Application of magnoflorine in preparation of products for preventing and / or treating liver injury

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of magnoflorine in preparation of a product for preventing / treating liver injury. The invention discusses the protective effect and mechanism of magnoflorine on MTX hepatotoxicity for the first time, and magnoflorine regulates ROS accumulation and mPTP permeability through targeting GSK3beta to inhibit pyroptosis of hepatocytes so as to reduce MTX hepatotoxicity. A new target and a new choice are provided for the prevention and treatment of MTX hepatotoxicity, and a research foundation is laid for the research of a new magnoflorine component liver-protecting drug.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of magnolia alkaloids in the preparation of products for preventing and / or treating liver damage. Background Art

[0002] Methotrexate (MTX) is a commonly used anti-tumor drug and immunosuppressant in clinical practice, and is listed as a first-line treatment for diseases such as acute lymphoblastic leukemia, lymphoma, osteosarcoma, psoriasis, and rheumatoid arthritis. The effectiveness of high-dose shock and low-dose long-term use of MTX has been widely verified in clinical practice, but the incidence of adverse hepatotoxic reactions induced by MTX is high and the risk of progression is high, which seriously affects the treatment effect of the disease. If it is not treated promptly and effectively, it will progress to liver fibrosis, cirrhosis, and even liver failure. The problem of hepatotoxicity has become the main reason for interruption of MTX administration in clinical treatment and a public health issue that cannot be ignored, which seriously limits its clinical application.

[0003] The mechanism of MTX hepatotoxicity is complex and is generally believed to be related to antagonizing folic acid metabolism, oxidative stress damage, and inhibiting adenosine degradation. However, the mechanism of MTX hepatotoxicity has not yet been fully elucidated, which has led to the lack of effective preventive and therapeutic drugs for MTX hepatotoxicity in clinical practice. The prevention and treatment of MTX hepatotoxicity has become a clinical problem that needs to be solved urgently. The project team found that NLRP3 / Caspase-1 / GSDMD-mediated cell pyroptosis is an important biological process in the complex pathological mechanism of MTX hepatotoxicity. The accumulation of ROS interacts with the continuous opening of mPTP, which plays a key regulatory role in the pyroptosis of hepatocytes caused by MTX. Therefore, it is urgent to find suitable targets and drugs for intervening in ROS accumulation and continuous opening of mPTP, so as to provide new options for the prevention and treatment of MYX hepatotoxicity. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned prior art, the inventors, after long-term technical and practical exploration, provide the application of magnolia alkaloids in the preparation of products for the prevention / treatment of liver damage. The present invention first discovered and confirmed through research that magnolia alkaloids can reduce MTX hepatotoxicity by targeting GSK3β to regulate ROS accumulation and mPTP permeability to inhibit hepatocyte pyroptosis. This application is disclosed for the first time and is different from the known clinical drug uses.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides use of a substance that inhibits the expression of GSK3β and / or reduces the activity of GSK3β in the preparation of a product for preventing or treating liver damage.

[0007] Among them, the substance that inhibits GSK3β expression and / or reduces GSK3β activity alleviates liver injury by targeting GSK3β to regulate ROS accumulation and mPTP permeability and inhibiting hepatocyte pyroptosis, and the liver injury is the liver injury caused by methotrexate.

[0008] The product can be a drug or a test reagent, and the test reagent is for basic research, so it can be used to construct relevant cell or animal models, and thus can be used for basic research on the pathogenesis of drug-induced liver injury-related diseases.

[0009] The substance that inhibits GSK3β expression and / or reduces GSK3β activity includes, but is not limited to, RNA interference molecules or antisense oligonucleotides targeting GSK3β, compounds, siRNAs, substances for implementing lentiviral infection or gene knockout, and specific antibodies against the GSK3β polypeptide itself or its upstream and downstream molecules.

[0010] In a specific embodiment of the present invention, the substance that inhibits GSK3β expression and / or reduces GSK3β activity is magnoflorine.

[0011] Magnoflorine (MAG for short) is an isoquinoline alkaloid, mainly present in Magnoliaceae plants such as Magnolia flowers and their congeners, and has various pharmacological effects, including anti-inflammatory, antihypertensive, anti-fertility, antioxidant, and neuroprotective effects. However, it is unknown whether magnoflorine can be used to treat MTX hepatotoxicity. The present invention proves for the first time through research that GSK3β is an effective target for alleviating MTX liver toxicity, and magnoflorine alleviates MTX liver toxicity by targeting GSK3β to regulate ROS accumulation and mPTP permeability and inhibiting hepatocyte pyroptosis.

[0012] In a second aspect of the present invention, there is provided a composition, and the active ingredient of the composition at least includes a substance that inhibits GSK3β expression and / or reduces its activity.

[0013] In a specific embodiment of the present invention, the substance that inhibits GSK3β expression and / or reduces GSK3β activity includes RNA interference molecules or antisense oligonucleotides targeting GSK3β, compounds, siRNAs, substances for implementing lentiviral infection or gene knockout, and specific antibodies against the GSK3β polypeptide itself or its upstream and downstream molecules;

[0014] In a specific embodiment of the present invention, the substance that inhibits GSK3β expression and / or reduces GSK3β activity is magnoflorine.

[0015] In a third aspect of the present invention, there is provided the use of the above composition in the preparation of a product for preventing and / or treating liver injury, and the liver injury is the liver injury caused by methotrexate.

[0016] The product can be a drug or an experimental reagent.

[0017] According to the present invention, when the product is a drug, the drug further comprises at least one pharmaceutically inactive ingredient.

[0018] The pharmaceutically inactive ingredient can be a pharmaceutically acceptable excipient or carrier. The carrier can be liquid or solid. The pharmaceutical preparation can be an oral preparation and a parenteral administration preparation, and can be tablets, pills, capsules, injections, etc.

[0019] The compounds of the present invention can be formulated into pharmaceutical compositions or preparations using techniques well known to those skilled in the art. Suitable pharmaceutical excipients are known in the art, for example, see the Handbook of Pharmaceutical Excipients (Fourth Edition, original work) in 2005.

[0020] In another specific embodiment of the present invention, the subjects to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, and humans are preferred.

