Application of silibinin A in treatment of idiopathic fibrosis diseases

Abstract

The invention discloses application of silybin A in preparation of a medicine for treating idiopathic pulmonary fibrosis, and the technical scheme provided by the invention shows that silybin A inhibits a TGF-beta1 signal transduction pathway by degrading TGF-betaRII, can reduce the collagen content of the lung of an IPF mouse, improve the alveolar structure of the mouse and reduce the levels of HYP, TGF-beta1 and TNF-alpha, and can be used for preparing a medicine for treating idiopathic pulmonary fibrosis. Finally, the anti-fibrosis treatment effect on the IPF mouse is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field, and particularly relates to an application of silybin A in treating idiopathic fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by inflammation and fibrosis of the alveolar wall, leading to gradual loss of lung function. The clinical prognosis for IPF patients is poor, with a median survival of approximately 2 to 5 years. Although organ transplantation and chemotherapy are the current mainstays of treatment, the US Food and Drug Administration (FDA)-approved drugs nintedanib and pirfenidone (PFD) can only partially slow disease progression without significantly improving patient survival. Pirfenidone reduces the synthesis of transforming growth factor-β1 (TGF-β1) by inhibiting overexpression of TGF-β1, suppressing lipid peroxidation, and scavenging reactive oxygen species (ROS), thereby reducing the severity of pulmonary fibrosis.

[0003] However, these drugs are typically administered orally, resulting in low bioavailability and, due to their lack of selectivity, are prone to systemic toxic side effects. Therefore, the development of novel, highly effective drugs and their delivery systems is urgently needed. The pathogenesis of IPF remains incompletely understood, but TGF-β1 is considered a key molecule in promoting the fibrotic process. TGF-β1 promotes fibroblast activation and proliferation, as well as excessive deposition of extracellular matrix (ECM) components such as collagen, laminin, and fibronectin, by activating members of the Smad protein family, particularly Smad2 / 3.

[0004] To more effectively intervene in this pathological process, researchers have begun to focus on therapeutic strategies targeting the TGF-β1 signaling pathway. TGF-β1 signaling relies on the interaction of type I (TGFβRII) and type II (TGFβRII) receptors. TGFβRII, as the initial binding receptor for TGF-β1, is crucial for controlling fibrosis. Heat shock protein 90 (HSP90), a key molecular chaperone, maintains TGFβRII stability by binding to it. Studies have shown that the HSP90 N-terminal inhibitor 17-AAG disrupts the interaction between HSP90 and TGFβRII, leading to its degradation and thus inhibiting TGF-β1-mediated fibrotic responses. However, because HSP90 is also involved in the stabilization of multiple other proteins, comprehensive HSP90 inhibition may lead to a wide range of side effects, including hepatotoxicity.

[0005] Given these challenges, researchers have begun exploring more specific and safe HSP90 inhibition strategies. Silybin, a natural flavonoid extracted from milk thistle (Silybum marianum (L.) Gaertn.), is clinically used to treat liver fibrosis due to its significant hepatoprotective effects. Recent studies have shown that silybin is also a potential HSP90 C-terminal inhibitor, effectively inhibiting HSP90 function without interfering with certain key client proteins, demonstrating the potential for reducing side effects. Silybin consists of two diastereoisomers, silybin A and silybin B, which exhibit distinct pharmacodynamic and pharmacokinetic properties in vivo. Currently, research on the independent roles of these two isomers in the treatment of IPF remains unresolved, providing new avenues for future research.

[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0007] The purpose of the present invention is to provide a use of silibinin A in the treatment of idiopathic fibrosis, so as to overcome various uncertainties arising from the treatment of silibinin mixtures in the prior art.

[0008] To achieve the above object, the present invention discloses an application of silybin A in preparing medicines for specific diseases, wherein the specific disease is idiopathic pulmonary fibrosis.

[0009] Preferably, in the above technical solution, silybin A is obtained by separating the diastereoisomers silybin A and silybin B contained in silybin.

[0010] Preferably, in the above technical solution, silybin is extracted from the Chinese herbal medicine milk thistle.

[0011] A drug for inhibiting idiopathic pulmonary fibrosis, comprising silybin A and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, an isotonic agent, a thickener, a preservative, a solid binder or a lubricant.

[0012] Preferably, in the above technical solution, the drug is silybin A nanosuspension.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The technical solution provided in the present disclosure shows that silibinin A can inhibit the TGF-β1 signaling pathway by degrading TGFβRII, thereby reducing the collagen content in the lungs of IPF mice, improving the alveolar structure of mice, and reducing the levels of HYP, TGF-β1 and TNF-α, ultimately achieving an anti-fibrotic therapeutic effect on IPF mice. The effect of silibinin A is stronger than that of silibinin B. Compared with PFD, silibinin A has higher safety and can avoid liver and kidney toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A) Image of milk thistle. Figure 1 B) Silybin A. Figure 1 C) Molecular formula of silybin B.

[0015] Figure 2A: An IPF mouse model was established in C57BL / 6J mice by intratracheal instillation of BLM. Silibinin A and silibinin B were administered orally 10 days later. Mice were sacrificed 14 days after administration, and lung tissue was analyzed. Figure 2B: Morphological, H&E, and Masson's staining of lung tissue sections from mice in each group 14 days after administration (Scale bar = 100 μm). Figure 2 (C) Lung coefficient (n = 3-6). Figure 2D Ashcroft scoring results of H&E-stained sections (n=9). Figure 2E Quantification of blue collagen in Masson-stained sections of mice in each group (n=9). Figure 2F Hydroxyproline content in mouse lung homogenate (n=3-6). Figure 2G TGF-β1 content in mouse lung homogenate (n=3-6). Figure 2H TNF-α levels in mouse lung homogenates (n = 3-6). Shown are mean ± SD. Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, ###P < 0.001. Note: Oral administration of silybin A is more effective than silybin B in preventing idiopathic pulmonary fibrosis.

