Evodiamine toxicity target confirmation method and evodiamine hepatotoxicity evaluation method

Abstract

The invention relates to the technical field of medicinal chemistry and pharmacotherapeutics, in particular to a technology and a method for confirming toxic targets of evodiamine (EVO) and accurately evaluating hepatotoxicity of evodiamine. The method comprises the following steps: determining that EVO can be directly combined through biotin labeling, pulldown, mass spectrum combination, small molecule transfection and the like, and up-regulating liver zo-1 protein to cause liver injury, while up-regulating the intestinal tract target is a key mechanism for treating the inflammatory bowel disease by EVO. By comparing the protein levels in the liver-intestine axes of animals in different states, the fact that the down-regulation of the ZO-1 protein level in the liver-intestine axis under the inflammatory bowel disease state can partially antagonize the EVO to the up-regulation of the liver ZO-1 is found, so that the liver toxicity is reduced, and only the therapeutic activity of the EVO is retained. The discovery provides an accurate target spot for understanding an EVO toxicity mechanism, and is beneficial to realizing accurate regulation and control in drug research and development and toxicity prevention in the future; meanwhile, the drug effects and toxic mechanisms of EVO in organisms in different states are compared from the aspect of ZO-1 protein expression in the liver-intestine axis, and direct evidence is provided for the theory of combating poison with poison in toxic traditional Chinese medicine.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry and pharmacotherapy, and in particular to a method for confirming the toxicity target of evodiamine and evaluating the hepatotoxicity of evodiamine. Background Art

[0002] Evodiamine is a natural indolequinoline alkaloid with a quinazoline and carboline skeleton. It is mainly found in the traditional Chinese medicine Evodiamine. It is a key quality control component of Evodiamine and one of the main components of Evodiamine for its pharmacological activity. Modern pharmacological studies have shown that evodiamine has multiple pharmacological activities such as anti-inflammatory, analgesic, anti-tumor, and anti-neurodegenerative activities, and has great development potential. However, with the in-depth exploration of evodiamine, some reports of adverse reactions have begun to appear. Recent research data show that the main toxic organ of evodiamine is the liver.

[0003] In terms of liver toxicity, researchers used L02, HepG2 and other in vitro cells to find that evodiamine can upregulate the expression levels of p-p38 / p38, Caspase-9 cleavage protein and Caspase-3 cleavage protein, downregulate the expression levels of BSEP and MRP2 bile acid transporters, and then induce lipid peroxidation damage, cell apoptosis and cholestasis. The researchers explored the mechanism of action of evodiamine through a rat experimental model and found that it may induce mitochondrial oxidative damage, mitochondrial permeability change and mitochondrial potential reduction. This series of changes ultimately led to ATP depletion and CytC release, ultimately activating the death signaling pathway in hepatocytes and triggering the hepatocyte death signaling pathway;

[0004] However, current studies on the hepatotoxic characteristics of evodiamine all rely on traditional toxicity evaluation models, using single cells or normal animals to evaluate the effects of evodiamine on in vitro hepatocytes and in vivo animal liver tissue cell structure and biochemical indicators, and then confirm the hepatotoxic effects of evodiamine. The evaluation conditions of the existing evaluation model are ultra-high dose treatment, which is not combined with the actual clinical use environment of Evodiamine and cannot systematically and accurately reflect the hepatotoxic nature of evodiamine. In addition, the direct hepatotoxic target of evodiamine has not yet been confirmed, and the hepatotoxic mechanism of evodiamine cannot be fully explained. For this reason, we propose the confirmation of the toxic target of evodiamine and the hepatotoxic evaluation method of evodiamine. Summary of the invention

[0005] The purpose of the present invention is to provide a method for confirming the toxicity target of evodiamine and evaluating the hepatotoxicity of evodiamine, so as to solve the problems raised in the above-mentioned background technology.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The method for confirming the toxicity target of evodiamine comprises the following steps:

[0008] S1: Quantification of tissue protein. After 24 hours of cell administration, the culture medium was discarded, the cells were washed twice with pre-cooled PBS, and then lysed at 4°C with lysis buffer containing 1× cocktail protease inhibitor. The cells were vortexed once every half an hour for 2 hours. The protein extract obtained by lysis was centrifuged at 4°C and 13000rpm for 15 minutes. The supernatant was transferred to a new 1.5mL EP tube and centrifuged again under the same conditions to collect the supernatant. After partial dilution, the concentration was determined by BCA method.