[0021] The above one or more technical solutions have the following beneficial effects:

[0022] The present invention has confirmed that magnoflorine can reduce MTX hepatotoxicity through a rat MTX liver injury model and an L02 hepatocyte injury model. Through further research, it is found that GSK3β is an effective target for magnoflorine to reduce MTX liver toxicity. Magnoflorine reduces MTX liver toxicity by targeting GSK3β to regulate ROS accumulation and mPTP permeability and inhibit hepatocyte pyroptosis, providing a new target and new option for the prevention and treatment of MTX hepatotoxicity, and laying a research foundation for the research of new hepatoprotective drugs containing magnoflorine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0024] Figure 1 Shows the effects of MAG on the changes in body weight and liver index of rats induced by MTX; wherein, n = 6, *: compared with the Control group, P < 0.05; **: compared with the Control group, P < 0.01; #: compared with the MTX group, P < 0.05; ##: compared with the MTX group, P < 0.01;

[0025] Figure 2 Shows the results of HE and ultrastructural staining of the liver induced by MAG in MTX; wherein, A: morphological changes of the liver, B: HE staining results of liver tissue, C: transmission electron microscopy observation results of liver tissue;

[0026] Figure 3 Effect of MAG on serum AST, ALT, and LDH in rats with MTX-induced liver injury. Here, n = 6, **: compared with the Control group, P < 0.01; ##: compared with the MTX group, P < 0.01; △△: compared with the DGC group, P < 0.01;

[0027] Figure 4 Effect of MAG on the ROS level in hepatocytes of rats with MTX-induced liver injury. Here, n = 3, A: representative images of ROS detection in hepatocytes of different treatment groups; B: statistical results of ROS levels in hepatocytes of different treatment groups; **: compared with the Control group, P < 0.01; ##: compared with the MTX group, P < 0.01;

[0028] Figure 5 Effect of MAG on the opening of mPTP in hepatocytes of rats with MTX-induced liver injury. Here, n = 3, A: representative images of mPTP opening in hepatocytes of different treatment groups; B: statistical analysis results of cell fluorescence intensity in each treatment group after adding solution A; C: statistical analysis results of cell fluorescence intensity in each treatment group after adding solution B; **: compared with the Control group, P < 0.01; ##: compared with the MTX group, P < 0.01;

[0029] Figure 6 Effect of MAG on the mitochondrial membrane potential of hepatocytes in rats with MTX-induced liver injury;

[0030] Figure 7 Effect of MAG on the protein expression of NLRP3, ASC, GSDMD, and GSDMD-N in the liver of rats induced by MTX. Here, n = 3, **: compared with the Control group, P < 0.01; #: compared with the MTX group, P < 0.05; ##: compared with the MTX group, P < 0.01;

[0031] Figure 8 Kinetics curve of specific binding of MAG to GSK3β. Here, A: sensorgram of binding of different concentrations of MAG to GSK3β protein; B: fitting curve of binding of different concentrations of MAG to GSK3β protein;

[0032] Figure 9 CETSA verification results of the interaction between MAG and GSK3β; Here, A: apparent melting curve; B: isothermal dose-response curve;

[0033] Figure 10 Effect of MAG on the proportion of pyroptosis in L02 hepatocytes induced by MTX;

[0034] Figure 11Effect of MAG on MTX-induced ROS accumulation in L02 hepatocytes; where n = 3, A: Representative images of ROS detection in hepatocytes of different treatment groups; B: Statistical results of ROS levels in hepatocytes of different treatment groups; **: Compared with the Control group, P < 0.01; ##: Compared with the MTX group, P < 0.01;

[0035] Figure 12 Effect of MAG on the ultrastructural changes of mitochondria in MTX-induced L02 hepatocytes;

[0036] Figure 13 Effect of MAG on the opening degree of mPTP in MTX-induced L02 hepatocytes; where n = 3, A: Representative images of mPTP opening in hepatocytes of different treatment groups; B: Statistical analysis results of fluorescence intensity of cells in each treatment group after adding solution A; C: Statistical analysis results of fluorescence intensity of cells in each treatment group after adding solution B; **: Compared with the Control group, P < 0.01; ##: Compared with the MTX group, P < 0.01;

[0037] Figure 14 Effect of MAG on the change of mitochondrial relative membrane potential in MTX-induced L02 hepatocytes;

[0038] Figure 15 Effect of MAG on the distribution of CytC in MTX-induced L02 hepatocytes;

[0039] Figure 16 Effect of MAG on the protein expression of NLRP3, ASC, GSDMD, and GSDMD-N in MTX-induced L02 hepatocytes; where n = 3, *: Compared with the Control group, P < 0.05; **: Compared with the Control group, P < 0.01; ##: Compared with the MTX group, P < 0.01;

[0040] Figure 17 Effect of MAG on the GSK3β target and downstream key proteins in MTX-induced L02 hepatocytes; where n = 3, A: Effects on the protein expression of GSK3β, p-GSK3β, Nrf2, NQO1, and HO-1; B: Effects on GSH content and SOD enzyme activity; **: Compared with the Control group, P < 0.01; ##: Compared with the MTX group, P < 0.01;

[0041] Figure 18Co-IP detection results of the effect of p-GSK3β target on the interaction between ANT1 and CypD; where n = 3, A: Representative WB bands of input detection of different proteins; B: Statistical analysis results of input detection of different proteins; C: Representative WB bands of IP detection of different proteins; D: Statistical analysis results of IP detection of different proteins; *: Compared with the Control group, P < 0.05; **: Compared with the Control group, P < 0.01; #: Compared with the MTX group, P < 0.05; ##: Compared with the MTX group, P < 0.01;

[0042] Figure 19 Effect of GSK3β silencing on the proportion of pyroptosis of L02 hepatocytes induced by MTX intervened by MAG;

[0043] Figure 20 Effect of GSK3β silencing on ROS accumulation in L02 hepatocytes induced by MTX intervened by MAG; where n = 3, A: Representative images of ROS detection in hepatocytes of different treatment groups; B: Statistical results of ROS levels in hepatocytes of different treatment groups; **: Compared with the Control group, P < 0.01; △: Compared with the MTX + siRNA group, P < 0.05;

[0044] Figure 21 Effect of GSK3β silencing on the opening degree of mPTP in L02 hepatocytes induced by MTX intervened by MAG; where n = 3, A: Representative images of mPTP opening in hepatocytes of different treatment groups; B: Statistical analysis results of fluorescence intensity of cells in each treatment group after adding solution A; C: Statistical analysis results of fluorescence intensity of cells in each treatment group after adding solution B; **: Compared with the Control group, P < 0.01;

[0045] Figure 22 Effect of GSK3β silencing on the protein expression of NLRP3, ASC, GSDMD, and GSDMD-N in L02 hepatocytes induced by MTX intervened by MAG; where n = 3, **: Compared with the Control group, P < 0.01; ##: Compared with the MTX + siRNA group, P < 0.01;

[0046] Figure 23 Effect of GSK3β silencing on key proteins downstream of the GSK3β target in L02 hepatocytes induced by MTX intervened by MAG; where n = 3, **: Compared with the Control group, P < 0.01; #: Compared with the MTX group, P < 0.05; △△: Compared with the MTX + siRNA group, P < 0.01. Specific implementation method

[0047] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application pertains.

