Use of ginkgolic acids in the preparation of a medicament for the treatment of acute lung injury

Abstract

The application belongs to the technical field of medicine application, and provides application of ginkgolic acid in preparation of medicine for treating acute lung injury. The chemical structure general formula of the ginkgolic acid is as follows, wherein the R group is a straight chain alkane or a straight chain alkene; the number of carbon atoms in the straight chain alkane and the straight chain alkene is 13-17; the number of double bonds in the straight chain alkene is 1 or 2; the ginkgolic acid has higher Caspase affinity and can be used as a broad-spectrum inhibitor of Caspase; the cell activity and in-vivo activity of the ginkgolic acid are evaluated by establishing a BMDM cell pyroptosis model and a mouse acute lung injury model caused by LPS. The results show that the ginkgolic acid has in-vitro and in-vivo anti-pyroptosis activity, and has a good improvement effect on lung injury, and has a potential application in preparation of medicine for treating acute lung injury.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug application, and particularly relates to application of ginkgoic acid in preparation of a drug for treating acute lung injury. BACKGROUND

[0002] Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are respiratory diseases with high morbidity and mortality. Severe inflammatory response is a typical feature of ALI / ARDS patients. Uncontrolled inflammatory response can destroy the epithelial and endothelial barrier, and eventually lead to high mortality. At present, there is no effective treatment to significantly reduce the mortality of ALI / ARDS patients and improve the quality of life of patients. Alveolar macrophages, which are directly in contact with pathogens, are the main immune cell population involved in regulating lung inflammation. The pro-inflammatory factors, inflammatory mediators and cytokines produced by them can damage or impair the function of endothelial cells and epithelial cells. It has been reported that pyroptosis of macrophages is associated with the progression of ALI. In LPS-induced ALI, activation of Caspase-1 promotes pyroptosis of alveolar macrophages. Therefore, precise treatment based on cell pyroptosis provides a direction for the development of drugs for ALI / ARDS.

[0003] Cell pyroptosis, also known as inflammatory necrosis, is a kind of programmed cell death. Compared with apoptosis, pyroptosis occurs more quickly and is accompanied by the release of a large amount of pro-inflammatory factors. In the Caspase-1-dependent classical pyroptosis pathway, activated Caspase-1 leads to cleavage of Gasdermin D (GSDMD) to form GSDMD-N, thereby inducing cell membrane perforation. At the same time, activated Caspase-1 cleaves pro-IL-1β and pro-IL-18 to form active IL-1β and IL-18, which are released into the extracellular space and recruit inflammatory cells. More and more evidence shows that pro-inflammatory cytokines IL-1β and IL-18 are potential molecules for predicting the morbidity and mortality of ALI / ARDS, and the increase in IL-1β and IL-18 concentration is associated with increased mortality. Therefore, Caspase-1 has the potential to be a target for ALI treatment.

[0004] Natural small molecule compounds have the advantages of low immune response, good pharmacokinetics, etc. in drug development, and have irreplaceable value. Many approved and clinical trial drugs are extracted from natural products, such as artemisinin and curcumin. However, the reported Caspase-1 inhibitors are mainly artificial synthetic peptidomimetic compounds and peptide-based compounds. Therefore, it is of great significance to discover Caspase-1 small molecule inhibitors from natural products and apply them to the treatment of acute lung injury.

[0005] Chinese invention patent CN108014119B discloses a new use of natural product phenylpropanoid glycoside Smiglaside A, specifically relates to the application of compound phenylpropanoid glycoside Smiglaside A in preparing drugs for improving or treating sepsis, mainly in that Smiglaside A can significantly reduce lipopolysaccharide-induced macrophage inflammation, Smiglaside A can significantly alleviate lipopolysaccharide-induced sepsis mouse death, can significantly improve lipopolysaccharide-induced acute lung injury, and can significantly reduce inflammation in blood and lung tissue, and is expected to become a new drug for treating sepsis and other inflammation-related diseases.

[0006] Chinese invention patent application CN114949034A discloses the application of Perilla frutescens and its extract in preparing drugs for preventing and treating acute pneumonia, and the drugs are drugs for inhibiting inflammatory cell infiltration and protein exudation of bronchial alveolitis of lung tissue, inhibiting expression of related inflammatory factors in lung, inhibiting expression of related inflammatory factors in plasma, and improving pathological damage caused by lung inflammation.

