Application of Noregenin in treatment of acute lung injury or acute respiratory distress syndrome

Abstract

The invention relates to the field of medicines, in particular to application of Noregenin to treatment of acute lung injury or acute respiratory distress syndrome. According to the application disclosed by the invention, NLRP3 inflammasome activation and downstream inflammatory factor release are remarkably reduced by utilizing an inhibition effect of Noregenin on Nur77, so that lung inflammatory response, macrophage proinflammatory polarization and tissue damage in acute lung injury and acute respiratory distress syndrome are effectively relieved; and the pharmaceutical composition shows a definite lung protection effect in various dosage forms and administration modes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of Noreugenin in the treatment of acute lung injury or acute respiratory distress syndrome. BACKGROUND

[0002] Acute lung injury (ALI) is characterized by diffuse alveolar damage, leading to excessive lung inflammation and alveolar epithelial cell apoptosis, and can develop into more severe acute respiratory distress syndrome, showing respiratory distress and refractory hypoxemia, and lung imaging shows heterogeneous exudative lesions. A study covering ICU patients in 50 countries found that the prevalence of ALI / ARDS in hospitalized ICU patients was about 10.4%, and the total mortality of severe ARDS patients was as high as 46.1%. Previous studies on ALI / ARDS patients found that there was excessive neutrophil infiltration and pro-inflammatory cytokine secretion in lung tissue, and macrophages participated in mediating endothelial and epithelial barriers, but its pathogenesis, risk factors, diagnostic markers and potential therapeutic targets still need further research to be clear.

[0003] Nur77 (also known as NR4A1) is an immediate early gene and an orphan nuclear receptor, which is known to participate in the regulation of inflammatory response by activating the NF-κB signaling pathway, and to regulate the inflammatory response of macrophages. In addition, studies have shown that Nur77 responds to disease-related inflammation and other biological processes, and can regulate the function of target proteins by changing subcellular localization. Among its target points, Nur77 in macrophages can recognize pathogen-associated molecular patterns (PAMPs) - lipopolysaccharide (LPS), thereby activating the non-canonical NLRP3 inflammasome signaling pathway, which is also considered a potential target for the treatment of inflammatory diseases such as sepsis and ALI / ARDS.

[0004] NLRP3 inflammasome is an intracellular multi-protein complex composed of NLRP3, ASC and Caspase-1, and is the core executor of the body's natural immune defense. During the development of ARDS, various dangerous signals (such as LPS, ATP, etc.) can lead to excessive activation of NLRP3 inflammasome, thereby driving the self-cleavage of Caspase-1 and the activation of potent pro-inflammatory factors such as interleukin-1β (IL-1β) and IL-18, inducing "cytokine storm", and ultimately leading to extensive damage to alveolar epithelium and vascular endothelium and pulmonary edema Noreugenin is a natural small molecule compound containing a flavone nucleus structure, which has antioxidant and preliminary anti-inflammatory activity. Studies have shown that Noreugenin can effectively scavenge free radicals and show inhibition of nitric oxide production in a simple cell model. Using 2-methyl-5,7-dihydroxyflavone treatment can alleviate the oxidative stress damage caused by some stimulants to a certain extent. In addition, studies have shown that the compound has micromolar level inhibitory activity on targets such as acetylcholinesterase, suggesting its potential research value in the field of nervous system diseases. SUMMARY

[0005] The present application covers the following technical solutions: One aspect of the present application relates to the non-therapeutic and diagnostic use of Noreugenin as a Nur77 protein inhibitor.

[0006] Another aspect of the present application relates to the use of Noreugenin in the preparation of a medicament for treating acute lung injury or acute respiratory distress syndrome.