[0016] Figure 3 A: Morphology, H&E, and Masson staining of lung tissue sections of mice in each group 14 days after administration (Scale bar = 100 μm). Figure 3 B: Lung coefficient (n = 3-6). Figure 3C Ashcroft scoring results of H&E-stained sections (n=9). Figure 3D Quantification of blue collagen in Masson-stained sections of mice in each group (n=9). Figure 3E TGF-β1 content in mouse lung homogenate (n=3-6). Figure 3F HYP content in mouse lung homogenate (n=3-6). Figure 3G TNF-α content in mouse lung homogenate (n=3~6). Figure 3H TGF-β1 in mouse serum. Figure 3I HYP content in mouse serum (n=3~6). Figure 3J Levels of TGF-β1, HYP, and TNF-α in mouse serum (n = 3-6). Shown are mean ± SD. Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, ###P < 0.001. This suggests that nebulized silibinin A nanosuspension has superior anti-idiopathic pulmonary fibrosis efficacy compared with silibinin B.

[0017] Figure 4A Appearance and TEM characterization of A-Lip, Cm, and Cm-A-Lip. (Scale bar = 100 nm) Figure 4B Particle size distribution of A-Lip, Cm, and Cm-A-Lip. Figure 4C Protein profiles of Cm and Cm-A-Lip. Figure 4D Zeta potential of A-Lip, Cm, and Cm-A-Lip. Figure 4E Colocalization of cell membranes and liposomes after cellular uptake. Figure 4F Particle size changes of A-Lip, Cm, and Cm-A-Lip after storage at 4°C for 14 days. Figure 4G Changes in PDI of A-Lip, Cm, and Cm-A-Lip after storage at 4°C for 14 days. Figure 4H Release profiles of A-Lip and Cm-A-Lip in PBS and SLF.

[0018] Figure 5A shows the uptake of C6-Lip and Cm-C6-Lip by L929 cells after 4-hour treatment. Figure 5B shows the uptake of C6-Lip and Cm-C6-Lip by RAW264.7 cells after 4-hour treatment. Figure 5C shows the mean fluorescence intensity of C6-Lip and Cm-C6-Lip uptake by L929 cells after 4-hour treatment. Figure 5D shows the mean fluorescence intensity of C6-Lip and Cm-C6-Lip uptake by RAW264.7 cells after 4-hour treatment. Figure 5E) shows the uptake of C6-Lip and Cm-C6-Lip by L929 cells as measured by flow cytometry. Figure 5F RAW264.7 uptake of C6-Lip and Cm-C6Lip was measured by flow cytometry. Figure 5G Fluorescence imaging of IPF mice following inhalation and tail vein injection of DiR-loaded Cm-Lip. Figure 5H Fluorescence distribution in isolated organs of IPF mice 12 hours after inhalation and tail vein injection of DiR-loaded Cm-Lip. Figure 5I Fluorescence intensity in the lungs of IPF mice 48 and 72 hours after inhalation of DiR-loaded drugs. Compared to the A-Lip group, the data are shown as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. This demonstrates that cell membrane mixed liposomes target fibroblasts and evade macrophages in vitro.

[0019] Figure 6A: An IPF mouse model was established by intratracheal instillation of BLM in C57BL / 6J mice. Ten days after model establishment, A-Lip and Cm-A-Lip were inhaled via nebulized inhalation. Fourteen days after administration, the mice were sacrificed, and serum and tissue samples were collected for analysis. Figure 6B: Lung tissue morphology, H&E staining, and Masson's staining of sections from mice in each group 14 days after administration (scale bar = 100 μm). Figure 6C: Lung coefficient of mice (n = 4-6). Figure 6D: Ashcroft score of H&E-stained sections from mice (n = 9). Figure 6E: Quantification of blue collagen in Masson-stained sections from mice (n = 9). Figure 6F: Hydroxyproline content in lung homogenates (n = 4-6). Figure 6G: TGF-β1 content in lung homogenates (n = 4-6). Figure 6H: TNF-α content in lung homogenates (n = 4-6). Figure 6 (I): Hydroxyproline content in serum (n = 4-6). Figure 6J: TGF-β1 content in serum (n = 4-6). Figure 6 K Serum TNFα levels (n = 4-6). Figure 6 L H&E staining results of heart, liver, spleen, and kidney sections in each group. Shown are mean ± SD. Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, ###P < 0.001. This demonstrates the anti-IPF effect of Cm-A-Lip in vivo. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0021] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1

[0028] 1. Experimental Materials and Equipment 1.1 Experimental reagents and consumables Sodium carboxymethyl cellulose (Sinopharm Chemical Reagent Co., Ltd., 20221025); Silybin 98% (Shanghai Aladdin Biochemical Technology Co., Ltd., C23081351); Bleomycin Hydrochloride for Injection (Hanhui Pharmaceutical Co., Ltd., 22037911); Normal saline (Hubei Kangsheng Pharmaceutical Co., Ltd., 20240303); Universal Tissue Fixative (Wuhan Saiweier Technology Biological Co., Ltd., CR2302145); Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., 20231123); Mouse Tumor Necrosis Factor α (TNF-α) ELISA Kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400); Mouse Transforming Growth Factor β1 (TGF-β1) ELISA Kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400); Mouse Hydroxyproline (HYP) ELISA Kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400).

[0029] Silybin A / B was prepared as follows: 10 g of silybin and 0.9 g of Novozym were weighed into a 250 mL stoppered Erlenmeyer flask, 50 mL of acetone and 5 mL of vinyl acetate were added, and the mixture was reacted at 37°C for 2 h. After completion of the reaction, the filtrate was filtered, dried, and dissolved in an appropriate amount of mobile phase (dichloromethane:acetone:formic acid = 80:20:1) to prepare the pre-column sample.

