A pharmaceutical composition for treating pulmonary fibrosis and use thereof

Abstract

The application discloses a kind of pharmaceutical composition for treating pulmonary fibrosis and application thereof, belong to biological medicine technical field.The ginkgolide K (GinK) raw material source of the present application is easy, preparation cost is low, with lower toxicity, as natural industrialization small molecule.Small molecule STING inhibitor C176 has the ability to inhibit STING signal and have positive effect on fibrosis endpoint, can clearly target inhibition STING signal.Ginkgolide K (GinK) and small molecule STING inhibitor C176 are combined to use, can improve histological lesions and down-regulate the expression of several fibrosis-related proteins, have obvious synergistic effect on histological improvement, fibrosis-related protein expression inhibition and other endpoints, so as to achieve the synergistic effect that single drug is difficult to achieve, and reduce the potential toxicity and cost problem brought by single drug high dose.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a pharmaceutical composition for treating pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF) is a chronic, progressive disease characterized by abnormal repair following damage to alveolar epithelial and interstitial cells. Clinical manifestations often include progressive dyspnea, chronic cough, and irreversible decline in lung function, which can lead to hypoxia, respiratory failure, and even death in severe cases. Its main pathological features are the overactivation and proliferation of fibroblasts and myofibroblasts, abnormal deposition of the extracellular matrix, especially various types of collagen, and the gradual destruction and remodeling of alveolar structure, ultimately resulting in irreversible thickening of the pulmonary interstitium and ventilation or perfusion imbalance. Clinically, PF often presents with progressive dyspnea, chronic dry cough, and irreversible decline in lung function. Severe cases can lead to hypoxemia, respiratory failure, and systemic complications, significantly negatively impacting patient survival and quality of life. Although extensive research has been conducted on the molecular and cellular mechanisms of PF, current clinically available treatments still fall short of meeting the demand for highly effective, low-toxicity, and widely applicable therapies. Currently available drugs and treatments face several challenges: First, dose-dependent adverse reactions and tolerability issues. To achieve the drug exposure levels required to inhibit fibrosis in vivo, relatively high doses are often necessary, leading to gastrointestinal, liver function, bleeding tendency, or immunosuppression-related adverse reactions, limiting the feasibility of long-term maintenance therapy. Second, cost and accessibility issues. The high cost of preparing some novel biological agents or complex formulations, coupled with stringent cold chain and administration conditions, hinder large-scale clinical promotion and application in resource-limited environments. Third, efficacy fluctuations due to indications and individual differences. The etiology and progression of pulmonary fibrosis are highly heterogeneous, and the efficacy of single drugs varies greatly among different patients and at different stages of the disease, lacking broad-spectrum and stable efficacy. In addition to drugs, other treatment approaches also have their shortcomings. For example, supportive care and long-term oxygen therapy can improve quality of life but cannot change the course of the disease; lung transplantation can cure advanced cases, but due to donor shortages, high surgical risks, postoperative immunosuppressive complications, and high costs, it is only applicable to a small number of patients; and cutting-edge solutions based on cell or gene therapy, although they have potential translational advantages, are limited by the lack of safety, compliance, cost, and long-term efficacy data, making it difficult to promote them on a large scale in the short term.

[0003] Monomeric active ingredients from natural products show promising potential in anti-fibrotic research. Ginkgolide K (GinK), an active small molecule isolated and identified from Ginkgo biloba leaf extract, has demonstrated activity in reducing inflammation and improving lesion phenotypes in previous in vitro and animal studies. Our own and related previous studies have provided evidence that GinK can improve histological lesions and downregulate the expression of several fibrosis-related proteins in a pulmonary fibrosis model. GinK's advantages also include its availability, low preparation cost, and low toxicity demonstrated in this preclinical study, making it a candidate industrially viable natural small molecule.

[0004] In recent years, STING (STimulator of INterferon Genes) signaling has been recognized as an important pathological regulatory node in various injuries, inflammation-related diseases, and fibrosis processes. STING activation has been shown to be associated with the upregulation of pro-inflammatory factors and pro-fibrotic signals. Inhibiting STING can alleviate tissue inflammation and fibrosis in several experimental models, thus making STING a potential therapeutic target. However, current STING treatments have several practical limitations: existing small-molecule inhibitors require further optimization in terms of in vivo pharmacokinetics, tissue selectivity, and toxicity; while blocking STING alone can inhibit pro-inflammatory signaling, it often falls short in promoting tissue repair or in terms of cost and toxicity control. The small-molecule STING inhibitor C176 has been reported to have the ability to inhibit STING signaling and positively influence fibrosis endpoints.

[0005] In drug development, several candidate compounds have shown anti-fibrotic potential in animal models of pulmonary fibrosis. However, overall, their efficacy stability and application prospects remain insufficient. On the one hand, while some candidate drugs can improve certain pathological indicators in animal experiments, their inhibitory effect is limited when administered alone, making it difficult to achieve sustained and significant improvement at the histological level. On the other hand, obtaining observable efficacy often requires increasing the dosage, thereby increasing the risk of toxicity or adverse reactions and limiting further application. Therefore, relying solely on a single-drug strategy often makes it difficult to achieve ideal anti-fibrotic effects while ensuring safety.

[0006] In summary, the technical problem to be solved by the present invention is to enhance the efficacy and reduce the cost through combination therapy without increasing the toxicity burden, which is expected to make up for the limited efficacy of monotherapy and provide more feasible experimental evidence for the treatment of pulmonary fibrosis. Summary of the Invention

[0007] This invention proposes a pharmaceutical composition for treating pulmonary fibrosis, which overcomes the shortcomings of existing technologies in terms of efficacy, safety, reproducibility and feasibility of application, and constructs a systematic solution for pulmonary fibrosis.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: The first object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, the pharmaceutical composition comprising at least a STING inhibitor and an active ingredient containing ginkgolide K.

[0009] Furthermore, the STING inhibitor is C176.

[0010] Furthermore, the active ingredient containing ginkgolide K includes any one or more of ginkgolide K, ginkgolide K stereoisomers, crystal forms of ginkgolide K, pharmaceutically acceptable ginkgolide K salts, and extracts containing ginkgolide K.

[0011] Furthermore, the mass ratio of the STING inhibitor to ginkgolide K is 3-5:1-2, preferably 5:1.

[0012] Furthermore, the pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia.

