Use of isowildtangshenoside in preparation of drugs for treating pulmonary fibrosis

Abstract

The application discloses application of isowuchukuerin in preparation of a drug for treating pulmonary fibrosis. Due to different treatment mechanisms, the isowuchukuerin has a better treatment effect on pulmonary fibrosis than wuchukuerin, and provides a safer and more effective selection for clinical treatment of pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of isanoside in the preparation of drugs for the treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is an end-stage manifestation of interstitial lung disease characterized by fibroblast proliferation and extensive extracellular matrix deposition, leading to the destruction of lung tissue structure. Because pulmonary fibrosis is a chronic disease, its effects can last for a long period. A variety of factors can trigger pulmonary fibrosis, including occupational or home environment factors, medications, radiation therapy, high-concentration oxygen therapy, smoking, disease-related factors, and genetic factors.

[0003] One common pulmonary fibrosis disease is idiopathic pulmonary fibrosis (IPF), a chronic, progressive pulmonary fibrosis that typically leads to death within 3-5 years of diagnosis. IPF is generally characterized by epithelial damage and aggressive fibroblast differentiation. The differentiation of fibroblasts into myofibroblasts plays a crucial role in the fibrotic process and shapes the histological features of IPF lung tissue. Although treatment with antifibrotic drugs such as pirfenidone and / or nintedanib can slow the decline in lung function and disease progression to some extent, it is difficult to reverse existing pulmonary fibrosis, and long-term treatment still has limitations in terms of efficacy and tolerability.

[0004] Type II alveolar epithelial cells are crucial for maintaining alveolar homeostasis and repairing lung injury. Persistent damage, abnormal aging, and decreased proliferation and repair capacity of type II alveolar epithelial cells can lead to impaired alveolar epithelial repair and further promote fibroblast activation and extracellular matrix deposition. Therefore, improving type II alveolar epithelial cell damage, mitigating abnormal aging, and restoring their repair function are important potential strategies for the treatment of pulmonary fibrosis.

[0005] Therefore, there is an urgent need to develop a safe and effective treatment for pulmonary fibrosis. Summary of the Invention

[0006] Given the limitations of existing pulmonary fibrosis treatments in terms of efficacy, the inventors have discovered that isorhoifolin possesses significant anti-pulmonary fibrosis effects, exhibiting superior therapeutic efficacy compared to rhoifolin under the experimental conditions described in this invention. Further research indicates that the anti-pulmonary fibrosis effect of isorhoifolin is related to the inhibition of abnormal activation of the alveolar type II epithelial cell (ATM) and the downstream p53 / p21 signaling pathway.

[0007] Furthermore, the molecular formula of the isostatin is C 27 H30 O 14 It has the structure shown in the following formula:

[0008]

[0009] Therefore, this invention provides the application of isanosine in the preparation of pulmonary fibrosis treatment drugs.

[0010] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0011] Preferably, the drug is administered via local lung administration, and more preferably via nebulized inhalation or intratracheal administration.

[0012] Furthermore, through in vitro studies on mouse alveolar type II epithelioid cell line (MLE-12 cells), this invention found that isanoside can alleviate alveolar epithelial cell damage and abnormal aging, improve cell viability, migration and repair capabilities, and inhibit abnormal activation of the p53 signaling pathway and abnormal expression of p21.

[0013] Furthermore, transcriptomic analysis revealed that isonoside treatment significantly altered p53 signaling pathway activity. Further molecular biology experiments showed that isonoside could inhibit aberrant p53 activation and reduce aberrant p21 expression. Additionally, isonoside inhibited aberrant activation of ATM (Ataxia-telangiectasia mutated) protein, accompanied by decreased downstream p53 signaling pathway activity and p21 expression. Molecular docking results showed that isonoside could form a stable predicted binding conformation with the inhibitory binding region of ATM protein, suggesting that ATM may be involved in the regulation of the p53 / p21 signaling pathway by isonoside.

[0014] Furthermore, this invention demonstrates through animal experiments that isanoside can reduce the abnormal activation of ATM and p53 and the abnormal expression of p21 in alveolar type II epithelial cells in pulmonary fibrosis lung tissue, while increasing the proportion of proliferating alveolar type II epithelial cells and improving the alveolar epithelial repair status.