[0030] Add an appropriate amount of 200-300 mesh silica gel to the chromatography column. Soak the silica gel in petroleum ether and stir to remove air bubbles. Add one column volume of mobile phase to equilibrate the column, then add quartz sand. When the liquid level reaches the same level as the quartz sand, add the pre-column sample. When the sample level drops to the same level as the quartz sand, add the mobile phase and immediately collect 10 mL of the effluent into a test tube. During column chromatography, spot the sample using a G254 silica gel thin layer plate (with the mobile phase as the developing solvent). After development in a developing cylinder, observe the sample in a darkroom UV analyzer at a wavelength of 254 nm. This serves as the basis for separating acetylated and unacetylated silybin. The sample that flows first from the silica gel column is 23-O-acetyl silybin, while the sample that flows later is unacetylated silybin. Solvents with identical results from all spots are combined and concentrated to dryness by rotary evaporation to obtain the acetylated and unacetylated silybin products.

[0031] After the acetylated silybin was dried, the solid powder was collected and deacetylated. Weigh 1.5 g of 23-O-acetylsilybin and 0.15 g of Novozym 435 into a stoppered Erlenmeyer flask. Add 20 mL of methyl tert-butyl ether and 2 mL of n-butanol. Seal with parafilm and incubate on a shaker at 30°C and 200 rpm for 4 days.

[0032] After the unacetylated and deacetylated silybin was dried, the resulting solid powder was collected and weighed. A 12-fold amount of anhydrous ethanol was added, dissolved in a hot water bath, and concentrated to 2 mL. The solution was cooled at room temperature and recrystallized for 24 hours before filtration. The filtrate was collected and dried to obtain silybin A and B powders (Figure 1).

[0033] 1.2 Experimental instruments and equipment Agilent 1260 infinity (Agilent Technologies, Inc.); ELISA analyzer Infinite F50 (TECAN, Switzerland); upright microscope DM3000 (Leica Microsystems); paraffin slicer RM2235 (Leica); biological tissue embedding machine TB-718D (Taiwei Technology); automatic tissue dehydrator TKD-TSF (Hubei Kangqiang Medical Equipment Co., Ltd.).

[0034] 1.3 Experimental animals Fifty-four C57BL / 6J male mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0035] 2. Experimental Methods 2.1 Experimental grouping and establishment of IPF mouse model 54 C57BL / 6J male mice were adaptively bred for one week. Considering the high mortality rate of the IPF model, the mice were randomly divided into groups after modeling. 48 mice were randomly selected and the IPF mouse model was established by tracheal instillation of bleomycin (BLM) (2 U / kg). Ten days after modeling, the surviving mice were randomly divided into 4 groups, namely the model group, the positive drug group (PFD 200 mg / kg), the silybin A group (50 mg / kg), and the silybin B group (50 mg / kg). The drugs were suspended in 0.5% sodium carboxymethylcellulose and administered orally once a day; the model group was given an equal amount of 0.5% sodium carboxymethylcellulose suspension by gavage for 14 consecutive days ( Figure 2A ).

[0036] 2.2 Sample Collection Fourteen days after administration, mice were sacrificed by cervical dislocation. Lung tissue was collected and washed with normal saline. The mice were weighed and the wet weight of the lung tissue was calculated. The lung coefficient was calculated as (lung wet weight (mg)) / (body weight (g)) × 100%. The left lung of the mice was fixed with 4% paraformaldehyde and embedded in paraffin. The lung tissue was sectioned and stained with H&E and Masson staining. The remaining lung tissue was immediately transferred to liquid nitrogen for storage.

[0037] 2.3 Lung Histomorphometric Analysis and Ashcroft Score The left middle lobe of the lung was immersed in 4% paraformaldehyde fixative for 24 hours, after which the specimens were paraffin-embedded, 4 μm sections were obtained, and dehydrated. The sections were stained with hematoxylin and eosin (H&E) and Masson's stain to examine the extent of fibrosis in the lung specimens. The sections were photographed under a microscope. The degree of pulmonary fibrosis was assessed using the semiquantitative Ashcroft method and collagen volume fraction score. Changes in H&E-stained fibrotic lung specimens were semiquantitatively assessed on a scale of 0 to 8 according to a modified Ashcroft method, using three sections per lung.

[0038] 2.4 Determination of TGF-β1, HYP, and TNF-α Contents in Lung Tissue Lungs were extracted and 40 mg of right lung was homogenized in 360 μL of PBS (pH 7.4). The levels of TGF-β1, HYP, and TNF-α in lung tissue and serum were measured using commercially available ELISA kits. An appropriate amount of lung tissue was weighed and homogenized with PBS (pH 7.4) at a ratio of 1:9. The lung tissue sample was thoroughly homogenized using a homogenizer, centrifuged at 3000 rpm, and the supernatant was collected after 20 minutes. TGF-β1, HYP, and TNF-α levels were then measured using a microplate reader according to the ELISA kit instructions.

[0039] 2.5 Statistical methods All results are expressed as mean ± standard deviation (SD). Analysis of variance was performed using a two-tailed t-test for comparisons between two groups or a nonparametric one-way analysis of variance (ANOVA) for comparisons between three or more groups. A p-value less than 0.05 indicates a statistically significant difference. Compared with the blank group, *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05; compared with the model group, ### indicates p < 0.001, ## indicates p < 0.01, and # indicates p < 0.05.