[0009] S2: Biotin-labeled evodiamine, labeling biotin at the appropriate site of evodiamine to obtain Biotin-evodiamine;

[0010] S3: Balance the fixed avidin gel, resuspend the gel solution upside down, and pipette 50 μL of the suspension into the labeled SpinColumn tube;

[0011] S4: fixation of biotin and biotin-evodiamine, followed by blocking of biotin and capture of biotin-evodiamine, and then cleaning of the spin column tube and electrophoresis;

[0012] S5: Protein pretreatment and sample loading, followed by protein identification and bioinformatics analysis. During the protein identification process, the mass spectrometry data obtained are searched through the Proteome Discoverer2.4 software to identify the protein type.

[0013] Preferably, during the process of equilibration and fixation of the avidin gel, the gel solution is inverted and resuspended, 50 μL of the suspension is aspirated into a marked Spin column tube, the bottom stopper is plugged and placed in a collection tube, 250 μL of TBS is added, the top cover is tightened, mixed, the top cover and the bottom stopper are removed, the Spin Column tube is centrifuged at 1250 g for 50 s, TBS is discarded, and the tube is reinserted into the collection tube, 250 μL of TBS is added and the subsequent operation is repeated twice, and the bottom stopper is plugged at the bottom of the Spin Column tube.

[0014] Preferably, during the fixation of biotin and Biotin-evodiamine, 300 μL of 5 mM biotin and Biotin-evodiamine are added to the Spin Column tube, the top cover and the bottom plug are tightened, and the tube is gently shaken and incubated at 4°C on a rotating platform for 60 minutes. After the incubation, the top cover and the bottom plug of the Spin column tube are removed, placed in a collection tube, centrifuged at 1250g for 60 seconds, and placed back in the collection tube.

[0015] Preferably, in the biotin blocking and biotin-evodiamine capture process, 250 μL of biotin blocking solution is added to the Spin column tube, mixed by inverting, and incubated at room temperature for 5 minutes. After the incubation, the top cover of the Spin Column tube is removed, and the tube is placed in a collection tube, centrifuged at 1250g for 50s, and the above operation is repeated once, and 250 μL of TBS is added to the Spin Column tube, the top cover is tightened, mixed by inverting, the top cover is removed, and the tube is placed in a collection tube, centrifuged at 1250g for 50s, and the above operation is repeated once, and the tube is placed in the collection tube again;

[0016] Add 300 μL of captured protein sample solution to the Spin Column tube, tighten the top cover, and incubate overnight at 4°C on a rotating platform. After the incubation, remove the top cover and bottom plug of the Spin Column tube, place it in a collection tube, centrifuge at 1250 g for 60 seconds, remove the Spin Column tube from the collection tube, cover the collection tube, place it on ice, and place the Spin Column tube in a new collection tube for elution.

[0017] Preferably, 250 μL Wash Buffer is added to each Spin Column tube, and the tube is gently inverted to mix, and then rinsed and incubated at room temperature for 1 min. After the incubation, the top cover and bottom plug of the Spin Column tube are removed, and the tube is placed in a collection tube, and centrifuged at 1250g for 50 s. The above steps are repeated three times. During the last rinse, after adding Wash Buffer, the liquid in the tube and the beads are transferred to a 1.5 mL centrifuge tube, and the supernatant is discarded after centrifugation. 50 uL 2× Loading buffer is added to resuspend the beads, and the mixture is shaken and placed in a boiling water bath for 5 min. After cooling on ice, the sample is loaded and electrophoresed at 80 V with 10% separation gel. When the marker bands are clearly separated, the voltage is increased to 120 V. After the electrophoresis is completed, Coomassie brilliant blue staining and gel cutting are performed.