[0015] Furthermore, this invention has found that isonostatin can alleviate abnormal senescence of type II alveolar epithelial cells and / or improve the proliferative capacity of type II alveolar epithelial cells, and further improve the damage and repair status of alveolar epithelium. Therefore, preferably, the drug is used to alleviate abnormal senescence of type II alveolar epithelial cells and / or improve the proliferative capacity of type II alveolar epithelial cells.

[0016] In some embodiments, the pulmonary fibrosis is accompanied by abnormal senescence and / or decreased proliferative capacity of type II alveolar epithelial cells. In some embodiments, the drug alleviates abnormal senescence of type II alveolar epithelial cells by inhibiting abnormal activation of the p53 signaling pathway and / or reducing abnormal expression of p21 in type II alveolar epithelial cells.

[0017] In some embodiments, the drug inhibits the abnormal activation of the p53 signaling pathway and / or reduces the abnormal expression of p21 by inhibiting the abnormal activation of ATM protein in type II alveolar epithelial cells.

[0018] In some embodiments, the drug is used to improve alveolar epithelial damage and / or promote alveolar epithelial repair; wherein, alveolar epithelial repair includes promoting the proliferation of type II alveolar epithelial cells.

[0019] This invention reveals that isanolyl glycoside is significantly more effective than rutin in treating pulmonary fibrosis, exhibiting significant efficacy at lower doses (while rutin did not show comparable efficacy under the same experimental conditions). This result demonstrates an unexpected difference in pharmacological activity. Further mechanistic studies show that the anti-pulmonary fibrosis effect of isanolyl glycoside is related to the inhibition of the ATM-p53 / p21 signaling axis.

[0020] Previous studies have shown that the anti-pulmonary fibrosis effect of isouracil can involve the TGF-β / Smad signaling pathway, the Nrf2 / HO-1 antioxidant pathway, and epithelial-mesenchymal transition (EMT). In contrast, this study found that isouracil can significantly inhibit abnormal ATM activation in type II alveolar epithelial cells and further inhibit the p53 / p21 signaling pathway, alleviating abnormal senescence of type II alveolar epithelial cells, improving their proliferative capacity, and enhancing alveolar epithelial repair. Therefore, although isouracil and rhubarb have highly similar chemical structures, they exhibit significantly different anti-pulmonary fibrosis pharmacological activities under the experimental conditions of this invention, and isouracil shows superior therapeutic effects compared to rhubarb. This result provides new pharmacological evidence for the application of isouracil in the treatment of pulmonary fibrosis and offers a new candidate drug for the treatment of pulmonary fibrosis.

[0021] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the grouping and administration process for animal experiments. On day 0, C57BL / 6 mice were given bleomycin or saline via airway infusion. On day 14, they were given Rhoifolin, Isorhoifolin (low / medium / high dose) or corresponding control treatments. They were sacrificed on day 28 for sample collection.

[0023] Figure 2 The change in body weight of each group of mice during the experiment is expressed as a percentage of body weight at each time point relative to body weight on day 0.

[0024] Figure 3 The lung function parameter FEV1 of mice in each group was measured using a small animal lung function testing system. 0.1 result;

[0025] Figure 4 The results of the lung function parameter FVC of each group of mice were measured using a small animal lung function testing system.

[0026] Figure 5 The results of the lung function parameter PEF of each group of mice were measured using a small animal lung function testing system.

[0027] Figure 6 The results of lung function parameters (RV) of mice in each group were measured using a small animal lung function testing system.

[0028] Figure 7 This is a quantitative analysis of the hydroxyproline content in the lung tissue of mice in each group;

[0029] Figure 8 These are representative images of paraffin-embedded lung tissue sections stained with hematoxylin and eosin (HE) from each experimental group. The experimental groups include the normal control group, the Isorhoifolin monotherapy group, the BLM group, the BLM combined with Rhoifolin group, and the BLM combined with low, medium, and high dose Isorhoifolin groups. The scale bar is 1 mm.

[0030] Figure 9 The Ashcroft score was determined for each group of lung tissue based on HE staining results.

[0031] Figure 10 These are representative images of Masson's trichrome staining of lung tissue from each group of mice. The blue areas represent collagen deposition. Scale bar: 100 µm.

[0032] Figure 11 This is a quantitative analysis of the area of ​​collagen-positive tissue stained with Masson staining in the lung tissue of mice in each group, expressed as the proportion of collagen-positive area to the total area;

[0033] Figure 12 Immunofluorescence staining was performed on lung tissue sections from each group of mice. Cell nuclei were labeled with DAPI in blue, E-cadherin in red, and α-SMA in green. The top row shows low-power fields of view, and the magnified images of the areas indicated by the dashed boxes are shown in the bottom row. The scale bars are 100 μm and 25 μm, respectively.