[0040] 3. Experimental Results 3.1 Oral administration of silybin A improves lung morphology and histopathology in mice with pulmonary fibrosis Lung morphology dissected from mice 14 days after drug administration revealed a normal, rosy color in the control group. However, after bleomycin treatment, the lungs of mice developed significant edema, resulting in a dark red appearance. Comparison of H&E staining between the various groups revealed that the control group displayed typical normal alveolar structure, lacked inflammation, and maintained normal lung structure. The lungs of bleomycin-treated mice exhibited significant alveolar structural destruction, with diffuse, severe alveolar damage and interstitial inflammatory cell infiltration, and a significant increase in the lung index. However, compared with the BLM group, the alveolar structure in the silibinin A and silibinin B groups was significantly improved, with a modest decrease in the lung index. The BLM group exhibited combined fibrous masses, while the silibinin A and silibinin B groups showed primarily single fibrous masses. Inflammatory infiltration and edema were also significantly reduced, and the lung morphology of the silibinin A-treated group was superior to that of the silibinin B-treated group, consistent with the Ashcroft score. Masson staining revealed larger blue areas and fibrous proliferation in the alveolar walls of the BLM group, indicating significant pulmonary fibrosis. The blue collagen area decreased in the silybin A and silybin B treatment groups, indicating a certain alleviation effect on pulmonary fibrosis, and silybin A was more effective than silybin B. According to our research results, oral administration of silybin A and B effectively reduced pulmonary fibrosis, and silybin A was more effective ( Figure 2B -E).

[0041] 3.2 Oral administration of silybin A reduces the levels of TGF-β1, HYP, and TNF-α in lung tissues of mice with pulmonary fibrosis TGF-β1 is a potent profibrotic cytokine that promotes the growth and differentiation of fibroblasts and plays an important role in the fibrotic process. TGF-β1 levels were significantly elevated in lung tissue homogenates of mice in the BLM group. Treatment with silybin A and silybin B significantly decreased TGF-β1, with silybin A having a more pronounced regulatory effect on TGF-β1 (Figure 2G).

[0042] Various studies have shown that excessive collagen deposition is a key characteristic of fibrotic diseases. Hydroxyproline (HYP), an amino acid unique to collagen, is one of the most commonly used methods for assessing the severity of fibrosis. HYP levels were significantly elevated in mice treated with BLM. However, these levels decreased after treatment with silybin A and silybin B, indicating a decrease in collagen content and ameliorative effects on pulmonary fibrosis. Furthermore, silybin A had a greater effect on reducing HYP than silybin B (Figure 2F).

[0043] Pulmonary fibrosis is a lung disease characterized by inflammation leading to persistent alveolar damage and the repetitive destruction, repair, reconstruction, and excessive deposition of the extracellular matrix. Therefore, the level of inflammation is crucial for assessing the severity of pulmonary fibrosis. Following BLM modeling, TNF-α levels were significantly upregulated in the lung tissues of mice. Treatment with silybin A and silybin B downregulated TNF-α levels, with silybin A exhibiting a stronger downregulation effect than silybin B (Figure 2H).

[0044] The present invention determined that oral administration of silibinin A has a therapeutic effect on mice with bleomycin-induced pulmonary fibrosis, and that silibinin A is more effective than silibinin B. Oral administration of silibinin A significantly improved alveolar structure, reduced collagen content, inflammatory infiltration, and edema in mice with pulmonary fibrosis, while also lowering TGF-β1, HYP, and TNF-α levels in the mouse lung tissue.

[0045] Example 2

[0046] 1. Experimental Materials and Equipment 1.1 Experimental reagents and consumables Silybin 98% (Shanghai Aladdin Biochemical Technology Co., Ltd., C23081351); Bleomycin Hydrochloride for Injection (Hanhui Pharmaceutical Co., Ltd., 22037911); Normal saline (Hubei Kangsheng Pharmaceutical Co., Ltd., 20240303); Universal Tissue Fixative (Wuhan Saiweier Technology Biological Co., Ltd., CR2302145); Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., 20231123); Mouse Tumor Necrosis Factor α (TNF-α) ELISA Kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400); Mouse Transforming Growth Factor β1 (TGF-β1) ELISA Kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400); Mouse Hydroxyproline (Hyp) ELISA Kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400).

[0047] The preparation method of Silybin A / B is the same as that of Example 1 1.2 Experimental instruments and equipment Agilent 1260 infinity (Agilent Technologies, Inc.); ELISA analyzer Infinite F50 (TECAN, Switzerland); upright microscope DM3000 (Leica Microsystems); paraffin slicer RM2235 (Leica); biological tissue embedding machine TB-718D (Taiwei Technology); automatic tissue dehydrator TKD-TSF (Hubei Kangqiang Medical Devices Co., Ltd.); APV-2000 laboratory-grade high-pressure homogenizer (Spike Fluid Technology, Germany); medical compressed air nebulizer (Haier Medical Devices Co., Ltd.).

[0048] 1.3 Experimental animals Fifty-four C57BL / 6J male mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0049] 2. Experimental Methods 2.1 Experimental grouping and establishment of IPF mouse model Fifty-four C57BL / 6J male mice were acclimated for one week. Given the high mortality rate of the IPF model, mice were randomly divided into groups after model establishment. Forty-eight mice were randomly selected and bleomycin (BLM) (2 U / kg) was administered via tracheal instillation. Ten days after model establishment, the surviving mice were randomly divided into four groups: the model group, the positive drug group (PFD 200 mg / kg), the silibinin A inhalation group, and the silibinin B inhalation group. Silibinin A and B were suspended in normal saline and homogenized using a high-pressure homogenizer at 1000 bar for 12 cycles to prepare a silibinin A / B nanosuspension. Mice were placed in a sealed nebulizer chamber and aerosolized with the silibinin A / B nanosuspension. The positive drug group received oral administration. The model group received aerosolized normal saline. Dosing was continued for 14 consecutive days, once daily.