[0007] Ginkgolic acid is a derivative of hexaalkyl or hexaalkenyl salicylic acid, and is one of the components with important biological activities in ginkgo leaves except lactones, proanthocyanidins and flavones, and has the effects of anti-tumor, anti-HIV, antibacterial, neurotoxicity, pro-apoptosis, pro-autophagy, etc. Chinese invention patent CN107184617B discloses the application of ginkgolic acid in preparing products for preventing and / or treating allergic diseases, and finds that active compound ginkgolic acid C15:1 or ginkgolic acid C13:0 has Syk tyrosine kinase Syk inhibitory activity and inhibits mast cell shedding, and the target is clear. Therefore, as a component with biological activity, the effect of ginkgolic acid on Caspase-1 needs to be studied. SUMMARY

[0008] The present application aims at the problems existing in the prior art, and screens Caspase-1 inhibitors in a natural product library to find a highly active cell pyroptosis inhibitor ginkgolic acid, and provides the application of ginkgolic acid in preparing drugs for treating acute lung injury.

[0009] The application relates to application of ginkgo acid in preparation of a medicine for treating acute lung injury, and a structure general formula of the ginkgo acid is as follows:

[0010]

[0011] The R group in the general formula of the ginkgo acid is a straight-chain alkane or a straight-chain alkene.

[0012] Preferably, the number of carbon atoms in the straight-chain alkane is 13-17.

[0013] Preferably, the number of carbon atoms in the straight-chain alkene is 13-17, and the number of double bonds is 1-2.

[0014] More preferably, the straight-chain alkene is a straight-chain alkene with one double bond, and the configuration is Z configuration or E configuration.

[0015] More preferably, the straight-chain alkene is a straight-chain alkene with two double bonds, and the configuration is Z configuration+Z configuration, E configuration+E configuration or Z structure+E structure.

[0016] Preferably, the ginkgo acid is C13:0, C15:0, C15:1, C17:1 or C17:2.

[0017] More preferably, the ginkgo acid is C15:1 and C15:0.

[0018] The chemical structural formula of the ginkgo acid C15:1 is as follows:

[0019]

[0020] The structural formula of the ginkgo acid C17:1 is as follows:

[0021]

[0022] The structural formula of the ginkgo acid C17:2 is as follows:

[0023]

[0024] The application also relates to application of the ginkgo acid as an inhibitor of a key target Caspase to inhibit pyroptosis.

[0025] Further, the ginkgo acid is a broad-spectrum inhibitor of the key target Caspase, and has inhibiting effects on the key target Caspase-1, Caspase-3, Caspase-4, Caspase-5 and Caspase-6.

[0026] Further, the active group of the ginkgo acid which has an effect on the activity of Caspase is a carbon chain and a carboxyl group on a benzene ring.

[0027] Further, the activity of the ginkgolic acid on Caspase requires the presence of both the carboxyl group and the carbon chain on the benzene ring.

[0028] The present application also relates to a preparation containing ginkgolic acid for treating acute lung injury.

[0029] Preferably, the dosage form of the preparation is any one of tablets, capsules, powders, injections.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1. Compared with the Caspase-1 inhibitor VX-765 artificially synthesized in the prior art, the ginkgolic acid has higher Caspase-1 affinity; and whether it is the improvement effect on lung injury or the detection results of Caspase-1 and pyroptosis-related indicators, it is shown that the activity of the ginkgolic acid is better than that of the Caspase-1 inhibitor VX-765 and the positive drug dexamethasone for lung injury.

[0032] 2. The present application shows that the activity of the ginkgolic acid C15:1 requires the presence of both the carboxyl group and the carbon chain on the benzene ring through the structure-activity relationship study of the ginkgolic acid Ginkgolic acid C15:1, the ginkgolic acid Ginkgolic acid C15:0, the cardanol Cardanol C15:0 and the salicylic acid Salicylic acid and Caspase-1; and at the same time, based on the fact that the activities of the ginkgolic acid C15:1 and the ginkgolic acid C15:0 are equivalent, it is shown that the ginkgolic acid C13:0, the ginkgolic acid C17:1 and the ginkgolic acid C17:2 also have the potential as Caspase-1 inhibitors.

[0033] 3. The ginkgolic acid has a broad spectrum on the Caspase family, and has the potential to simultaneously inhibit the classical pathway and the non-classical pathway pyroptosis; compared with the exclusive inhibitor of Caspase-1, the ginkgolic acid has a wider application in inhibiting pyroptosis.

[0034] 4. The ginkgolic acid is a pyroptosis inhibitor with in vitro and in vivo activities taking Caspase-1 as a target, which can significantly improve the acute lung injury of mice caused by LPS. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The enzyme activity curve and IC value of the ginkgolic acid Ginkgolic acid C15:1 on Caspase-1 50 ;

[0036] Figure 2The in vitro detection results of the antipyroptosis activity of Ginkgolic acid C15:1 and VX-765 were obtained by detecting GSDMD cleavage using Coomassie Brilliant Blue staining.

[0037] Figure 3 SPR results for Ginkgolic acid C15:1 and VX-765; time-response curve (A) and concentration-response curve (B) of Ginkgolic acid C15:1 with Caspase-1; time-response curve (C) and concentration-response curve (D) of VX-765 with Caspase-1.