[0013] A second objective of this invention is to provide the use of a pharmaceutical composition for treating pulmonary fibrosis in the preparation of a medicament for treating pulmonary fibrosis.

[0014] A third objective of this invention is to provide a medicament for treating pulmonary fibrosis, the medicament comprising at least a pharmaceutical composition containing an active ingredient of a STING inhibitor and ginkgolide K, and further comprising a pharmaceutically acceptable excipient carrier or excipient.

[0015] Furthermore, the dosage form of the drug includes oral dosage forms and / or non-oral dosage forms, wherein the oral dosage forms include any one or more of capsules, tablets or oral liquids; and the non-oral dosage forms include nebulized liquids or dry powder inhalers suitable for inhalation administration, or injections.

[0016] The pharmaceutical composition for treating pulmonary fibrosis of the present invention and its application have the following beneficial effects: (1) Ginkgolide K (GinK) is readily available, has low preparation costs, and exhibits low toxicity, making it a natural, industrially viable small molecule. The small molecule STING inhibitor C176 has the ability to inhibit STING signaling and positively influence fibrosis endpoints, and can clearly target and inhibit STING signaling. Combining Ginkgolide K (GinK) and the small molecule STING inhibitor C176 can improve histological lesions and downregulate the expression of several fibrosis-related proteins, showing a significant synergistic effect on endpoints such as histological improvement and inhibition of fibrosis-related protein expression. This achieves a synergistic effect that is difficult to achieve with a single drug and reduces the potential toxicity and cost issues associated with high doses of a single drug.

[0017] (2) The pharmaceutical composition of the present invention combines Ginkgolide K (GinK) and STING inhibitor C176 to enhance efficacy and reduce cost without increasing toxicity burden, making up for the limited efficacy of monotherapy and providing feasibility for the treatment of pulmonary fibrosis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The cell survival of the CCK8 experiment with different concentrations of GinK in Example 6 is shown.

[0020] Figure 2This section describes the effect of GinK treatment on the expression of fibrosis-related proteins in a TGF-β-induced 3T3 cell fibrosis model, as shown in Example 6. A shows the expression of α-SMA, Vimentin, and the internal control β-actin protein in 3T3 cells under different treatments using Western blotting. B shows the quantitative analysis of the Western blotting bands (*: P < 0.05; **: P < 0.01, n = 5). C shows the cell viability of different treatment groups in the CCK8 assay (***: P < 0.01, n = 5). D shows the expression of Collagen I protein in 3T3 cells under different treatments using laser confocal microscopy. Collagen I is labeled in red, and the cell nucleus is labeled in blue with DAPI. Scale bar: 5 μm. E shows the quantitative analysis of immunofluorescence Collagen I protein (**: P < 0.01, n = 5). F shows the expression of Fibronectin protein in 3T3 cells under different treatments observed by laser confocal microscopy. Fibronectin is labeled in red, and the cell nucleus is labeled in blue with DAPI. Scale bar: 5 μm. G shows the bar chart showing the quantitative analysis of immunofluorescence Fibronectin protein (****: P<0.01, n=5). Figure 2 The GK group in D, E, and F is the GinK group. Figure 2 In this context, GK is an abbreviation for GinK; the GT group is the TGF-β+GinK group. Figure 2 GT is an abbreviation for GinK+TGF-β.

[0021] Figure 3 This section describes the effects of GinK treatment on the pathology and expression of fibrosis-related proteins in a mouse BLM fibrosis model, as shown in Example 5. A shows the morphological changes in lung tissue of mice in each group as indicated by HE and Masson staining (scale bar: 500 μm). B shows the quantitative analysis of pathological staining using a bar chart (**: P < 0.01, n = 5). C shows the expression of α-SMA, Vimentin, and internal control β-actin and gapdh proteins in mice with different treatments using Western blots. D shows the quantitative analysis of Western blots using a bar chart (*: P < 0.05; **: P < 0.01, n = 5; **P < 0.01). Figure 3 The group gk that appears in B is GinK.

[0022] Figure 4This section describes the effects of C176 treatment on the pathology and expression of fibrosis-related proteins in a mouse BLM fibrosis model, as shown in Example 7. A shows the morphological changes in lung tissue of mice in each group as indicated by HE and Masson staining (scale bar: 200 μm). B shows the quantitative analysis of pathological staining using a bar chart (**: P < 0.01, n = 5; ***: P < 0.01, n = 5; ****: P < 0.01, n = 5). C shows the expression of α-SMA protein in mice under different treatments observed by laser confocal microscopy; α-SMA is labeled in red, and cell nuclei are labeled with DAPI in blue (scale bar: 10 μm). D shows the quantitative analysis of immunofluorescence α-SMA protein using a bar chart (***: P < 0.01, n = 5; ****: P < 0.01, n = 5). E shows the expression of Collagen I protein in mice under different treatments observed by laser confocal microscopy; Collagen I is labeled in green, and cell nuclei are labeled with DAPI in blue (scale bar: 10 μm). F is a bar chart showing the quantitative analysis of immunofluorescence Collagen I protein (*: P<0.05; **: P<0.01, n=5).

[0023] Figure 5 This section describes the effects of combined GinK and C176 treatment on the pathology and fibrosis-related protein expression in a mouse BLM fibrosis model, as shown in Example 8. A shows the morphological changes in lung tissue of mice in each group as indicated by HE and Masson staining (HE scale bar: 200 μm; Masson scale bar: 500 μm). B shows the quantitative analysis of pathological staining using a bar chart (****: P < 0.01, n = 5). C shows the expression of α-SMA protein in mice under different treatments as observed by laser confocal microscopy; α-SMA is labeled red, and cell nuclei are labeled blue with DAPI (scale bar: 10 μm). D shows the quantitative analysis of immunofluorescence α-SMA protein using a bar chart (***: P < 0.01, n = 5; ****: P < 0.01, n = 5). E shows the expression of Collagen I protein in mice under different treatments as observed by laser confocal microscopy; Collagen I is labeled green, and cell nuclei are labeled blue with DAPI (scale bar: 10 μm). F is a bar chart showing the quantitative analysis of immunofluorescence Collagen I protein (**: P<0.01, n=5). Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0025] This invention provides a pharmaceutical composition for treating pulmonary fibrosis, comprising at least a STING inhibitor and an active ingredient containing ginkgolide K. The active ingredient containing ginkgolide K includes any one or more of ginkgolide K, ginkgolide K stereoisomers, crystalline forms of ginkgolide K, pharmaceutically acceptable ginkgolide K salts, and extracts containing ginkgolide K; the STING inhibitor is C176; the mass ratio of the STING inhibitor to ginkgolide K is 3-5:1-2. Preferably, the mass ratio of the STING inhibitor to ginkgolide K is 5:1.

