Application of 2-methoxycarbonylferric shimmone in the preparation of anti-pulmonary fibrosis drugs

Abstract

This invention belongs to the field of biomedicine, specifically disclosing the application of β-carbon alkaloid derivative 2-methoxycarbonylferritin in the preparation of anti-pulmonary fibrosis drugs. Through a series of experiments, it has been demonstrated that 2-methoxycarbonylferritin can regulate the TGF-β / SMAD signaling pathway by inhibiting the phosphorylation of SMAD2 / 3, thereby inhibiting the transformation of fibroblasts into myofibroblasts and reducing the production of extracellular matrix proteins such as COL1, thus alleviating pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of a β-carbon alkaloid derivative, 2-methoxycarbonylferricimone, in the preparation of anti-pulmonary fibrosis drugs. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic respiratory disease of unknown etiology. Its development is highly complex, and its pathogenesis remains incompletely understood. Based on human genetic studies and mouse models, the prevailing view is that IPF is caused by chronic alveolar damage and the failure of normal adaptive repair mechanisms. IPF is the most common form of pulmonary fibrosis, and its incidence has been rising in recent years. It is estimated that the annual incidence of IPF is 0.57–4.51 cases per 100,000 people in Asia, 0.33–2.51 cases per 100,000 people in Europe, and 2.4–2.98 cases per 100,000 people in North America. There is no specific treatment for IPF; currently, only pirfenidone (PFD) and nintedanib are approved for clinical use. Pirfenidone is a broad-spectrum anti-fibrotic drug with multiple targets, inhibiting the TGF-β signaling pathway, although its specific mechanism of action is not yet clear. Nintedanib is a multi-target tyrosine kinase inhibitor that can inhibit VEGF, FGF, and PDGFR for anti-fibrotic effects. However, none of these drugs can reverse fibrosis; they can only moderately delay the progression of pulmonary fibrosis, with limited improvement in patient survival and quality of life. Lung transplantation is the only effective treatment for end-stage disease, but clinical lung donors are scarce, and the surgery carries extremely high risks, making it unsuitable for routine application. Therefore, finding effective anti-fibrotic treatments is of great significance.

[0003] TGF-β is considered a major regulator of pulmonary fibrosis. Currently, fibrosis is defined as the abnormal excessive deposition of extracellular matrix (ECM) in specific tissues (also known as scarring). Activation of TGF-β signaling can lead to the transdifferentiation of pulmonary interstitial fibroblasts into myofibroblasts. These myofibroblasts not only exhibit significantly enhanced anti-apoptotic capabilities but also secrete large amounts of ECM, suggesting that TGF-β may be a core regulatory factor in the development and progression of pulmonary fibrosis. Type I collagen (COL1) is the most abundant type of collagen in the human body and a core component of ECM. COL1 not only provides structural support but also participates in cell signaling, tissue repair, and disease progression. During pulmonary fibrosis, continuous activation of TGF-β signaling leads to excessive synthesis and deposition of COL1; COL1 deposition can alter matrix composition and stiffness, promote fibroblast activation, and create a vicious cycle, leading to progressive disease development and ultimately organ failure.

[0004] Canthin-6-one (CO) is a potentially biologically active compound belonging to the flavonoid family. Its main biological effects are concentrated in anti-inflammatory, antioxidant, antitumor, neuroprotective, and antimicrobial properties. However, CO has poor water solubility and low antiproliferative activity, which limits its clinical application. Summary of the Invention

[0005] To solve the above problems, the present invention adopts the following technical solution: Application of iron shammonone in the preparation of anti-pulmonary fibrosis drugs.

[0006] Application of iron shamidone in the preparation of drugs for treating idiopathic pulmonary fibrosis.

[0007] The application manifests itself as follows: Iron shamone inhibits the phosphorylation of SMAD2 / 3, thus regulating the TGF-β / SMAD signaling pathway.

[0008] Iron shampoosone inhibits the transformation of fibroblasts into myofibroblasts.

[0009] Iron shimmone reduces the production of extracellular matrix proteins.

[0010] In some specific implementations, the iron shamone is obtained by inserting a methyl ester at position 2 of Canthin-6-one.

[0011] In some specific implementations, the iron shamone is a solid powder.

[0012] In some specific implementations, the solvent used for the iron shamone is dimethyl sulfoxide.

[0013] In some specific implementations, the molecular weight of the iron shamone is 278.