[0034] Figure 13This is a quantitative analysis of the E-cadherin-positive area in the lung tissue of mice in each group;

[0035] Figure 14 This is a quantitative analysis of the α-SMA-positive area in the lung tissue of mice in each group;

[0036] Figure 15 Immunofluorescence staining was performed on lung tissue sections from each group of mice. Cell nuclei were labeled with DAPI in blue, RAGE in red, Ki67 in green, and SFTPC in white. The dashed box area shows a magnified local image with scale bars of 100 μm and 10 μm, respectively.

[0037] Figure 16 This is a quantitative analysis of the RAGE-positive area in the lung tissue of each group;

[0038] Figure 17 This is a quantitative analysis of the proportion of Ki67⁺SFTPC⁺ double-positive cells to SFTPC⁺ cells in lung tissue of each group;

[0039] Figure 18 The method employed CCK-8 assay to quantitatively analyze the cell viability of MLE-12 cells treated with different concentrations of BLM (1, 3, 5, 10, and 30 μg / mL).

[0040] Figure 19 The method employed CCK-8 assay to quantitatively analyze the cell viability of MLE-12 cells after treatment with different concentrations of Isorhoifolin (0.3, 1, 3, 10 and 30 μM) alone.

[0041] Figure 20 This study involved quantitative analysis of cell viability after MLE-12 cells were induced with 1 μg / mL BLM and treated with different concentrations of Isorhoifolin (0.3, 1, 3, 10, and 30 μM).

[0042] Figure 21 The proportion of TUNEL-positive cells in each group of MLE-12 cells was detected by TUNEL staining combined with flow cytometry.

[0043] Figure 22 This is a quantitative analysis of the proportion of TUNEL-positive cells in each group;

[0044] Figure 23 The cell scratch assay was used to observe the scratch closure of MLE-12 cells in each group at 0 h, 24 h and 48 h.

[0045] Figure 24 This is a quantitative analysis of the scratch closure rate of each group of cells at 24 h and 48 h;

[0046] Figure 25 SA-β-gal staining was used to detect the senescence status of MLE-12 cells in each group. The scale bar is 25 μm.

[0047] Figure 26 This is a quantitative analysis of the proportion of SA-β-gal positive cells in each group;

[0048] Figure 27 The relative mRNA expression levels of Col1a1 (A), Cdh1 (B), and Acta2 (C) in MLE-12 cells of each group were detected by real-time quantitative PCR.

[0049] Figure 28 Western blot was used to detect the expression of Collagen I, E-cadherin and α-SMA proteins in cells of each group, with GAPDH as an internal control.

[0050] Figure 29 This is a gray-scale quantitative analysis of the relative expression levels of Collagen I (A), E-cadherin (B), and α-SMA (C) proteins;

[0051] Figure 30 Principal component analysis of MLE-12 cell transcriptome data from the BLM group and the BLM combined with Isorhoifolin treatment group;

[0052] Figure 31 PROGENY analysis compared the activity of p53, PI3K, JAK-STAT, NF-κB, Hypoxia and TRAIL signaling pathways in the two groups of cells;

[0053] Figure 32 It is based on pathway enrichment analysis of differentially expressed genes using the WikiPathways database;

[0054] Figure 33 It is a GSEA enrichment analysis of the p53 signaling pathway;

[0055] Figure 34 This is a volcano diagram of differentially expressed genes between the two groups;

[0056] Figure 35 The relative mRNA expression levels of Trp53 (A) and Cdkn1a (B) in MLE-12 cells of each group were detected by real-time quantitative PCR.

[0057] Figure 36 Western blot was used to detect the expression of p-p53, p53 and p21 proteins in cells of each group, with GAPDH as an internal control.

[0058] Figure 37 This is a gray-scale quantitative analysis of the relative protein expression levels of p-p53 / p53 ratio (A) and p21 (B);

[0059] Figure 38 This is a quantitative analysis of the proportion of p-p53⁺SFTPC⁺ double-positive cells in each group;

[0060] Figure 39 This is a quantitative analysis of the proportion of p21⁺SFTPC⁺ double-positive cells to SFTPC⁺ cells in lung tissue of each group;

[0061] Figure 40 This describes the overall molecular docking conformation of Isorhoifolin with ATM and its local binding mode at the ATM inhibitor binding site.