[0007] By utilizing the inhibitory effect of Noreugenin on Nur77, the present application achieves a significant down-regulation of NLRP3 inflammasome activation and the release of its downstream inflammatory factors, thereby effectively reducing lung inflammation, macrophage pro-inflammatory polarization and tissue damage in acute lung injury and acute respiratory distress syndrome, and showing clear lung protection in various dosage forms and administration methods. BRIEF DESCRIPTION OF DRAWINGS

[0008] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed in the specific embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0009] Figure 1 Molecular docking and fluorescence titration experiments were used to verify the targeting of NRG to Nur77. A is a schematic diagram of molecular docking; B is the nonlinear curve fitted by fluorescence spectrophotometry test of Nur77 and EJF-06; C is the graph of the curve after each titration; the abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity (a.u.); D is the RT-qPCR experiment to verify the targeting of NRG to Nur77 (n=3); E is the WB experiment to verify the targeting of NRG to Nur77 (n=3). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0010] Figure 2 : Effect of NRG on cell viability and LPS-induced pro-inflammatory phenotype of macrophages. A, effect of NRG on cell viability of MH-S (n=4); B-G, RT-qPCR experiments to verify the effect of NRG on the expression level of inflammation-related genes induced by LPS (n=3); H-P, flow cytometry to verify the effect of NRG on the change of pro-inflammatory polarization phenotype induced by LPS (n=3). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0011] Figure 3 : Effect of Noreugenin on cell injury induced by LPS. WB experiments to detect the inhibitory effect of Noreugenin on the activation of NLRP3 inflammatory signaling pathway induced by LPS (n=3).

[0012] Figure 4 : Therapeutic effect of NRG on LPS-induced acute lung injury mice. A, schematic diagram of drug administration and modeling of experimental mice; B-D, RT-qPCR experiments to verify the expression level of inflammation-related genes in the lung tissue of mice (n=5); E-F, ELISA experiments to detect the levels of IL-6 and TNF-a in the BALF supernatant of mice (n=3); G, BCA experiment to detect the protein level in the BALF supernatant of mice (n=3); H-I, flow cytometry to detect the pro-inflammatory polarization phenotype of macrophages in the lung tissue of mice (n=3); J-K, HE staining and lung injury score of the lung tissue of mice (n=5). Figure 4 ** p<0.01, *** p<0.001, **** p<0.0001.

[0013] Figure 5 : Schematic diagram of the signal pathway and mechanism of action involved in the drug of the present application. DETAILED DESCRIPTION

[0014] Reference will now be made in detail to the embodiments of the present application, one or more examples of which are set forth below. Each example is provided as an explanation and not as a limitation of the present application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0015] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and experimental procedures of organic chemistry, protein and nucleic acid chemistry, molecular biology, and related fields are those generally used by those skilled in the respective fields. In addition, the definitions and explanations of the relevant terms are provided below for better understanding of the present application.

[0016] The alternative of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or all related listed items.

[0017] The terms "containing", "including", and "comprising" used in the present application are synonymous and are inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

[0018] The numerical ranges in the present application expressed in endpoints include all numbers and fractions subsumed within the range, and the recited endpoints.

[0019] The term "about" or "approximately" used in the present application means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It also includes the specific number, for example, about 20 includes 20.

[0020] Further, in describing representative embodiments of the present application, the specification can have presented the method and / or procedures of the present application as a particular sequence of steps. However, to the extent that the method or procedures depends on the performance of such steps in the sequence presented, the method or procedures should not be limited to the specific order of steps described. Other sequences of steps can also be possible, and are intended to be subsumed within the scope of the present application. Therefore, the particular order of steps presented in the specification should not be construed as a limitation on the claims. In addition, the claims of the present application should not be limited to the steps of the methods and / or procedures in their written order of execution, and one of ordinary skill in the art will readily recognize that the sequences can be varied and still remain within the spirit and scope of the present application.

[0021] In the present application, the concentration values involved include fluctuations within a certain range.

[0022] As used in the present application, the singular forms "a", "an" and "the" include plural referents unless otherwise specified.

[0023] In the present application, the descriptions of "a plurality of", "a plurality of kinds", and the like, if not specifically limited, refer to greater than or equal to 2 in number.

[0024] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0025] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0026] In this invention, "Noreugenin" (also known as 2-methyl-5,7-dihydroxychromone) refers to a natural small molecule compound belonging to the chromone class, with the molecular formula C0. 10 H8O4, CAS Registry Number: 1013-69-0; can exist in free form, crystalline form, amorphous form, pharmaceutically acceptable salt, solvate, eutectic or mixture thereof, unless otherwise specified, and is included in the Noreugenin described in this invention.