[0026] The structural formula of ginkgolide K (GinK) is as follows: .

[0027] The present invention also provides the use of a pharmaceutical composition for treating pulmonary fibrosis in the preparation of a medicament for treating pulmonary fibrosis.

[0028] This invention also provides a medicament for treating pulmonary fibrosis, comprising a STING inhibitor and an active ingredient containing ginkgolide K, and further comprising a pharmaceutically acceptable excipient carrier or excipient. The medicament can be prepared in various dosage forms, including oral and / or non-oral dosage forms, according to methods conventionally used by those skilled in the art. Oral dosage forms include any one or more of capsules, tablets, or oral solutions; non-oral dosage forms include nebulized solutions or dry powder inhalers suitable for inhalation administration, or injections.

[0029] Example 1

[0030] A pharmaceutical composition for treating pulmonary fibrosis includes the STING inhibitor C176 and ginkgolide K in a 5:1 ratio, wherein the STING inhibitor is 20 mg / kg, the ginkgolide K is 4 mg / kg, and the ginkgolide K is 5 mg / kg.

[0031] Example 2

[0032] A pharmaceutical composition for treating pulmonary fibrosis includes a STING inhibitor C176 and ginkgolide K in a ratio of 3:1, wherein the STING inhibitor is 15 mg / kg and the ginkgolide K is 5 mg / kg.

[0033] Example 3

[0034] A pharmaceutical composition for treating pulmonary fibrosis includes a STING inhibitor C176 and ginkgolide K in a ratio of 5:1, wherein the STING inhibitor is 25 mg / kg and the ginkgolide K is 5 mg / kg.

[0035] Example 4

[0036] A pharmaceutical composition for treating pulmonary fibrosis includes the STING inhibitor C176 and ginkgolide K in a ratio of 5:1, wherein the STING inhibitor is 15 mg / kg and the ginkgolide K is 5 mg / kg.

[0037] Example 5

[0038] Application of pharmaceutical compositions in the preparation of drugs for treating pulmonary fibrosis 5.1 Construction of a mouse model of lung fibrosis Experimental materials and methods: Commercially available 6-8 week old male C57BL / 6J mice; bleomycin (BLM, MedChemExpress, HY-17565); paraffin sections of mouse lung tissue; hematoxylin and eosin (H&E) staining kit (catalog number: G1120, Beijing Solarbio Science & Technology Co., Ltd.); Masson's trichrome staining kit (catalog number: G1340, Beijing Solarbio Science & Technology Co., Ltd.); fully automated multispectral scanning microscopy system (VS200, Olympus). Antibodies: FN1 antibody (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 15613-1-AP); VIMENTIN antibody (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 10366-1-AP); α-SMA antibody (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 10366-1-AP), anti-collagen type I (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 10366-1-AP), β-catenin (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 51067-2-AP), GAPDH (Wuhan Sanying Biotechnology Co., Ltd., mouse source, 60004-1-Ig); Secondary antibody: HRP-labeled goat anti-mouse / anti-rabbit (Cell Signaling); Secondary antibody (fluorescent label): Alexa Fluor 488 and 594 antibodies (Invitrogen); DMEM medium (C11330500BT, Gibco, USA). C57BL / 6 wild-type mice were randomly assigned to five groups (n=5): a control group, a BLM group, a GinK group, and a BLM + GinK group. Control group: C57BL / 6 wild-type mice were given an equal volume of physiological saline; Model group (BLM group): C57BL / 6 wild-type mice were given bleomycin (BLM, 5.0 mg / kg body weight) via intratracheal injection to induce a mouse model of pulmonary fibrosis. Before the operation, the mice were anesthetized with isoflurane, fixed in a supine position, and the trachea was exposed. An appropriate amount of bleomycin solution was slowly dripped into the tracheal tube using a 1 mL syringe.

[0039] GinK+BLM group: Mice in the model group were injected with GinK injection solution, 200ul / g via intraperitoneal injection, once every other day, from day 1 to day 28.

[0040] GinK group: C57BL / 6 wild-type mice were injected with GinK injection solution via intraperitoneal injection at a rate of 200 μL / g, once every other day, from day 1 to day 28.

[0041] The preparation of GinK injection (sterile solution) is as follows: Weigh an appropriate amount of GinK (purity = 98.59%), add a small amount of DMSO (<1% by volume) to dissolve it, and then slowly dilute it with PBS to the required concentration. After aliquoting, store at -20°C to avoid repeated freeze-thaw cycles.

[0042] On day 28, mice were euthanized after intraperitoneal anesthesia. Blood from the entire lungs and heart was rapidly extracted via thoracotomy. Samples were taken from the upper lobe of the left lung, flash-frozen in liquid nitrogen, and stored at -80°C for Western blot and biochemical analysis; the remaining portion of the left lung was used for protein analysis; the right lung was slowly injected with 10% neutral formalin for fixation for 24 hours, then embedded in paraffin for sectioning and staining.

[0043] 5.2 Western blot analysis, the specific method is as follows: A portion of mouse left lung cells were harvested, and cells were completely lysed using RIPA lysis buffer containing protease / phosphatase inhibitors. After incubation on ice for 30 minutes, the cells were centrifuged, and the supernatant was collected to obtain total protein lysis buffer. Quantification was then performed using the BCA method, followed by denaturation by boiling with loading buffer. 10 μg aliquots of protein were separated by SDS-PAGE, transferred to PVDF membranes, and blocked at room temperature with 5% skim milk powder or 5% BSA for 1 hour. Primary antibodies (α-SMA, Vimentin, Fibronectin, all diluted 1:1000, incubated overnight at 4°C) were used; subsequently, HRP-labeled secondary antibody (1:5000, 1 hour) was incubated at room temperature. ECL chemiluminescence imaging was performed, and grayscale values ​​were analyzed using ImageJ software. GAPDH or β-Actin was used as an internal control.

[0044] 5.3 Immunofluorescence detection, the specific method is as follows: The paraffin sections of the right lung were baked in a 67°C oven for 60-120 minutes to enhance tissue adhesion. They were then soaked in xylene I and xylene II for 10 minutes each to completely remove the paraffin.