[0062] Figure 41 The relative mRNA expression level of Atm in MLE-12 cells of each group was detected by real-time quantitative PCR.

[0063] Figure 42 Western blot was used to detect the expression of p-ATM and ATM proteins in MLE-12 cells of each group, with GAPDH as an internal control.

[0064] Figure 43 It is a quantitative analysis of the p-ATM / ATM ratio in grayscale;

[0065] Figure 44 This is a quantitative analysis of the proportion of p-ATM⁺SFTPC⁺ double-positive cells to ATM⁺SFTPC⁺ double-positive cells in each group;

[0066] Statistical notation: ns, no statistically significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are expressed as mean ± standard deviation (mean ± SD). Detailed Implementation

[0067] Example 1: Effects of bleomycin on the overall phenotype and lung function of pulmonary fibrosis in mice.

[0068] like Figure 1As shown, this invention uses C57BL / 6 mice. A pulmonary fibrosis model was established on day 0 of the experiment using bleomycin (BLM) via airway infusion. The model included a normal control group, an Isorhoifolin monotherapy group, a BLM group, a BLM combined with Rhoifolin group, and BLM combined with low, medium, and high doses of Isorhoifolin. The dose of Isorhoifolin monotherapy was 1.4 mg / kg, the dose of Rhoifolin in the BLM combined with Rhoifolin group was 0.14 mg / kg, and the doses in the low, medium, and high dose groups of BLM combined with Isorhoifolin were 0.14 mg / kg, 0.7 mg / kg, and 1.4 mg / kg, respectively. On day 14 after modeling, mice were administered the corresponding drugs or saline via airway infusion as a control. Mice were sacrificed on day 28 for tissue sampling.

[0069] Experimental results showed that, compared with the normal control group, BLM treatment led to a significant decrease in the body weight of mice. Figure 2 Forced expiratory volume in 0.1 seconds (FEV1) 0.1 Forced vital capacity (FVC), peak expiratory flow (PEF), and residual volume (RV) all decreased, while the hydroxyproline content in lung tissue significantly increased. Figure 3-7 Rhoifolin intervention failed to produce a stable improvement in the above indicators. After administration of low, medium, and high doses of Isorhoifolin, the trend of weight loss in mice was alleviated to varying degrees, and FEV1 decreased. 0.1 FVC, PEF, and RV were all significantly elevated and approached normal levels, while the hydroxyproline content in lung tissue was significantly decreased. Figure 3-7 ).

[0070] Furthermore, the body weight, lung function, and hydroxyproline levels of mice in the Isorhoifolin monotherapy group were similar to those in the normal control group, with no significant lung tissue damage or abnormalities in related indicators. These results indicate that Isorhoifolin can effectively alleviate bleomycin-induced overall physiological damage in mice, improve lung ventilation, and inhibit collagen deposition in lung tissue, demonstrating a potential role in improving and treating pulmonary fibrosis. Moreover, under the same low-dose conditions, it exhibits a more significant anti-pulmonary fibrosis effect than Rhoifolin.

[0071] Example 2: Effects on improving the pathological structure and degree of fibrosis in lung tissue

[0072] like Figure 8 As shown, the pathological changes in the lung tissue of mice in each group were observed by hematoxylin and eosin (HE) staining.

[0073] The results showed that the lung tissue of mice in the normal control group was intact, with open alveolar cavities and thinner alveolar septa. The lung tissue morphology of the Isorhoifolin-only group was consistent with the normal control group, with no obvious tissue damage. The BLM group showed extensive consolidation in the lung tissue, significant alveolar structural damage or collapse, thickened alveolar septa, and obvious inflammatory cell aggregation. Compared with the BLM group, the lung tissue still showed significant structural damage and interstitial thickening after Rhoifolin intervention. After low, medium, and high doses of Isorhoifolin intervention, the extent of lung consolidation was significantly reduced, alveolar structure was restored to varying degrees, and the degree of alveolar septal thickening and tissue structural disorder was alleviated.

[0074] Ashcroft scoring analysis was performed based on HE staining results to quantitatively assess the severity of pulmonary fibrosis. The results are as follows: Figure 9 As shown in the figure. Compared with the BLM group, there was no statistically significant difference in Ashcroft scores in the Rhoifolin group, while Ashcroft scores were significantly lower in the low, medium, and high dose Isorhoifolin groups.