[0014] In some specific implementations, the iron shamnone is dissolved in dimethyl sulfoxide to form a drug that can be used in in vitro experiments; the drug is dissolved in a mixed solution of polyethylene glycol and physiological saline that can be used in animal experiments.

[0015] The beneficial effects of this invention are: Iron shimead, a β-carbon alkaloid derivative, can inhibit fibrotic cell models in vivo and in vitro, showing significant targeted inhibition of COL1 and alleviating the symptoms of pulmonary fibrosis.

[0016] The mechanism by which β-carbon alkaloid derivative iron shimegone can alleviate pulmonary fibrosis is closely related to the inhibition of the TGF-β / SMAD signaling pathway in fibroblasts. Attached Figure Description

[0017] Figure 1The effect of TGF-β stimulation on the viability of 3T3 cells; Figure 2 The effect of different concentrations of Cant on the viability of 3T3 cells; Figure 3 The effect of cant on TGF-β-induced apoptosis in 3T3 cells; Figure 4 The effect of Cant on the morphology of 3T3 cells; Figure 5 To detect the mRNA of fibrosis markers in 3T3 cells stimulated by TGF-β. Figure 6 To detect the mRNA of TGF-β-induced fibrosis markers in 3T3 cells using Cand; Figure 7 Western blot analysis of TGF-β-stimulated fibrosis markers in 3T3 cells; Figure 8 Western blot analysis of Cant as an indicator of TGF-β-induced fibrosis in 3T3 cells; Figure 9 Western blot analysis of proteins related to the TGF-β-induced SMAD signaling pathway in 3T3 cells using Cand; Figure 10 GO pathway enrichment analysis for control group vs. Cant group; Figure 11 Hallmarker pathway enrichment analysis for control group vs. Cant group; Figure 12 KEGG pathway enrichment analysis for the control group vs. the Cant group; Figure 13 Volcano plot of differentially expressed genes between the control group and the Cant group; Figure 14 Enrichment analysis of the GO pathway in the TGF-β group vs. the TGF-β+Cant group; Figure 15 Hallmarker pathway enrichment analysis for the TGF-β group vs. the TGF-β+Cant group; Figure 16 KEGG pathway enrichment analysis for the TGF-β group vs. the TGF-β+Cant group; Figure 17 Volcano plot of differentially expressed genes in the TGF-β group vs. the TGF-β+Cant group; Figure 18 A volcano map showing the genetic differences between pulmonary fibrosis and normal individuals; Figure 19 KEGG enrichment analysis of differentially expressed genes between pulmonary fibrosis and normal populations; Figure 20GSEA analysis of pulmonary fibrosis and normal populations; Figure 21 This is a single-sample gene enrichment analysis. Detailed Implementation

[0018] The invention will now be described in detail with reference to specific research projects.

[0019] This embodiment constructs an in vivo and in vitro model of pulmonary fibrosis and uses methods such as tissue HE staining, Masson staining, mRNA, and WB to detect the effects of the β-carbon alkaloid derivative 2-methoxycarbonyl iron scutellarin (Cant) on the pulmonary fibrosis model.

[0020] Cant is a solid powder synthesized by the School of Pharmacy, Nantong University. In cell experiments, DMSO (dimethyl sulfoxide) is used as the solvent for dissolution, while an equal dose of DMSO is used as a control in drug experiments. Cant has a molecular weight of 278. It is directly dissolved in DMSO to a concentration of 20 mM. If a small amount of precipitate forms, it is sonicated for approximately 5 minutes to obtain a clear solution. Cells can be treated with concentrations of 10 μM, 20 μM, and 40 μM as described below.

[0021] Experiments showed that excessively high DMSO concentrations may cause toxic reactions in the body. After multiple adjustments, it was finally determined that the drug could be dissolved to 1 mg / ml using 50% PEG300 (polyethylene glycol 300) + 50% physiological saline. The dissolved liquid could be stored at 4℃ for one week. Before use (e.g., gavage), it should be bathed in a 40 to 50℃ water bath for 10 to 20 minutes to promote complete dissolution. The amount of drug was drawn according to the mouse's weight and administered by gavage (generally around 0.1 ml). The dosage for mice was 5 mg / kg, administered once every other day (QOD).

[0022] A mouse pulmonary fibrosis model induced by bleomycin (BLM) was established. Mouse fibroblast 3T3 cells were pretreated with TGF-β (5 ng / ml) to explore the therapeutic effect of Cant on pulmonary fibrosis and its possible molecular mechanism.