[0027] In this invention, "Nur77" refers to Nuclear Receptor Subfamily 4 Group A Member 1 (also known as NR4A1, NGFI-B, or TR3), a ligand-dependent transcription factor that participates in various biological processes such as inflammatory responses, apoptosis, and metabolic regulation. The Nur77 described in this invention includes its naturally occurring amino acid sequence, functionally equivalent variants, homologs, splice isoforms, and recombinant protein forms usable in vitro, unless otherwise specified, all of which are included in the term "Nur77".

[0028] In this invention, "inhibitor" refers to a compound, component, or molecular entity that can directly or indirectly reduce the activity, function, or related signaling pathway level of a target protein (e.g., Nur77) under in vitro conditions. This includes, but is not limited to, any substance that reduces activity by binding to the target protein, interfering with its conformation, blocking its interaction with ligands or substrates, reducing its upstream activation signals, or weakening its downstream effects. The degree of inhibition can be partial or complete, and is not limited to a specific inhibitory mechanism or site of action.

[0029] In this invention, "treatment" refers to any measure that prevents, delays, inhibits, improves, alleviates, or partially or completely relieves a subject's disease or pathological state, including reducing the risk of disease occurrence, preventing disease progression, reducing the severity of disease, improving disease symptoms, or preventing disease from causing further tissue damage.

[0030] In this invention, "acute lung injury" (ALI) refers to a pathological state caused by factors such as infection, toxic exposure, excessive release of inflammatory factors, diffuse alveolar damage, impaired gas exchange, lung tissue inflammation and edema, including but not limited to acute inflammatory lung damage induced by endotoxins, pathogens, smoke or other damaging stimuli.

[0031] In this invention, "acute respiratory distress syndrome" (ARDS) is a severe form of acute lung injury, characterized by a more severe inflammatory response, increased alveolar-capillary barrier permeability, diffuse pulmonary edema, and significant oxygenation impairment. The ARDS described in this invention is not limited to specific triggers, but includes various types of acute inflammatory lung injury caused by infectious or non-infectious factors.

[0032] The first aspect of the invention relates to the use of Noreugenin as an inhibitor of the Nur77 protein, particularly for non-therapeutic and diagnostic purposes.

[0033] In this invention, Noreugenin is defined as an inhibitor capable of reducing the activity or functional level of Nur77 protein under in vitro conditions. Its core non-therapeutic use lies in its application for in vitro analysis, detection, and pathway studies related to Nur77. Noreugenin possesses a well-defined chemical structure and molecular formula, and can be used in its purified form, crystalline form, amorphous form, or pharmaceutically acceptable salts and solvates. Those skilled in the art can apply it under routine biochemical experimental conditions to in vitro systems containing mammalian, such as human or mouse, Nur77, to conduct research related to Nur77 inhibition by observing changes in Nur77 activity or interactions. In these applications, Noreugenin can be directly added to in vitro reaction systems containing recombinant Nur77 protein, cell lysates, or constructed signaling pathway models. Utilizing its ability to bind to or inhibit Nur77 activity, it induces detectable changes in Nur77-mediated downstream signals, thereby enabling in vitro detection, mechanism analysis, or use as a standard inhibitor.

[0034] The non-therapeutic use of this invention enables direct intervention on the activity of Nur77 protein under in vitro conditions, resulting in detectable regulation of Nur77-mediated signaling pathway responses. This provides a clear, reliable, and reproducible chemical tool for Nur77-related protein interaction studies, inhibitor screening, pathway construction, and downstream molecular change analysis, and offers an important means for in-depth research on Nur77-related biological mechanisms.