[0048] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0049] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the sales company; the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through commercial channels.

[0050] Among them, the chemical formula structure of magnoflorine used in the examples is shown as follows:

[0051]

[0052] Example

[0053] Experimental method

[0054] 1. Evaluation of the effect of magnoflorine on MTX-induced pyroptotic injury of rat hepatocytes

[0055] 1.1 Experimental animals and construction of experimental models

[0056] Adult male Wistar rats aged 6 - 7 weeks (body weight 180 - 210 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., production license number: SCXK(Shandong)20220006. Breeding environment: Animal Experiment Center of Affiliated Hospital of Shandong University of Traditional Chinese Medicine, barrier environment breeding, use license number: SYXK(Shandong)20230031. Before the study, the rats were acclimated to the laboratory environment for 7 days. The experiment was approved by the Experimental Animal Ethics Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine (approval number: AWE-2022-020).

[0057] Wistar rats were randomly divided into a blank group, an MTX group, a diammonium glycyrrhizinate (DGC) group, a low-dose MAG group, and a high-dose MAG group (n = 6); DGC was administered continuously for 9 days, once a day, at a dose of 45 mg / kg; the low dose of MAG was set at 20 mg / kg and the high dose at 40 mg / kg, and they were administered continuously for 9 days, once a day. The rats in the blank group and the MTX group were intragastrically administered with 0.9% normal saline. On the 6th and 8th days, 1 hour after intragastric administration, the MTX group, the low-dose MAG group, and the high-dose MAG group were intraperitoneally injected with MTX at 20 mg / kg, and the blank group was intraperitoneally injected with 0.9% normal saline. On the 9th day, the rats were fasted for 12 h. On the 10th day, the animals were anesthetized with 20% urethane, sacrificed, and blood was collected. -1 It was centrifuged at 3000 r·min

[0058] 1.2 Detection of body weight and liver index

[0059] The rats in each group were weighed on the 1st and 9th days of the experiment. The liver was washed with pre-cooled normal saline, wiped with filter paper, and the liver weight was measured. Liver index (%) = liver weight (g) / body weight (g) × 100%

[0060] 1.3 Histological analysis

[0061] The liver tissues of rats fixed with 4% paraformaldehyde were taken for HE staining of liver tissues to observe the effect of MAG on the pathological structural changes of the liver induced by MTX.

[0062] The liver tissues of rats fixed with 2.5% glutaraldehyde were taken for uranium-lead double staining, and transmission electron microscopy was used to observe the effect of MAG on the ultrastructural pathological changes of the liver induced by MTX.

[0063] 1.4 Detection of serum AST, ALT, and LDH

[0064] The sera of rats in each group were taken, thawed, and the levels of AST, ALT, and LDH in the sera were detected according to the instructions of the ELISA kit.

[0065] 1.5 Detection of ROS level

[0066] The fresh liver tissues of each group were taken and washed clean with pre-cooled PBS. The tissue blocks were cut into small pieces, rinsed with pre-cooled PBS, appropriate trypsin digestion solution was added to the tissue, and it was digested in a water bath at 37°C for 20 - 30 min. During this period, it was intermittently pipetted, the digestion was terminated with the culture medium, and it was filtered through a 300-mesh nylon mesh. The filtered cells were collected and centrifuged at 500 r·min -1After centrifugation for 10 min, remove the supernatant. Wash the precipitate with PBS 1 - 2 times to prepare a single - cell suspension. Operate according to the instructions of the DCFH - DA fluorescence probe kit. Observe the fluorescence intensity of cells in each group with a fluorescence microscope, and calculate and perform statistical analysis using Image J software.

[0067] 1.6 Detection of the opening degree of mPTP

[0068] Take fresh liver tissues from each group, prepare a single - cell suspension with reference to the method of "1.5 Detection of ROS level", operate according to the instructions of the calcein - AM kit, and observe the fluorescence intensity of cells in each group with a fluorescence microscope. Calculate and perform statistical analysis using Image J software.

[0069] 1.7 Detection of changes in mitochondrial relative membrane potential

[0070] Take fresh liver tissues from each group, prepare a single - cell suspension with reference to the method of "1.5 Detection of ROS level", operate according to the instructions of the JC - 1 fluorescence probe kit, and observe the change in the fluorescence color of cells in each group. Measure the change in mitochondrial membrane potential according to the relative ratio of red and green fluorescence.

[0071] 1.8 Detection of the levels of IL - 1β, IL - 18 and the activity of Caspase - 1.

[0072] Take the serum of rats in each group, thaw it, and detect the levels of IL - 1β and IL - 18 in the serum according to the instructions of the ELISA kit; take the fresh liver tissues of rats in each group, thaw them, lyse them according to the instructions of the ELISA kit, and detect the activity of Caspase - 1 in the lysate.

[0073] 1.9 Detection of pyroptosis - related proteins NLRP3, ASC, GSDMD, GSDMD - N

[0074] Use Western blot to detect the changes in the expression levels of pyroptosis - related proteins NLRP3, ASC, GSDMD, GSDMD - N in the livers of rats in each group, and evaluate the protective effect of magnoflorine on MTX - induced pyroptotic injury of rat hepatocytes.