[0075] The above results indicate that Isorhoifolin can effectively alleviate bleomycin-induced alveolar structural damage, interstitial thickening, and pulmonary fibrosis under different dosage conditions.

[0076] Example 3: Inhibition of collagen deposition in mouse lung tissue

[0077] like Figure 10 As shown, Masson's trichrome staining was used to evaluate collagen deposition in the lung tissue of mice in each group. The results showed that the alveolar structure of the normal control group was intact. The lung tissue morphology and collagen distribution in the Isorhoifolin-only group were basically consistent with those of the normal control group. Large areas of blue-stained collagen fiber deposition were observed in the lung tissue of the BLM group, accompanied by alveolar structural damage and significant thickening of the pulmonary interstitium. Rhoifolin intervention did not significantly reduce collagen deposition in the lung tissue. After intervention with low, medium, and high doses of Isorhoifolin, the extent and degree of blue-stained collagen fiber deposition in the lung tissue were significantly reduced, and the thickening of the pulmonary interstitium and alveolar structural disorder were also improved to varying degrees.

[0078] Further quantitative analysis of the collagen-positive area in the Masson staining images yielded the following results: Figure 11As shown, there were no significant differences between the Isorhoifolin monotherapy group and the normal control group, and between the BLM combined with Rhoifolin group and the BLM group. The proportion of collagen-positive area in the low, medium, and high dose Isorhoifolin treatment groups was significantly lower than that in the BLM group.

[0079] The above results indicate that Isorhoifolin inhibits collagen deposition associated with pulmonary fibrosis at different dosages.

[0080] Example 4: Regulatory effects on lung epithelial cell phenotype and myofibroblast activation

[0081] like Figure 12 As shown, immunofluorescence staining was used to detect the expression of epithelial cell marker E-cadherin and myofibroblast activation-related marker α-SMA in the lung tissue of mice in each group.

[0082] The results showed that E-cadherin expression was abundant in the normal control group, while α-SMA was mainly confined to the airways and perivascular area. The expression characteristics of the Isorhoifolin monotherapy group were basically consistent with those of the normal control group. After BLM treatment, the positive signal of E-cadherin in lung tissue was significantly reduced, while the positive signal of α-SMA was significantly enhanced. Compared with the BLM group, after low, medium, and high doses of Isorhoifolin intervention, the positive signal of E-cadherin in lung tissue was significantly restored, while the positive signal of α-SMA was significantly reduced.

[0083] Further quantitative analysis of the positive staining area showed no statistically significant difference between the Isorhoifolin monotherapy group and the normal control group. The E-cadherin positive area in the low, medium, and high dose Isorhoifolin treatment groups was significantly increased compared to the BLM group. Figure 13 The positive area of ​​α-SMA was significantly lower in the group than in the BLM group. Figure 14 ).

[0084] The above results indicate that Isorhoifolin can effectively improve the phenotype of lung epithelial cells and inhibit abnormal activation of myofibroblasts at different dosages, thereby playing a role in alleviating pulmonary fibrosis.

[0085] Example 5: Promoting the repair of alveolar epithelial cells after bleomycin-induced lung injury

[0086] like Figure 15 As shown, immunofluorescence staining was used to detect the expression of type I alveolar epithelial cell marker RAGE, type II alveolar epithelial cell marker SFTPC, and proliferation marker Ki67 in the lung tissue of mice in each group.

[0087] The results showed that the normal control group and the Isorhoifolin-only group had abundant RAGE positive signals and fewer Ki67 and SFTPC co-positive cells. Compared with the BLM group, after low, medium and high doses of Isorhoifolin intervention, RAGE positive signals were significantly restored, and the number of Ki67 and SFTPC co-positive cells increased significantly.

[0088] Further quantitative analysis showed no statistically significant difference between the Isorhoifolin monotherapy group and the normal control group. The RAGE positive area ( ) in the low, medium, and high dose Isorhoifolin treatment groups was significantly different. Figure 16 ) and the proportion of Ki67 and SFTPC double-positive cells to SFTPC positive cells ( Figure 17 Both were significantly increased compared to the BLM group. These results indicate that Isorhoifolin can promote the proliferation of type II alveolar epithelial cells and restore the phenotype of type I alveolar epithelial cells, thereby promoting alveolar epithelial repair.