[0035] In some specific implementations, the use is selected from at least one of the following: i) Active ingredients used for in vitro detection of changes in Nur77 activity; ii) Active ingredients used for screening drugs that regulate the Nur77 pathway; iii) Active ingredients used to evaluate the interaction between Nur77 and its ligands; iv) The active ingredient used to establish an in vitro regulatory model of the Nur77 signaling pathway; v) Used to prepare active ingredients for in vitro detection of downstream molecular changes of Nur77; vi) Standard control active ingredient used in Nur77 ligand competition experiments; vii) An in vitro analytical reagent used to assess changes in inflammatory signaling pathways induced by Nur77 inhibition.

[0036] According to a second aspect of the invention, there is a use of Noreugenin in the preparation of a medicament for treating acute lung injury or acute respiratory distress syndrome.

[0037] Noreugenin has demonstrated clear technical efficacy in treating acute lung injury or acute respiratory distress syndrome (ARDS). It inhibits inflammatory signaling pathways driven by Nur77 activity, reduces the production of inflammatory factors, decreases the intensity of pulmonary inflammatory responses, and alleviates lung tissue damage, thereby improving pulmonary dysfunction caused by ARDS. The present invention provides a novel chemical entity and drug preparation route for the treatment of related diseases, offering a new solution for the intervention of acute inflammatory lung diseases.

[0038] In some embodiments, the medicament is a pharmaceutical composition comprising Noreugenin and pharmaceutically acceptable excipients.

[0039] Pharmaceutical compositions can be formulated using methods known to those skilled in the art. For example, they can be administered parenterally as a sterile solution or suspension of water or a pharmaceutically acceptable solution other than water. For example, suitable combinations of pharmacologically acceptable carriers or media, specifically sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, mediators, preservatives, binders, etc. It is considered to be formulated by mixing in a commonly accepted unit dosage form required for pharmaceutical administration. The amount of the active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0040] Sterile compositions for injection can be formulated using excipients such as distilled water for injection, following conventional formulation methods.

[0041] Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, lactose, glucose, and other supplements (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Appropriate dissolving aids, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80, etc.), can be used in combination.

[0042] As an oily liquid, sesame oil and soybean oil can be examples, and benzyl benzoate and / or benzyl alcohol can be used in combination as a solubilizing agent. The composition can be mixed with buffers (e.g., phosphate buffer solutions and sodium acetate buffer solutions), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is typically packaged in appropriate ampoules.

[0043] In some embodiments, the drug is a dosage form administered via oral, intravenous, intraperitoneal, or airway routes.

[0044] Those skilled in the art can dissolve or disperse Noreugenin in pharmaceutically acceptable carriers or excipients using conventional formulation processes, giving it physicochemical properties suitable for different routes of administration. For example, it can be absorbed in the gastrointestinal tract in the form of an oral solution, directly enter the circulatory system in the form of an injection, achieve rapid systemic exposure through intraperitoneal injection, or allow the active ingredient to act directly on lung tissue through airway administration methods such as sprays or powder mists.

[0045] In some embodiments, the pharmaceutical composition is selected from one or more of liposome formulations, nanoparticle formulations, oral solutions, injections, sprays, nasal drops, aerosols, powder inhalers, tablets, capsules, and granules.

[0046] These dosage forms are all well-known in the art and can be achieved through conventional formulation methods. Liposomes or nanoparticles can enhance the stability and distribution efficiency of Noreugenin in lung tissue by encapsulating it, making them more suitable for direct contact with inflamed areas; sprays, nasal drops, aerosols, and powders can be used for airway delivery, allowing the active ingredient to act directly on damaged alveolar areas; oral solutions, tablets, capsules, and granules are suitable for non-invasive administration, facilitating patient compliance; injections, once administered intravenously or intraperitoneally, can rapidly reach effective concentrations in the circulatory system.

[0047] In the medicament prepared according to the present invention, in addition to Noreugenin as the main active ingredient, the medicament may further contain at least one other active pharmaceutical ingredient for treating acute lung injury or acute respiratory distress syndrome, depending on the pathological characteristics of the disease and clinical needs.