[0075] 2. Analysis and verification of the key targets of the protective effect of magnoflorine on MTX - induced liver injury

[0076] Studies have shown that GSK3β belongs to the serine / threonine kinase of the glycogen synthase kinase subfamily and plays important functions in regulating glycogen metabolism, mitochondrial function, cell proliferation and differentiation, oxidative stress, and the body's inflammatory response. Among them, phosphorylated GSK3β (Ser9) can promote the activation and expression of nuclear transcription-related factor 2 (Nrf2), and then promote the expression of a series of downstream antioxidant genes such as superoxide dismutase (SOD), glutathione (GSH), quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1), thereby reducing the ROS level. At the same time, phosphorylated GSK3β (Ser9) can also inhibit the opening of mPTP by promoting the binding of the mitochondrial outer membrane protein voltage-dependent anion-selective channel 1 (VDAC1) to hexokinase 2 (HK2) and competitively binding to adenine nucleotide translocase (ANT) to reduce the affinity of ANT for the mitochondrial matrix protein cyclophilin D (CypD), resulting in a decrease in cytochrome C and regulating cell death. From the above studies, it can be seen that the Gsk3β target is closely related to MTX-induced oxidative stress, inflammatory response, and mitochondrial dysfunction in liver injury. Combining the key pathological mechanism of MTX-induced liver pyroptosis injury mediated by ROS accumulation and continuous opening of mPTP revealed in the previous period, GSK3β has multiple regulatory effects on regulating ROS levels and mPTP opening. Targeting GSK3β (Ser9) phosphorylation may be a potentially effective strategy to inhibit cell pyroptosis mediated by ROS accumulation and mPTP opening. This study focused on the key GSK3β target and used surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) techniques. Based on human liver L02 cells (product number SNL-141 purchased from Wuhan Shangen Biotechnology Co., Ltd.), the interaction between MAG and GSK3β was verified by experiments to further clarify the effect of MAG targeting GSK3β.

[0077] 3. Evaluation of the effect of magnoflorine targeting GSK3β on protecting MTX-induced hepatocyte pyroptosis injury

[0078] 3.1 Cell culture

[0079] Human liver L02 cells (product number SNL-141, purchased from Wuhan Shangen Biotechnology Co., Ltd.). L02 cells were cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 mg / mL streptomycin in a 37°C, 5% CO2 incubator.

[0080] The L02 cells in the logarithmic growth phase were washed twice with PBS, 1 mL of 0.25% trypsin was added for digestion for 2 min, and then DMEM high-glucose complete medium was added and pipetted evenly. According to 5×10 3Density plate with [number] cells / well. Place the 96-well plate in an incubator at 37°C for incubation. After the cell density reaches 70%, treat with different concentrations of MAG (0, 9.375, 18.75, 37.5, 75, 150, 200, 300 μM) for 24 h respectively to determine the appropriate concentration, and select MAG 75 and 150 μM as low and high concentrations for subsequent experiments.

[0081] 3.2 Detection of the proportion of pyroptosis

[0082] Seed L02 cells in the logarithmic growth phase at a density of 1×10 5 cells / well in a 24-well plate. After the cell density reaches 70%, divide them into a blank group, an MTX (5 mM) model group, an MTX + MAG low-dose (75 μM) group, and an MTX + MAG high-dose (150 μM) group. Each group has 3 replicate wells and culture for 24 h. After 24 h, collect the cells, wash them twice with PBS, incubate each well with 5 μL of Hoechst 33342 and 5 μL of PI staining solution in an ice bath for 30 min, rinse the cells with PBS again, and observe the proportion of red fluorescence and blue fluorescence cells by fluorescence microscopy after smearing.

[0083] 3.3 Detection of intracellular ROS level

[0084] Cell culture and grouping are the same as in "3.1 Cell culture and 3.2 Detection of the proportion of pyroptosis". Collect the cells in each treatment group, wash them twice with PBS, operate according to the DCFH-DA fluorescence probe kit instructions, and observe the fluorescence intensity of each group of cells by fluorescence microscopy. Randomly select 3 fields of view, and use Image J software to statistically analyze the average fluorescence intensity of cells in the fields of view.

[0085] 3.4 Detection of the opening degree of mPTP

[0086] Cell culture and grouping are the same as in "3.1 Cell culture and 3.2 Detection of the proportion of pyroptosis". Collect the cells in each treatment group, wash them twice with PBS, operate according to the calcein-AM kit instructions, and observe the fluorescence intensity of each group of cells by fluorescence microscopy. Randomly select 3 fields of view, and use Image J software to statistically analyze the average fluorescence intensity of cells in the fields of view.

[0087] 3.5 Observation of mitochondrial ultrastructure

[0088] The cell culture and grouping were the same as those in "3.1 Cell culture and 3.2 Detection of the proportion of pyroptotic cells". Cells in each group were collected into 1.5 mL EP tubes, and the changes in the ultrastructure of cell mitochondria were observed by transmission electron microscopy. The specific procedures for cell treatment and observation were as follows: (1) Fixation: Gently fill the EP tube wall with 2.5% glutaraldehyde fixative, let it stand at room temperature for 3 h, and then place it at 4 °C for fixation for 24 h. Take the cell samples fixed with 2.5% glutaraldehyde, aspirate the fixative, rinse with PBS 5 times, 15 min each time, centrifuge at 500 rpm for 5 min, aspirate the supernatant, and gently fill the tube wall with 1% osmium tetroxide fixative for fixation for 1.5 h; (2) Dehydration: Aspirate the osmium tetroxide fixative, rinse with PBS 3 times, 15 min each time, then rinse with ultrapure water 3 times, 15 min each time, centrifuge at 500 rpm for 5 min, aspirate the supernatant, and dehydrate the remaining cell mass successively with 50% ethanol → 70% ethanol → 80% ethanol → 90% ethanol → 90% acetone → pure acetone 3 times, 15 min each time; (3) Infiltration: The dehydrated cell mass was treated with a mixture of epoxy resin embedding agent and acetone (V / V = 1 / 1) for 1 h; then treated with a mixture of epoxy resin embedding agent and acetone (V / V = 1 / 2) overnight, and finally treated with pure epoxy resin embedding agent 2 times, 4 h each time; (4) Embedding: Add pure resin to the sample that has been infiltrated with the embedding agent, and then place it in a polymerization instrument for a program of 37 °C for 24 h → 45 °C for 24 h → 60 °C for 48 h to form a resin block embedding the sample by thermal polymerization; (5) Block trimming and sectioning: After trimming the surface of the resin block, perform ultra-thin sectioning with a thickness of 65 nm and pick up the sections onto 3 grids; (6) Staining: The obtained sections were stained with 2% uranyl acetate solution for 25 min, washed with water, and then stained with lead citrate staining solution for 7 min, and washed thoroughly with water; (7) Baking the sections and observing and taking pictures: Bake the sections under an infrared lamp for 10 min until dry, observe under a transmission electron microscope at 80 KV, and take pictures at a magnification of 30 K.

