Application of GKT137831 in preparation of medicine for relieving toxicity of aflatoxin AFB1

Abstract

The invention discloses application of GKT137831 in preparation of a medicine for relieving toxicity of aflatoxin AFB1, and belongs to the technical field of biological medicine. The NOX4 inhibitor GKT137831 is proved to be capable of relieving spleen injury induced by aflatoxin AFB1, and particularly, weight loss, spleen weight reduction and spleen coefficient reduction caused by AFB1 recovery are shown; the spleen structure damage caused by AFB1 is repaired, and inflammatory factor imbalance is reversed; the anomalies of oxidative stress markers are improved; and NOX4, Akt and GSK3 beta signals are regulated and controlled. The compound is expected to be used for preparing medicines for relieving spleen organ injury induced by aflatoxin AFB1.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of GKT137831 in the preparation of drugs to alleviate the toxicity of aflatoxin AFB1. Background Technology

[0002] Aflatoxin AFB1 is a Group 1 carcinogen produced by Aspergillus flavus and enters the human body through the food chain, including contaminated grains and nuts. Its primary target organ is the spleen. Recent studies have shown that the spleen, as an important peripheral immune organ, is highly sensitive to AFB1 and can suffer significant damage. AFB1-induced spleen damage involves complex pathological mechanisms, affects immune function, and may ultimately lead to decreased immunity and increased susceptibility to disease.

[0003] Exposure to aflatoxin AFB1 significantly inhibited growth in mice, and this inhibitory effect increased in a time-dependent manner with prolonged exposure. Long-term exposure induced splenic atrophy or functional inhibition, accompanied by apoptosis and cell cycle arrest, leading to a significant decrease in the spleen index. Notably, long-term low-dose AFB1 exposure caused progressive structural damage and functional loss of the spleen, with the degree of damage showing a clear dose-dependent effect. AFB1 exposure significantly increased the content of malondialdehyde (MDA), a lipid peroxidation marker, in spleen tissue, indicating damage to splenic cell membrane structure and exacerbated oxidative stress. Simultaneously, AFB1 significantly reduced the activities of key antioxidant enzymes catalase (CAT) and glutathione peroxidase (GSH-Px). Furthermore, AFB1 led to a significant reduction in splenic glutathione (GSH) content, further weakening the body's antioxidant defense capabilities and exacerbating oxidative damage. AFB1 regulates the balance between pro-inflammatory and anti-inflammatory factors through multiple pathways, and the inflammatory factor changes it induces are dose-dependent and exhibit bidirectional immunomodulatory characteristics. Specifically, AFB1 can activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, significantly upregulating the expression of tumor necrosis factor-alpha (TNF-α). Studies in the kidneys have shown that it upregulates the expression of CXC chemokine receptor type 4 in glomerular podocytes, promotes the formation of the thioredoxin-NOD-like receptor thermoprotein domain-associated protein 3 complex, leading to increased interleukin-1β (IL-1β) secretion. Chronic AFB1 exposure also inhibits the secretion of the splenic anti-inflammatory factor interleukin-10 (IL-10), ultimately causing an imbalance in the anti-inflammatory / pro-inflammatory balance, resulting in a suppressive state of "high inflammation - low defense."

[0004] Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is a key constitutive enzyme that generates intracellular reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2). Under physiological conditions, the low levels of H2O2 produced by NOX4 are crucial for maintaining redox signaling balance and are widely involved in various fundamental cellular processes, including the regulation of inflammatory responses and activation of the protein kinase B (Akt) / glycogen synthase kinase 3β (GSK3β) signaling pathway. However, under various pathological conditions, the expression or activity of NOX4 is abnormally increased, leading to excessive production of ROS (mainly H2O2). This excessive ROS production triggers significant oxidative stress and activates key pro-inflammatory transcription factors, such as NF-κB and activator protein-1. As a result, the expression of downstream pro-inflammatory genes (such as TNF-α and IL-1β genes) is upregulated, exacerbating the inflammatory response. Further studies have shown that NOX4 overexpression can promote renal tubular epithelial cell apoptosis, and its derived ROS also participate in driving fibrosis processes such as collagen deposition. The Akt / GSK3β signaling pathway is a key hub regulating core biological functions such as cell survival, proliferation, and metabolism. Akt kinases transmit signals by phosphorylating their downstream substrates (including GSK3β). Inhibition of GSK3β is one of the key downstream effects of Akt. As a constitutively active multifunctional kinase, GSK3β's activity is regulated by Akt phosphorylation, thus profoundly affecting glycogen metabolism, Wnt signaling, cell cycle progression, differentiation, and the pathogenesis of various diseases. The role of NOX4 activity in this process and its impact on downstream signaling pathways (such as Akt / GSK3β) have not been fully elucidated, and there is currently a lack of effective strategies to target and regulate NOX4 activity to intervene in the process of aflatoxin AFB1-induced spleen injury.