[0038] Figure 4 Enzyme activity curves and Coomassie brilliant blue staining results for Ginkgolic acid C15:1 (A), Ginkgolic acid C15:0 (B), Cardanol C15:0 (C), and Salicylic acid (D);

[0039] Figure 5 The enzyme activity curves of Ginkgolic acid C15:1 against Caspase-4(A), Caspase-5(B), Caspase-3(C), and Caspase-6(D) are shown.

[0040] Figure 6 The results of Caspase-1 detection in the BMDM cell pyroptosis model are as follows: (A) Caspase-1p20 protein level in cell supernatant was detected by Western blotting; (B) Statistical analysis of three Western blotting results of Caspase-1p20. ### P<0.001 vsControl group, * P<0.05, ** P<0.01, *** P<0.001 vs Model group, (C) Caspase-1 activity in cells was detected by FAM-FLICA Caspase-1;

[0041] Figure 7 The results of GSDMD detection in the BMDM cell pyroptosis model are shown in the following: (A) Levels of GSDMD and GSDMD-N in cells were detected by Western blotting (WB); (B) Statistical analysis of GSDMD-N and GSDMD (C) results from three Western blotting experiments. ### P<0.001 vs Control group * P<0.05, ** P<0.01, ***P < 0.001 vs Model group, ns indicates no statistical difference compared with the Control group;

[0042] Figure 8 The release of IL-1β and IL-18 in the BMDM cell pyroptosis model, (A) IL-1β level, (B) IL-18 level. ### P<0.001 vs Control group * P<0.05, ** P<0.01, *** P<0.001 vs Model group;

[0043] Figure 9 HE staining results for lung tissue;

[0044] Figure 10 The results show the detection of total protein, albumin, and LDH in bronchoalveolar lavage fluid. Total protein (A), albumin (B), and LDH (C) are also listed. ### P<0.001 vs Control group * P<0.05, *** P<0.001 vs Model group;

[0045] Figure 11 The results of Caspase-1 activation in bronchoalveolar lavage fluid and lung tissue are as follows: (A) Western blotting (WB) detection of Caspase-1 p45 and p20 in bronchoalveolar lavage fluid; (B) Statistical analysis of three WB results for Caspase-1 p45 in bronchoalveolar lavage fluid; (C) Statistical analysis of three WB results for Caspase-1 p20 in bronchoalveolar lavage fluid; (D) Western blotting (WB) detection of Caspase-1 p45 and p20 in lung tissue; (E) Statistical analysis of three WB results for Caspase-1 p45 in lung tissue; (F) Statistical analysis of three WB results for Caspase-1 p20 in lung tissue. ### P<0.001 vs Control group * P<0.05, ** P<0.01, *** P<0.001 vs Model group;

[0046] Figure 12 Immunohistochemical results of Caspase-1p20 in lung tissue;

[0047] Figure 13The results show the activation of GSDMD in bronchoalveolar lavage fluid and lung tissue. (A) The protein levels of GSDMD-N in bronchoalveolar lavage fluid and GSDMD in lung tissue were detected by Western blotting (WB). (B) Statistical analysis of three WB results of GSDMD-N in bronchoalveolar lavage fluid. (C) Statistical analysis of three WB results of GSDMD in lung tissue. ### P<0.001 vs Control group ** P<0.01, *** P<0.001 vs Model group;

[0048] Figure 14 The results show the detection of IL-1β and IL-18 in bronchoalveolar lavage fluid. ### P<0.001 vs Control group * P<0.05, ** P<0.01, *** P<0.001 vs Model group. Detailed Implementation

[0049] The following specific experiments further illustrate the content of this invention. It should be particularly noted that these descriptions are merely exemplary and do not constitute a limitation on the scope of this invention. Based on the discussion in this specification, many variations and modifications of this invention will be apparent to those skilled in the art.

[0050] In this invention, ginkgolic acid has the following general structural formula:

[0051]

[0052] The structure-activity relationship between ginkgolic acid and Caspase was studied using ginkgolic acid C15:1 and C15:0 as active compounds.

[0053] The levels of ginkgolic acid in cells and in vivo were evaluated using ginkgolic acid C15:1 as the active compound.

[0054] The chemical structure of ginkgolic acid C15:1 is as follows:

[0055]

[0056] Example 1: Verification of the enzyme activity of Ginkgolic acid C15:1 against Caspase-1

[0057] (1) To detect the activity of ginkgolic acid C15:1 against caspase-1, an enzyme activity assay of caspase-1 was performed. A series of concentrations of ginkgolic acid C15:1 were prepared: 200, 100, 50, 25, 12.50, 6.25, 3.13, 1.56, and 0.78 μM. The inhibition rate at each concentration was measured, and the IC50 was calculated using Graphpad software. 50 Value. Result as follows Figure 1 As shown, ginkgolic acid C15:1 exhibits high activity against Caspase-1, with an IC50 value of [missing information]. 50 The value is 4.223 ± 0.219 μM.