[0048] Other active pharmaceutical ingredients may be compounds commonly used to control inflammatory responses or improve lung damage in acute lung injury or acute respiratory distress syndrome, such as one or more of anti-inflammatory drugs, glucocorticoids, IL-1β blockers, and NLRP3 inhibitors. Those skilled in the art can, based on the physicochemical properties and characteristics of different drugs, use conventional formulation processes to co-formulate these active ingredients with Noreugenin into a single pharmaceutical composition, or design multi-component treatment regimens that can be administered simultaneously or sequentially, so that the effects of each component in the body can complement each other, thereby more effectively alleviating lung inflammation, reducing cell damage, and improving lung function impairment caused by acute lung injury or acute respiratory distress syndrome as a whole.

[0049] In the application of this invention, the drug is applicable to mammals, which may include mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats commonly used for pharmacodynamic and toxicological evaluation, as well as pigs, monkeys, etc., which can be used for higher-level pharmacological studies. Their lung structure, inflammatory response pathways, and immune regulation mechanisms are comparable to those of humans in the overall framework, so that the administration of the drug composition prepared by this invention to these animals can more realistically reflect the biological effects of Noreugenin in inhibiting Nur77 and regulating its downstream inflammatory pathways.

[0050] Among the target animals for the drugs described in this invention, primates are the preferred group of mammals, as they have lung tissue structure, immune response patterns and inflammatory pathway characteristics that are more similar to those of humans, making them an important target for evaluating the efficacy and safety of the drugs of this invention.

[0051] In the application of this invention, humans are the most preferred subjects and the ultimate recipients of the drug of this invention.

[0052] According to a third aspect of the invention, there is a method for treating acute lung injury or acute respiratory distress syndrome, comprising administering an effective amount of Noreugenin to a subject requiring the treatment to reduce the Nur77-mediated inflammatory response and alleviate lung tissue damage.

[0053] In some embodiments, the Noreugenin is administered orally, intravenously, intraperitoneally, via airway spray, nebulized inhalation, or via nasal drops.

[0054] In some embodiments, the Noreugenin is administered in the form of a solution, injection, liposome formulation, nanoparticle formulation, spray, aerosol, powder, tablet, capsule, or granule.

[0055] In some embodiments, the dosage of said Noreugenin is 0.1–100 mg / kg.

[0056] In some embodiments, the dosage of the Noreugenin is 5–30 mg / kg, preferably 10–20 mg / kg.

[0057] In some embodiments, the acute lung injury or acute respiratory distress syndrome is caused by overactivation of the NLRP3 inflammasome.

[0058] In some embodiments, the Noreugenin is used in combination with at least one other drug for treating acute lung injury or acute respiratory distress syndrome, the other drug being selected from anti-inflammatory drugs, glucocorticoids, IL-1β blockers, or NLRP3 inhibitors.

[0059] In some embodiments, the subject is a mammal, preferably a primate, and more preferably a human.

[0060] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0061] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0062] Experimental data were analyzed using Graphpad Prism 10.0 statistical software, and the statistical results are expressed as Mean ± SD. When the samples conformed to a normal distribution and had homogeneity of variance, analysis of variance was used to compare the continuous data among multiple groups, with α = 0.05 as the significance level, and P < 0.05 indicating statistical significance.

[0063] Example 1: In vitro cell culture and construction of an in vitro model of acute lung injury Mouse alveolar macrophages (MH-S) cells were cultured in vitro using RPMI 1640 (Seville) medium supplemented with 10% fetal bovine serum (Penosex) and 1% penicillin-streptomycin (Penosex). The medium was changed the day after seeding, and cell density was increased to approximately 90% before passage or plating. In in vitro experiments, the MH-S macrophage line was stimulated with 1 μg / mL LPS. After 24 hours of stimulation, significantly elevated levels of inflammatory cytokines TNF-α, IL-6, and IL-1β were detected, and MH-S cells were observed to transform into a pro-inflammatory phenotype (M1 type), thus confirming the successful establishment of the in vitro inflammation model.