[0005] The NOX4 inhibitor GKT137831 was purchased from Aladdin Company, product code: S413718-1g. GKT137831 is a highly selective NOX4 inhibitor. Its core mechanism of action is to significantly reduce the abnormal production of reactive oxygen species such as hydrogen peroxide in cells by targeting and inhibiting NOX4 activity, thereby intervening in multiple pathological processes driven by oxidative stress and chronic inflammation. GKT137831 exhibits potent and specific inhibitory activity against NOX4 subtypes. By inhibiting NOX4, it directly blocks the source of excessive ROS production, thereby alleviating oxidative stress damage, reducing the release of downstream pro-inflammatory factors TNF-α and IL-1β, and regulating the Akt / GSK3β signaling pathway related to metabolic disorders.

[0006] Currently, there are no reports of the NOX4 inhibitor GKT137831 being able to combat spleen damage caused by aflatoxin AFB1. Summary of the Invention

[0007] The purpose of this invention is to provide the application of GKT137831 in the preparation of drugs that alleviate the toxicity of aflatoxin AFB1, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] One of the technical solutions of this invention is the application of GKT137831 in the preparation of drugs that alleviate the toxicity of aflatoxin AFB1.

[0010] The second technical solution of the present invention is a drug for alleviating the toxicity of aflatoxin AFB1, including GKT137831.

[0011] The third technical solution of the present invention is a pharmaceutical preparation for alleviating the toxicity of aflatoxin AFB1, comprising GKT137831 or the drug, and a pharmaceutically acceptable carrier.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] This invention demonstrates that the NOX4 inhibitor GKT137831 can alleviate aflatoxin AFB1-induced spleen damage, specifically by restoring AFB1-induced weight loss, spleen weight reduction, and decreased spleen coefficient; repairing AFB1-induced spleen structural damage; reversing inflammatory cytokine imbalance; improving abnormal oxidative stress markers; and regulating NOX4, Akt, and GSK3β signaling. This compound holds promise for use in the preparation of drugs to alleviate aflatoxin AFB1-induced spleen organ damage. Attached Figure Description

[0014] Figure 1GKT137831 improved the pathological damage to the spleen induced by aflatoxin AFB1 in mice.

[0015] Figure 2 The effect of GKT137831 on aflatoxin AFB1-induced inflammation in mice was investigated. In the table, A represents plasma TNF-α levels, B represents plasma IL-1β levels, C represents the relative expression level of TNF-α mRNA in the spleen, D represents the relative expression level of IL-1β mRNA in the spleen, and E represents the relative expression level of IL-10 mRNA in the spleen.

[0016] Figure 3 GKT137831 was used to improve aflatoxin AFB1-induced oxidative stress in the spleen of mice. In the figures, A represents H2O2 content, B represents CAT activity, C represents MDA content, D represents SOD activity, and E represents GSH content.

[0017] Figure 4 GKT137831 inhibited NOX4-activated Akt and GSK3β phosphorylation, thus improving aflatoxin AFB1-induced spleen damage in mice. In the figures, A represents NOX4 immunohistochemical staining, B represents relative spleen mRNA expression, C represents relative NOX4 protein expression, D represents relative Akt protein expression, E represents relative p-Akt protein expression, F represents relative GSK3β protein expression, and G represents relative p-GSK3β protein expression. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides the application of GKT137831 in the preparation of drugs that alleviate the toxicity of aflatoxin AFB1.