[0058] (2) To verify the in vitro antipyroptosis activity of ginkgolic acid C15:1, the effect of the compound on GSDMD cleavage was detected using a "Caspase-1 + GSDMD + compound" system. The compound (experimental group: ginkgolic acid C15:1; positive control group: VX-765; blank control group: DMSO solvent) at a final concentration of 50 μM was pre-incubated with Caspase-1 at a final concentration of 0.5 μM at 37 °C for 30 min. Then, GSDMD at a final concentration of 20 μM was added, and incubation was continued for 30 min. Samples were then run on a gel and stained with Coomassie Brilliant Blue. The results are as follows: Figure 2 As shown, it can be seen that ginkgolic acid C15:1 inhibits the cleavage of GSDMD by Caspase-1.

[0059] (3) SPR was used to study the affinity of the compound for Caspase-1: SPR experiments were performed using a Biacore 8K high-throughput molecular interaction detection system with a CM5 chip. The running buffer used in the experiment consisted of 20 mM HEPES (pH 8.0), 150 mM NaCl, and 0.05% Tween 20. The experimental procedure was as follows: The CM5 chip was activated by passing a 1:1 mixture of 0.1 M N-hydroxysuccinimide (NHS) and 0.1 M N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide (EDC) through the chip. Then, Caspase-1 was diluted with sodium acetate at pH 5.5 to a final concentration of 50 μg / mL. The protein solution was then passed through the chip, and the protein was immobilized on the chip surface by amino coupling (response value greater than 8000). Finally, the remaining binding sites on the chip were blocked with 1 M ethanolamine (pH 8.5).

[0060] The results are as follows Figure 3 As shown, where, Figure 3A's time-response curve and Figure 3 The concentration-response curve of B was fitted to determine the K values ​​of ginkgolic acid C15:1 and Caspase-1. D The value is 4.83 μM; Figure 3 The time-response curve of C and Figure 3 The concentration-correlation curve of D was fitted to the K of VX-765. D The value was 11.3 μM, indicating that ginkgolic acid C15:1 has a higher affinity for Caspase-1 compared to VX-765.

[0061] (4) The structure-activity relationship between ginkgolic acid C15:1 and Caspase-1 was studied: Ginkgolic acid C15:0, cardanol C15:0 and salicylic acid are analogs of ginkgolic acid C15:1. The activity of the four compounds was determined by enzyme activity experiment, and the cleavage of GSDMD was detected by Coomassie brilliant blue staining.

[0062] See results Figure 4 It can be seen that, compared with ginkgolic acid C15:1, ginkgolic acid C15:0 has no double bonds on its carbon chain, IC 50 The activity values ​​were 4.959 ± 0.419 μM, indicating that the double bonds had little effect on the activity of ginkgolic acid C15:1. Compared to ginkgolic acid C15:1, cashew phenol C15:0 lacked a carboxyl group on the benzene ring, and salicylic acid lacked a carbon chain on the benzene ring. The IC50 values ​​of the two compounds for Caspase-1 were [not specified in the original text]. 50 All values ​​were greater than 200 μM, and their activities were much lower than those of ginkgolic acid C15:1. The results of Coomassie brilliant blue staining were consistent with the enzyme activity results. This indicates that the activity of ginkgolic acid C15:1 requires the simultaneous presence of both the carboxyl group and the carbon chain on the benzene ring. Furthermore, based on the comparable activities of ginkgolic acid C15:1 and ginkgolic acid C15:0, it is suggested that ginkgolic acid C13:0, ginkgolic acid C17:1, and ginkgolic acid C17:2 also possess the potential to act as Caspase-1 inhibitors.

[0063] (5) Verification of the broad-spectrum activity of ginkgolic acid C15:1 for Caspases: Enzyme activities of Caspase-1, Caspase-4, Caspase-5, Caspase-3, and Caspase-6 were measured using corresponding substrates to investigate the selectivity of ginkgolic acid C15:1 for the Caspase family. In the Caspase-1, Caspase-4, and Caspase-5 enzyme activity systems, the protein concentration was 0.25 μM and the substrate Ac-WEHD-AMC concentration was 100 μM. In the Caspase-3 enzyme activity reaction system, the Caspase-3 concentration was 0.01 μM and the substrate Ac-DEVD-AMC concentration was 50 μM. In the Caspase-6 enzyme activity reaction system, the Caspase-6 concentration was 0.05 μM and the substrate AC-VEID-AMC concentration was 100 μM. Detection was performed immediately after substrate addition, and fluorescence values ​​were recorded within 10 min.