[0045] Soak the tissue sections sequentially in a gradient of alcohols (e.g., anhydrous ethanol, 95% ethanol, 80% ethanol) for 5 minutes each, and finally rinse with distilled water or PBS to ensure complete hydration. Immerse the sections in sodium citrate buffer, place the retrieval solution containing the sections in an autoclave or pressure cooker, heat to boiling, maintain for 2 minutes, immediately stop heating, and allow the retrieval solution to cool to room temperature naturally. Wash three times with PBS, 5 minutes each time.

[0046] Add PBS solution containing 0.1% Triton X-100 and incubate at room temperature for 10 minutes. Then wash three times with PBS. Aspirate the liquid, add blocking buffer (usually normal serum of the same host species as the secondary antibody or PBS containing BSA), and incubate at room temperature for 60 minutes.

[0047] Dilute the primary antibody (Collagen I, 1:200) with antibody dilution buffer. Remove the blocking buffer and add the diluted primary antibody. Incubate overnight in a humidified chamber at 4°C. Wash three times with PBS to thoroughly remove unbound primary antibody. Dilute the fluorescently labeled secondary antibody with antibody dilution buffer, ensuring it matches the host species of the primary antibody. Add Alexa Fluor 488 fluorescent secondary antibody and incubate at room temperature in the dark for 60 minutes. Wash three times with PBS in the dark, 5 minutes each time.

[0048] Add nuclear dye (DAPI) and incubate at room temperature in the dark for 5–10 minutes. Wash 2–3 times with PBS or ultrapure water in the dark. Aspirate excess liquid from the slide, add a small amount of anti-fluorescence quenching mounting medium to the slide, and gently invert a coverslip containing cells onto the mounting medium. Observe and photograph using a laser confocal microscope. The mounted slide should be stored at 4°C in the dark.

[0049] 5.4 H&E staining, the specific steps are as follows: (1) Place the paraffin sections of the right lung in a constant temperature oven at 67℃ and bake for 60-120 minutes to enhance tissue adhesion. Soak in xylene I and xylene II for 10 minutes each to completely remove the paraffin.

[0050] (2) Soak the tissue in a series of alcohols (such as anhydrous ethanol, 95% alcohol, 80% alcohol) for 5 minutes, and finally rinse with distilled water or PBS to fully hydrate the tissue.

[0051] (3) Immerse the hydrated sections in hematoxylin staining solution for 5-10 minutes. Hematoxylin is a basic dye that will turn the cell nuclei blue-purple. After a brief rinse with running water, use 0.5% hydrochloric acid alcohol for differentiation to remove excess hematoxylin dye, and control the depth of cell nucleus staining by microscopic observation.

[0052] (4) Place the slides in a weakly alkaline solution to restore the cell nuclei to a stable blue-purple color, then rinse thoroughly with running water. Stain the slides in a 0.5% eosin alcohol solution for 5 minutes. Eosin is an acidic dye that will turn the cytoplasm, muscle fibers, and extracellular matrix pink. After staining, the slides need to be dehydrated using a gradient of alcohols, and then cleared with xylene. Remove the slides, add neutral resin, cover with a coverslip for sealing, and then observe under a microscope.

[0053] 5.5 Masson's trichrome staining (1) Place the paraffin sections of the right lung in a constant temperature oven at 67℃ and bake for 60-120 minutes to enhance tissue adhesion. Soak in xylene I and xylene II for 10 minutes each to completely remove the paraffin.

[0054] (2) Soak the tissue in a series of alcohols (such as anhydrous ethanol, 95% alcohol, 80% alcohol) for 5 minutes, and finally rinse with distilled water or PBS to fully hydrate the tissue.

[0055] (3) Immerse the sections in Bouin's solution and place them in a 37°C incubator for 1 hour for mordation. Rinse the sections thoroughly with running water. Stain with Weigert's iron hematoxylin working solution for 5 minutes until the cell nuclei are black or dark brown. Rinse thoroughly with running water.

[0056] (4) Immerse the sections in a Ponceau S-acid fuchsin solution for 10 minutes. At this point, the cytoplasm, muscle fibers, and collagen fibers are stained red. Immerse in a phosphomolybdic acid / phosphotungstic acid solution for 5 minutes. This solution selectively removes most of the red dye, especially from the collagen fibers. Quickly transfer the sections to an aniline blue or Fast Green solution for 5 minutes. The aniline blue will bind to the collagen fibers that have been destained in the previous step, turning them blue. Finally, perform gradient alcohol dehydration, xylene clearing, and mounting with neutral resin.

[0057] The results are as follows Figure 3 As shown, the control group had intact lung tissue structure, thin alveolar septa, and no obvious inflammatory infiltration; the BLM model group showed extensive inflammatory cell infiltration, alveolar wall thickening, and fibrin in the alveolar lumen, with Masson staining revealing abundant blue collagen deposition. After GinK treatment, the alveolar structure of mice improved, the degree of inflammatory infiltration and fibrosis was significantly reduced, and the blue-stained collagen area in the lung tissue was significantly decreased; the fibrosis score was significantly lower than that of the model group. Quantitative imaging showed that the GinK group had a decreased degree of inflammatory cell infiltration and a decreased proportion of collagen area. Figure 3 C Figure 3 The results showed that the pulmonary fibrosis-related markers VIM and α-SMA decreased after GinK treatment compared to the BLM group, indicating that GinK significantly alleviated BLM-induced pulmonary fibrosis.

[0058] Example 6

[0059] Application of pharmaceutical compositions in the preparation of drugs for treating pulmonary fibrosis 6.1 Ginkgolide K (GinK) intervention in a TGF-β-induced 3T3 fibroblast fibrosis model Experimental materials and methods: CCK8 cell proliferation assay kit (Beyotime). Approximately 2000 cells were seeded into 96-well plates with cell suspension (100 u / well). After culturing at 37℃ and 5% CO2 for 24 hours, drug-treated groups, control groups, and blank control groups were established. In the treatment group, different concentrations of GinK (0uM, 1uM, 2.5uM, 5uM, 7.5uM, 10uM) were added. After 2 hours of pretreatment, TGF-β1 at a concentration of 10 ng / mL was added for stimulation, and the treatment lasted for 24 hours. Control group: GinK was replaced with an equal volume of cell culture medium, and other treatments were the same as in the drug-treated group; Control group: No cells were inoculated, and other treatments were the same as those in the drug-treated group.