[0089] 3.6 Detection of changes in mitochondrial relative membrane potential

[0090] The cell culture and grouping were the same as those in "3.1 Cell culture and 3.2 Detection of the proportion of pyroptotic cells". Cells in each treatment group were collected and operated according to the JC-1 fluorescent probe kit instructions, and the changes in the fluorescence color of cells in each group were observed with a fluorescence microscope. The changes in mitochondrial membrane potential were measured according to the relative ratio of red and green fluorescence.

[0091] 3.7 Detection of CytC distribution

[0092] The cell culture and grouping were the same as those in "3.1 Cell culture and 3.2 Detection of the proportion of pyroptosis cells". Cells in each treatment group were collected; the cells were washed with PBS, and after the washing was completed, a certain volume of 4% paraformaldehyde for fixation was added for fixation at room temperature for 30 min; after the fixation was completed, the cells were washed 3 times with pre-cooled PBS, and permeabilized with 0.1% Triton X-100 at 1 ml / well on ice for 15 min, and then washed 3 times with pre-cooled PBS, 5 min each time; the washing solution was aspirated completely, and the slides of the same group were transferred to a 60 mm dish, and 5% BSA was added to the dish and slowly shaken on a shaker at room temperature for 1 h; the blocking solution was aspirated completely, and the slides of the same group were transferred to a new dish, and immediately added with the incubation solution of CytC primary antibody and placed overnight in a 4 °C refrigerator; the primary antibody was recovered, the dish was washed thoroughly with PBS, and then the incubation solution of secondary antibody was added and shaken on a shaker for 1 h under light-proof conditions; the secondary antibody was removed, washed 3 times with PBS, DAPI was added, and left to stand at room temperature for 5 min, DAPI was removed, and washed 3 times with PBS; a drop of mounting medium was added to the glass slide, the slide was slowly buckled on the glass slide, the excess mounting medium was blotted with absorbent paper, and left to stand slightly and then placed in a light-proof box for observation; the green fluorescence was detected under a fluorescence microscope. When the green fluorescence was distributed in dots, it represented that it was distributed in the mitochondria; when the green fluorescence was diffusely distributed, it represented that it was released into the cytoplasm.

[0093] 3.8 Detection of the activities of AST, ALT, LDH, Caspase-1 and the levels of IL-1β and IL-18

[0094] L02 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1×10 6 cells / well. After the cell density reached 90%, they were divided into a blank group, an MTX (5 mM) model group, an MTX+MAG low-dose (75 μM) group, and an MTX+MAG high-dose (150 μM) group, with 3 replicate wells in each group, and cultured for 24 h. After 24 h, the cell supernatant in each well was collected, and the activities of AST, ALT, LDH and the levels of IL-1β and IL-18 in the supernatant were detected according to the instructions of the ELISA kit; the cells in each group were collected, lysed according to the instructions of the ELISA kit, and the activity of Caspase-1 in the lysate was detected.

[0095] 3.9 Detection of NLRP3, ASC, GSDMD, GSDMD-N proteins

[0096] The cell culture and grouping were the same as those in "3.8 Detection of the activities of AST, ALT, LDH, Caspase-1 and the levels of IL-1β and IL-18". Cells in each treatment group were collected, and the expression levels of each protein were detected by Western blot.

[0097] 3.10 Detection of GSK3β target and downstream key proteins

[0098] Cell culture and grouping were the same as those in "3.8 Detection of AST, ALT, LDH, Caspase-1 activities and IL-1β, IL-18 levels". Cells from each treatment group were collected, and the protein expression levels of GSK3β, p-GSK3β (Ser9), Nrf2, HO-1, and NQO1 in each group were detected by Western blot. Cells from each group were collected and lysed, and the GSH content and SOD enzyme activity in the cell lysate were detected according to the instructions of the ELISA kit.

[0099] 3.11 Co-IP detection of the effect of GSK3β target on the interaction between ANT1 and CyP-D

[0100] After phosphorylation of GSK3β (SER9), it can promote the binding to mitochondrial adenine nucleotide translocase ANT1, change the conformation of ANT1, reduce the interaction with CyP-D, and decrease the opening degree of mPTP.

[0101] (1) Cell culture, grouping and lysis: Cell culture and grouping were the same as those in "3.8 Detection of AST, ALT, LDH, Caspase-1 activities and IL-1β, IL-18 levels". Cells from each treatment group were collected, and the cells in each group were lysed with the IP lysis buffer in the Co-IP kit. The BCA protein concentration of each cell lysate was measured, and the protein content of each group was adjusted to be uniform according to the protein concentration. (2) Preparation of magnetic bead-antibody complex: Take 25 μL of immunomagnetic beads, add them to 175 μL of immunoprecipitation lysis buffer, vortex and mix well, then use a magnetic stand for adsorption. After discarding the supernatant, repeat the washing once. Add 200 μL of immunoprecipitation lysis buffer to the magnetic bead precipitate, mix well, and add 2 μg of IgG and p-GSK3β (Ser9) antibodies, and incubate at room temperature on a rotary shaker for 1 h. (3) Co-incubation of cell lysate-magnetic bead antibody complex: Take a part of the cell lysates with uniform protein content from each group as Imput; the remaining cell lysate samples (containing 500 μg of protein) were mixed evenly with the incubated magnetic bead-antibody complex, incubated overnight at 4°C, and then the magnetic beads were adsorbed with a magnetic stand, and the supernatant was discarded and saved for later use. (4) Washing of immune complex-magnetic beads: Add 500 μL of immunoprecipitation lysis buffer to the magnetic bead mixture, mix well, adsorb the magnetic beads with a magnetic stand, discard the supernatant, and repeat the washing twice. Then add 500 μL of deionized water to wash once, adsorb the magnetic beads with a magnetic stand, add 100 μL of 1× protein loading buffer, boil at 100°C for 10 min to elute the magnetic beads, and collect the samples from each group for standby. (5) Detect the protein levels of p-GSK3β (Ser9), ANT1, and Cyp-D in each group by Western blot.

[0102] 4. Salvage experiment on magnoflorine targeting GSK3β to protect MTX-induced hepatocyte pyroptosis injury

[0103] 4.1 Cell culture is the same as "3.1 Cell culture".

[0104] 4.2 Cell transfection

[0105] In L02 cells, small interfering RNA (siRNA) was used to conduct a salvage test on the liver toxicity of MTX intervened by MAG, and it was clarified that MAG exerts a protective effect on MTX-induced pyroptosis injury by targeting GSK3β. The siRNA or negative control was transfected into L02 cells using the mate plus transfection reagent. The expression of GSK3β protein in the cells was detected by Western blot. Finally, the GSK3β-siRAN-1802 interference fragment was selected for subsequent research. The sequence information of the designed and synthesized RNA interference fragments of GSK3β is shown in Table 1.