[0064] The results are as follows Figure 5 As shown, the IC50 of ginkgolic acid C15:1 against Caspase-1, Caspase-4, Caspase-5, Caspase-3, and Caspase-6 is... 50 The values ​​were 4.278±0.301μM, 9.722±0.767μM, 10.04±1.168μM, 8.169±0.45μM, and 6.636±0.122μM, respectively. This indicates that ginkgolic acid C15:1 is a broad-spectrum caspase inhibitor.

[0065] Example 2: Evaluation of cellular activity of ginkgolic acid

[0066] (1) Cell viability assay methods:

[0067] (1-1) Extraction of BMDM cells:

[0068] Six- to eight-week-old C57BL / 6J mice were euthanized by dislocation and immersed in 75% ethanol for 3 minutes. Bilateral femurs and tibias were harvested, excess muscle was removed, and the bones were immersed in culture dishes containing 75% ethanol. Pre-chilled PBS containing 2% fetal bovine serum was prepared in a laminar flow hood and poured into the culture dish. The mouse femurs and tibias were then transferred to this dish. The ends of the femurs and tibias were cut off with ophthalmic scissors. A 5 mL syringe was used to draw pre-chilled PBS (containing 2% PBS), and the needle was inserted into the medullary cavity to flush out the bone marrow. The bone marrow was repeatedly pipetted to disperse the cells, and the cells were filtered through a 70 μm sterile filter. The cells were centrifuged at 4°C (300 g, 5 min) to obtain a cell pellet. Red blood cell lysis buffer was added at a ratio of 3 mL per mouse, and the cells were resuspended and lysed on ice for 10 min. Centrifugation at 4°C (300 g, 5 min) was repeated to obtain another cell pellet, and the cells were resuspended in pre-chilled PBS (containing 2% PBS). Centrifuge at 4℃ (300g, 5min) to obtain cell pellet. Resuspend in culture medium (DMEM + 10% FBS + 1% penicillin antibiotics + 20ng / mL M-CSF) and seed into culture dishes (3 10cm dishes for two mice), 10mL of culture medium per dish, and incubate in a CO2 incubator. On the third day, add 5mL of culture medium and continue culturing until the sixth day, when the cells mature and are ready for experiments.

[0069] (1-2) Establishing a BMDM cell pyroptosis model:

[0070] BMDM cells were seeded into 12-well plates and cultured overnight. 500 μL of medium containing 100 ng / mL LPS was added, and the cells were incubated for 3 h. The medium was discarded, and 500 μL of medium containing different concentrations of ginkgolic acid (C15:1) was added, and the cells were incubated for 0.5 h. The medium was then discarded, and medium containing 5 mM ATP was added, and the cells were induced for 0.5 h. Cell supernatant and cell pellet were collected for Western blotting (WB) and ELISA experiments, respectively.

[0071] (1-3) Preparation of WB samples from cell lysates:

[0072] BMDM cells were seeded in 12-well plates at 500,000 cells / well, and experiments were performed on day 6. The culture medium was changed to serum-free medium containing 100 ng / mL LPS (0.5 mL / well), and incubated for 3 h. The original medium was discarded, and the medium was replaced with serum-free medium containing different concentrations of ginkgolic acid C15:1 (0.5 mL / well), and incubated for 30 min. The medium was then discarded, and the medium was replaced with serum-free medium containing 5 mM ATP. After culturing for another 30 min, the supernatant was collected for Western blotting (WB) and ELISA experiments. Cells were washed twice with 1 mL PBS, and 80 μL LRIPA was added directly to the wells to prepare WB samples.

[0073] (1-4) Preparation of WB samples from cell supernatant:

[0074] Western blotting was used to detect the level of Caspase-1p20 in the supernatant. The supernatant was collected and centrifuged at 1000g for 5 min. 500 μL of supernatant was added to 500 μL of methanol and 125 μL of chloroform, vortexed for at least 30 s, and centrifuged (13000g, 5 min). The protein was positioned between the aqueous and organic phases. The aqueous phase was discarded, and 500 μL of methanol was added again. The mixture was vortexed and centrifuged (13000g, 5 min) to form a protein precipitate. The precipitate was dried at 55℃ for 5-10 min, then 40 μL of 1× loading buffer was added. The mixture was vortexed vigorously for 1 min, centrifuged at 13000g for a few seconds, and then heated at 95℃ for denaturation for 5 min. The mixture was then vortexed vigorously for 1 min and cooled on ice for later use.

[0075] (1-5) Western blotting experiment:

[0076] ① Electrophoresis: Use 12% separating gel for cell supernatant and 10% separating gel for cell lysis buffer. Run the gel at 80V constant voltage for 30 minutes, then adjust to 120V and continue running for 50 minutes.

[0077] ② Transfer: Using a 0.45μm PVDF membrane, transfer the membrane at a constant current of 300mA for 40min under ice bath conditions.