[0023] First, the effect of different concentrations of TGF-β on 3T3 cells was tested. Specifically, cells were seeded in 96-well plates, and after stable adhesion, 5 ng / ml or 10 ng / ml of TGF-β was added and incubated for 24 h, 48 h, and 72 h. Cell proliferation was then detected using the CCK-8 assay. The results showed that 5 ng / ml of TGF-β did not significantly affect the proliferation of 3T3 cells, but long-term treatment with high concentrations of TGF-β (10 ng / ml, 72 h) had a certain inhibitory effect on cell proliferation. Figure 1The effect of Cant on 3T3 cell viability was also evaluated. Similarly, cells were treated with different concentrations (0, 20, 40, 80 μM) of Cant for 24, 48, and 72 hours, and cell proliferation was assessed using CCK8 assay. 10-20 μM of Cant significantly inhibited 3T3 cell proliferation. The results indicate that Cant has a significant inhibitory effect on fibroblast growth. Figure 2 ).

[0024] The effect of TGF-β (0, 5 ng / ml) on apoptosis of 3T3 cells was investigated. Additionally, the effect of Cant (10, 20, 40 μM) on apoptosis of 3T3 cells pretreated with TGF-β (5 ng / ml) (for ease of description, TGF-β-activated cells will be referred to as model cells hereafter) was analyzed, with Nint (0.5 μM) used as a positive control. The specific experimental procedure involved pretreating each group of cells with TGF-β for 2 hours, followed by continuous treatment with different drugs for 24 hours. Adherent cells were collected by trypsin digestion, stained with Annexin V-PE / 7-AAD reagent, and the percentage of apoptotic cells was detected by flow cytometry. Finally, the results were analyzed using Flowjo_v10.8.1 software. 5 ng / ml of TGF-β had little effect on apoptosis in model cells, but Cant at 20 μM significantly promoted apoptosis in 3T3 model cells, with the degree of apoptosis similar to that in the Nint (0.5 μM) group, suggesting that Cant can selectively induce apoptosis in 3T3 model cells. Treatment with 40 μM Cant appeared to decrease the apoptosis rate in the 3T3 model cells. This may be due to increased cell death after high-concentration drug treatment and insufficient cell number collected after trypsin digestion. Figure 3 ).

[0025] Apoptosis results indicated that Cant promoted apoptosis in activated fibroblasts, with an effect at 20 μM similar to that of the therapeutic dose of nintedanib (0.5 μM). To further understand the drug's effect on cells, morphological changes in model cells were observed after treatment with different concentrations of the drug for 24 h and 48 h. Microscopic comparison revealed that 3T3 cells showed significant morphological changes at a Cant concentration of 40 μM, with increased cell size and more granules. Figure 4 This is consistent with the apoptosis results obtained by flow cytometry. Based on CCK8, apoptosis, and morphological observations, 20 μM Cant was ultimately selected as the concentration for in vitro cell experiments to more clearly observe changes in cell phenotype and fibrosis indicators under various treatments.

[0026] Fibroblast activation was induced by 5 ng / ml TGF-β. Cells were collected after 24 and 48 hours to extract mRNA. The expression levels of type I collagen (Collagen 1A1, COL1), fibronectin (FN), and α-smooth muscle actin (α-SMA) were detected by RT-PCR. The results showed that these fibrosis markers generally showed a significant upward trend after TGF-β stimulation. In fibroblasts, TGF-β stimulation at 24 hours led to a peak in COL1 transcription, and also increased the expression of FN and α-SMA. FN transcription peaked at 48 hours of stimulation. Figure 5 COL1 and FN are known to be major components of extracellular matrix proteins and important indicators of the severity of pulmonary fibrosis, while increased expression of α-SMA is closely related to the transformation of fibroblasts into myofibroblasts. These results confirm that stimulation with 5 ng / ml TGF-β can successfully induce fibrosis-like changes in cells. Based on the magnitude of the increase in COL1 expression, it can be seen that the response of fibroblasts to TGF-β stimulation is more significant after 24 hours, which will be the main indicator for assessing fibrosis in subsequent experiments.

[0027] After confirming that TGF-β can induce fibrosis-like changes in cells, we further investigated the effects of Cant (0, 20, 40 μM) on fibrosis markers in model cells, using Nint (0.5 μM) as a positive control. Twenty-four hours after drug treatment, mRNA was extracted, and the expression of COL1, FN, and α-SMA was detected by RT-PCR to assess whether Cant could inhibit fibrosis-like transformation in model cells. The results showed that both Nint and Cant treatment improved fibrosis markers in the model cells, especially significantly decreasing COL1 expression. α-SMA also showed a decreasing trend, although its decrease was slightly less than that of COL1. However, the change in FN within 24 hours was not significant, which may be related to the choice of observation time point. Figure 6 ).