[0020] In some specific implementations, GKT137831 alleviates aflatoxin AFB1 toxicity by improving aflatoxin AFB1-induced weight loss and spleen damage, inhibiting aflatoxin AFB1-induced inflammatory response and oxidative stress, and adjusting the levels of NOX4, Akt, and GSK3β proteins in the spleen.

[0021] This invention also provides a drug for alleviating aflatoxin AFB1 toxicity, including GKT137831.

[0022] This invention also provides a pharmaceutical formulation for alleviating aflatoxin AFB1 toxicity, comprising GKT137831 or the aforementioned drug, and a pharmaceutically acceptable carrier.

[0023] In some specific implementations, the pharmaceutically acceptable carrier is suitable for liquid dosage forms, solid dosage forms, or ointment dosage forms.

[0024] This invention discloses the use of the NOX4 inhibitor GKT137831 in the preparation of drugs for alleviating spleen damage caused by aflatoxin AFB1 exposure, particularly in the preparation of drugs for alleviating spleen damage-related symptoms, spleen inflammation, spleen oxidative stress and regulating NOX4 / Akt / GSK3β caused by AFB1.

[0025] Experimental and testing methods:

[0026] The specific operational steps of hematoxylin-eosin staining, quantitative reverse transcription polymerase chain reaction, oxidative stress level detection, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining in this invention are as follows:

[0027] Hematoxylin-eosin staining:

[0028] Fresh spleen tissue was fixed in 4% paraformaldehyde phosphate buffer at room temperature for 24 hours, rinsed with running water overnight, and then dehydrated sequentially with a gradient of ethanol (70%-100%) for 25 minutes each, followed by clearing with xylene I / II for 30 minutes each. After paraffin embedding, 4μm thick sections were cut and baked at 60℃ for 2 hours. During dewaxing, the sections were rehydrated with xylene I / II for 10 minutes each, followed by a gradient of ethanol. After rinsing with distilled water, the sections were stained with hematoxylin for 5 minutes, rinsed with running water for 10 minutes, differentiated with 1% hydrochloric acid ethanol for 3 seconds, and then blued with running water for 15 minutes. The sections were counterstained with 0.5% eosin aqueous solution for 1 minute, rinsed quickly with running water, dehydrated sequentially with a gradient of ethanol for 30 seconds each, cleared with xylene for 5 minutes each, and mounted with neutral resin.

[0029] Quantitative reverse transcription polymerase chain reaction:

[0030] RNA was extracted from the spleen and reverse transcribed to generate cDNA. The pretreated nucleic acid template was mixed with SYBR Green I-containing reaction mixture, and an amplification program was run in a real-time quantitative PCR instrument, consisting of 40 replicate cycles (denaturation: 95℃, 5 seconds; annealing: 55℃, 10 seconds; extension: 72℃, 15 seconds), with fluorescence signals acquired at the end of the extension phase of each cycle. Relative quantification was performed using the ΔΔCt method in conjunction with an internal reference gene by analyzing the number of cycles in which the fluorescence signal reached a set threshold.

[0031] Oxidative stress level detection:

[0032] Using the Solarbio Hydrogen Peroxide Content Detection Kit, fresh spleen tissue was homogenized in 1 mL of pre-cooled acetone in an ice bath at a concentration of 8000 × g. Centrifuge at 4℃ for 10 minutes and collect the supernatant for testing; prepare the colorimetric working solution by preheating Reagent II (concentrated hydrochloric acid to dissolve the powder, in a fume hood), Reagent III and Reagent IV at 37℃ for 10 minutes; dilute H2O2 standard (1 mmol / mL) with acetone to 2 μmol / mL; add the reaction system to EP tubes in sequence: test tube (250 μL sample supernatant + 25 μL Reagent II + 50 μL Reagent III), standard tube (250 μL standard + 25 μL Reagent II + 50 μL Reagent III), blank tube (250 μL acetone + 25 μL Reagent II + 50 μL Reagent III), centrifuge at 4000×g for 10 minutes and discard the supernatant; wash the precipitate three times with acetone and add 250 μL Reagent IV and shake to dissolve; let the solution stand at room temperature for 5 minutes, transfer 200 μL to a 96-well plate, and measure the absorbance (A) at 415 nm using a microplate reader, zeroing the plate with the blank tube; calculate the H2O2 content according to the formula.