[0078] ③ Sealing: 5% skim milk powder, sealed at room temperature for 1 hour. Wash once with TBST;

[0079] ④ Incubation with primary antibody: Dilute the antibody with PBS (1:1000), incubate at room temperature for 30 min, then place in a 4°C refrigerator and incubate overnight. The next day, remove and incubate at room temperature for 30 min.

[0080] ⑤ Washing: TBST washing, 3×10min;

[0081] ⑥ Incubate the secondary antibody: Dilute the antibody with PBS (1:2000) and incubate at room temperature for 1 hour;

[0082] ⑦ Washing membrane: TBST washing, 3×10min;

[0083] ⑧ Development.

[0084] (1-6) ELISA experiment:

[0085] IL-1β (catalog number CSB-E08054m) and IL-18 (catalog number CSB-E04609m) were both purchased from Wuhan Huamei and were used in accordance with the kit instructions.

[0086] (1-7) FLICA Experiment:

[0087] The FAM-FLICA Caspase 1YVAD Assay (catalog number 98) was purchased from immunochemistry and used according to the instructions.

[0088] (2) Ginkgo biloba acid C15:1 inhibits the activation of Caspase-1.

[0089] Western blotting (WB) was used to detect the protein levels of Caspase-1 p45 and p20 in cell supernatant and cell lysate. The antibodies used were Anti-Caspase-1 (p20) (purchased from AdipoGen, catalog number AG-20B-0042-C100, diluted 1:1000) and Anti-Cleaved Caspase-1 (Asp296) (purchased from CST, catalog number 89332, diluted 1:1000). The results are as follows: Figure 6 As shown in A and 6B, in cell lysates and cell supernatants, the normal group cells showed almost no Caspase-1p20, while the model group showed a significant increase in Caspase-1p20 due to LPS and ATP induction. After treatment with Ginkgo biloba extract C15:1, the level of Caspase-1p20 in the cell supernatant was significantly reduced. To further verify the inhibitory effect of Ginkgo biloba extract C15:1 on Caspase-1 activity, the Caspase-1 activity was detected using the FAM-FLICACaspase-1 kit. The results are as follows: Figure 6 As shown in Figure C, the fluorescence of cells treated with 5 μM ginkgolic acid C15:1 was significantly reduced, indicating that ginkgolic acid C15:1 inhibits the activation of Caspase-1 in the BMDM cell pyroptosis model.

[0090] (3) Ginkgo biloba acid C15:1 inhibits the activation of GSDMD.

[0091] GSDMD and GSDMD-N in cell lysates were detected by Western blotting (WB). The antibodies used were Gasdermin D (E9S1X) (purchased from CST, catalog number 39754, dilution 1:1000) and Cleaved Gasdermin D (Asp276) (purchased from CST, catalog number 10137, dilution 1:1000). Results are as follows: Figure 7 As shown, the normal group cells had almost no GSDMD-N, while in the model group, due to the induction of LPS and ATP, a large amount of GSDMD was cleaved by Caspase-1. In the 1.25μM, 2.5μM and 5μM ginkgolic acid C15:1 administration groups, the activation of GSDMD was significantly inhibited.

[0092] (4) Ginkgo biloba extract C15:1 inhibits the production of IL-1β and IL-18 in pyroptosis cells.

[0093] IL-1β and IL-18 were detected by ELISA, and the results were as follows Figure 8 shown. Compared with the model group, the levels of IL-1β and IL-18 in the cell culture medium of the ginkgolic acid C15:1 administration group were significantly reduced and showed a dose-dependent manner, further demonstrating the inhibition of ginkgolic acid C15:1 on pyroptosis.

[0094] Example 3: In vivo activity evaluation of ginkgolic acid

[0095] (1) In vivo activity experimental method:

[0096] Experimental animals: C57BL / 6J mice, 7 weeks old, purchased from Beijing Huafukang Biotech Co., Ltd., animal license: SYXK(Beijing)2019-0003, ethics number: 2023B004, housed in the animal house of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The animals were in a suitable environment with constant temperature and humidity, free to eat and drink, and maintained a 12-hour circadian rhythm. After one week of adaptive feeding, the experiment was carried out.

[0097] Establish a mouse acute lung injury (ALI) model: 1 hour after the mice were administered, the mice were anesthetized with 1% pentobarbital sodium, a retention needle (22G) was inserted into the trachea of the mice, and LPS was infused through the retention needle, so that LPS entered the lungs with the normal breathing of the mice. LPS was dissolved in sterile water to 2.5 mg / mL and modeled at a dose of 5 mg / kg, that is, 25 g mice were given 50 μL LPS. The normal group mice were infused with an equal amount of sterile water. 24 hours after LPS modeling, the mice were sacrificed and samples were taken.

