Method for preparing VA-ECMO lung injury treatment medicine by regulating YARS1 through ginkgolide A

Abstract

The invention discloses a method for preparing a VA-ECMO lung injury treatment medicine by using ginkgolide A to regulate YARS1, and relates to the technical field of biological medicine, the method comprises the following steps: by using ginkgolide A as a YARS1 protein regulator, preparing the medicine for treating the VA-ECMO lung injury through virtual screening, binding affinity confirmation and cell efficacy confirmation; wherein the virtual screening is based on a protein structure model of YARS1, and bilobalide A with high affinity binding energy with YARS1 is screened out through molecular docking. It is proved that ginkgolide A can effectively improve the lung ventilation function by regulating YARS1, repair the alveolar epithelial barrier structure and inhibit the inflammatory oxidative stress reaction, and the lung injury treatment effect and clinical transformation safety under the support of VA-ECMO are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with ginkgolide A. Background Technology

[0002] While providing effective gas exchange, ECMO also performs the heart's pumping function, and is widely used in the treatment of cardiogenic shock, cardiac arrest, and severe mixed respiratory and circulatory failure. With the advancement of medical technology, ECMO is not only used as a short-term "bridge" support method, but its application is also increasing during organ transplant waiting periods and the waiting period for cardiopulmonary function recovery. However, the parallel circulation characteristics of VA-ECMO alter the body's inherent hemodynamic state, leading to a significant increase in left ventricular afterload, which in turn causes or aggravates pulmonary congestion and alveolar damage. This complication severely restricts the patient's final recovery prognosis. Therefore, the prevention and treatment of VA-ECMO-related lung injury has become an important research direction in the field of extracorporeal life support.

[0003] Although strategies such as left ventricular unloading or pharmacological support are commonly used in clinical practice to alleviate hemodynamic disturbances caused by VA-ECMO, specific drug interventions targeting alveolar epithelial cell damage, inflammatory cascades, and oxidative stress remain relatively scarce. Traditional broad-spectrum anti-inflammatory or antioxidant therapies often lack precise molecular targets, making it difficult to achieve effective lung protection under complex mechanical circulatory support conditions. There is an urgent need to identify drug targets and corresponding candidate molecules that can precisely intervene in the pathological process of lung injury caused by VA-ECMO, in order to address problems such as pulmonary congestion, edema, and barrier function disruption due to increased afterload. Therefore, establishing a modifiable molecular target closely related to this pathological process and screening for modulators with high affinity and specificity is key to overcoming current technological bottlenecks and improving the safety of ECMO treatment. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A, which solves the problem in the prior art that it is difficult to effectively alleviate alveolar epithelial cell damage, inflammatory cascade and oxidative stress through specific drug intervention due to the lack of precise molecular targets for VA-ECMO-induced lung injury.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a VA-ECMO lung injury treatment drug by regulating YARS1 with ginkgolide A, comprising, Using ginkgolide A as a regulator of YARS1 protein, a drug for treating lung injury caused by VA-ECMO was prepared through virtual screening, binding affinity confirmation, and cellular efficacy confirmation. The virtual screening was based on the protein structure model of YARS1, and ginkgolide A with high affinity binding energy to YARS1 was screened through molecular docking.

[0007] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A according to the present invention, the specific steps of the virtual screening include: Construct a compound library containing natural products; Potential binding pockets were identified based on the crystal structure of YARS1, and blind screening docking was performed using AutoDock Vina software. Candidate molecules were screened based on binding energy scoring thresholds, and ginkgolide A was selected through conformational visualization.

[0008] Furthermore, a library of natural product compounds with structural diversity and drug-like properties was constructed to provide a rich chemical space for subsequent discovery of lead compounds. Secondly, based on the high-resolution crystal structure of the YARS1 protein, computational chemistry methods were used to determine its potential active binding pockets on its surface. Blind docking of the entire protein surface was then performed using AutoDock Vina molecular docking software to comprehensively explore the interaction possibilities between small molecules and target proteins without pre-setting binding sites. Finally, a screening threshold was set based on binding affinity to initially screen high-affinity candidate molecules. The binding conformations were then visualized and analyzed using specialized molecular graphics software to examine key interactions (such as hydrogen bonds and hydrophobic interactions), ultimately leading to the selection of the most promising natural product—ginkgolide A.