[0033] Using the Sangon Biotech Catalase Activity Assay Kit, fresh spleen tissue was homogenized in 1 mL of pre-cooled tissue lysis buffer in an ice bath, centrifuged at 8000×g at 4℃ for 10 minutes, and the supernatant was collected for testing. The working solution was prepared by mixing 25 μL of enzyme activator with 5 mL of reaction substrate buffer after preheating at 37℃ for 10 minutes. The reaction system was added to a 96-well plate with 10 μL of sample supernatant and 190 μL of working solution, mixed immediately, and the timing was started. The initial absorbance A1 (0 minutes) and the absorbance A2 after 1 minute of reaction were measured at 240 nm using a microplate reader. ΔA = A1 - A2 was calculated. The CAT activity was calculated according to the formula.

[0034] Using the Sangon Biotech Superoxide Dismutase (SOD) activity assay kit, fresh spleen tissue was homogenized in 1 mL of pre-chilled tissue lysis buffer in an ice bath, centrifuged at 8000×g at 4℃ for 10 minutes, and the supernatant was collected for testing. The enzyme reaction solution was prepared by diluting 10 times with distilled water using 100 μL of xanthine oxidase solution. The chromogenic solution was prepared by dissolving 0.25 mL of NBT solution in a 37℃ water bath and then diluting it 5 times with distilled water. The reaction system was added sequentially to the following wells in a 96-well plate: assay wells (20 μL sample supernatant + 45 μL substrate buffer + 20 μL enzyme reaction solution + 35 μL auxiliary reaction solution + 70 μL distilled water + 10 μL chromogenic solution) and control wells (20 μL sample supernatant + 45 μL substrate buffer + 35 μL auxiliary reaction solution + 90 μL distilled water + 10 μL chromogenic solution). Blank well 1 (45 μL substrate buffer + 20 μL enzyme reaction solution + 35 μL auxiliary reaction solution + 90 μL distilled water + 10 μL chromogenic solution), blank well 2 (45 μL substrate buffer + 35 μL auxiliary reaction solution + 110 μL distilled water + 10 μL chromogenic solution), mixed well, and incubated at 37°C in the dark for 30 minutes; absorbance (A) was measured at 560 nm using an ELISA reader, and ΔAmeasured = Ameasured well - Acontrol well, ΔAblank = Ablank well 1 - Ablank well 2, inhibition rate (%) = (ΔAblank - ΔAmeasured) ÷ ΔAblank × 100%; SOD activity was calculated according to the formula.

[0035] Using the Sangon Biotech malondialdehyde (MDA) content detection kit, fresh spleen tissue was homogenized in 1 mL pre-cooled tissue lysis buffer at 8000 × g. Centrifuge at 4℃ for 10 minutes and collect the supernatant for testing; the MDA detection working solution is prepared by dissolving TBA powder in 20mL working solution buffer per bottle, heated at 70℃ or sonicated to aid dissolution; the reaction system is added to the EP tube as follows: test tube (300μL working solution + 100μL sample supernatant + 100μL acidifier) ​​and blank tube (300μL working solution + 100μL distilled water + 100μL acidifier), mix well, tighten the cap, and incubate precisely in a 100℃ water bath for 60 minutes; after cooling the reaction solution in an ice bath, centrifuge at 10000×g for 10 minutes, take 200μL of supernatant and transfer it to a 96-well plate, and measure the absorbance at 532nm and 600nm using a microplate reader; calculate ΔA532 = A532 assay - A532 blank, ΔA600 = A600 assay - A600 blank, ΔA = ΔA532 - ΔA600; calculate the MDA content according to the formula.