[0028] Proteins were extracted 24 hours after TGF-β treatment, and the protein levels of fibrosis-related molecules in cells were observed. In 3T3 cells, COL1 expression was significantly increased; compared with the control group, treatment with 5 ng / ml TGF-β showed a significant difference, and expression further increased at 10 ng / ml. Figure 7 This indicates that the degree of fibrosis in 3T3 cells continuously worsens under TGF-β stimulation. In TGF-β-induced 3T3 model cells, different concentrations of Cant (0, 20, 40 μM) were used for treatment, with 0.5 μM Nint serving as a positive control. The results showed that Cant significantly inhibited the expression levels of COL1 and FN in 3T3 cells, with significant differences observed at a concentration of 20 μM. Figure 8 Therefore, in subsequent experiments, the research group still chose 20 μM Cant treatment for 24 hours as the main observation point.

[0029] Previous studies have confirmed that TGF-β can activate downstream signaling pathways (the classical SMAD pathway) through its receptor, promoting the transformation of fibroblasts in the lungs into myofibroblasts. Activated myofibroblasts secrete large amounts of collagen and other ECM components, leading to excessive accumulation of fibrous tissue and ultimately fibrosis. Therefore, we first used Western blotting to examine the effect of candidium (Cant) on the SMAD signaling pathway. The results showed that in 3T3 model cells, treatment with 20 μM or 40 μM Cant significantly reduced the protein levels of SMAD2 / 3, its activated form p-SMAD2 / 3, and SMAD4; similar effects were observed in the Nint treatment group. This suggests that Cant may inhibit the activity of the SMAD pathway in myofibroblasts, thereby inhibiting the expression of fibrosis-related genes such as COL1. Figure 9 ).

[0030] In vitro experimental results suggest that Cant may alleviate fibrosis by inhibiting the activation of lung fibroblasts and reducing the production of extracellular matrix proteins, primarily COL1. A mouse model of lung fibrosis induced by BLM (forming the BLM model group) was labeled and treated with Cant to observe the drug's effect on lung fibrosis in vivo.

[0031] After CANT treatment, the mRNA and protein expression levels of COL1 in lung tissue decreased.

[0032] Cant may improve the condition of pulmonary fibrosis by inhibiting the TGF-β / SMAD pathway and reducing COL1 expression.

[0033] A key milestone in the progression of pulmonary fibrosis is the transformation of fibroblasts into myofibroblasts. To further explore the molecular mechanism by which cant alleviate pulmonary fibrosis, 3T3 cells were selected as the research subject, and RNA sequencing was used to analyze the effect of cant treatment on the transcriptome of fibroblasts. Cells were divided into four groups: control group (no treatment), TGF-β group (TGF-β 5 ng / ml), cant group (Cant 20 μM), and TGF-β+Cant group (TGF-β 5 ng / ml + Cant 20 μM). 3T3 cells were treated with the aforementioned drugs for 24 hours, and then RNA-seq was performed. Differentially expressed genes between the cant-treated group and the control group were obtained, and GO, Hallmark, and KEGG pathway enrichment analyses were performed on these differentially expressed genes. The results showed that compared with the control group, the TGF-β, SMAD, and p-SMAD signaling pathways were inhibited in the cant group. Differential gene analysis also indicated that after Cant treatment, the expression of genes involved in ECM deposition and fibrosis progression, such as Ccn2, Lum, and Col3a1, was downregulated, while the expression of genes with antioxidant properties or those that inhibit fibroblast activation, such as Hmox1 and Gsta4, was upregulated. Figure 10 , 11 12, 13). Similarly, comparing the transcriptomes of TGF-β-activated 3T3 cells and those treated with Cant, it was found that the TGF-β+Cant group inhibited the TGF-β and SMAD signaling pathways compared to the TGF-β group. Differential gene analysis also revealed that in model cells, after TGF-β+Cant treatment, the expression of genes related to fibroblast activation, such as Postn and Col3a1d, was downregulated, while the expression of genes with antioxidant stress or inhibitory fibroblast activation functions, such as Hmox1 and Gsta4, was upregulated. Figure 14 , 15 16, 17). To further elucidate the relationship among pulmonary fibrosis, TGF-β, and COL1, correlation analysis was performed. Using the GSE199949 dataset (including 8 normal lung tissues and 13 fibrotic lung tissues), 1976 upregulated genes and 682 downregulated genes were identified in patients with pulmonary fibrosis. Figure 18 KEGG pathway analysis revealed that the five most significantly enriched pathways were neuroactive ligand-receptor interaction, cytokine-cytokiner-receptor interaction, cytoskeletonin in muscle cells, PI3K-Akt signaling pathway, and calcium signaling pathway. Figure 19The TGF-β signaling pathway was also among the upregulated enriched pathways. For downregulated genes, the five most significantly enriched KEGG pathways were biosynthesis of amino acids, carbon metabolism, glutathione metabolism, PPAR signaling pathway, and glycine, serine, and threonine metabolism. Furthermore, GSEA enrichment analysis also confirmed that the TGF-β signaling pathway was upregulated in patients with pulmonary fibrosis. Figure 20 The ssGSEA algorithm was used to calculate the TGF-β signaling pathway score for each sample, and the correlation between this score and the gene expression of COL1A1 and COL1A2 was calculated. It was found that the TGF-β signaling pathway score was positively correlated with the gene expression of COL1A1 and COL1A2. Figure 21 Type I collagen consists of a triple helix composed of two α1 chains and one α2 chain. COL1A1 encodes the α1 chain, while COL1A2 encodes the α2 chain, consistent with experimental results, suggesting that the TGF-β signaling pathway may promote COL1 synthesis.