[0018] Preferably, the cut strips are decolorized, reduced and alkylated, enzymatically hydrolyzed, and enzymatically hydrolyzed polypeptides are extracted, and the Ziptip C18 column is desalted and then vacuum dried. The dried sample is re-dissolved by adding 0.1% FA, and 1 to 2 ug of sample is loaded on the machine. The mobile phases A and B are 0.1% formic acid-water and 0.1% formic acid-acetonitrile respectively; the elution gradient is 0-3 min, 3-23 min, 23-42 min, 42-47 min, 47-48 min; 48-60 min; the flow rate is 200 nL / min, and the tandem mass spectrometry detection adopts data-dependent scanning mode, the full scan resolution is 60000, the mass-to-charge ratio range is set to m / z350-1800, the HCD fragmentation mode, and the collision energy is 30 eV.

[0019] Preferably, during the bioinformatics analysis, the differential proteins of the experimental group and the control group are compared according to the grouping information, and the differential proteins of the evodiamine group excluding the intersection of the two groups are considered to be evodiamine-specific binding proteins. The results are displayed using a Venn diagram, and KEGG enrichment analysis is performed on the evodiamine-specific binding proteins.

[0020] Preferably, after the bioinformatics analysis, the mRNA sequence of Z0-1 is obtained from the NCBI database, purified by HPLC, and FITC-labeled Z0-1 siRNA is synthesized by conventional chemical synthesis, and a negative control siRNA is designed and synthesized by a random shuffle method.

[0021] Preferably, the Z0-1 siRNA is synthesized and then transfected with Z0-1 siRNA, and the effects of evodiamine on the proliferation toxicity of Z0-1 knockdown L02 cells and the effects of evodiamine on the apoptosis of Z0-1 knockdown L02 cells are verified by experiments.

[0022] The hepatotoxicity evaluation method of evodiamine is applicable to the toxicity target confirmation method of evodiamine, and comprises the following steps:

[0023] S1: C57 mice were adaptively raised for one week, during which the weight, water intake, appetite, and mental state of the mice were observed to ensure that the experimental mice were normal;

[0024] S2: C57 mice were divided into blank group, 4% DDS treatment group, evodiamine treatment group, and DDS treatment group + evodiamine treatment group;

[0025] S3: Drug treatment: The blank group was not treated with any treatment, and only provided with normal drinking water and food; the model group was given 4% DSS aqueous solution every day for seven consecutive days; after the model was successfully established, the drug-treated group was given intragastric administration once a day for one week, during which the weight, appetite, water intake, and mental state of the mice were observed and recorded; and the model group was given the same amount of CMC-Na solution;

[0026] S4: After the experiment, serum, liver, and intestinal tissues were sampled;

[0027] The liver and intestinal tissues of mice in different treatment groups were then placed in 4% paraformaldehyde for treatment, and then the slices were placed under a slice scanner for observation, photography, and analysis;

[0028] Liver and intestinal tissues were collected, and western blot technology was used to determine the effect of evodiamine on Z0-1 protein.

[0029] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0030] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0031] The present invention exhibits beneficial effects in many aspects, mainly including:

[0032] Through biotin labeling technology, pulldown, mass spectrometry and other methods, we successfully confirmed that the direct target of evodiamine in the liver is zo-1. This discovery provides a precise molecular target for understanding the toxic mechanism of evodiamine, which will help to more accurately regulate in future drug development and toxicity prevention.

[0033] Based on the idea of ​​toxicity-effect conversion, a hepatotoxicity evaluation model of evodiamine was established. This model not only takes into account the dose factor, but also the impact of the animal's body state on drug safety. This will help to more comprehensively evaluate the safety of evodiamine in drug development and clinical applications, and reduce potential drug risks.

[0034] Mechanistic studies have found that evodiamine-induced liver injury is associated with upregulated zo-1 protein expression levels in the liver. However, compared with healthy animals, the Z0-1 protein in the liver-gut axis is downregulated overall under inflammatory bowel disease conditions. Therefore, the upregulation of Z0-1 in the liver-gut axis by evodiamine may only manifest as a therapeutic effect on inflammatory bowel disease under inflammatory bowel disease conditions, and will no longer cause liver injury. This discovery provides a new perspective for understanding the mechanism of action of evodiamine in the liver-gut axis, and can also provide a deeper understanding of the mechanism of action of evodiamine under different physiological and pathological conditions, thereby providing a scientific basis for personalized drug treatment. By regulating specific targets, the efficacy and safety of drugs can be achieved more accurately.