[0009] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A according to the present invention, in the preparation process, the binding relationship between Ginkgolide A and YARS1 is biophysically confirmed using surface plasmon resonance technology; specifically, recombinant rat YARS1 protein is immobilized on the surface of a CM5 sensor chip, and the binding kinetic parameters and equilibrium dissociation constant of Ginkgolide A and YARS1 are measured using a Biacore 8K instrument.

[0010] Furthermore, during the preparation process, surface plasmon resonance (SPR) technology was used to conduct label-free, real-time, and dynamic biophysical confirmation of the binding relationship between ginkgolide A and YARS1. Specifically, using the standard amine coupling method, high-purity recombinant rat YARS1 protein hydrolysis domains were stably immobilized on the surface of a CM5 sensor chip as ligands to construct a protein functionalized chip. Subsequently, using a Biacore 8K high-precision instrument, ginkgolide A at different concentration gradients was passed through the chip surface as analytical streams, and the changes in response signals during the binding and dissociation processes were monitored in real time. By fitting the sensor data to a 1:1 Langmuir adsorption model, the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) between ginkgolide A and YARS1 were accurately calculated, providing quantitative evidence of a direct and specific interaction between the two at the molecular level, confirming the feasibility of ginkgolide A as a YARS1 modulator.

[0011] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in this invention, the cellular efficacy confirmation is achieved by using an oxygen-glucose deprivation model to simulate an ischemic hypoxic environment and verifying the protective effect of Ginkgolide A on alveolar epithelial cells; specifically, A549 cells are placed in a glucose-free culture medium and a 1% O2 hypoxic environment to establish an OGD model, and Ginkgolide A at a concentration of 5-20 μM is used for intervention, preferably at a concentration of 15 μM.

[0012] Furthermore, the cellular efficacy was confirmed using an oxygen-glucose deprivation (OGD) model to simulate the ischemic and hypoxic microenvironment supported by VA-ECMO in vitro, thereby verifying the direct protective effect of ginkgolide A on alveolar epithelial cells. Specifically, human alveolar basal epithelial cell line (A549) was selected as the research subject. Normal culture medium was discarded and replaced with glucose-free culture medium, and the cells were placed in a hypoxic incubator containing 1% O2 to establish an OGD cell model simulating ischemic and hypoxic injury. Based on this, ginkgolide A was used to treat the cells at concentration gradients of 5-20 μM, and cell viability was detected using methods such as the CCK-8 assay. The experimental results showed that ginkgolide A significantly improved the cell survival rate after OGD injury, exhibiting a certain dose-dependent effect. The optimal concentration of 15 μM showed the best cell protective effect, providing effective in vitro data support for subsequent in vivo experiments.

[0013] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A according to the present invention, wherein: the lung injury caused by VA-ECMO is pulmonary congestion and pulmonary edema caused by increased left ventricular afterload; the method of applying the drug includes prophylactic administration before or within 0-2 hours after VA-ECMO initiation, or use in parallel with a left ventricular unloading strategy.

[0014] Furthermore, the lung injury caused by VA-ECMO is mainly due to the increased left ventricular afterload caused by the parallel circulation of VA-ECMO, which in turn leads to pulmonary congestion, increased pulmonary capillary hydrostatic pressure, and pulmonary edema. Therefore, the administration of the drug includes prophylactic administration within the "golden window" of 0-2 hours before or after VA-ECMO initiation, aiming to intervene at the early stage of lung injury and block or delay the pathological process. In addition, the drug can also be used in parallel with clinical left ventricular unloading strategies (such as IABP, Impella, etc.) to form a synergistic treatment plan of "mechanical assistance + pharmacological protection," which reduces lung injury from both hemodynamic and molecular biological perspectives and improves the overall treatment effect.

[0015] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drug by regulating YARS1 with Ginkgolide A according to the present invention, the drug is an injectable preparation administered via intravenous injection; the effective concentration in vitro is 1-30 μM, and the in vivo dose in animals is 0.1-50 mg / kg, with a preferred dose of 10 mg / kg.

[0016] Furthermore, to meet the clinical dosing needs of critically ill patients, the drug is formulated into injectable preparations suitable for intravenous administration, such as lyophilized powder injections or solutions, to ensure rapid and stable entry into the bloodstream. Regarding dosage, based on the effective dose range from in vitro cell experiments and subsequent animal studies, the effective concentration in in vitro cell culture is determined to be 1-30 μM, while the effective dose range in in vivo animal experiments is 0.1-50 mg / kg. Considering both efficacy and safety, the preferred dose is 10 mg / kg. This dose ensures therapeutic efficacy while minimizing potential toxic side effects, providing an important dosimetric reference for future clinical translation.