[0098] Grouping and Administration: C57BL / 6J mice were randomly divided into 7 groups of 12 mice each, as follows: Control group, ALI model group, low-dose Ginkgolic Acid C15:1 group L (10 mg / kg), medium-dose Ginkgolic Acid C15:1 group M (20 mg / kg), high-dose Ginkgolic Acid C15:1 group H (30 mg / kg), VX-765 group (50 mg / kg), and dexamethasone group (5 mg / kg). Ginkgolic Acid C15:1 was dissolved in DMSO (10%), PEG300 (40%) was added, vortexed and mixed, Tween-80 (5%) was added, vortexed and mixed, and finally physiological saline (45%) was added, mixed, and administered intraperitoneally. VX-765 was prepared with 20% Cremophor EL solution and administered via intraperitoneal injection. Dexamethasone injection (5 mg / mL) was diluted 10 times with physiological saline and administered via intraperitoneal injection. The medication was administered once daily for 6 consecutive days prior to modeling, with the final dose given on the seventh day, and modeling initiated 1 hour after administration.

[0099] (1-1) Detection of total protein:

[0100] The protein concentration was measured using the BCA protein quantification kit (purchased from Beyotime Biotechnology, catalog number: P0010). The procedure was as follows: 10 μL of bronchoalveolar lavage fluid and 10 μL of PBS were added to each well of a 96-well plate, and standard curve wells were set up according to the manufacturer's instructions. 200 μL of BCA working solution was added to each well, and the plate was incubated at 37°C for 30 min. The absorbance was measured at 570 nm, and the protein concentration of each sample was calculated using the standard curve.

[0101] (1-2) Albumin detection:

[0102] The albumin content was detected using a kit (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number: ml095003). The procedure was as follows: 20 μL of bronchoalveolar lavage fluid was added to each well of a 96-well plate for detection, followed by 200 μL of reagent 1. The mixture was then incubated at 25°C for 1 min. Blank and standard wells were also prepared. The absorbance was measured at 630 nm.

[0103] (1-3) Detection of LDH:

[0104] The detection was performed using a lactate dehydrogenase (LDH) assay kit (purchased from Nanjing Jiancheng Biotechnology, catalog number: A020-2-2). The procedure was as follows: Add 20 μL of bronchoalveolar lavage fluid, 25 μL of matrix buffer, and 5 μL of coenzyme I to each well of a 96-well plate, mix well, and incubate at 37°C for 15 min. Add 25 μL of 2,4-dinitrophenylhydrazine to each well, mix well, and incubate at 37°C for 15 min. Add 250 μL of NaOH, mix well, and incubate at room temperature for 5 min. Measure the absorbance at 450 nm.

[0105] (1-4) HE staining:

[0106] ① Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min in sequence, then wash with water;

[0107] ② Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with water, differentiate with differentiation solution, wash with water, blue back solution, and rinse with running water.

[0108] ③ Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin staining solution for 5 min.

[0109] ④ Dehydration and mounting: The sections are sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min, and then cleared and mounted with neutral resin.

[0110] ⑤ Microscopic examination, image acquisition and analysis.

[0111] (1-5) Immunohistochemistry:

[0112] ① Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with water.

[0113] ② Antigen retrieval: Place tissue sections in a retrieval chamber containing EDTA antigen retrieval buffer (pH 9.0) and microwave on medium heat for 8 minutes until boiling, then turn off the heat and keep warm for 8 minutes, followed by medium-low heat for 10 minutes. During this process, prevent excessive evaporation of the buffer and avoid drying the slides. After natural cooling, place the slides in PBS (pH 7.4) and wash three times on a destaining shaker for 5 minutes each time.

[0114] ③ Blocking endogenous peroxidase: Place the slide in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0115] ④ Serum blocking: Add 3% BSA to the histochemistry zone, cover the tissue evenly, and block at room temperature for 30 minutes;

[0116] ⑤ Incubation with primary antibody: Gently shake off the blocking solution, add primary antibody to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight;

[0117] ⑥ Incubation with secondary antibody: Place the slide in PBS (pH 7.4), wash 3 times on a decolorizing shaker for 5 min each time, add secondary antibody, and incubate at room temperature for 50 min;

[0118] ⑦ DAB staining: Place the slide in PBS (pH 7.4), wash it 3 times on a decolorizing shaker for 5 minutes each time, add freshly prepared DAB staining solution, control the staining time under a microscope, the positive color is brownish-yellow, rinse the slide with tap water to stop the staining.

[0119] ⑧ Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, use hematoxylin blue solution to blue, and rinse with running water.

[0120] ⑨ Dehydration and mounting: Place the sections in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene I for 5 min in sequence to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0121] ⑩ Microscopic examination, image acquisition and analysis.