[0035] Evodiamine is toxic to the mouse liver under normal conditions, but under the IBD model, its liver toxicity is significantly reduced and it shows an improving effect on intestinal inflammation; this study provides important basis for the potential application of evodiamine in the treatment of IBD and suggests that it may have different pharmacological effects under different pathological conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0037] Figure 1 is the chemical formula of Biotin-evodiamine of the present invention;

[0038] Figure 2 It is the electrophoresis gel diagram of the present invention;

[0039] Figure 3 The Venn diagram of the evodiamine and Biotin specific binding protein of the present invention;

[0040] Figure 4 It is the enrichment analysis result diagram of the present invention;

[0041] Figure 5 This is a KEGG enrichment analysis result diagram of the present invention;

[0042] Figure 6 This is a fluorescence expression diagram after Z0-1 siRNA transfection of the present invention;

[0043] Figure 7 This is a comparison chart of the expression changes after knocking down Z0-1 in the present invention and the model group;

[0044] Figure 8 Schematic diagram of the effect of evodiamine of the present invention on the proliferation-toxicity of L02 cells transfected with siZ0-1;

[0045] Fig. 9 This is a graph showing changes in apoptotic proteins after knocking down Z0-1 in the present invention;

[0046] Fig.10 This is a graph verifying the protein expression after overexpression transfection of Z0-1 of the present invention;

[0047] Fig.11 This is a graph showing the effect of evodiamine of the present invention on the proliferation-toxicity of L02 cells transfected with OE-Z0-1;

[0048] Fig.12 This is a diagram showing changes in apoptotic proteins after overexpression of Z0-1 in the present invention;

[0049] Fig.13 This is a diagram showing changes in the liver, intestines and other tissues of the C57 mouse of the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0051] The present invention includes the following steps: confirming that EV0 can directly bind to and upregulate the liver zo-1 protein to cause liver damage through biotin labeling, pulldown, mass spectrometry, small molecule transfection, etc., and the upregulation of this target in the intestine is the key mechanism for EV0 to treat inflammatory bowel disease. Comparing the levels of this protein in the liver-gut axis of animals in different states, it was found that the downregulation of the Z0-1 protein level in the liver-gut axis under the inflammatory bowel disease state can partially antagonize the upregulation of EV0 on liver Z0-1, thereby reducing its liver toxicity and retaining only its therapeutic activity; it can be understood that evodiamine and "EV0" are synonymous in the present invention.

[0052] Example 1

[0053] refer to Figure 1-12 , a method for confirming the toxicity target of evodiamine:

[0054] SM pull-down uses the principle of specific binding between small molecule compounds and target proteins, uses chemical probes to fish for proteins bound to drug molecules in cell lysates, and combines proteomics to achieve high-throughput screening and discover targets with small molecule binding specificity. The present invention uses SM pull-down to screen out the target of evodiamine, and uses transfection technology to detect the correlation between the expression level of the target and the liver toxicity caused by evodiamine.

[0055] 1. Experimental Materials of the Present Invention

[0056] Instruments and equipment: mass spectrometer Orbitrap Fusion Lumos; EASY-nLC1000 liquid phase system; ZiptipC18 desalting column; vortex mixer; low-speed centrifuge; refrigerated centrifuge; gel preparation rack, electrophoresis electrode, electrotransfer electrode and other WB experimental equipment.