[0017] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drug by regulating YARS1 with Ginkgolide A according to the present invention, the preparation method further includes constructing a VA-ECMO combined with TAC (transverse aortic coarctation) rat model for in vivo efficacy verification; specifically: establishing a rat VA-ECMO system and placing a 1.4 mm constriction ring at the aortic arch to simulate the increased left ventricular afterload state under clinical VA-ECMO support, followed by treatment with Ginkgolide A.

[0018] Furthermore, the preparation method also includes constructing a rat model of VA-ECMO combined with TAC (Transverse Aortic Constriction) that highly simulates clinical pathological conditions for in vivo efficacy verification. Specifically, a rat VA-ECMO circulatory support system is established surgically to simulate the operation of clinical ECMO. To accurately reproduce the key pathological link of increased left ventricular afterload unique to VA-ECMO, a constriction ring with an inner diameter of 1.4 mm is placed between the second and third branches of the aortic arch to artificially create aortic stenosis, thereby simulating the pathological state of increased left ventricular ejection resistance and elevated pulmonary circulation pressure under ECMO support. After the model was successfully constructed, ginkgolide A was immediately administered to comprehensively evaluate its protective effect against VA-ECMO-related lung injury under complex in vivo conditions.

[0019] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A according to the present invention, wherein: in the in vivo efficacy verification, a multi-dimensional efficacy evaluation system is used to assess the drug effect, including: Functional indicator: Monitors improvements in arterial partial pressure of oxygen (PaO2); Structural indicators: H&E staining was used to observe the decrease in lung tissue pathological scores and TEM was used to observe the recovery of the ultrastructure of type II alveolar epithelial cells; Molecular markers: Detect changes in the levels of alveolar epithelial injury markers SP-D, inflammatory factors IL-1β and IL-6, and oxidative stress markers MPO and MDA in plasma or lung tissue.

[0020] Furthermore, in the in vivo efficacy validation, a multi-dimensional efficacy evaluation system covering functional, structural, and molecular levels was employed to comprehensively and objectively assess the drug's effects. At the functional level, changes in arterial oxygen partial pressure (PaO2) were monitored through arterial blood gas analysis, directly reflecting the improvement in pulmonary gas exchange function. At the structural level, H&E staining was used to perform pathological scoring of lung tissue, and transmission electron microscopy (TEM) was used to observe the morphological recovery of type II alveolar epithelial cells at the ultrastructural level. At the molecular level, techniques such as ELISA and Western blotting were used to systematically detect changes in the levels of alveolar epithelial injury markers SP-D, pro-inflammatory factors IL-1β and IL-6, and oxidative stress indicators MPO and MDA in plasma or lung tissue, elucidating the anti-inflammatory, antioxidant, and cytoprotective effects of the drug from a molecular mechanism perspective.

[0021] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in this invention, wherein: in the multidimensional efficacy evaluation system, the reduction of SP-D level is used as a specific indicator for evaluating the repair of alveolar epithelial barrier structure.

[0022] Furthermore, within the aforementioned multidimensional efficacy evaluation system, the reduction in SP-D (alveolar surfactant protein D) levels is specifically used as a specific indicator for evaluating the repair of the alveolar epithelial barrier structure. SP-D is primarily secreted by type II alveolar epithelial cells. When alveolar epithelial cells are damaged, large amounts of SP-D are released into the bloodstream. Therefore, its plasma concentration is a highly sensitive and specific biomarker reflecting the integrity of the alveolar-capillary barrier. Monitoring a significant decrease in SP-D levels directly demonstrates that the therapeutic effect of Ginkgolide A is not merely limited to improving gas exchange, but extends to the fundamental level of repairing alveolar epithelial cell structure and maintaining the integrity of the lung barrier, providing strong molecular evidence for the drug's efficacy.

[0023] As a preferred embodiment of the method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in this invention, the drug prepared by the method establishes YARS1 as an interventional molecular target for VA-ECMO afterload-related lung injury during the preparation process, expanding the new applications of the aminoacyl-tRNA synthase family in mechanotransduction and inflammation regulation.

