure 7 (D in the text). The above results suggest that 428 may bind to the GPX4 protein.

[0054] 7.428 may compete with RSL3 for GPX4.

[0055] This invention purified human recombinant GPX4 protein for CETSA experiments, and the results were consistent with those of cellular-level CETSA experiments. Figure 7 428 can improve the thermal stability of GPX4. Consistent with literature reports, RSL3 can covalently bind to GPX4. This invention found that 428 can inhibit the covalent binding of RSL3 and GPX4, which means that 428 and RSL3 may compete for binding to GPX4. Figure 8 (A) Therefore, this invention further investigated at the cellular level, first treating BEAS-2B cells with different concentrations of RSL3 or FIN56 (indirectly degrading GPX4), and then adding 428 at different time points. CCK8 assay results showed that 428 could time-dependently inhibit RSL3-induced cell death (…). Figure 8 (B in the original text), but the ferroptosis protection effect on FIN56 is not time-dependent. Figure 8 (C in the middle).

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of compound 428 in the preparation of medicaments for the prevention, control, or treatment of idiopathic pulmonary fibrosis, wherein the chemical structural formula of compound 428 is shown in Formula 1: Formula 1.

2. Use according to claim 1, characterized in that, The drug is in the form of an injection.

3. Use according to claim 1 or 2, characterized in that, The drug is administered in unit doses; the drug contains compound 428 which is effective in preventing, controlling or treating idiopathic pulmonary fibrosis, in the range of 30 to 60 mg / kg of compound 428 per day.

4. Use according to claim 1, characterized in that, The idiopathic pulmonary fibrosis mentioned is bleomycin-induced idiopathic pulmonary fibrosis.

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