[0057] Reagents and materials: RIPA strong lysis buffer; protease inhibitor cocktail; BCA protein quantification kit; biotin; pulldown kit; protein marker; Coomassie brilliant blue; DTT; trypsin enzyme solution: lodoacetimide; evodiamine; transfection kit; cck8; 1640 basic medium; L02 special medium; trypsin; RIPA strong lysis buffer; protease inhibitor cocktail; BCA protein quantification kit; tetramethylethylenediamine; ammonium persulfate; 5× protein loading buffer; ColorPrestained Protein Marker; 10× blocking-washing buffer; skim milk powder; methanol; 30% acrylamide-bisacrylamide solution, 4× separation gel buffer, 4× stacking gel buffer, 10× SDS-PAGE electrophoresis buffer; ECL ultrasensitive developer, universal antibody diluent, protein blot membrane regeneration solution / antibody stripping solution. 1.3 siRNA and overexpression plasmid ZO-1 siRNA and ZO-1 overexpression plasmid.

[0058] 2. Experimental Methods

[0059] 1. Quantification of tissue protein: 24 hours after cell administration, discard the culture medium, wash the cells twice with pre-cooled PBS, add lysis solution containing 1× cocktail protease inhibitor and lyse at 4°C, vortex and shake once every half an hour, and the lysis time is 2 hours. The protein extract obtained by lysis was centrifuged at 4°C 13000rpm for 15 minutes, and the supernatant was transferred to a new 1.5mL EP tube, and the supernatant was collected by centrifugation again under the same conditions. After partial dilution, the concentration was determined by BCA method.

[0060] 2. Biotin-labeled evodiamine: label the biotin at the appropriate site of evodiamine to obtain Biotin-evodiamine.

[0061] 3. Balance the fixed avidin gel, resuspend the gel solution upside down, draw 50μL of the suspension into the marked Spin Column tube, plug the bottom stopper and place it in the collection tube, add 250μLTBS, tighten the top cover, and mix well. Remove the top cover and bottom stopper, centrifuge the Spin Column tube at 1250g for 50s, discard TBS, and reinsert it into the collection tube. Repeat the subsequent operation from "add 250μLTBS" twice, and plug the bottom stopper at the bottom of the Spin Column tube.

[0062] 4. Fixation of biotin and biotin-evodiamine: add 300 μL of 5 mM biotin and biotin-evodiamine to the Spin Column tube, tighten the top cover and bottom plug, and gently shake and incubate at 4°C on a rotating platform for 60 minutes. After the incubation, remove the top cover and bottom plug of the Spin Column tube, place it in a collection tube, centrifuge it at 1250g for 60 seconds, and put it back into the collection tube.

[0063] 5. Blocking of biotin and capture of biotin-evodiamine: Add 250 μL of biotin blocking solution to the Spin Column tube, mix by inverting, and incubate at room temperature for 5 minutes. After incubation, remove the top cover of the Spin Column tube, place it in the collection tube, centrifuge at 1250g for 50s, and repeat the above steps once. Add 250 μL of TBS to the Spin Column tube. Tighten the top cover, mix by inverting. Remove the top cover, place it in the collection tube, centrifuge at 1250g for 50s, repeat the above steps once, and put it back into the collection tube.

[0064] Add 300 μL of captured protein sample solution to the Spin Column tube and tighten the top cover. Incubate overnight at 4°C on a rotating platform. After the incubation, remove the top cover and bottom plug of the Spin Column tube, place it in a collection tube, centrifuge at 1250g for 60s, remove the Spin Column tube from the collection tube, cover the collection tube, and place it on ice. Place the Spin Column tube in a new collection tube and prepare for elution.

[0065] 6. Washing and electrophoresis of the Spin Column tubes: Add 250 μL Wash Buffer to each Spin Column tube, gently invert to mix, rinse, and incubate at room temperature for 1 minute. After incubation, remove the top cover and bottom plug of the Spin Column tube, place it in a collection tube, centrifuge at 1250g for 50 seconds, and repeat the above steps three times. During the last rinse, add Wash Buffer, transfer the liquid in the tube and the beads to a 1.5mL centrifuge tube, and discard the supernatant after centrifugation. Add 50uL 2× Loading buffer to resuspend the beads, shake and mix, place in a boiling water bath for 5 minutes, and cool on ice before loading. Perform electrophoresis at 80V with 10% separation gel. When the marker bands are clearly separated, increase the voltage to 120V. After electrophoresis, perform Coomassie Brilliant Blue staining and gel cutting.