[0089] Example 6: Improvement of bleomycin-induced MLE-12 cell damage

[0090] like Figure 18-20 As shown, the cell viability of MLE-12 cells treated with different concentrations of BLM and Isorhoifolin was detected using the CCK-8 assay. Figure 18 As shown, with increasing BLM concentration, the viability of MLE-12 cells significantly decreased, with treatment at 1 μg / mL BLM causing a significant decrease in cell viability, indicating that this concentration can effectively establish an MLE-12 cell damage model. Figure 19 As shown, treatment with different concentrations of Isorhoifolin (0.3, 1, 3, 10, and 30 μM) alone did not significantly alter the viability of MLE-12 cells compared to the normal control group, indicating that Isorhoifolin did not exhibit significant cytotoxicity within the aforementioned concentration range. Further treatment involved inducing MLE-12 cell damage with 1 μg / mL BLM, followed by intervention with different concentrations of Isorhoifolin. The results are as follows... Figure 20 As shown, compared with the BLM group, treatment with 0.3 μM Isorhoifolin did not significantly change cell viability, while treatments with 1, 3, 10, and 30 μM Isorhoifolin all significantly improved MLE-12 cell viability. These results indicate that Isorhoifolin can effectively improve BLM-induced MLE-12 cell viability decline and alleviate alveolar epithelial cell damage within a concentration range with no significant cytotoxicity.

[0091] Subsequent in vitro experiments used 1 μg / mL BLM to establish an MLE-12 cell injury model, and 1 μM Sorhoifolin was selected as the intervention concentration to further evaluate its effect on alveolar epithelial cell injury and related phenotypic changes.

[0092] Example 7: Inhibition of bleomycin-induced apoptosis in MLE-12 cells

[0093] like Figure 21 As shown, the apoptosis level of MLE-12 cells in each group was detected by TUNEL staining combined with flow cytometry. The results showed that the proportion of TUNEL-positive cells in the normal control group and the Isorhoifolin-only treatment group was low, at approximately 0.46% and 0.55%, respectively. After BLM treatment, the proportion of TUNEL-positive cells increased significantly to approximately 12.5%, while it decreased to approximately 5.3% after Isorhoifolin intervention.

[0094] Further quantitative analysis, such as Figure 22 As shown, there was no statistically significant difference between the Isorhoifolin-treated group and the normal control group. The proportion of TUNEL-positive cells in the BLM group was significantly higher than that in the normal control group, while the proportion of cells treated with Isorhoifolin was significantly lower than that in the BLM group.

[0095] The above results indicate that Isorhoifolin can significantly reduce BLM-induced apoptosis in MLE-12 cells, thereby exerting a protective effect on alveolar epithelial cells.

[0096] Example 8 Inhibition of Bleomycin-Induced Abnormal Migration of MLE-12 Cells

[0097] like Figure 23 As shown, the migration ability of MLE-12 cells in each group was detected using a cell scratch assay. The results showed that the scratch closure rates were similar in the normal control group and the Isorhoifolin-only treatment group at 24 h and 48 h. After BLM treatment, the scratch closure of MLE-12 cells was significantly accelerated, and cell migration ability was significantly enhanced, exhibiting functional changes consistent with epithelial-mesenchymal transition (EMT). Isorhoifolin intervention significantly inhibited BLM-induced scratch closure.

[0098] Further quantitative analysis, such as Figure 24As shown, there was no statistically significant difference between the Isorhoifolin-only treatment group and the normal control group. The scratch closure rate was significantly increased in the BLM group at both 24 h and 48 h, while the scratch closure rate was significantly decreased in the BLM combined with Isorhoifolin treatment group compared to the BLM group. These results indicate that Isorhoifolin can inhibit the BLM-induced enhancement of MLE-12 cell migration.

[0099] Example 9: Alleviating bleomycin-induced senescence in MLE-12 cells

[0100] like Figure 25 As shown, senescence-associated β-galactosidase (SA-β-gal) staining was used to detect the cellular senescence level in each group. The results showed that no SA-β-gal positive cells were found in the normal control group and the Isorhoifolin-only treatment group. BLM treatment significantly increased the number of SA-β-gal positive cells, while Isorhoifolin intervention significantly reduced the number of SA-β-gal positive cells.

[0101] Further quantitative analysis, such as Figure 26 As shown, the BLM combined with Isorhoifolin treatment significantly reduced the proportion of BLM-induced SA-β-gal positive senescent cells. These results indicate that Isorhoifolin can significantly alleviate BLM-induced senescence in MLE-12 cells.

[0102] Example 10: Maintaining the epithelial phenotype of MLE-12 cells and inhibiting bleomycin-induced profibrotic phenotype acquisition.