[0036] Using the Nanjing Jiancheng Reduced Glutathione Assay Kit, fresh spleen tissue was added to physiological saline at a weight (g):volume (mL) ratio of 1:9 and homogenized on ice. The mixture was centrifuged at 3500×g for 10 minutes, and the supernatant was collected for testing. A 20 μmol / L GSH standard solution was prepared by serially diluting the standard powder (3.07 mg) with standard solvent. The reaction system was set up in a 96-well plate as follows: assay wells (100 μL sample supernatant + 25 μL DTNB chromogenic solution + 100 μL reaction buffer), standard wells (100 μL GSH standard solution + 25 μL DTNB chromogenic solution + 100 μL reaction buffer), and blank wells (100 μL protein precipitant + 25 μL DTNB chromogenic solution + 100 μL reaction buffer). The samples were gently shaken to mix and then incubated at room temperature in the dark for 5 minutes. The absorbance (A) was measured at 405 nm using a microplate reader. The GSH content was calculated according to the formula.

[0037] Western blot of proteins:

[0038] Spleen samples were lysed with a lysis buffer containing protease and phosphatase inhibitors. After centrifugation to remove insoluble substances, the supernatant was analyzed for protein concentration using the BCA method. The supernatant was then mixed with a loading buffer containing reducing agent and SDS and denatured at approximately 100°C for 10 minutes. 30 μg of the denatured protein sample was subsequently loaded onto a polyacrylamide gel and electrophoresed at a constant voltage of approximately 60V / 120V in the appropriate electrode buffer until the indicator reached the predetermined position. The separated proteins were then transferred to a nitrocellulose membrane using a wet transfer method at a constant current of 300mA for approximately 180 minutes in a transfer buffer. The transferred membrane was then blocked at room temperature for 1 hour in a wash buffer containing 5% skim milk powder. The blocked membrane was then subjected to a specific reaction with a specific target... The primary antibodies specific to the target protein (NOX4, Beyotime, AF1498, 1:1000; Akt, Seville, GB15689, 1:1000; p-Akt, Seville, GB150002, 1:1000; GSK3β, Seville, GB11099, 1:1000; p-GSK3β, Seville, GB114582, 1:500) were incubated at 4°C for approximately 16 hours. After washing five times with washing buffer (approximately 6 minutes each time), the protein was incubated with the corresponding secondary antibody at room temperature for approximately 1 hour, followed by another wash. Finally, signal detection was performed using the corresponding chemiluminescent substrate, and the signal was captured using an imaging system. Parallel assays were performed using an internal control protein antibody (GAPDH, Seville, GB11002, 1:2000).

[0039] ELISA:

[0040] The double-antibody sandwich assay was performed according to the instructions of the mouse tumor necrosis factor α ELISA kit and the mouse interleukin 1β ELISA kit. Plasma was centrifuged to remove particulate matter, and the supernatant was diluted 2-fold. 20× wash buffer / diluent was diluted with distilled water at a volume ratio of 1:20. The standard stock solution (20 ng / mL) was serially diluted to prepare seven concentration gradient standards (approximately 15.625–1000 pg / mL) and a zero-concentration control (0 pg / mL). 100× biotinylated antibody and 100× SA-HRP were diluted with 1× diluent at a ratio of 1:100 for later use. 100 μL of diluted sample or standard was added to the wells of a pre-coated ELISA plate, covered with a sealing film, and incubated at approximately 37°C for approximately 1.5 hours. The solution was discarded, and the plate was washed three times with approximately 300 μL of 1× wash buffer. 100 μL of biotinylated antibody working solution was added, and the plate was incubated at approximately 37°C for approximately 1 hour, followed by four washes. 100 μL of... SA-HRP working solution was incubated at approximately 37°C for about 30 minutes, followed by 4 washes. Under light-protected conditions, 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B were added to each well sequentially, and the mixture was incubated at approximately 37°C for about 15 minutes. The reaction was terminated by adding 50 μL of stop solution, and the absorbance was measured at 450 nm within 5 minutes. The concentration was calculated based on the standard curve.