[0024] Furthermore, the drug prepared by the aforementioned method not only validated the efficacy of a single drug during its preparation process, but more importantly, through a complete research chain of "virtual screening - biophysical confirmation - in vivo and in vitro efficacy verification," it established YARS1 as an interventional molecular target for VA-ECMO afterload-related lung injury for the first time. This discovery expands the traditional understanding of the aminoacyl-tRNA synthase family, revealing that in addition to its classic protein synthesis role, it also participates in regulating non-classical functions such as mechanotransduction, inflammatory response, and oxidative stress. This provides a new perspective for understanding the molecular mechanisms of ECMO-related complications and opens up a completely new technical path for developing novel lung-protective drugs targeting this pathway, possessing significant scientific importance and clinical application prospects.

[0025] The beneficial effects of this invention are as follows: Through virtual screening based on the YARS1 protein structure model and surface plasmon resonance biophysical confirmation, the high affinity binding relationship between ginkgolide A and the YARS1 target was accurately identified and verified from the natural product library, establishing its molecular basis as a specific regulator and solving the problem of lack of precise drug targets in the existing VA-ECMO lung injury treatment; combined with the oxygen-glucose deprivation cell model and the VA-ECMO combined with TAC animal model verification steps simulating increased left ventricular afterload, multi-dimensional efficacy evaluation was achieved under the simulated complex hemodynamic environment of clinical practice, confirming that ginkgolide A can effectively improve lung gas exchange function, repair alveolar epithelial barrier structure and inhibit inflammatory oxidative stress response by regulating YARS1, thereby improving the treatment effect and clinical translation safety of lung injury under VA-ECMO support. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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.

[0027] Figure 1 This is a docking model between YARS1 and Ginkgolide A.

[0028] Figure 2 This diagram illustrates the protective effect of Ginkgolide A in the OGD hypoxic-ischemic A549 cell model.

[0029] Figure 3 This is a schematic diagram illustrating the effect of Ginkgolide A on the partial pressure of arterial oxygen in VA-TAC-AM rats.

[0030] Figure 4This is a schematic diagram illustrating the effect of Ginkgolide A on lung tissue damage in VA-TAC-AM rats.

[0031] Figure 5 This is a schematic diagram illustrating the effect of Ginkgolide A on MPO in VA-TAC-AM rats.

[0032] Figure 6 The effect of Ginkgolide A on IL-6 in VA-TAC-AM rats. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Reference Figures 1-6 Example 1: Verification of the protective effect of ginkgolide A in an OGD cell model This embodiment aims to verify the protective effect of ginkgolide A on hypoxic-ischemic A549 cells (human alveolar epithelial cells) and its optimal dose window through in vitro cell experiments.

[0037] 1. Cell preparation Cell line: A549 cells (human alveolar epithelial cells).

[0038] Inoculation: Dilute the cell suspension with complete culture medium containing 10% fetal bovine serum (FBS) and inoculate into 96-well plates.

[0039] Pre-culture: Incubate overnight in a 37℃, 5% CO2 cell culture incubator to allow cells to adhere and grow.

[0040] 2. Model Building (OGD Processing) Washing: Discard the original complete culture medium and wash the cells once with room temperature PBS.

[0041] Modeling: The experimental group was replaced with sugar-free culture medium and placed in a hypoxic cell culture incubator (simulating a hypoxic environment) for 2 hours.

[0042] Control group: The control group was replaced with fresh complete culture medium and placed back into a 37℃, 5% CO2 incubator for continued incubation.

[0043] 3. Drug intervention Drug administration: While performing oxygen-glucose deprivation treatment, different concentrations of ginkgolide A were added to the cells of the experimental group.

[0044] Concentration gradient: Set to 5, 10, 15, 20 Incubation was performed using four concentration gradients (M).

[0045] Control group: No drug or equal amount of solvent added to the control group.

[0046] 4. Result Detection Detection method: CCK-8 assay.

[0047] Indicator: Calculate cell survival rate under different modeling conditions and different drug concentrations.

[0048] Conclusion: The optimal effective concentration of ginkgolide A was determined (preferred concentration mentioned in the document is 15%). M).

[0049] Example 2: Establishment and efficacy verification of VA-ECMO rat model This embodiment aims to verify the lung-protective effect of ginkgolide A in vivo by establishing a simulated VA-ECMO post-load increase SD rat model.

[0050] 1. Laboratory animals and grouping Animals: 8-10 weeks old male SD rats, weighing 300-350 g.

[0051] Preoperative preparation: Fasting for 12 hours and abstaining from water for 4 hours before surgery.