[0103] The expression of fibrosis and epithelial phenotype-related molecules in MLE-12 cells of each group was detected by real-time quantitative PCR and Western blot. PCR results ( Figure 27 The results showed that Isorhoifolin intervention could reverse BLM-induced changes in profibrotic gene expression, reduce Col1a1 and Acta2 mRNA expression, and restore Cdh1 mRNA expression. Western blot results ( Figure 28 , 29 Further studies showed that, compared with the BLM group, Isorhoifolin reduced the expression of Collagen I and α-SMA proteins while restoring the expression of E-cadherin protein.

[0104] The above results indicate that Isorhoifolin can alleviate BLM-induced loss of epithelial phenotype in MLE-12 cells, while inhibiting Collagen I expression and the acquisition of α-SMA-related profibrotic phenotypes, thereby maintaining alveolar epithelial cell phenotype stability.

[0105] Example 11 Regulation of the p53 signaling pathway in MLE-12 cells

[0106] like Figure 30 As shown, transcriptome sequencing analysis was performed on MLE-12 cells from the BLM group and the BLM combined with Isorhoifolin treatment group. Principal component analysis showed that the two groups were clearly separated at the transcriptional level, and the intragroup repeats were well clustered, indicating that Isorhoifolin intervention can significantly alter the overall gene expression profile of MLE-12 cells after BLM treatment.

[0107] like Figure 31 The pathway activity analysis shown indicates that, compared with the BLM group, the overall activity of signaling pathways such as p53, PI3K, JAK-STAT, and NF-κB was reduced after Isorhoifolin treatment. Enrichment analysis results are as follows... Figure 32 As shown, the p53 pathway was prominent in the enrichment analysis. GSEA further suggests that the p53 signaling pathway was generally suppressed after Isorhoifolin treatment. Figure 33 Meanwhile, differential gene clustering and volcano plots ( Figure 34 The results showed that the classic downstream target gene Cdkn1a of p53 was significantly downregulated after treatment with Isorhoifolin. These results suggest that Isorhoifolin may alleviate alveolar epithelial cell damage by inhibiting the overactivation of the p53–Cdkn1a / p21 signaling axis induced by BLM.

[0108] Example 12 Inhibition of bleomycin-induced activation of the p53 / p21 signaling axis in MLE-12 cells

[0109] like Figure 35 As shown, real-time quantitative PCR was used to detect changes in the p53 / p21 signaling axis in MLE-12 cells of each group at the mRNA level. The results showed that, compared with the normal control group, the expression of Trp53 and Cdkn1a mRNA was significantly increased after BLM treatment. After Isorhoifolin intervention, the expression of both was significantly decreased.

[0110] Figure 36 and 37Western blot results further showed that BLM treatment significantly increased the p-p53 / p53 ratio and p21 protein expression, while Isorhoifolin intervention significantly reduced these changes. These results indicate that Isorhoifolin can inhibit BLM-induced p53 activation and downstream p21 expression.

[0111] Example 13 Inhibition of bleomycin-induced abnormal p53 activation in type II alveolar epithelial cells

[0112] Immunofluorescence staining was used to detect the expression of p-p53, p53, and the alveolar type II epithelial cell marker SFTPC in the lung tissue of mice in each group. The results showed that Isorhoifolin intervention could significantly reduce the double positive signal of p-p53 and SFTPC in BLM-induced lung tissue.

[0113] Further quantitative analysis results, such as Figure 38 As shown, the proportion of p53⁺SFTPC⁺ double-positive cells in the BLM group was significantly increased, while this proportion was significantly reduced after treatment with different doses of Isorhoifolin. These results indicate that Isorhoifolin can inhibit BLM-induced abnormal p53 activation in type II alveolar epithelial cells in vivo.

[0114] Example 14: Inhibition of bleomycin-induced aberrant p21 expression in type II alveolar epithelial cells

[0115] Immunofluorescence staining was used to detect the expression of p21 and SFTPC, a marker of type II alveolar epithelial cells, in the lung tissue of mice in each group. After BLM treatment, p21 expression in SFTPC-positive type II alveolar epithelial cells was significantly increased, while it was significantly reduced after low, medium, and high doses of Isorhoifolin intervention.

[0116] Further quantitative analysis results, such as Figure 39 As shown, the proportion of p21⁺SFTPC⁺ double-positive cells among SFTPC⁺ cells was significantly increased in the BLM group, while this proportion was significantly reduced after treatment with different doses of Isorhoifolin. These results indicate that Isorhoifolin can inhibit bleomycin-induced abnormal p21 expression in type II alveolar epithelial cells.