[0064] Example 2: Investigating the effect of noreugenin on cell viability and its alleviating effect on LPS-induced inflammation. Cell viability was assessed using the CCK-8 assay kit (Elabscience) according to the manufacturer's instructions. MH-S cells (1×10⁶) were then... 4 MH-S cells were seeded in 96-well plates (number of cells / well) and cultured for 12 hours to allow them to adhere. Different concentration gradients (0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM) of Noreugenin-containing medium were prepared and used to treat MH-S cells for 24 hours. Cell viability was assessed according to the CCK-8 assay. The results showed that when the Noreugenin concentration did not exceed 2 μM, the survival rate of MH-S cells was above 80%, with no significant cytotoxicity. Finally, concentration gradients of 0.25 μM, 0.5 μM, 1 μM, and 2 μM were selected for subsequent experiments.

[0065] MH-S cells (1×10) 6 MH-S cells were seeded per well in 6-well plates and cultured for 12 hours to allow them to adhere. Medium containing Noreugenin (0.25 μM, 0.5 μM, 1 μM, and 2 μM) was prepared and used to pretreat MH-S cells for 6 hours, followed by stimulation with 1 μg / mL medium for 24 hours. Cells were gently washed with 1 mL of PBS (Seville) and the PBS was discarded; the liquid was then aspirated as dry as possible. For RNA extraction, 1 mL of pre-chilled RNAiso Plus (Takara) was added to the cells, and RNA was extracted using the PrimeScript reverse transcription reagent. TMReverse transcription was performed using RT Master Mix (Perfect Real Time) (Takara) under the following conditions: 37°C for 15 min, 85°C for 5 sec, and 4°C incubation. This step yields cDNA, which can be used for subsequent amplification experiments. 10 μL of the TB Green® Premix Ex Taq quantitative PCR kit was added to the PCR reaction plate (axygen). TM (TliRNaseH Plus) (Takara) was added to the PCR reaction plate. 0.4 μL of forward and reverse primers (10 pmol / μL), 2 μL of cDNA working solution, and 7.2 μL of DEPC water were used. The plate was then sealed with a sealing film, ensuring it was firmly pressed against the surrounding area to prevent evaporation at high temperatures. The plate was centrifuged using a dedicated centrifuge and then placed in an RT-qPCR instrument for detection. The reaction conditions were: 95 ℃ for 30 sec, 95 ℃ for 5 sec, repeated 40 times, and then 60 ℃ for 30 sec. The mRNA expression level of the target gene was normalized using 18S. The experimental results are shown below. Figure 2 As shown in the table. The specific primer sequences are shown in Table 1.

[0066] Table 1 - Mouse RT-qPCR Primer Sequences

[0067] Figure 2 As shown, LPS stimulation significantly increased the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and CD80 mRNA in MH-S cells, while intervention with Norreugenin significantly decreased their expression. Cells after stimulation were collected in 1.5 mL centrifuge tubes for subsequent flow cytometry analysis to determine the pro-inflammatory polarization phenotype. Before staining, samples were prepared, and blank and single-positive tubes were set up. After centrifugation of EP tubes, the supernatant was discarded, leaving approximately 100 mL of liquid at the bottom of each tube for subsequent staining. CD80 flow cytometry antibody (Thermo Fisher Scientific) was used to stain each sample and corresponding single-positive tube. 1 μL of antibody was added to each tube, and the tubes were incubated on ice for 10 min. Staining was terminated with 1 mL of PBS, and the tubes were centrifuged again, with 100 μL of liquid remaining at the bottom. Samples were fixed using a membrane-breaking fixative for at least 2 hours. After fixation, centrifuge the cells and resuspend the cell pellet in a cell permeabilization buffer. Repeat this process twice. Then discard the supernatant and keep 100 μL of the liquid. Add CD206 flow cytometry antibody (Thermo Fisher Scientific) to the liquid and incubate on ice for 30 min. After stopping the incubation with PBS, centrifuge the pellet and resuspend it in 500 μL of PBS. Filter the pellet and prepare it for flow cytometry analysis.Figure 2 As shown, LPS stimulation significantly increased the pro-inflammatory polarization level of MH-S cells, while LPS intervention significantly decreased the pro-inflammatory polarization phenotype of MH-S cells. These results indicate that Noreugenin helps alleviate LPS-induced cellular inflammatory responses.