[0034] Specific embodiments may include, but are not limited to: Example 1: Construction of pulmonary fibrosis cells and detection of fibrosis markers In this invention, the fibroblast cell line 3T3 was selected. The method for inducing a lung fibrosis cell model was as follows: induction with 5 ng / ml TGF-β for 24 h. After induction, cells were collected after trypsin digestion and proteins were extracted. The levels of fibrosis-related indicators, mainly including α-SMA, COL1, and FN, were detected by Western blotting and RT-PCR.

[0035] Example 2: Detection of fibrosis markers after Cant treatment of fibrotic cells Mouse fibroblast 3T3 cells were pretreated with TGF-β (5 ng / ml) for 2 hours, followed by the addition of Cant (0, 20, 40 μM), with Nint (0.5 μM) used as a positive control. After 24 hours of drug treatment, cells were collected after trypsin digestion, and proteins were extracted. The levels of fibrosis-related markers, including α-SMA, COL1, and FN, were detected using Western blotting and RT-PCR.

[0036] Example 3: Detection of the effect of TGF-β stimulation on 3T3 cell viability Cells were seeded in 96-well plates and incubated with 5 ng / ml or 10 ng / ml TGF-β for 24 h, 48 h, and 72 h after stable adhesion. Cell proliferation was then detected using CCK-8 reagent.

[0037] Example 4: Detection of the effect of different concentrations of Cant on the activity of TGF-β-induced 3T3 cells Cells were seeded in 96-well plates and, after stable adhesion, were treated with different concentrations (0, 20, 40, 80 μM) of Cant for 24, 48, and 72 hours. Cell proliferation was then detected using CCK-8 reagent.

[0038] Example 5: Detection of TGF-β-induced apoptosis in 3T3 cells using Cant 3T3 cells were pretreated with TGF-β (5 ng / ml) for 2 hours, followed by Cant (10, 20, 40 μM) and Nint (0.5 μM) as a positive control for 24 hours. Adherent cells were collected by trypsin digestion, stained with Annexin V-PE / 7-AAD reagent, and the percentage of apoptotic cells was detected by flow cytometry. The results were then analyzed using Flowjo_v10.8.1 software.

[0039] Example 6: Detection of 3T3 cell morphology using Cant The morphological changes of 3T3 cells induced by TGF-β for 24 h and 48 h were observed and photographed using an inverted microscope. 3T3 cells were treated with Nint (0.5 μM) or Cant (0-80 μM) for 24 h or 48 h, and the changes in cell morphology were dynamically observed and photographed.

[0040] Example 7: Western blot detection of TGF-β-induced SMAD signaling pathway-related proteins in Cant cells. Mouse fibroblast 3T3 cells were pretreated with TGF-β (5 ng / ml) for 2 hours, followed by the addition of Cant (0, 20, 40 μM), with Nint (0.5 μM) as a positive control. After 24 hours of drug treatment, cells were collected after trypsin digestion, and proteins were extracted. Western blotting was used to detect the levels of proteins related to the SMAD signaling pathway, primarily p-SMAD2 / 3 and SMAD4.