[0050] 2.2 Sample Collection Fourteen days after administration, blood was collected from the orbital cavity, centrifuged to obtain serum, and then rapidly transferred to liquid nitrogen for storage. Mice were sacrificed by cervical dislocation, and lung tissue was collected and washed with normal saline. The mice were weighed and the wet weight of the lung tissue was calculated. The lung coefficient was calculated as (wet lung weight (mg)) / (body weight (g)) × 100%. The left lung of the mice was fixed with 4% paraformaldehyde and embedded in paraffin. The lung tissue was sectioned and stained with H&E and Masson's staining. The remaining lung tissue was rapidly transferred to liquid nitrogen for storage.

[0051] The lung tissue morphological analysis and Ashcroft score, the determination of TGF-β1, HYP and TNF-α levels in lung tissue and serum, and the statistical analysis methods were the same as those in Example 1.

[0052] 3. Experimental Results 3.1 Inhalation of silybin A nanosuspension improves lung morphology and histopathology in mice with pulmonary fibrosis Lung morphology of mice dissected 14 days after administration showed a normal rosy hue in the control group. However, after bleomycin induction, the mice developed significant edema, with a dark red appearance. Fourteen days after aerosol inhalation of silibinin A and silibinin B nanosuspensions, pulmonary edema was alleviated, lung morphology showed an improvement trend, and the rosy luster of the lower lung apex was restored, indicating that inhalation of silibinin A / B nanosuspensions can ameliorate fibrotic damage caused by BLM. Comparison of H&E images of sections revealed that the lungs of bleomycin-treated mice exhibited significant alveolar structural destruction, diffuse severe alveolar damage, interstitial inflammatory cell infiltration, and a significant increase in the lung function coefficient. The lung function coefficient did not improve significantly after aerosol inhalation of silibinin A / B, possibly due to the extremely poor water solubility of silibinin. Even after high-pressure homogenization to smaller particles, the low bioavailability of silibinin still caused some irritation to the lungs, leading to pulmonary edema. However, H&E images show that inhalation of silibinin A / B nanosuspension significantly improved alveolar structure, with a significant reduction in inflammatory infiltration and fibrous masses. Silibinin A was more effective than silibinin B in improving lung structure, consistent with the Ashcroft score. Masson staining revealed a significant increase in blue collagen in the lungs of the BLM group, which decreased after treatment, indicating a reduction in fibrosis. Quantification of blue collagen revealed that silibinin A had a superior inhibitory effect on collagen production compared to silibinin B (Figure 3A, B, C, D).

[0053] 3.2 Nebulized silybin A nanosuspension reduces TGF-β1, HYP, and TNF-α levels in lung tissue and serum of mice with pulmonary fibrosis TGF-β1 is a potent profibrotic cytokine that promotes fibroblast growth and differentiation and plays a crucial role in the fibrotic process. TGF-β1 levels were significantly elevated in lung tissue homogenates from mice in the BLM group. TGF-β1 levels decreased significantly after nebulized treatment with silybin A and silybin B, with silybin A having a more pronounced regulatory effect on TGF-β1 (Figure 3E). Serum TGF-β1 levels were consistent with those in lung tissue homogenates (Figure 3H).

[0054] Hydroxyproline (HYP) content is an important indicator of pulmonary fibrosis severity and indicates the collagen content in lung tissue. HYP content in lung tissue homogenates of mice in the BLM group was significantly elevated. However, HYP content decreased after nebulized treatment with silybin A and silybin B, indicating a decrease in collagen content and an alleviating effect on pulmonary fibrosis. Silybin A had a greater HYP-lowering effect than silybin B (Figure 3F). In serum, both nebulized silybin A and silybin B downregulated HYP content, but the downregulation effect of silybin B was slightly stronger than that of silybin A (Figure 3I).

[0055] Tumor necrosis factor (TNF)-α is a key inflammatory factor, and its level represents the degree of inflammation in lung injury. The level of inflammation is also crucial for assessing the severity of pulmonary fibrosis. Following BLM modeling, TNF-α levels were significantly upregulated in mouse lung tissue. Nebulized silybin A and silybin B downregulated TNF-α levels, with silybin A exhibiting a stronger downregulation effect than silybin B (Figure 3G). In serum, both nebulized silybin A and silybin B downregulated TNF-α levels, but silybin B had a slightly stronger downregulation effect than silybin A (Figure 3J).

[0056] The present invention demonstrates that aerosolized inhalation of a silibinin A nanosuspension has a therapeutic effect on mice with bleomycin-induced pulmonary fibrosis, with silibinin A exhibiting superior efficacy compared to silibinin B. Aerosolized silibinin A significantly improves alveolar structure, reduces collagen content, and decreases inflammatory infiltration in mice with pulmonary fibrosis, while also lowering levels of TGF-β1, HYP, and TNF-α in the mouse lung tissue. Although silibinin B is slightly more effective than silibinin A in downregulating HYP and TNF-α in serum, silibinin A is superior to silibinin B in improving lung structure and in lung tissue homogenate across all indicators. Therefore, aerosolized inhalation of a silibinin A nanosuspension demonstrates superior anti-pulmonary fibrosis efficacy compared to silibinin B.

[0057] Example 3

[0058] 1. Experimental Materials and Equipment 1.1 Experimental reagents and consumables Silybin 98% (Shanghai Aladdin Biochemical Technology Co., Ltd., C23081351); Bleomycin Hydrochloride for Injection (Hanhui Pharmaceutical Co., Ltd., 22037911); Normal Saline (Hubei Kangsheng Pharmaceutical Co., Ltd., 20240303); DPPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd., C10722); POPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd., U0812); Cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd., C20894); DMEM (Nanjing Senbeijia Biotechnology Co., Ltd. Technology Co., Ltd., BC20240129); fetal bovine serum (Nanjing Senbeijia Biotechnology Co., Ltd., BC20240524); universal tissue fixative (Wuhan Saiweier Technology Biological Co., Ltd., CR2302145); anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., 20231123); mouse tumor necrosis factor α (TNF-α) ELISA kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400); mouse transforming growth factor β1 (TGF-β1) ELISA kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400); mouse hydroxyproline (HYP) ELISA kit (Nanjing Yifeixue Biotechnology Co., Ltd., 20240400).