[0060] After treatment, the supernatant of each group was aspirated, and CCK 8 reagent was added to each well at a mass ratio of (CCK 8 reagent: serum-free DMEM = 1:10). The cells were incubated in the cell culture incubator for another 2 hours. After 2 hours, the culture plates were removed and detected by an ELISA reader. The absorbance (OD) at 450 nm was measured by the ELISA reader, and cell viability was calculated. .

[0061] The results are as follows Figure 1 As shown, from Figure 1 As can be seen, 3T3 cells exhibit the highest cell viability at a GinK concentration of 5 μM.

[0062] 6.2 Evaluation of GinK's antifibrotic activity using a TGF-β1-induced fibrosis model Experimental materials: TGF-β1 (USA, MCE), FN1 antibody (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 15613-1-AP); VIMENTIN antibody (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 10366-1-AP); α-SMA antibody (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 10366-1-AP), anti-collagen type I (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 10366-1-AP), β-catenin (Wuhan Sanying Biotechnology Co., Ltd., rabbit source, 51067-2-AP), GAPDH (Wuhan Sanying Biotechnology Co., Ltd., mouse source, 60004-1-Ig); secondary antibody: HRP-labeled goat anti-mouse / anti-rabbit (Cell Signaling); secondary antibody (fluorescent label): Alexa Fluor 488 and 594 antibodies (Invitrogen); DMEM medium (C11330500BT, Gibco, USA). RIPA lysis buffer, protease phosphatase inhibitor, sodium dodecylbenzenesulfonate, N,N-methylenebisacrylamide, skim milk powder, etc., are products of Solarbio; polyvinylidene fluoride (PVDF) membrane is a product of Merck; ECL luminescence solution is purchased from Millipore, USA. PageRuler™ prestained protein molecular weight standard (26617, Thermo Fisher Scientific, USA), Pierce™ BCA protein assay kit (23227, Thermo Fisher Scientific, USA); Bio-Rad vertical electrophoresis apparatus (Bio-Rad Scientific, USA); electronic pellet imaging system, e-blot product.

[0063] Group processing: Cell culture: 3T3 mouse fibroblasts were used in DMEM culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and cultured at 37°C and 5% CO2. Cells were seeded into 6-well plates at 1.0 × 10^5 cells / well, and processed after reaching 60–70% confluence.

[0064] The group was set as follows: control group ( Figure 2 The middle control group), TGF-β treatment group ( Figure 2 (tgfb group), GinK group) Figure 2 The GinK group and the TGF-β+GinK group ( Figure 2 (The tgfb+gink group); among which... Control group (contrl group): Cells were not treated with GinK and TGF-β, but were treated with an equal volume of DMEM medium; TGF-β treatment group (tgfb group): Cells were treated with recombinant TGF-β1 at a concentration of 10 ng / mL for 24 hours to induce upregulation of fibrosis-related markers; TGF-β+GinK group (tgfb+gink group): The treatment was the same as the TGF-β treatment group, except that before adding TGF-β1, the cells were pretreated with 5uM GinK for 2 hours. GinK group: Cells were pretreated with 5 μM GinK for 2 hours; Detection method: (a) Western blot detection, the specific steps are as follows: (1) Cells from the control group, TGF-β treatment group, TGF-β+GinK group and GinK group were completely lysed using RIPA lysis buffer containing protease / phosphatase inhibitors. After incubation on ice for 30 minutes, the cells were centrifuged and the supernatant was collected to obtain total protein lysis buffer. Quantification was then performed using the BCA method, and the total protein lysis buffer was denatured by boiling with loading buffer. (2) Aliquot 10 μg of protein sample for SDS-PAGE separation, transfer to PVDF membrane, and block with 5% skim milk powder or 5% BSA at room temperature for 1 hour. (3) Incubation with primary antibody: α-SMA, Vimentin (both diluted 1:1000, incubated overnight at 4℃); followed by incubation with HRP-labeled secondary antibody (1:5000, 1 hour) at room temperature. ECL luminescence imaging was performed, and the grayscale value was analyzed using ImageJ software. GAPDH or β-Actin was used as an internal control.

[0065] The results obtained are as follows Figure 2 A and Figure 2 As shown in Figure B, 3T3 cells exhibited a typical fibrotic phenotype after TGF-β1 stimulation. Western blot results showed significantly increased expression levels of fibrosis-related marker proteins such as α-SMA and Vimentin. Compared with the TGF-β1-only treatment group, GinK pretreatment significantly reduced the expression of these proteins, suggesting that GinK can effectively inhibit TGF-β1-induced molecular changes in fibrosis.

[0066] (II) Cell immunofluorescence staining detection, the specific steps are as follows: (1) Cells treated with the control group, TGF-β treatment group, TGF-β+GinK group and GinK group were gently washed with PBS (phosphate buffer) 2-3 times to remove the culture medium.

[0067] (2) Add fixative (4% paraformaldehyde) and fix at room temperature for 15 minutes; paraformaldehyde can cross-link proteins, maintaining cell morphology and antigenicity. Wash three times with PBS for 5 minutes each time to remove residual fixative; (3) Add PBS solution containing permeabilizer (0.3% Triton X-100) and incubate at room temperature for 5–10 minutes. Wash 3 times with PBS to remove the permeabilizer; (4) Add blocking buffer (1%–10% BSA) and incubate at room temperature for 30–60 minutes. Dilute the primary antibody, Collagen I, and anti-Fibronectin (1:200 dilution) with antibody dilution buffer. Remove the blocking buffer and add the diluted primary antibody. Incubate overnight in a humidified chamber at 4°C. Wash three times with PBS to thoroughly remove unbound primary antibody. Dilute the fluorescently labeled secondary antibody with antibody dilution buffer, ensuring it matches the host species of the primary antibody. Add Alexa Fluor 488 / 594 fluorescent secondary antibody and incubate at room temperature in the dark for 60 minutes. Wash three times with PBS in the dark, 5 minutes each time. (5) Add nuclear dye (DAPI) and incubate at room temperature in the dark for 5–10 minutes. Wash 2–3 times with PBS or ultrapure water in the dark. Aspirate excess liquid from the slide, add a small amount of anti-fluorescence quenching mounting medium to the slide, and gently invert the coverslip containing cells onto the mounting medium. Observe and photograph using a laser confocal microscope. The mounted slide should be stored at 4°C in the dark.