[0106] Table 1 Designed and synthesized RNA interference fragments of GSK3β

[0107]

[0108] 4.3 Cell proliferation experiment

[0109] The L02 cells in the logarithmic growth phase were digested, added to DMEM medium, and evenly pipetted. They were inoculated into 96-well plates at a density of 5×10 3 cells / well, repeated 3 times, and placed in a constant temperature incubator at 37°C and 5% CO2 for incubation. After the cell density reached 70%, they were divided into a blank group, a GSK3β-siRNA + MTX (5 mM) group, and a GSK3β-siRNA + MTX + MAG (75 μM) group. The growth of the cells was detected daily using the Cell Counting Kit-8 reagent kit.

[0110] 4.4 Detection of the proportion of cell pyroptosis

[0111] The L02 cells in the logarithmic growth phase were inoculated into 6-well plates at a density of 2×10 5 cells / well. After transfection with the screened GSK3β-siRNA sequence for 48 h, they were divided into a blank group, a GSK3β-siRNA + MTX (5 mM) group, and a GSK3β-siRNA + MTX + MAG (75 μM) group. Each group had 3 replicate wells and was cultured for 24 h. After 24 h, the cells were collected, washed 2 times with PBS, and incubated with 5 μL of Hoechst33342 and 5 μL of PI staining solution in an ice bath for 30 min in each well. The cells were rinsed again with PBS, smeared, and observed for red fluorescence and blue fluorescence using a fluorescence microscope.

[0112] 4.5 Detection of intracellular ROS level

[0113] The cell treatment and grouping were the same as those in "4.4 Detection of the pyroptosis ratio". Cells in each treatment group were collected, rinsed twice with PBS, and operated according to the instructions of the DCFH-DA fluorescence probe kit. The fluorescence intensity of cells in each group was observed under a fluorescence microscope. Three fields of view were randomly selected, and the average fluorescence intensity of cells in the fields of view was statistically analyzed using Image J software. Statistical analysis was performed by calculating with Image J software.

[0114] 4.6 Detection of the opening degree of mPTP

[0115] The cell treatment and grouping were the same as those in "4.4 Detection of the pyroptosis ratio". Cells in each treatment group were collected, rinsed twice with PBS, and operated according to the instructions of the calcein-AM kit. The fluorescence intensity of cells in each group was observed under a fluorescence microscope. Three fields of view were randomly selected, and the average fluorescence intensity of cells in the fields of view was statistically analyzed using Image J software.

[0116] 4.7 Detection of the activities of AST, ALT, LDH, Caspase-1 and the levels of IL-1β, IL-18

[0117] The cell treatment and grouping were the same as those in "4.4 Detection of the pyroptosis ratio". The supernatant of each well of cells was collected, and the activities of AST, ALT, LDH and the levels of IL-1β, IL-18 in the supernatant were detected according to the instructions of the ELISA kit; cells in each group were collected, lysed according to the instructions of the ELISA kit, and the activity of Caspase-1 in the lysate was detected.

[0118] 4.8 Detection of NLRP3, ASC, GSDMD, GSDMD-N pyroptosis proteins

[0119] Cell culture and grouping were the same as those in "4.4 Detection of the pyroptosis ratio". Cells in each treatment group were collected, and the expression levels of each pyroptosis protein were detected by Western blot.

[0120] 4.9 Detection of key proteins downstream of the GSK3β target

[0121] Cell culture and grouping were the same as those in "4.4 Detection of the pyroptosis ratio". Cells in each treatment group were collected, and the expression levels of Nrf2, HO-1, NQO1 proteins in each group were detected by Western blot; the GSH content and SOD enzyme activity in the cell lysate of each group were detected using an ELISA kit.

[0122] 5. Data processing

[0123] The experimental data were statistically analyzed and graphed using Graphpad Prism 7.0 software. The data of each group were expressed as mean ± standard deviation (X±SD). The t-test was used for comparison between two groups, and one-way analysis of variance (One-Way ANOVA) was used for comparison among multiple groups. The LSD method (homogeneous variance) or Dunnett's T3 method (heterogeneous variance) was used for pairwise multiple comparisons between groups. P<0.05 was considered significant, and P<0.01 was considered highly significant.

[0124] Test results

[0125] 1. Evaluation of the effect of magnoflorine on MTX-induced pyroptosis injury of rat hepatocytes