[0059] The preparation method of Silybin A / B is the same as that of Example 1 1.2 Experimental instruments and equipment Agilent 1260 Infinity (Agilent Technologies, Inc.); Hitachi-7800 transmission electron microscope (Hitachi, Ltd., Japan); AE001 liposome extruder (Suzhou Nalotech Instrument Co., Ltd.); Zetasizer Nano ZS90 nanoparticle size analyzer (Malvern Instruments, Ltd., UK); SM-1000D ultrasonic cell disruptor (Nanjing Shunma Equipment Co., Ltd.); RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); ELISA analyzer Infinite F50 (TECAN, Switzerland); STELLARIS 8 DIVE laser confocal microscope (Leica Microsystems); DM3000 upright microscope (Leica Microsystems); RM2235 paraffin slicer (Leica); TB-718D biological tissue embedding machine (Taiwei Technology); TKD-TSF automatic tissue dehydrator (Hubei Kangqiang Medical Devices Co., Ltd.); and medical compressed air nebulizer (Haier Medical Devices Co., Ltd.).

[0060] 1.3 Experimental animals Fifty-four C57BL / 6J male mice and 24 ICR mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0061] 2. Experimental Methods 2.1 Preparation and Characterization of L929 Cell Membrane Hybrid Silybin A Liposomes (Cm-A-LiP) 2.1.1 Cell membrane extraction L929 cells in the logarithmic growth phase were obtained, and the cell suspension was collected. After hypotonic lysis and ultrasonic disruption, the supernatant was collected by centrifugation at 1000 x g for 10 min, the supernatant was collected by centrifugation at 3000 x g for 30 min, and the precipitate was collected by centrifugation at 20,000 x g for 70 min. After resuspending in PBS, the cell membrane was washed by centrifugation at 20,000 x g for 70 min. The precipitate was collected and extruded through 800 nm, 400 nm, 200 nm, and 100 nm polycarbonate membranes in sequence to form uniform cell membrane vesicles (Cm).

[0062] 2.1.2 Preparation of Cm-A-Lip hybrid vesicles Silybin A liposomes (A-Lip) were prepared using a thin film dispersion method. Specific proportions of DPPC, POPC, cholesterol, and silybin A were dissolved in anhydrous ethanol and a rotary evaporator was used to form a uniform film on the bottom of an eggplant-shaped flask. An appropriate amount of PBS was then added to uniformly disperse the film in the PBS. Ultrasonication was then performed and the particle size was homogenized using an extruder to form a uniform and stable liposome suspension. The liposomes and extracted cell membranes were mixed in a specific ratio and ultrasonically disrupted. The mixture was then extruded through 800 nm, 400 nm, 200 nm, and 100 nm polycarbonate membranes, sequentially. Both the cell membranes and the liposomes possess a certain degree of fluidity due to their phospholipid bilayer structure. Ultrasonication and high-pressure extrusion forces facilitated fusion of the cell membrane with the liposome membrane.

[0063] 2.1.3 Characterization The particle size, PDI, and zeta potential of A-Lip, Cm, and Cm-A-Lip were measured using a Malvern particle size analyzer, and their morphology was characterized by transmission electron microscopy (TEM). Extraction of cell membrane proteins and their transfer into liposomes were observed by SDS-PAGE gel electrophoresis. Transmission electron microscopy was used to observe the morphology of cell membranes and liposomes. TEM samples were stained with 1% phosphotungstic acid and then dropped onto copper grids. A-Lip and Cm-A-Lip were stored at 4°C, and their particle size, PDI, and zeta potential were measured at 0, 3, 7, and 14 days to assess drug stability. Drug release rates were measured by shaking A-Lip and Cm-A-Lip in 3.5 kD dialysis bags using PBS and simulated alveolar fluid, respectively, at 37°C at 100 rpm / min. Drug concentrations were determined by high-performance liquid chromatography.

[0064] 2.1.4 Indicates successful hybridization Liposomes and cell membranes were stained with DiO and DiD, respectively, and then fused to form biomimetic nanoparticles. L929 cells in the logarithmic growth phase were plated in 12-well plates. After the cells attached, the stained biomimetic nanoparticles were added. After 4 hours, the culture medium was discarded, and the cells were fixed with 4% PFA. The cell nuclei were counterstained with Dapi, and the colocalization of the two fluorescent signals was observed under a confocal laser scanning microscope (CLSM) to indicate successful membrane fusion.

[0065] 2.2 Targeting studies of lung fibroblasts 2.2.1 Cellular uptake C6-Lip and Cm-C6-Lip were prepared using coumarin-6 instead of silybin A. Considering some C6 retention during the extrusion process, the C6 concentration in the formulations was determined, and the final dose concentration was controlled at 300 ng / ml. L929 and RAW264.7 cells in the logarithmic growth phase were plated in 48-well plates. After the cells attached, blank culture medium and drug-containing culture medium containing C6-Lip and Cm-C6-Lip were added, respectively. The cells were cultured in an incubator for 4 hours, then removed and the culture medium discarded. After fixation with 4% PFA, the nuclei were counterstained, and cellular uptake was qualitatively analyzed by fluorescence microscopy. After digestion, the cells were collected and the cell suspension was analyzed quantitatively by flow cytometry to measure fluorescence intensity.