[0068] The results obtained are as follows Figure 2 D、 Figure 2 E, Figure 2 F and Figure 2 As shown in Figure G, the immunofluorescence detection results further indicate that TGF-β1 treatment significantly enhanced the positive signal and distribution range of Collagen I and Fibronectin in cells, while the fluorescence intensity and positive area of ​​ECM-related proteins were significantly reduced after GinK treatment, indicating that GinK has an inhibitory effect on excessive deposition of extracellular matrix.

[0069] (III) CCK8 experimental verification, the specific methods and steps are as follows: (1) Seed each group of cell suspension (100u / well) into a 96-well plate, approximately 2000 cells. Discard the original solution and add 10% 100u CCK-8 solution to each well. Incubate for 2 hours in a cell culture incubator. After 2 hours, detect the absorbance (OD) at 450 nm using a microplate reader.

[0070] The results obtained are as follows Figure 2As shown in Figure C, the CCK-8 results showed that the cell survival rate in the TGF-β1 + GinK group was higher than that in the TGF-β1 group, suggesting that while inhibiting the fibrotic phenotype, GinK did not have a significant adverse effect on cell viability.

[0071] Example 7

[0072] Efficacy validation of the single-drug STING inhibitor C176 in an in vivo BLM-induced mouse pulmonary fibrosis model Experimental materials: Male C57BL / 6 mice, 6-8 weeks old; bleomycin (BLM, MedChemExpress, HY-17565); paraffin sections of mouse lung tissue; hematoxylin and eosin (H&E) staining kit (catalog number: G1120, Beijing Solarbio Science & Technology Co., Ltd.); Masson's trichrome staining kit (catalog number: G1340, Beijing Solarbio Science & Technology Co., Ltd.); fully automated multispectral scanning microscopy system (VS200, Olympus). Antibody information as previously stated.

[0073] Experimental grouping and treatment: Mouse model establishment: C57BL / 6 wild-type mice were randomly divided into four groups (n=5): control group, BLM group, GinK group, and BLM + GinK group. Control group: C57BL / 6 wild-type mice were given an equal volume of physiological saline; Model group (BLM group): C57BL / 6 wild-type mice were given bleomycin (BLM, 5.0 mg / kg body weight) via intratracheal injection to induce a mouse model of pulmonary fibrosis. Before the operation, the mice were anesthetized with isoflurane, fixed in a supine position, and the trachea was exposed. An appropriate amount of bleomycin solution was slowly dripped into the tracheal tube using a 1 mL syringe.

[0074] BLM + C176 group: Mice in the model group were injected with C176 injection solution, 200ul / g, via intraperitoneal injection, once every two days, from day 1 to day 28.

[0075] C176 group: C57BL / 6 wild-type mice were injected with C176 injection solution, 200ul / g, via intraperitoneal injection, once every two days, from day 1 to day 28.

[0076] On day 28, mice were euthanized after intraperitoneal anesthesia. Blood from the entire lungs and heart was rapidly extracted via thoracotomy. Samples were taken from the upper lobe of the left lung, flash-frozen in liquid nitrogen, and stored at -80°C for Western blot and biochemical analysis; the remaining portion of the left lung was used for protein analysis; the right lung was slowly injected with 10% neutral formalin for fixation for 24 hours, then embedded in paraffin for sectioning and staining.

[0077] For immunofluorescence, paraffin sections are baked in a 67°C incubator for 60-120 minutes to enhance tissue adhesion. They are then soaked in xylene I and xylene II for 10 minutes each to completely remove paraffin. Next, they are soaked in a gradient of alcohols (e.g., anhydrous ethanol, 95% ethanol, 80% ethanol) for 5 minutes each, and finally rinsed with distilled water or PBS to ensure complete tissue hydration. The sections are then immersed in sodium citrate buffer. The retrieval solution containing the sections is placed in an autoclave or pressure cooker and heated to boiling, held for 2 minutes, and then immediately stopped. The retrieval solution is allowed to cool to room temperature. The sections are washed three times with PBS for 5 minutes each time. A PBS solution containing 0.1% Triton X-100 is added, and the sections are incubated at room temperature for 10 minutes. This is followed by three washes with PBS. The liquid is aspirated, and blocking buffer (usually normal serum of the same host species as the secondary antibody or PBS containing BSA) is added. The sections are incubated at room temperature for 60 minutes. Dilute the primary antibody (α-SMA, Collagen I, 1:200) with antibody dilution buffer. Remove the blocking buffer and add the diluted primary antibody. Incubate overnight at 4°C in a humidified chamber. Wash three times with PBS to thoroughly remove unbound primary antibody. Dilute the fluorescently labeled secondary antibody with antibody dilution buffer, ensuring it matches the host species of the primary antibody. Add Alexa Fluor 488 fluorescent secondary antibody and incubate at room temperature in the dark for 60 minutes. Wash three times with PBS for 5 minutes each time. Add nuclear dye (DAPI) and incubate at room temperature in the dark for 5–10 minutes. Wash 2–3 times with PBS or ultrapure water in the dark. Remove excess liquid from the slide, add a small amount of anti-fluorescence quenching mounting medium to the slide, and gently invert a coverslip containing cells onto the mounting medium. Observe and photograph using a laser confocal microscope. Store the mounted slide at 4°C in the dark.

[0078] For H&E staining, place the prepared paraffin sections in an oven at 60℃-70℃ for 1 hour to ensure the tissue adheres firmly to the slide. Remove the paraffin by immersion in xylene, then hydrate using a gradient of alcohols, and finally rinse with tap water to restore hydration. Stain the hydrated sections in hematoxylin solution for 5-10 minutes. Hematoxylin is a basic dye that turns cell nuclei blue-purple. After a brief rinse with running water, differentiate using 0.5% hydrochloric acid-alcohol solution to remove excess hematoxylin dye and control the staining depth of the nuclei under a microscope. Place the sections in a weakly alkaline solution to restore the cell nuclei to a stable blue-purple color, then rinse thoroughly with running water. Stain the sections in 0.5% eosin-alcohol solution for 5 minutes. Eosin is an acidic dye that turns cytoplasm, muscle fibers, and extracellular matrix pink. After staining, dehydrate the sections using a gradient of alcohols, then clear them with xylene. Remove the slide, add a drop of neutral resin, cover with a coverslip to seal, and then observe under a microscope.