[0068] Example 3: Investigating the effect of the chromogen ketone natural small molecule compound Noreugenin on LPS-induced cell damage. MH-S cells (1×10) 6 Cells were seeded per well in 6-well plates and cultured for 12 hours to allow them to adhere. MH-S cells were pretreated with 0.25 μM, 0.5 μM, 1 μM, and 2 μM Noreugenin-containing medium for 6 hours, followed by CSE stimulation for 24 hours. After collection, 200 μL of protein lysis buffer was added to each well. The protein lysis buffer was prepared in the following ratio: RIPA protein lysis buffer (Solepro):cocktail protease inhibitor (Roche):PMSF (Solepro):phosphatase inhibitor (Solepro) = 88:10:1:1. After lysis on ice for 20 minutes, protein concentration was determined using the BCA method. After homogenization, 1 / 4 volume of loading buffer (Solepro) was added, and the samples were sonicated for 5 minutes. Then, the samples were denatured in a metal bath at 95-100 °C for 5 minutes. After denaturation, the protein samples were stored at -80 °C for gel electrophoresis and Western blotting. After preparing the PAGE gel, samples were loaded for electrophoresis, transfer, blocking, antibody incubation, and development. Figure 3 As shown, LPS stimulation significantly increased the expression levels of cell damage-related proteins in MH-S cells, while Noreugenin intervention significantly downregulated their expression levels. These results indicate that Noreugenin helps alleviate LPS-induced cell damage.

[0069] Example 4: Investigating the therapeutic effect of the chromogen ketone natural small molecule compound Noreugenin on LPS-induced acute lung injury in mice. To verify the in vivo effects of NRG, mice were intraperitoneally injected daily with 10 mg / kg or 20 mg / kg Noreugenin or corn oil containing 5% DMSO for 7 consecutive days; subsequently, 1 mg / mL LPS or PBS was instilled via the airway to establish an acute lung injury model or a non-acute lung injury control, respectively. Figure 4As shown, LPS stimulation significantly increased the mRNA expression levels of inflammatory factors in mouse lung tissue, the secretion levels of inflammatory factors in BALF, and the pro-inflammatory polarization level of macrophages, while intervention with Noreugenin significantly downregulated all of these factors. These results indicate that Noreugenin can treat LPS-induced acute lung injury in mice.

[0070] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Non-therapeutic and diagnostic uses of Noreugenin as an inhibitor of Nur77 protein.

2. The use according to claim 1, wherein the use is selected from at least one of the following: i) Active ingredients used for in vitro detection of changes in Nur77 activity; ii) Active ingredients used for screening drugs that regulate the Nur77 pathway; iii) Active ingredients used to evaluate the interaction between Nur77 and its ligands; iv) The active ingredient used to establish an in vitro regulatory model of the Nur77 signaling pathway; v) Used to prepare active ingredients for in vitro detection of downstream molecular changes of Nur77; vi) Standard control active ingredient used in Nur77 ligand competition experiments; vii) An in vitro analytical reagent used to assess changes in inflammatory signaling pathways induced by Nur77 inhibition.

3. Use of Noreugenin in the preparation of medicines for the treatment of acute lung injury or acute respiratory distress syndrome.

4. The use according to claim 3, wherein the medicament is a pharmaceutical composition comprising Noreugenin and pharmaceutically acceptable excipients.

5. The use according to claim 4, wherein the drug is a dosage form administered via oral, intravenous, intraperitoneal, or airway route.

6. The use according to claim 4, wherein the form of the pharmaceutical composition is selected from one or more of liposome formulations, nanoparticle formulations, oral solutions, injections, sprays, nasal drops, aerosols, powder inhalers, tablets, capsules, and granules.

7. The use according to any one of claims 3-6, wherein the medicament further comprises at least one other pharmaceutically active ingredient for treating acute lung injury or acute respiratory distress syndrome, such as an anti-inflammatory drug, a glucocorticoid, an IL-1β blocker, and an NLRP3 inhibitor.

8. The use according to any one of claims 3-6, wherein the subject of the drug is a mammal.

9. The use according to claim 8, wherein the subject of the drug is a primate.

10. The use according to claim 9, wherein the subject of the drug is a human being.

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