[0052] 2. Anesthesia and intubation Anesthesia: Induction anesthesia with 5% isoflurane for 3 minutes, then fixation to the operating table; endotracheal intubation and connection to a ventilator, with anesthesia maintained by 2% isoflurane.

[0053] Respiratory parameters: respiratory rate 70-75 breaths / min, tidal volume 6-8 ml / kg.

[0054] 3. Surgery and Catheter Placement Skin preparation and disinfection: Surgical preparation of the surgical site.

[0055] Arterial catheterization: The right common carotid artery was freed, and a 22 G intravenous catheter was inserted to connect to the ECMO arterial perfusion end.

[0056] Intravenous catheterization: The right jugular vein is freed, surgically cut open, and a venous drainage tube is inserted (the tip reaches the level of the inferior vena cava entering the right atrium), and connected to the ECMO venous drainage end.

[0057] Fixation: All blood vessels are ligated distally and fixed proximally with silk sutures.

[0058] 4. ECMO system establishment and TAC modeling Pre-filling: The ECMO system (peristaltic pump + silicone tubing + specially made rat membrane lung) was pre-filled with 15ml of 6% hydroxyethyl starch and 5ml of 5% sodium bicarbonate injection without blood.

[0059] Operating parameters: Flow rate: 10–15 ml / (kg·min).

[0060] Gas source: a mixture of oxygen and nitrogen, with a flow rate of 1 L / min.

[0061] Time: 4 hours of operation.

[0062] Afterload Increase (TAC): A circumduction (TAC) is performed at the second and third branches of the aortic arch, with the degree of circumduction controlled at 1.4 mm, to simulate the left ventricular afterload increase and pulmonary congestion caused by VA-ECMO.

[0063] 5. Drug intervention and detection Administration: Ginkgolide A was administered during model establishment (the document mentions a preferred dose of 10 mg / kg, administered intravenously).

[0064] Monitoring: Monitor mean arterial pressure and heart rate in real time, and administer vasoactive drugs if necessary.

[0065] Endpoint Indicator: Blood gas analysis: measures arterial oxygen partial pressure (PaO2).

[0066] Histopathology: Lung tissue was taken for H&E staining and scoring, and ultrastructure was observed by transmission electron microscopy (TEM).

[0067] Biochemical indicators: detection of MPO (myeloperoxidase), IL-6 (interleukin-6), SP-D (surfactant protein D), etc.

[0068] Example 3: Detection and molecular docking of the YARS1 target This embodiment aims to demonstrate at the molecular level that the target of ginkgolide A is YARS1 and to confirm its binding ability.

[0069] 1. In-Silico Screening Database: The Natural Product Compound Library (3057 compounds) was downloaded from the Selleck China website, and 1989 compounds were obtained after screening according to the five rules of drug-likeness.

[0070] Software and tools: AutoDock Vina software, AutoDock Tools (ADT).

[0071] process: Receptor preparation: Water molecules, ligands and ions were removed from the structure of the YARS1 protein (PDB ID: A0A0S2Z4R1), polar hydrogen was added, and Kollman charge was calculated.

[0072] Ligand preparation: Save the ligands in PDBQT format.

[0073] Molecular docking: A blind screening method is used, with the docking box encapsulating the entire protein. Docking parameters are generated based on an internal script.

[0074] Results analysis: Based on docking scores (binding energy), the Top N candidate molecules were screened, and Ginkgolide A was ultimately selected as the best candidate.

[0075] 2. Combining affinity verification (SPR - surface plasmon resonance) Instrument: Biacore 8K (GE Healthcare).

[0076] Immobilization: The hydrolytic domain of recombinant rat YARS1 protein was immobilized on the surface of the CM5 sensor chip (amine coupling method).

[0077] Buffer solution: HBS-N buffer (containing 0.05% Tween-20).

[0078] Testing conditions: Temperature: 25℃.

[0079] Flow rate: 30 L / min.

[0080] Binding / dissociation time: 120 seconds for injection, 150 seconds for dissociation.

[0081] Sample processing: Analyte: Ginkgolide A was serially diluted twofold to a concentration range of 0.39 mM to 25 mM.

[0082] DMSO control: The buffer solution contains 2% DMSO, and the effect of DMSO is eliminated through solvent correction.

[0083] Data analysis: A 1:1 binding kinetic model was fitted using Biacore Evaluation Software to calculate the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD).