[0041] Immunohistochemical staining:

[0042] 4μm sections were cut from paraffin-embedded spleen tissue fixed with 4% paraformaldehyde and attached to poly-L-lysine slides. The slides were baked overnight at 60°C. After dewaxing with xylene I / II for 10 minutes each and rehydration with graded ethanol, the slides were microwaved at 92–95°C for 15 minutes with 0.01M sodium citrate buffer and allowed to cool naturally. After rinsing with PBS, the slides were incubated with 3% hydrogen peroxide methanol solution at room temperature in the dark for 15 minutes to block endogenous enzymes. The slides were then rinsed with PBS. 5% normal goat serum was added for blocking at room temperature for 30 minutes. The target protein-specific primary antibody (NOX4, Beyotime, AF1498, 1:1000) was added and incubated overnight in a humidified chamber at 4°C. The slides were then rinsed with PBS. HRP-labeled polymer secondary antibody was added and incubated at room temperature for 30 minutes. The slides were then rinsed with PBS. DAB chromogenic solution was used for controlled staining under a microscope for 1–3 minutes, and the staining was stopped with distilled water. The slides were counterstained with hematoxylin for 1 minute, then blued with running water for 15 minutes. The slides were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin.

[0043] Example 1

[0044] GKT137831 can reverse AFB1-induced weight loss and spleen damage:

[0045] Male BALB / c mice weighing 18–22 g were randomly divided into five groups (n = 8 / group) and administered the drug by gavage for 30 consecutive days.

[0046] The specific grouping and processing are as follows:

[0047] (1) Control group: The same volume of solvent as each drug administration group was administered by gavage;

[0048] (2) GKT137831 monotherapy group (GKT): GKT137831 (60 mg / kg / d) was administered by gavage;

[0049] (3) AFB1 model group: AFB1 (0.75 mg / kg / d) was administered by gavage;

[0050] (4) AFB1+GKT137831 (30mg / kg) combined treatment group (AFB1+GKT-L): AFB1 (0.75mg / kg / d) and GKT137831 (30mg / kg / d) were administered by gavage simultaneously;

[0051] (5) AFB1+GKT137831 (60mg / kg) combined treatment group (AFB1+GKT-H): AFB1 (0.75mg / kg / d) and GKT137831 (60mg / kg / d) were administered by gavage at the same time.

[0052] Twenty-four hours after the last administration, all animals were euthanized, and blood samples and spleen tissue were rapidly collected. After rinsing with physiological saline, the spleen tissue was blotted dry, weighed, and recorded. Subsequently, some of the tissue was flash-frozen in liquid nitrogen and then transferred to -80°C for storage, while some was fixed in 4% paraformaldehyde solution for subsequent histological analysis.

[0053] Table 1 shows the changes in body weight, spleen weight, and spleen coefficient in each mouse group. The results indicate that 30 days of AFB1 exposure significantly inhibited mouse growth and damaged the spleen. Compared with the control group, mice in the AFB1 model group showed significantly lower body weight, spleen weight, and spleen coefficient at week 4. Combined administration of GKT137831 restored body weight, spleen weight, and spleen coefficient, demonstrating that GKT137831 can effectively antagonize the growth inhibition and spleen damage caused by AFB1.

[0054] Table 1. Changes in body weight, spleen weight, and spleen coefficient in mice of different groups.

[0055]

[0056]

[0057] Each value represents the mean ± SEM (n = 8). * P<0.05 ** P<0.01 and indicates * ** P<0.001 indicates a significant difference compared to the control group mice. # P<0.05 ## P<0.01 and### P<0.001 indicates a significant difference compared to the AFB1 model group. &&& P<0.001 indicates a significant difference compared to the AFB1+GKT-L group.

[0058] Example 2

[0059] Results of GKT137831 improving aflatoxin AFB1-induced spleen pathological damage in mice:

[0060] Hematoxylin-eosin staining was used to detect aflatoxin AFB1-induced pathological damage in the spleen of mice. Figure 1 As shown, the white pulp structure of the spleen in normal control mice was intact and the boundaries were clear. Administration of the NOX4 inhibitor GKT137831 alone had no effect on the spleen. 30 days of AFB1 exposure led to severe damage to the white pulp structure of the spleen in mice, with blurred boundaries between it and the red pulp region. After combined administration of GKT137831, the structure and boundary clarity of the white pulp were restored.

[0061] Example 3

[0062] Effects of GKT137831 on aflatoxin AFB1-induced inflammation in mice:

[0063] The levels of plasma inflammatory factors TNF-α and IL-1β were detected using an ELISA kit, and the expression of inflammatory factors TNF-α, IL-1β and IL-10 in the spleen was detected by quantitative reverse transcription polymerase chain reaction.