[0126] The final body weight of the MTX group was significantly lower than that of the blank group (P<0.05); compared with the MTX group, the DGC and MAG groups could significantly improve the decreasing trend of rat body weight (P<0.01 or P<0.05), and there was no significant difference in the effect between the DGC group (P>0.05). The results are shown in Figure 1 (A); both the DGC and MAG treatment groups could significantly reduce the increase in liver index induced by MTX (P<0.01), and there was no significant difference in the effect between the MAG group and the DGC group (P>0.05). The results are shown in Figure 1 (B). Compared with the blank group, the liver of rats in the MTX group was significantly congested and swollen, showing a dark red color, as shown in Figure 2 (A); the results of HE staining of liver sections showed that there were no abnormalities in the hepatic cords, hepatic sinusoids and hepatocytes in the blank group. However, the liver sections of rats in the MTX group showed disordered hepatic cords, dilated hepatic sinusoids, aggregation of inflammatory cells and swelling of hepatocytes. The DGC and MAG treatments significantly improved the organizational structure of MTX-induced liver injury, as shown in Figure 2 (B); the results of transmission electron microscopy observation showed that compared with the blank group, the nuclei in the MTX group were significantly distorted, the mitochondria were swollen, the cristae membranes were reduced, and the autophagolysosomes, lipid droplets and vacuoles were increased. Compared with the MTX group, the DGC and MAG treatments significantly improved the morphology of the nuclei and mitochondria, and the mitochondrial cristae were more dense, as shown in Figure 2 (C). The ELISA test results are as shown in Figure 3 Compared with the blank group, the activities of serum ALT, AST and LDH in the MTX group were significantly increased (P<0.01); compared with the MTX group, the DGC and MAG treatments significantly reduced the levels of ALT, AST and LDH (P<0.01). The low-dose MAG group had an equivalent effect to the DGC group in terms of AST and LDH, and the high-dose MAG group had an equivalent effect to the DGC group in terms of ALT, AST and LDH. This indicates that MAG has a significant protective effect on MTX-induced hepatocyte injury. The results of ROS level detection are as shown in Figure 4As shown, compared with the blank group, the fluorescence intensity of cells in the MTX group was significantly increased (P<0.01), indicating that MTX could induce ROS accumulation in rat hepatocytes; compared with the MTX group, the fluorescence intensity of cells in the high- and low-dose MAG groups was significantly decreased (P<0.01), indicating that MAG had the effect of scavenging ROS accumulation in hepatocytes induced by MTX. The results of calcein-AM / cobalt chloride staining were as Figure 5 shown. After adding solution A, the cells in each group showed strong green fluorescence; after adding solution B, compared with the blank group, the green fluorescence intensity of cells in the MTX group was significantly decreased (P<0.01), suggesting an increased opening degree of mPTP; compared with the MTX group, the green fluorescence intensity of cells in the MAG group was significantly increased (P<0.01), indicating that MAG had an inhibitory effect on the opening of mPTP in hepatocytes induced by MTX. The results of JC-1 fluorescence probe detection were as Figure 6 shown. Most of the cells in the blank group showed red fluorescence; compared with the blank group, the number of green fluorescence cells in the MTX group was significantly increased, indicating that MTX significantly decreased the mitochondrial membrane potential of hepatocytes and caused significant mitochondrial damage; compared with the MTX group, the number of green fluorescence cells in the high- and low-dose MAG groups was significantly decreased, suggesting that MAG had a certain protective effect on the decrease of mitochondrial membrane potential induced by MTX. The effects of MAG on the activities of Caspase-1 and the levels of serum IL-1β and IL-18 in MTX-induced rat hepatocytes are shown in Table 2. Compared with the control group, the activities of Caspase-1 and the levels of IL-1β and IL-18 in the MTX group were significantly increased (P<0.01); compared with the MTX group, high- and low-dose MAG could significantly decrease the activities of Caspase-1 and the levels of IL-1β and IL-18 (P<0.01).

[0127] Table 2 Effects of different doses of MAG on the activities of Caspase-1 and the levels of serum IL-1β and IL-18 in MTX-induced rat hepatocytes ( n = 6)

[0128]

[0129] Note: **: P<0.01 compared with the Control group; compared with the MTX group; ##: P<0.01 compared with the MTX group.

[0130] The expressions of NLRP3, ASC, GSDMD, and GSDMD-N pyroptosis-related proteins in the livers of rats in each group were detected by Western blot, and the results were as Figure 7As shown. Compared with the blank group, the levels of NLRP3, ASC, GSDMD, and GSDMD-N pyroptosis proteins in the MTX group were significantly increased (P < 0.01); compared with the MTX group, the protein expression levels of NLRP3, ASC, GSDMD, and GSDMD-N in the MAG group were significantly decreased (P < 0.01 or P < 0.05), indicating that MAG can significantly inhibit MTX-induced hepatocyte pyroptosis.

[0131] 2. Experimental verification of the interaction between magnoflorine and the GSK3β target

[0132] The Biacore T200 pre-enrichment system detected the highest response value of GSK3β under the condition of pH 4.0, and determined that pH 4.0 was the optimal protein coupling condition. Finally, the coupling amount of GSK3β was about 10,000 RU. The specific binding kinetic curves of different concentrations of MAG with GSK3β are shown in Figure 8 . The results showed that different concentrations of MAG all showed relatively strong binding response values with the GSK3β recombinant protein, and the binding response value increased with the increase of the drug concentration. Its KD value was (11.65 ± 0.20) μmol / L, indicating that there was a strong affinity between MAG and GSK3β.

[0133] The apparent melting curves of GSK3β protein before and after treatment with MAG at different temperatures are shown in Figure 9 (A). It can be seen that the melting curve shifted significantly to the right after treatment with MAG. In the DMSO blank group, as the temperature increased, the gray value of the GSK3β band gradually decreased, while at the same temperature, the gray value of the GSK3β band in the MAG group increased significantly, indicating that there was an interaction between MAG and the GSK3β target protein, and MAG played a certain stabilizing role on the GSK3β protein.

[0134] The isothermal dose-response curves of GSK3β protein treated with different concentrations of MAG are shown in Figure 9 (B). It can be seen that with the increase of the MAG concentration, the amount of soluble GSK3β protein gradually increased, further indicating the stabilizing effect of MAG on the GSK3β protein, suggesting that the GSK3β protein is the direct target of MAG. The intensity of the soluble protein band of the internal reference GAPDH did not change significantly, indicating that GAPDH is not the target of MAG.

[0135] 3. In vitro experiment of magnoflorine targeting GSK3β to protect against MTX-induced hepatocyte pyroptosis injury

[0136] The results of Hoechst33342 / PI double staining, calcein-AM / cobalt chloride staining, JC-1 fluorescent probe staining, DCFH-DA fluorescent probe staining, and CytC distribution immunofluorescence staining are as shown in Figures 10 to 15As shown, compared with the MTX group, the high- and low-dose MAG groups could significantly reduce the proportion of pyroptotic cells induced by MTX, inhibit the opening degree of mPTP (P<0.01), restore the relative mitochondrial membrane potential, reduce the ROS level (P<0.01), and significantly improve the distribution state of CytC. The effects of MAG on the activities of AST, ALT, LDH, Caspase-1 and the levels of IL-1β and IL-18 in MTX-induced L02 cells are shown in Table 3. Compared with the MTX group, the activities of AST, ALT, LDH, Caspase-1 and the levels of IL-1β and IL-1 in the high- and low-dose MAG groups were significantly decreased (P<0.05).

[0137] Table 3 Effects of MAG on the activities of AST, ALT, LDH, Caspase-1 and the levels of IL-1β and IL-18 in MTX-induced L02 cells ( n = 3)

[0138]

[0139] Note: **: P<0.01 compared with the Control group; #: P<0.05 compared with the MTX group; ##: P<0.01 compared with the MTX group.

[0140] The results of Western blot detection are as Figures 16 - 17 shown. Compared with the MTX group, the levels of NLRP3, ASC, GSDMD, and GSDMD-N in the low- and high-dose MAG groups were significantly decreased (P<0.01), and the levels of p-GSK3β(Ser9), Nrf2, NQO1, and HO-1 were significantly increased (P<0.05 or P<0.01). The content of GSH and the activity of SOD were significantly increased (P<0.01). The results of Co-IP detection are as Figure 18 shown. Compared with the MTX group, the MAG treatment group could significantly enhance the binding ability of p-GSK3β(SER9) and ANT1 (P<0.01) and reduce the interaction between CypD and ANT1 (P<0.01).