[0066] 7. Protein pretreatment and loading, the cut strips were decolorized, reduced alkylated, enzymatically hydrolyzed, enzymatic peptides were extracted, Ziptip C18 column was desalted and then vacuum dried, the dried samples were re-dissolved by adding 0.1% FA, and 1-2ug of samples were loaded on the machine. The mobile phases A and B were 0.1% formic acid-water and 0.1% formic acid-acetonitrile respectively; the elution gradient was 0-3min (2%→5%B), 3-23min (5%→8%B), 23-42min (8%→16%B), 42-47min (16%→28%B), 47-48min (28%→80%B); 48-60min (80%B); the flow rate was 200nL / min. Tandem mass spectrometry detection adopted data dependent acquisition (DDA) mode, full scan resolution of 60,000 (FWHM), mass-to-charge ratio range set to m / z 350-1800, HCD fragmentation mode, and collision energy of 30 eV.

[0067] 8. Protein identification: The mass spectrometry data were searched using Proteome Discoverer 2.4 (Sequent HT, Thermo Scientific, USA) software to identify the protein types.

[0068] 9. Bioinformatics analysis: Based on the grouping information, the differential proteins of the experimental group (evodiamine) and the control group (Biotin) were compared. It was considered that the differential proteins of the evodiamine group excluding the intersection of the two groups were evodiamine-specific binding proteins. The results were displayed using a Venn diagram. KEGG enrichment analysis was also performed on evodiamine-specific binding proteins.

[0069] 10. Changes in the toxic response of L02 cells to evodiamine after Z0-1 knockdown.

[0070] 11. Design of siRNA: The mRNA sequence of Z0-1 was obtained from the NCB1 database, purified by HPLC, and FITC-labeled Z0-1 siRNA was synthesized by conventional chemical synthesis. Negative control siRNA was designed and synthesized by random shuffling method. The sequence is shown in the following table:

[0071] siRNA sequences

[0072]

[0073] 12. Transfect Z0-1 siRNA. Centrifuge the siRNA slightly before opening the cap, collect all of it at the bottom of the centrifuge tube, add DEPC water to dissolve and dilute the siRNA to make a 20μM liquid, store it in aliquots at -20℃ to -80℃, and avoid repeated freezing and thawing. Take the L02 cells in the logarithmic growth phase, inoculate them in a 6-well plate at a cell density of 1.0×105 / 2.0mL / well, and culture them in a 37℃, 5% C02 incubator for 24h. When the cells reach 50%-70% fusion, aspirate the cell culture medium, replace it with 2mL of serum-free 1640 basal medium, transfect with 100nM siRNA, and set up a normal control group at the same time. The experimental group was transfected with Z0-1 siRNA, and the negative control group was transfected with Negative Control siRNA: 11 μL siRNA was pipetted with 200 μL Buffer and mixed by pipetting, vortexed for 10 seconds and centrifuged slightly, then 3 μL transfection reagent was added and mixed by pipetting, vortexed for 1 second and centrifuged slightly, and the mixed solution was transferred to a six-well plate after standing at room temperature for 10 minutes. Each well corresponds to a 1.5 mL centrifuge tube with a mixture of siRNA and transfection reagent. During the transfection process, siRNA was always placed on ice for operation. After 6 hours of transfection, the culture medium was replaced with complete culture medium and continued to be cultured in the cell culture incubator. After 24 hours of transfection, the fluorescence expression in the cells was observed, and the expression of ZO-1 protein was detected at 48 hours.

[0074] 13. The effect of evodiamine on the proliferation toxicity of ZO-1 knockdown L02 cells. The cells in logarithmic growth phase were inoculated in a 96-well culture plate at a density of 5×103 / 100μL / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. The cells were transfected after attachment, and 24 hours after transfection, they were treated and grouped into normal control group, L02-si-ZO-1 group, L02-si-NC group, L02+EV0 group, L02-si-ZO-1+EV0 group and L02-si-NC+EV0 group. After transfection, the cell culture medium was replaced with complete culture medium containing evodiamine, and the culture was continued for 24 hours. At the end of the experiment, the culture medium was aspirated, and 110 μL CCK-8 solution was added to each well (diluted at a ratio of cell culture medium: cCK-8 = 10:1, operated in the dark, and prepared before use). After continuing to culture for 2 hours, the OD value at a wavelength of 450 nm was measured by an ELISA instrument. The cell survival rate was calculated according to formula (6).