[0084] In summary, this invention, through virtual screening based on the YARS1 protein structure model and surface plasmon resonance biophysical confirmation, accurately identified and verified the high-affinity binding relationship between ginkgolide A and the YARS1 target from a natural product library, establishing its molecular basis as a specific regulator and solving the problem of lack of precise drug targets in existing VA-ECMO lung injury treatments. Combining the oxygen-glucose deprivation cell model and the VA-ECMO combined with TAC animal model simulating increased left ventricular afterload, this invention achieved multi-dimensional efficacy evaluation under a simulated complex clinical hemodynamic environment, demonstrating that ginkgolide A can effectively improve lung gas exchange function, repair alveolar epithelial barrier structure, and inhibit inflammatory oxidative stress response by regulating YARS1, thereby improving the therapeutic effect and clinical translation safety of lung injury under VA-ECMO support.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a VA-ECMO lung injury treatment drug by regulating YARS1 with Ginkgolide A, characterized in that: This includes using ginkgolide A as a regulator of YARS1 protein, and preparing a drug for treating lung injury caused by VA-ECMO through virtual screening, binding affinity confirmation, and cellular efficacy confirmation; wherein the virtual screening is based on the protein structure model of YARS1, and ginkgolide A with high affinity binding energy to YARS1 is screened through molecular docking.

2. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 1, characterized in that: The specific steps of the virtual screening include: Construct a compound library containing natural products; Potential binding pockets were identified based on the crystal structure of YARS1, and blind screening docking was performed using AutoDock Vina software. Candidate molecules were screened based on binding energy scoring thresholds, and ginkgolide A was selected through conformational visualization.

3. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 2, characterized in that: During the preparation process, the binding relationship between ginkgolide A and YARS1 was biophysically confirmed using surface plasmon resonance technology. Specifically, recombinant rat YARS1 protein was immobilized on the surface of a CM5 sensor chip, and the binding kinetic parameters and equilibrium dissociation constant of ginkgolide A and YARS1 were measured using a Biacore 8K instrument.

4. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 3, characterized in that: The cell efficacy confirmation was achieved by using an oxygen-glucose deprivation model to simulate an ischemic hypoxic environment and verify the protective effect of ginkgolide A on alveolar epithelial cells. Specifically, A549 cells were placed in a glucose-free culture medium and a 1% O2 hypoxic environment to establish an OGD model, and ginkgolide A at a concentration of 5-20 μM was used for intervention, with a preferred concentration of 15 μM.

5. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 4, characterized in that: The lung injury caused by VA-ECMO is pulmonary congestion and pulmonary edema caused by increased left ventricular afterload; the administration of the drug includes prophylactic administration before or within 0-2 hours after VA-ECMO initiation, or in parallel with a left ventricular unloading strategy.

6. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 5, characterized in that: The drug is an injectable formulation, administered via intravenous injection; the effective concentration in vitro is 1-30 μM, and the in vivo dose in animals is 0.1-50 mg / kg, with a preferred dose of 10 mg / kg.

7. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 6, characterized in that: The preparation method also includes constructing a rat model of VA-ECMO combined with TAC (transverse aortic coarctation) for in vivo efficacy verification; specifically: establishing a rat VA-ECMO system and placing a 1.4 mm constriction ring at the aortic arch to simulate the increased left ventricular afterload state under clinical VA-ECMO support, followed by treatment with ginkgolide A.

8. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 7, characterized in that: In the in vivo efficacy validation, a multi-dimensional efficacy evaluation system was used to assess the drug's effectiveness, including: Functional indicator: Monitors improvements in arterial partial pressure of oxygen (PaO2); Structural indicators: H&E staining was used to observe the decrease in lung tissue pathological scores and TEM was used to observe the recovery of the ultrastructure of type II alveolar epithelial cells; Molecular markers: Detect changes in the levels of alveolar epithelial injury markers SP-D, inflammatory factors IL-1β and IL-6, and oxidative stress markers MPO and MDA in plasma or lung tissue.

9. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 8, characterized in that: In the aforementioned multidimensional efficacy evaluation system, the reduction of SP-D level is used as a specific indicator for evaluating the repair of alveolar epithelial barrier structure.

10. The method for preparing VA-ECMO lung injury treatment drugs by regulating YARS1 with Ginkgolide A as described in claim 9, characterized in that: The drug prepared by the method establishes YARS1 as an interventional molecular target for VA-ECMO afterload-related lung injury during the preparation process, expanding the new applications of the aminoacyl-tRNA synthetase family in mechanotransduction and inflammation regulation.

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