[0079] Masson trichrome staining: Place the prepared paraffin sections in an oven at 60-70°C for 1 hour to ensure the tissue adheres firmly to the slide. Remove the paraffin by immersion in xylene, then hydrate with a gradient of alcohols, and finally rinse with tap water to restore hydration. Mordant the sections in Bouin's solution at 37°C for 1 hour. Rinse thoroughly with running water. Stain with Weigert's iron hematoxylin working solution for 5 minutes to make the nuclei black or dark brown. Rinse thoroughly with running water. Immerse the sections in Ponceau S-acid fuchsin solution for 10 minutes. At this point, the cytoplasm, myofibrils, and collagen fibers are stained red. Immerse in phosphomolybdic acid / phosphotungstic acid solution for 5 minutes. This solution selectively removes most of the red dye, especially from the collagen fibers. Quickly transfer the sections to aniline blue or Fast Green solution for 5 minutes. Aniline blue binds to the destained collagen fibers from the previous step, turning them blue. Finally, the slides were dehydrated with a gradient of alcohols, cleared with xylene, and then sealed with neutral resin.

[0080] The results are as follows Figure 4 As shown, C176 administration can improve the degree of pulmonary fibrosis in a BLM-induced mouse model of pulmonary fibrosis. Figure 4 A, Figure 4B. In pathological staining, HE and Masson staining results showed that the BLM group exhibited significant lung tissue lesions and collagen deposition. The BLM group showed significantly deeper inflammatory cell infiltration and abundant blue collagen deposition compared to the Control group. The lung tissue morphology and collagen content of the C176 monotherapy group were similar to the control group; while the BLM + C176 group showed reduced lesions compared to the BLM group, with decreased pathological lesion area and collagen area fraction, indicating that C176 can partially treat BLM-induced histological lesions. Figure 4 C Figure 4 D、 Figure 4 E and Figure 4 F indicates that the immunofluorescence results reflect fibrosis-related markers. α-SMA IF was significantly enhanced in the BLM group, with a significantly higher mean fluorescence intensity than the Control group. The α-SMA level in the C176 monotherapy group was similar to that in the Control group. In the BLM+C176 group, the α-SMA positive signal was significantly decreased, and the mean fluorescence intensity was significantly lower than that in the BLM group. Collagen I IF showed a similar trend: BLM significantly increased Collagen I positive signal, while C176 reduced its expression level; the mean fluorescence intensity of Collagen I in the BLM+C176 group was significantly lower than that in the BLM group. This suggests that C176 can downregulate fibrosis-related markers and may relatively slow the progression of pulmonary fibrosis.

[0081] Example 8

[0082] Application of pharmaceutical compositions in the preparation of drugs for treating pulmonary fibrosis Efficacy validation of combined GinK and C176 in a BLM-induced mouse pulmonary fibrosis model Experimental materials: Male C57BL / 6 mice, 6-8 weeks old; bleomycin (BLM, MedChemExpress, HY-17565); paraffin sections of mouse lung tissue; hematoxylin and eosin (H&E) staining kit (catalog number: G1120, Beijing Solarbio Science & Technology Co., Ltd.); Masson's trichrome staining kit (catalog number: G1340, Beijing Solarbio Science & Technology Co., Ltd.); fully automated multispectral scanning microscopy system (VS200, Olympus). Antibody information as previously stated.

[0083] Experimental grouping and treatment: Mouse model establishment: C57BL / 6 wild-type mice were randomly divided into four groups (n=5): control group, BLM group, GinK group, and BLM + GinK group. Control group: C57BL / 6 wild-type mice were given an equal volume of physiological saline; Model group (BLM group): C57BL / 6 wild-type mice were given bleomycin (BLM, 5.0 mg / kg body weight) via intratracheal injection to induce a mouse model of pulmonary fibrosis. Before the operation, the mice were anesthetized with isoflurane, fixed in a supine position, and the trachea was exposed. An appropriate amount of bleomycin solution was slowly dripped into the tracheal tube using a 1 mL syringe.

[0084] BLM + GinK + C176 group: Mice in the model group were injected with C176 injection and GinK injection. Both C176 injection and GinK injection were administered intraperitoneally at 200 μL / g. C176 injection was administered every two days from day 1 to day 28; GinK injection was administered every other day from day 1 to day 28.

[0085] C176 group: C57BL / 6 wild-type mice were injected with C176 injection solution, 200ul / g, via intraperitoneal injection, once every two days, from day 1 to day 28.

[0086] GinK group: C57BL / 6 wild-type mice were injected with GinK injection solution via intraperitoneal injection at a rate of 200 μL / g, once every other day, from day 1 to day 28.

[0087] The preparation of GinK injection (sterile solution) is as follows: Weigh an appropriate amount of GinK (purity = 98.59%), add a small amount of DMSO (<1% by volume) to dissolve it, and then slowly dilute it with PBS to the required concentration. After aliquoting, store at -20°C to avoid repeated freeze-thaw cycles.

[0088] On day 28, mice were euthanized after intraperitoneal anesthesia. Blood from the entire lungs and heart was rapidly extracted via thoracotomy. Samples were taken from the upper lobe of the left lung, flash-frozen in liquid nitrogen, and stored at -80°C for Western blot and biochemical analysis; the remaining portion of the left lung was used for protein analysis; the right lung was slowly injected with 10% neutral formalin for fixation for 24 hours, then embedded in paraffin for sectioning and staining.

[0089] Experimental methods: For immunofluorescence, paraffin sections are baked in a 67°C incubator for 60-120 minutes to enhance tissue adhesion. They are then soaked in xylene I and xylene II for 10 minutes each to completely remove paraffin. Next, they are soaked in a gradient of alcohols (e.g., anhydrous ethanol, 95% ethanol, 80% ethanol) for 5 minutes each, and finally rinsed with distilled water or PBS to ensure complete tissue hydration. The sections are then immersed in sodium citrate buffer. The retrieval solution containing the sections is placed in an autoclave or pressure cooker and heated to boiling, held for 2 minutes, and then immediately stopped. The retrieval solution is allowed to cool to room temperature. The sections are washed three times with PBS for 5 minutes each time. A PBS solution containing 0.1% Triton X-100 is added, and the sections are incubated at room temperature for 10 minutes. The sections are then washed three times with PBS. The liquid is aspirated, and blocking buffer is added, with incubation at room temperature for 60 minutes. The primary antibody (α-SMA and Collagen I, diluted 1:200) is diluted with antibody dilution buffer. The blocking buffer is removed, and the diluted primary antibody is added. Typically, incubation overnight in a humidified chamber at 4°C is chosen. Wash three times with PBS to thoroughly remove unbound primary antibody. Dilute the fluorescently labeled secondary antibody with antibody dilution buffer, ensuring it matches the host species of the primary antibody. Add Alexa Fluor 488 fluorescent secondary antibody and incubate at room temperature in the dark for 60 minutes. Wash three times with PBS in the dark, 5 minutes each time. Add nuclear dye (DAPI) and incubate at room temperature in the dark for 5–10 minutes. Wash 2–3 times with PBS or ultrapure water in the dark. Aspirate excess liquid from the slide, add a small amount of anti-fluorescence quenching mounting medium to the slide, and gently invert a coverslip containing cells onto the mounting medium. Observe and photograph using a laser confocal microscope. The mounted slide should be stored at 4°C in the dark.