[0141] 4. Rescue experiment of magnoflorine targeting GSK3β to protect MTX-induced hepatocyte pyroptosis injury

[0142] The results of Hoechst33342 / PI double staining to detect the effect of GSK3β silencing on the cell pyroptosis rate intervened by MAG in MTX-induced cells are as Figure 19 shown. Compared with the MTX group, the number of strongly blue + strongly red fluorescent cells in the MTX+siRNA+MAG group did not decrease significantly, indicating that GSK3β silencing hindered the inhibitory effect of MAG on MTX-induced cell pyroptosis, further suggesting that MAG plays a protective role against MTX-induced cell pyroptosis injury through the GSK3β target.

[0143] The results of the effect of GSK3β silencing on MAG intervention in MTX-induced ROS accumulation are as follows Figure 20 shown. Compared with the MTX group, there was no significant difference in the average fluorescence intensity of cells in the MTX+siRNA group and the MTX group (P>0.05); compared with the MTX+siRNA group, the average fluorescence intensity of cells in the MTX+siRNA+MAG group decreased (P<0.05), suggesting that there may be other targets for the role of MAG in reducing ROS accumulation. However, the ROS level in the MTX+siRNA+MAG group of cells was still at a relatively high level, indicating that GSK3β plays an important role in MAG scavenging MTX-induced ROS accumulation.

[0144] The results of the effect of GSK3β silencing on MAG intervention in MTX-induced mPTP opening degree are as follows Figure 21 shown. Compared with the MTX group, there was no significant difference in the average fluorescence intensity of cells in the MTX+GSK3β group and the MTX+siRNA+MAG group (P>0.05), indicating that GSK3β silencing hindered the inhibitory effect of MAG on MTX-induced mPTP opening.

[0145] The effects of GSK3β silencing on MAG intervention in MTX-induced AST, ALT, LDH, Caspase-1 activities and IL-1β, IL-18 levels are shown in Table 4. Compared with the blank group, the levels of AST, ALT, LDH, Caspase-1 activities and IL-1β, IL-18 in the MTX group and the MTX+siRNA group were significantly increased (P<0.05 or P<0.01); compared with the MTX+siRNA group, there was no significant difference in the levels of ALT, LDH, Caspase-1 activity and IL-18 in the MTX+siRNA+MAG group (p>0.05), and the AST activity and IL-1β level decreased (p<0.05).

[0146] Table 4 Effects of GSK3β silencing on MAG intervention in MTX-induced AST, ALT, LDH, Caspase-1 activities and IL-1β, IL-18 levels( n = 3)

[0147]

[0148] Note: **: Compared with the Control group, P<0.01; △: Compared with the MTX+siRNA group, P<0.05.

[0149] The effects of GSK3β silencing on MAG intervention in MTX-induced NLRP3, ASC, GSDMD, GSDMD-N protein expression are as follows Figure 22As shown, compared with the blank group, the protein levels of NLRP3, ASC, GSDMD, and GSDMD-N in the MTX group and the MTX+siRNA group were significantly increased (P < 0.01); compared with the MTX+siRNA group, there were no significant differences in the levels of NLRP3, GSDMD, and GSDMD-N in the MTX+siRNA+MAG group (P > 0.05), while the ASC level was significantly decreased (P < 0.01).

[0150] The effects of GSK3β silencing on the key proteins downstream of the GSK3β target intervened by MAG in MTX-induced are as Figure 23 shown. Compared with the blank group, the protein levels of Nrf2, NQO1, and HO-1 in the MTX group were significantly decreased (P < 0.01), and the GSH content and SOD activity were significantly decreased (P < 0.01); compared with the MTX group, the protein level of Nrf2 in the MTX+siRNA group was significantly decreased (P < 0.05), and there were no significant differences in the other protein levels (P > 0.05); compared with the MTX+siRNA group, there were no significant differences in the protein levels of Nrf2, NQO1, and HO-1 and the GSH content in the MTX+siRNA+MAG group (P > 0.05), but the SOD activity was significantly increased (P < 0.01).

[0151] Conclusion: Magnoflorine alleviates MTX hepatotoxicity by targeting GSK3β to regulate ROS accumulation and mPTP permeability and inhibit hepatocyte pyroptosis.

[0152] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a substance that inhibits GSK3β expression and / or reduces GSK3β activity in the preparation of a product for preventing and / or treating liver injury.

2. The application according to claim 1, characterized in that, The substance that inhibits GSK3β expression and / or reduces GSK3β activity alleviates liver injury by targeting GSK3β to regulate ROS accumulation and mPTP permeability and inhibiting hepatocyte pyroptosis.

3. The application according to claim 1, wherein The product is a drug or a laboratory reagent.

4. The application according to claim 1, characterized in that, The liver injury is liver injury caused by methotrexate.

5. The application according to claim 1, characterized in that, The substance that inhibits GSK3β expression and / or reduces GSK3β activity includes RNA interference molecules or antisense oligonucleotides against GSK3β, compounds, siRNA, substances for implementing lentiviral infection or gene knockout, and specific antibodies against the GSK3β polypeptide itself or its upstream and downstream molecules; Preferably, the substance that inhibits GSK3β expression and / or reduces GSK3β activity is magnoflorine.

6. A composition, characterized in that, The composition has an active ingredient that at least includes a substance that inhibits GSK3β expression and / or reduces GSK3β activity.

7. The composition according to claim 6, wherein The substance that inhibits GSK3β expression and / or reduces GSK3β activity includes RNA interference molecules or antisense oligonucleotides against GSK3β, compounds, siRNA, substances for implementing lentiviral infection or gene knockout, and specific antibodies against the GSK3β polypeptide itself or its upstream and downstream molecules; Preferably, the substance that inhibits GSK3β expression and / or reduces GSK3β activity is magnoflorine.

8. Use of the composition according to claim 6 or 7 in the preparation of a product for preventing and / or treating liver injury, wherein the liver injury is liver injury caused by methotrexate.

9. The application according to claim 8, wherein The product is a medicine or a laboratory reagent.

10. The application according to claim 9, characterized in that, When the product is a drug, the drug further includes at least one pharmaceutically inactive ingredient.

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