[0064] like Figure 2 As shown, compared with the control group, administration of GKT137831 alone did not affect the expression of inflammatory factors. In the aflatoxin AFB1 model group, the expression of inflammatory factors TNF-α and IL-1β was significantly increased, while the expression of IL-10 was decreased. However, compared with the aflatoxin AFB1 model group, GKT137831 treatment could reverse the changes in the expression of inflammatory factors.

[0065] The results showed that AFB1 could induce an inflammatory response in mice, while GKT137831 could inhibit AFB1 from inducing an inflammatory response in mice.

[0066] Example 4

[0067] GKT137831 improves aflatoxin AFB1-induced oxidative stress in mouse spleen:

[0068] The levels of H2O2, CAT, MDA, SOD, and GSH in the spleen were detected using an oxidative stress level detection kit.

[0069] like Figure 3As shown, compared with the control group, the aflatoxin AFB1 exposure group exhibited significantly increased H2O2 and MDA levels in spleen tissue, while significantly decreased SOD, CAT activities, and GSH levels, indicating that AFB1 induced significant oxidative stress damage. However, after intervention with compound GKT137831, H2O2 and MDA levels significantly decreased, while SOD, CAT activities, and GSH levels significantly recovered. Compound GKT137831 effectively inhibited AFB1-induced oxidative stress, suggesting that it antagonizes the spleen toxicity of AFB1 by inhibiting oxidative stress.

[0070] Example 5

[0071] GKT137831 inhibits NOX4-activated phosphorylation of Akt and GSK3β, thus improving aflatoxin AFB1-induced spleen damage in mice.

[0072] Immunohistochemical staining was used to detect NOX4 expression in the spleen. Quantitative reverse transcription polymerase chain reaction was used to detect changes in the RNA levels of NOX4, Akt, and GSK3β in the spleen. Western blotting was used to detect changes in the protein levels of NOX4, Akt, p-Akt, GSK3β, and p-GSK3β in the spleen.

[0073] like Figure 4 As shown, aflatoxin AFB1 exposure upregulated NOX4 protein expression in spleen tissue (increased brown granules on immunohistochemical staining). At the transcriptional level, AFB1 significantly increased NOX4 mRNA expression compared to the control group, while GKT137831 treatment inhibited its expression. At the protein level, Western blot analysis showed that NOX4 was significantly increased and the phosphorylation levels of Akt and GSK3β were significantly decreased in the AFB1 model group, while GKT137831 intervention reduced NOX4 and significantly restored p-Akt and p-GSK3β protein levels.

[0074] These results indicate that AFB1 treatment induces spleen damage in mice by activating NOX4 and inhibiting phosphorylation signaling of Akt and GSK3β, while GKT137831 treatment may improve spleen damage in mice by regulating these signals.

[0075] In summary, this invention demonstrates that the NOX4 inhibitor GKT137831 can alleviate aflatoxin AFB1-induced spleen damage, specifically by restoring AFB1-induced weight loss, spleen weight reduction, and decreased spleen coefficient; repairing AFB1-induced spleen structural damage; reversing inflammatory factor imbalance; improving abnormal oxidative stress markers; and regulating NOX4, Akt, and GSK3β signaling. This compound holds promise for use in the preparation of drugs to alleviate aflatoxin AFB1-induced spleen organ damage.

[0076] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of GKT137831 in the preparation of drugs to alleviate the toxicity of aflatoxin AFB1.

2. The application according to claim 1, characterized in that, GKT137831 alleviates aflatoxin AFB1 toxicity by improving aflatoxin AFB1-induced weight loss and spleen damage, inhibiting aflatoxin AFB1-induced inflammatory response and oxidative stress, and adjusting the levels of NOX4, Akt, and GSK3β proteins in the spleen.

3. A drug for alleviating aflatoxin AFB1 toxicity, characterized in that, Including GKT137831.

4. A pharmaceutical preparation for alleviating the toxicity of aflatoxin AFB1, characterized in that, Includes the drug as described in GKT137831 or claim 3, and a pharmaceutically acceptable carrier.

5. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutically acceptable carrier is suitable for liquid dosage forms, solid dosage forms, or ointment dosage forms.

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