[0090] H&E staining: Place the cut paraffin sections in an oven at 60℃-70℃ for 1 hour to ensure the tissue adheres firmly to the slide. Remove the paraffin by immersion in xylene, then hydrate using a gradient of alcohols, and finally rinse with tap water to restore hydration. Immerse the hydrated sections in hematoxylin staining solution for 5-10 minutes. Hematoxylin is a basic dye that will turn cell nuclei blue-purple. After a brief rinse with running water, differentiate using 0.5% hydrochloric acid-alcohol solution to remove excess hematoxylin dye and control the staining depth of the nuclei under a microscope. Place the sections in a weakly alkaline solution to restore the cell nuclei to a stable blue-purple color, then rinse thoroughly with running water. Immerse the sections in 0.5% eosin-alcohol solution for 5 minutes. Eosin is an acidic dye that will turn cytoplasm, muscle fibers, and extracellular matrix pink. After staining, the sections need to be dehydrated using a gradient of alcohols and then cleared with xylene. Remove the slide, add a drop of neutral resin, cover with a coverslip to seal, and then observe under a microscope.

[0091] Masson's trichrome staining: Place the prepared paraffin sections in an oven at 60-70°C for 1 hour to ensure the tissue adheres firmly to the slide. Remove the paraffin by immersion in xylene, then hydrate with a gradient of alcohols, and finally rinse with tap water to restore hydration. Mordant the sections in Bouin's solution at 37°C for 1 hour. Rinse thoroughly with running water. Stain with Weigert's iron hematoxylin working solution for 5 minutes until the nuclei are black or dark brown. Rinse thoroughly with running water. Immerse the sections in Ponceau S-acid fuchsin solution for 10 minutes. At this point, the cytoplasm, myofibrils, and collagen fibers are stained red. Immerse in phosphomolybdic acid / phosphotungstic acid solution for 5 minutes. This solution selectively removes most of the red dye, especially from the collagen fibers. Quickly transfer the sections to aniline blue or Fast Green solution for 5 minutes. Aniline blue binds to the destained collagen fibers from the previous step, turning them blue. Finally, the slides were dehydrated with a gradient of alcohols, cleared with xylene, and then sealed with neutral resin.

[0092] Experimental results: The combination of GinK and C176 significantly improved the degree of pulmonary fibrosis in mice compared with single-drug treatment.

[0093] like Figure 5 A and Figure 5 B showed that the BLM group mice exhibited obvious lung lesions and extensive collagen deposition, compared to... Figure 3 and Figure 4 In comparison, both the BLM+GinK and BLM+C176 monotherapy groups showed varying degrees of improvement in structural repair and collagen reduction. The BLM+GinK+C176 combination group exhibited the most significant pathological improvement, with a significantly reduced area of ​​pathological damage and collagen fraction compared to the BLM group. Its tissue structure was closer to the control group, and the pathological changes were more pronounced than in the monotherapy groups. Immunofluorescence analysis further supports these observations. Figure 5 C Figure 5 D、 Figure 5 E and Figure 5 F showed that α-SMA was strongly expressed in the lung tissue of the BLM group, suggesting significant activation of myoblasts and fibroblasts; compared with Figure 3 and 4 In comparison, treatment with GinK or C176 alone partially reduced the fluorescence intensity of α-SMA, while the combined treatment group showed a further decrease in α-SMA expression, with a significantly lower average fluorescence intensity than the individual single-drug treatment groups. The immunofluorescence trend of Collagen I was consistent with that of α-SMA. The corresponding semi-quantitative analysis results were consistent with histological observations, indicating that the combined treatment was superior to single-drug administration in inhibiting fibrosis-related phenotypes.

[0094] The results of this embodiment show that the combined administration of GinK and C176 has a better anti-fibrotic effect than single drug in a BLM-induced mouse pulmonary fibrosis model, manifested by more significant histological repair, lower collagen deposition and stronger targeting of fibrosis marker proteins.

[0095] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that: It includes at least a STING inhibitor and an active ingredient containing ginkgolide K.

2. The pharmaceutical composition for treating pulmonary fibrosis according to claim 1, characterized in that: The STING inhibitor is C176.

3. The pharmaceutical composition for treating pulmonary fibrosis according to claim 1, characterized in that: The active ingredient containing ginkgolide K includes any one or more of ginkgolide K, ginkgolide K stereoisomers, crystal forms of ginkgolide K, pharmaceutically acceptable ginkgolide K salts, and extracts containing ginkgolide K.

4. The pharmaceutical composition for treating pulmonary fibrosis according to claim 1, characterized in that: The mass ratio of the STING inhibitor to ginkgolide K is 3-5:1-2.

5. The pharmaceutical composition for treating pulmonary fibrosis according to claim 1, characterized in that: The pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia.

6. Use of a pharmaceutical composition for treating pulmonary fibrosis according to any one of claims 1-5 in the preparation of a medicament for treating pulmonary fibrosis.

7. A drug for treating pulmonary fibrosis, characterized in that: It includes at least the pharmaceutical composition for treating pulmonary fibrosis as described in any one of claims 1-5.

8. The medicament for treating pulmonary fibrosis according to claim 7, characterized in that: The drug also includes pharmaceutically acceptable excipients or carriers.

9. The medicament for treating pulmonary fibrosis according to claim 7, characterized in that: The dosage forms of the drug include oral dosage forms and / or non-oral dosage forms. The oral dosage forms include any one or more of capsules, tablets, or oral liquids. The non-oral dosage forms include nebulized liquids or dry powder inhalers suitable for inhalation administration, or injections.

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