[0066] 2.2.2 Lung tissue distribution of DIR-loaded liposomes in mice with pulmonary fibrosis ICR mice were given a single, high-dose endotracheal instillation of 2 u / kg bleomycin to establish a pulmonary fibrosis mouse model. Ten days later, free DiR, DiR-stained A-Lip and Cm-A-lip were administered via the lungs, and Cm-A-Lip was injected into the tail vein. Excised tissues were collected 48 and 72 hours later to observe the fluorescence intensity and tissue distribution of the drug and the lung retention time.

[0067] 2.3 Experimental grouping and establishment of IPF mouse model Fifty-four C57BL / 6J male mice were acclimated for one week. Given the high mortality rate of the IPF model, mice were randomly divided into groups after model establishment. Forty-eight mice were randomly selected and bleomycin (BLM) (2 U / kg) was administered via tracheal instillation. Ten days after model establishment, the surviving mice were randomly divided into four groups: the model group, the positive drug group (PFD 200 mg / kg), the A-Lip inhalation group, and the Cm-A-Lip inhalation group. Mice were placed in a sealed nebulizer chamber and received aerosolized A-Lip and Cm-A-Lip, respectively. The positive drug group received the drug via gavage. The model group received aerosolized saline. Drug administration was continued once daily for 14 days (Figure 6A).

[0068] 2.4 Sample Collection Fourteen days after administration, blood was collected from the orbital cavity, centrifuged to obtain serum, and then rapidly transferred to liquid nitrogen for storage. Mice were sacrificed by cervical dislocation, and lung tissue was collected and washed with normal saline. The mice were weighed and the wet weight of the lung tissue was calculated. The lung coefficient was calculated as (wet lung weight (mg)) / (body weight (g)) × 100%. The left lung of the mice was fixed with 4% paraformaldehyde and embedded in paraffin. The lung tissue was sectioned and stained with H&E and Masson's staining. The remaining lung tissue was rapidly transferred to liquid nitrogen for storage.

[0069] The lung tissue morphological analysis and Ashcroft score, the determination of TGF-β1, HYP and TNF-α levels in lung tissue and serum, and the statistical analysis methods were the same as those in Example 1.

[0070] 3. Experimental Results 3.1 Successful hybridization of cell membrane with liposomes and improved liposome stability Fibroblasts are considered the primary initiator of fibrosis and the primary target cell of TGF-β1. To enhance the specificity of silybin A for fibroblasts, we designed cell membrane-hybridized silybin A liposomes using L929 cell membranes. As shown in Figure 4A, the prepared A-Lip, Cm, and Cm-A-Lip were transparent with a blue opalescence. Transmission electron microscopy (TEM) revealed that A-Lip was spherical and had a lipid layer, resembling the overall structure of liposomes. After hybridization with Cm, the liposome morphology remained unchanged, confirming the fusion of the two vesicles. The hydrodynamic diameter of A-Lip was measured to be 161.87 ± 0.94 nm, the polydispersity index (PDI) was 0.14 ± 0.01, and the zeta potential was -17.43 ± 0.29 mV. Meanwhile, the hydrodynamic diameter of Cm was 197.73 ± 2.66 nm, the PDI was 0.24 ± 0.04, and the zeta potential was -8.60 ± 0.86 mV; whereas the diameter of Cm-A-Lip was 164.97 ± 2.15 nm. The larger hydrodynamic diameter of Cm-A-Lip may be attributed to the integration of the cell membrane into the liposome membrane, which increases the interaction with water molecules and forms a hydration layer ( Figure 4B and Figure 4D In addition, the PDI value of the mixed liposomes was lower than that of A-Lip and Cm alone, indicating that the liposomes were more uniformly dispersed after mixing.

[0071] Proteins extracted from L929 cells, Cm, and Cm-A-Lip were separated by SDS-PAGE and stained with Coomassie Brilliant Blue to confirm the transfer of proteins to the cell membrane during hybridization. The purified membrane protein profile showed changes compared to the original cell lysate; however, the ratio of membrane protein to pure cell membrane did not change significantly after hybridization, indicating successful transfer of membrane protein ( Figure 4C To further verify the fusion of liposomes with the cell membrane, A-Lip and Cm were stained with DiO and DiD, respectively. After hybridization, the cells took up the hybridized nanoparticles. Laser confocal microscopy showed that the liposomes were co-localized with the cell membrane, confirming that the membrane fusion in Cm-A-Lip was successful and remained intact under cellular endocytosis ( Figure 4E ).

[0072] The particle size of A-Lip gradually increased within 14 days of storage at 4°C, but remained stable within 14 days after hybridization with cell membranes. This may be due to the increased cholesterol content of the cell membrane inserted into the lipid bilayer structure, which led to a decrease in lipid bilayer fluidity. The PDI of A-Lip and Cm-A-Lip remained unchanged over 14 days, while the PDI of the cell membrane alone showed an increasing trend ( Figure 4F and Figure 4G ). Taking into account the high shear force of the subsequent inhalation drug nebulizer, we investigated the changes in encapsulation efficiency before and after nebulization. The encapsulation efficiency of silybin A liposomes was 80.73%, and the encapsulation efficiency after nebulization was 53.06%, indicating that high shear force would cause a certain degree of damage to the liposomes. The cell membrane hybrid liposomes changed from 89.76% to 84.75% after nebulization, indicating that the cell membrane hybrid liposomes are more stable and can deliver more drugs to the lesion site. In vitro release experiments showed that both A-Lip and Cm-A-Lip had a rapid release curve in PBS and a slower release in SLF. However, after the initial rapid release stage, A-Lip tended to release slowly, which may result in the drug not being effectively released at the disease site and failing to reach the effective drug concentration. In comparison, Cm-A-Lip maintained a uniform release rate in SLF for 24 hours, showing excellent release behavior ( Figure 4H ).