[0122] (2) Evaluation of the effect of ginkgolic acid C15:1 on acute lung injury in mice:

[0123] HE staining of the lungs and analysis of total protein, albumin, and LDH in bronchoalveolar lavage fluid are commonly used indicators for assessing the degree of lung injury. To demonstrate the ameliorative effect of ginkgolic acid C15:1 on lung injury, HE staining was performed on the lung tissue of mice in each group to examine lung tissue damage. The results are as follows: Figure 9 As shown, the lung tissue of mice in the ALI model group exhibited severe alveolar interstitial edema, alveolar wall thickening, and neutrophil infiltration. Compared with the ALI model group, the low-dose group L, the medium-dose group M, and the high-dose group H all improved the lesions in the lung tissue with ginkgolic acid C15:1. Meanwhile, the results of total protein, albumin, and LDH detection in the bronchoalveolar lavage fluid are as follows... Figure 10 As shown, all three indicators in the model group were significantly elevated. Ginkgo biloba extract (C15:1), VX-765, and dexamethasone significantly reduced the abnormal elevation of the three indicators in the low-dose group (L), medium-dose group (M), and high-dose group (H), showing a dose-dependent effect. Furthermore, the improvement effect of low, medium, and high doses of Ginkgo biloba extract (C15:1) on the three indicators was better than that of dexamethasone, and the high-dose Ginkgo biloba extract (C15:1) was better than that of VX-765.

[0124] (3) Ginkgo biloba acid C15:1 inhibits the activation of Caspase-1 in vivo:

[0125] Western blotting (WB) and immunohistochemistry were used to detect the levels of Caspase-1p45 and Caspase-1p20 in lung tissue and bronchoalveolar lavage fluid. Caspase-1p20 was almost undetectable in the bronchoalveolar lavage fluid of the control group mice, while the Caspase-1p20 protein level was significantly increased in the ALI model group, consistent with the results in the bronchoalveolar lavage fluid. The WB results of the lung tissue (…) Figure 11 ) and immunohistochemical results ( Figure 12 The study also showed high expression of Caspase-1p45 and Caspase-1p20 in the ALI model group mice, indicating that the established ALI model has obvious characteristics of Caspase-1 activation and is suitable for evaluating the activity of Caspase-1 inhibitors. Compared with the ALI model group, the levels of Caspase-1p45 and Caspase-1p20 in the bronchoalveolar lavage fluid and lung tissue of mice in the low-dose group L, medium-dose group M, high-dose group H (ginkgolic acid C15:1) and 50 mg / kg VX-765 administration group were significantly reduced; among them, the activity of ginkgolic acid C15:1 in the high-dose group H was significantly better than that in VX-765.

[0126] (4) Ginkgo biloba acid inhibits the activation of GSDMD:

[0127] Western blotting (WB) was used to detect the levels of GSDMD and GSDMD-N in bronchoalveolar lavage fluid and lung tissue. GSDMD-N was almost undetectable in the bronchoalveolar lavage fluid of the control group mice, while a sharp increase in GSDMD-N was detected in the ALI model group, indicating the presence of significant pyroptosis in the ALI model. Results are as follows... Figure 13 As shown, compared to the ALI model group, lower levels of GSDMD-N were detected in the bronchoalveolar lavage fluid of the low-dose group L, the medium-dose group M, the high-dose group H, the ginkgolic acid C15:1 group, and the VX-765 group. This indicates that ginkgolic acid C15:1 and VX-765 inhibited the activation of GSDMD.

[0128] (5) Ginkgo biloba extract C15:1 inhibits the release of IL-1β and IL-18:

[0129] To further evaluate the inhibitory effect of ginkgolic acid C15:1 on pyroptosis, IL-1β and IL-18 in bronchoalveolar lavage fluid were quantitatively detected by ELISA. The results are as follows: Figure 14As shown, compared with the control group, IL-1β and IL-18 were significantly increased in the ALI model group mice, while Ginkgo biloba extract C15:1, VX-765 and dexamethasone inhibited the production and release of IL-1β and IL-18. Moreover, the inhibitory effect of Ginkgo biloba extract C15:1 on IL-1β and IL-18 in the medium-dose group M and the high-dose group H was significantly better than that of VX-765 and dexamethasone.

[0130] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of ginkgolic acid in the preparation of a drug for treating acute lung injury, characterized in that, The ginkgolic acid is C13:0, C15:0, C15:1, C17:1 or C17:

2.

2. The application according to claim 1, characterized in that, The structural formula of ginkgolic acid C15:1 is as follows: 。 3. The application according to claim 1, characterized in that, The structural formula of ginkgolic acid C17:1 is as follows: 。 4. The application according to claim 1, characterized in that, The structural formula of ginkgolic acid C17:2 is as follows: 。 5. The application according to any one of claims 1-4, characterized in that, The application of ginkgolic acid as an inhibitor of the key target Caspase in inhibiting pyroptosis.

6. The application according to any one of claims 1-4, characterized in that, Ginkgolic acid is a broad-spectrum inhibitor of the key target Caspase.

7. The application according to claim 6, characterized in that, The key target Caspase includes at least one of Caspase-1, Caspase-3, Caspase-4, Caspase-5, and Caspase-6.

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