[0041] Example 8: RNA extraction, library preparation, and sequencing Total RNA was extracted from 3T3 cell samples treated with different drugs using TRIzol reagents according to the manufacturer's instructions. After RNA extraction, the samples were treated with DNase I to eliminate potential DNA contamination. The A260 / A280 ratio of the extracted RNA was measured using a Nanodrop™ OneC spectrophotometer to assess the purity of the extracted RNA. RNA integrity was verified using a LabChip GXTouch system (Revity). The acceptable RNA concentration was then determined using a Qubit 3.0 fluorometer and the Qubit™ RNA BroadRange Assay kit. mRNA sequencing libraries were prepared using the KC™ mRNA Library Prep Kit according to the kit's instructions. PCR products of 200 to 500 bp fragments were enriched during library preparation. After quantification of the enriched fragments, sequencing was performed on a DNBSEQ-T7 using the PE150 sequencing model.

[0042] Example 9: Data Processing and Software Usage In the data processing and analysis workflow of this study, the research group strictly followed scientific standards and used professional software and tools to systematically process and deeply analyze the raw sequencing data, aiming to fully explore the biological significance behind the data. The research group used FastQ (v.0.23.0) software for data quality control and alignment of the raw sequencing data, with the reference genome being GRCm38.102, to generate gene expression matrices, and used R language (v.4.3.1) for analysis and result presentation.

[0043] Example 10: Differential Gene Expression Analysis The gene expression matrix obtained from sequencing was normalized using edgeR (v.3.42.4) and limma (v.3.56.2) packages, a design matrix was constructed, and a linear model was fitted to obtain differentially expressed genes between groups. The screening thresholds were: |log2FoldChange|>0.25 and P<0.01. A volcano plot was generated using ggpubr (v.0.6.0) for visualization to show the relationship between gene expression changes and statistical significance.

[0044] Example 11 Enrichment Analysis To delve deeper into the potential functions and regulatory mechanisms of differentially expressed genes, we used the clusterProfiler (v.4.8.3) package to perform gene set enrichment analysis (GSEA) on differentially expressed genes based on the Hallmark, KEGG, and GOBP databases. Through in-depth interpretation of the enrichment analysis results, we clarified the enrichment status of differentially expressed genes in important biological processes such as cell proliferation, apoptosis, and metabolism, as well as in key signaling pathways such as PI3K-Akt and MAPK, thereby uncovering gene functions and potential regulatory mechanisms.

[0045] Those skilled in the art will appreciate that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

The application of 1,2-methoxycarbonylferric shimmone in the preparation of anti-pulmonary fibrosis drugs, characterized in that, A methyl ester was inserted at position 2 of ferroshione to form 2-methoxycarbonylferroshione.

2. The application of 2-methoxycarbonylferric shimmone according to claim 1 in the preparation of anti-pulmonary fibrosis drugs, characterized in that, The 2-methoxycarbonyl iron shimmone inhibits the phosphorylation of SMAD2 / 3 and regulates the TGF-β / SMAD signaling pathway.

3. The application of 2-methoxycarbonylferric shimmone according to claim 2 in the preparation of anti-pulmonary fibrosis drugs, characterized in that, The 2-methoxycarbonyl iron shimmone inhibits the transformation of fibroblasts into myofibroblasts.

4. The application of 2-methoxycarbonylferric shimmone according to claim 2 in the preparation of anti-pulmonary fibrosis drugs, characterized in that, The 2-methoxycarbonyl iron shimmone reduces the production of extracellular matrix proteins.

5. The application of 2-methoxycarbonylferric shimmone according to claim 1 in the preparation of anti-pulmonary fibrosis drugs, characterized in that, The 2-methoxycarbonyl iron shimmone is a solid powder.

6. The application of 2-methoxycarbonylferric shimmone according to claim 5 in the preparation of anti-pulmonary fibrosis drugs, characterized in that, The solvent used for the 2-methoxycarbonyl iron shimmone is dimethyl sulfoxide.

7. The use of 2-methoxycarbonylferricyanidone according to claim 6 in the preparation of anti-pulmonary fibrosis drugs, wherein, The molecular weight of the 2-methoxycarbonyl iron shimone is 278.

8. The use of 2-methoxycarbonylferricyanidone according to claim 7 in the preparation of anti-pulmonary fibrosis drugs, wherein, The 2-methoxycarbonyl iron shimmone is dissolved in dimethyl sulfoxide to form a drug, which is dissolved in a mixed solution of polyethylene glycol and physiological saline.

Images