[0117] Example 15: Molecular docking suggests that ATM may be a potential target for Isorhoifolin.

[0118] Molecular docking was used to screen potential targets of Isorhoifolin in regulating the p53 signaling pathway. Results showed that Isorhoifolin could form predicted binding conformations with candidate proteins including ATM, ATR, PTGS2, CHEK1, CHEK2, and MDM2. Further comparison of the binding sites and spatial conformations of these candidate proteins revealed that the predicted binding conformation of Isorhoifolin in ATM showed high consistency with the binding regions of classic ATM inhibitors, and it was able to occupy the corresponding binding pockets well. Figure 40 This suggests that ATM may be an important candidate target for Isorhoifolin to regulate the p53 signaling pathway.

[0119] Example 16 Inhibition of bleomycin-induced ATM expression and activation in MLE-12 cells

[0120] like Figures 41-43 As shown, the expression and activation levels of ATM in MLE-12 cells of each group were detected using real-time quantitative PCR and Western blot. Figure 41 As shown, compared with the normal control group, BLM treatment significantly increased Atm mRNA expression, while Isorhoifolin intervention significantly decreased it. Western blot results are shown below. Figure 42 and 43 As shown, compared with the normal control group, the p-ATM / ATM ratio was significantly increased after BLM treatment. After Isorhoifolin intervention, the p-ATM / ATM ratio was significantly lower than that in the BLM group.

[0121] The above results indicate that Isorhoifolin can inhibit BLM-induced increases in Atm transcription levels and aberrant ATM activation, further supporting the possibility that ATM may act as an upstream node in Isorhoifolin's regulation of the p53 signaling pathway.

[0122] Example 17 Inhibition of Bleomycin-Induced Abnormal Activation of ATM in Type II Alveolar Epithelial Cells

[0123] The expression of p-ATM, ATM, and the alveolar type II epithelial cell marker SFTPC in the lung tissues of mice in each group was detected by multiplex immunofluorescence staining. The results showed that after BLM treatment, the double positive signals of p-ATM and SFTPC in the lung tissue were significantly enhanced. After intervention with low, medium, and high doses of Isorhoifolin, the double positive signals were significantly weakened.

[0124] Further quantitative analysis, such as Figure 44As shown, the proportion of p-ATM⁺SFTPC⁺ double-positive cells to ATM⁺SFTPC⁺ double-positive cells was significantly increased in the BLM group, while this proportion was significantly reduced after treatment with different doses of Isorhoifolin. These results indicate that Isorhoifolin can inhibit BLM-induced abnormal ATM activation in type II alveolar epithelial cells in vivo.

[0125] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of isonostatin in the preparation of drugs for the treatment of pulmonary fibrosis, wherein the molecular formula of isonostatin is C 27 H 30 O 14 It has the structure shown in the following formula: 。 2. The application as described in claim 1, characterized in that, The pulmonary fibrosis mentioned is idiopathic pulmonary fibrosis.

3. The application as described in claim 1, characterized in that, The pulmonary fibrosis is accompanied by abnormal aging and / or decreased proliferative capacity of type II alveolar epithelial cells.

4. The application as described in claim 1, characterized in that, The drug is used to reduce abnormal aging of type II alveolar epithelial cells and / or improve the proliferative capacity of type II alveolar epithelial cells.

5. The application as described in claim 4, characterized in that, The drug alleviates abnormal senescence of type II alveolar epithelial cells by inhibiting the abnormal activation of the p53 signaling pathway and / or reducing the abnormal expression of p21 in type II alveolar epithelial cells.

6. The application as described in claim 1, characterized in that, The drug inhibits the abnormal activation of ATM protein in type II alveolar epithelial cells, thereby inhibiting the abnormal activation of the p53 signaling pathway and / or reducing the abnormal expression of p21.

7. The application as described in claim 1, characterized in that, The drug is used to improve alveolar epithelial damage and / or promote alveolar epithelial repair.

8. The application as described in claim 7, characterized in that, The alveolar epithelial repair includes promoting the proliferation of type II alveolar epithelial cells.

9. The application as described in claim 1, characterized in that, The drug is administered locally to the lungs.

10. The application as described in claim 9, characterized in that, The local lung administration method is nebulized inhalation or intratracheal administration.

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