[0073] 3.2 Cell membrane hybridized liposomes target fibroblasts in vitro and evade macrophage phagocytosis We speculated that liposomes hybridized with fibroblast membranes may target fibroblasts through a homing effect. The autofluorescent liposoluble drug coumarin 6 (C6) was used instead of silybin A to prepare liposomes (C6-Lip) and cell membrane hybrid liposomes (Cm-C6-Lip). After incubation with Cm-C6-Lip for 4 hours, the fluorescence intensity in fibroblasts was higher than that of C6-Lip, indicating that the cell membrane hybrid liposomes have a specific targeting effect on fibroblasts. In contrast, C6-Lip showed a higher fluorescence intensity than Cm-A-Lip after binding to macrophages, indicating that the hybridization of liposomes with cell membranes reduced the phagocytosis of macrophages. These observations indicate that after mixing with cell membranes, liposomes can achieve fibroblast targeting and evade immune cell clearance, thereby achieving sustained drug circulation in the body. The fluorescence intensity was quantified using flow cytometry, and the results obtained were consistent with those obtained by fluorescence microscopy ( Figure 5A -F).

[0074] 3.3 Nebulized inhalation of cell membrane hybrid liposomes targets the disease site and prolongs retention time The in vivo distribution of cell membrane hybrid liposomes after inhalation was studied using DiR-labeled Cm-Lip. After tracheal aerosol administration, inhaled Cm-C6-Lip showed high fluorescence distribution in the lungs, while Cm-Lip delivered via the tail vein was mainly distributed in the liver, indicating that inhaled administration can directly reach the lesion site and achieve lung-specific targeting ( Figure 5G and Figure 5H Interestingly, analysis of lung fluorescence intensity 48 and 72 hours after inhalation showed that the residence time of liposomes was significantly prolonged after hybridization with fibroblast membranes. The residence time of liposomes was not significantly improved compared with that of free drug ( Figure 5I ), which may be due to the inability of liposomes to penetrate the dense collagen tissue and reach the lesions of pulmonary fibrosis for passive targeting. However, after hybridization with the cell membrane, liposomes can target fibroblasts through proteins and accumulate in the lesions.

[0075] 3.4 Cell membrane hybrid liposomes enhance the anti-pulmonary fibrosis effect of silybin A Ten days after BLM modeling, mice were placed in a sealed chamber and nebulized with A-Lip or Cm-A-Lip daily for 14 consecutive days. The lung deposition rate of the selected nebulizer was approximately 75.23%. PFD was administered orally as a positive control. The mental state of mice in the nebulized group significantly improved, and their activity level increased. After 14 days of treatment, H&E analysis of lung tissue sections revealed significant alveolar damage, severe structural deformation, and increased fibrosis in the BLM + Saline group. In contrast, the BLM + A-Lip and BLM + Cm-A-Lip groups showed improved alveolar structure, reduced inflammatory infiltration, and decreased pulmonary edema. Masson trichrome staining revealed characteristic features of pulmonary fibrosis in the BLM + Saline group, while the BLM + A-Lip and BLM + Cm-A-Lip groups showed reduced blue areas in the alveolar walls, indicating decreased fibrosis. Compared with the control group, the lung coefficient in the BLM + Saline group was significantly increased, indicating severe pulmonary edema after BLM modeling. However, all three treatment groups showed improvement in lung histomorphology, reduction in lung coefficient, and improvement in pulmonary edema. The quantitative results of Ashcroft score and collagen volume fraction were consistent with this ( Figure 6B - Figure 6E ).

[0076] It is worth noting that the BLM + Cm-A-Lip group showed superior performance in all indicators compared with the BLM + A-Lip group. Lung morphology improved more significantly, and the alveolar tissue structure was closer to that of the control group. In addition, the volume fraction of blue collagen was also reduced. These findings suggest that inhaled Cm-A-Lip may alleviate IPF by preserving alveolar epithelial structure and reducing collagen deposition. After Cm-A-Lip treatment, HYP and TGF-β1, key biomarkers of pulmonary fibrosis, were significantly downregulated, while the inflammatory factor TNF-α also showed decreased levels. In addition, the efficacy of Cm-A-Lip treatment was better than that of A-Lip treatment, and the serum results were consistent with the tissue homogenate results ( Figure 6F - Figure 6K All animals were euthanized after the experiment, and H&E sections of the heart, liver, spleen, and kidney of each group were stained and examined. It was observed that there were no obvious organ or tissue lesions in the BLM + A-Lip group and the BLM + Cm-A-Lip group. In contrast, although the positive drug PFD group showed effective anti-pulmonary fibrosis activity, the liver tissue sections showed an increase in cell vacuoles, indicating that oral administration of PFD may produce hepatotoxicity ( Figure 6L ).

[0077] The present invention determines that aerosol inhalation of silibinin A liposomes and cell membrane hybridized silibinin A liposomes has a therapeutic effect on mice with bleomycin-induced pulmonary fibrosis, can increase cell uptake of the drug and has a certain immune escape effect, and the anti-pulmonary fibrosis efficiency of the cell membrane hybridized silibinin A liposomes is better than that of the silibinin A liposomes.

[0078] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A use of silibinin A in preparing medicines for specific diseases, characterized in that: The specific disorder is idiopathic pulmonary fibrosis.

2. The use according to claim 1, characterized in that Silybin A is obtained by separating the diastereoisomers silybin A and silybin B contained in silybin.

3. The use according to claim 2, characterized in that The silybin is extracted from the Chinese herbal medicine milk thistle.

4. A drug for inhibiting idiopathic pulmonary fibrosis, characterized in that: The drug comprises silybin A and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspension aid, a surfactant, an isotonic agent, a thickener, a preservative, a solid binder or a lubricant.

5. The drug for inhibiting idiopathic pulmonary fibrosis according to claim 4, characterized in that: The drug is silybin A nanosuspension.

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