[0075] 14. Effect of evodiamine on apoptosis of ZO-1 knockdown L02 cells. L02 cells in logarithmic growth phase were inoculated in 6-well plates at a cell density of 1.0×105 / 2.0mL / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. Cells were transfected after attachment. After 24 hours of transfection, evodiamine was administered and grouped into L02-si-NC group, L02-si-ZO-1+EVO group and L02-si-NC+EVO group. The cell culture medium after transfection was replaced with complete culture medium containing evodiamine and cultured for 24 hours. Cells were collected at the end of the experiment to detect the expression of apoptotic protein Caspase3.

[0076] 15. Changes in the toxic response of L02 cells to evodiamine after overexpression of ZO-1.

[0077] 16. Transfect ZO-1 plasmid at a concentration of 2 μg / well. The experiment set up a normal control group, an OE-ZO-1 transfection group, and a Negative Control transfection group. The transfection method was the same as that of ZO-1 siRNA transfection.

[0078] 17. The toxic effect of evodiamine on the proliferation of ZO-1 overexpressing L02 cells. The experimental grouping was normal control group, L02-OE-ZO-1 group, L02-OE-NC group, L02-EV0 group, L02-OE-ZO-1+EV0 group and L02-OE-NC+EV0 group. The determination method was the same as item 13 of the present invention.

[0079] 18. The effect of evodiamine on the apoptosis of L02 cells overexpressing ZO-1 was studied. The groups were divided into L02-OE-NC group, L02-OE-ZO-1+EV0 group and L02-OE-NC+EV0 group. The determination method was the same as item 14 of the present invention.

[0080] 19. Statistical analysis: GraphPad Prism 8 software was used for statistical analysis. Data were expressed as mean ± SD. One-way analysis of variance was used to compare multiple means. Student's test was used for pairwise comparisons between groups. p < 0.05 indicated that the results were statistically significant.

[0081] 3. Experimental Results

[0082] 1. Electrophoresis gel image

[0083] The electrophoresis gel images of each group are shown in Figure 2As shown, they are Marker, Biotin pulled-down protein, Biotin-evodiamine pulled-down protein and whole protein bands in cell lysate, respectively. The electrophoresis bands of the biotin group were compared, and the Biotin-evodiamine bands were cut from the gel for subsequent protein identification and analysis.

[0084] The overlapping relationship between the differentially expressed proteins in the control group and the experimental group is shown in Figure 2. Figure 3 As shown, by identifying the pulled-down proteins, 1970 Biotin-evodiamine binding proteins and 490 Biotin binding proteins were obtained. The two groups of proteins were compared and the overlapping proteins were eliminated, and 1551 evodiamine-specific binding proteins were obtained.

[0085] The differentially expressed proteins between groups were enriched by G0 and KEGG, and bioinformatics annotation and analysis were performed at two levels. The top 10 of the G0 analysis results of differentially expressed proteins between groups were selected to draw a barplot for display. In the barplot, the vertical axis is the number of targets and the horizontal axis is the entry, such as Figure 4 As shown. The enrichment results showed biological processes such as amide biosynthesis process, cell amide metabolism process, cell macromolecule localization, cell protein localization, intracellular protein transport, mRNA metabolism process, organic nitrogen compound biosynthesis process, polypeptide biosynthesis process, peptide metabolism process and protein translation; cell membrane, cytoplasm and some organelles were the main cell components enriched; catalytic activity, heterocyclic compound binding, nucleoside phosphate binding, nucleotide binding and nucleotide binding were the main molecular functions. It was suggested that the related targets may be cell membrane or cytoplasmic proteins involved in nucleotide signal transduction in the nucleus.

[0086] Different proteins are summarized in the form of directional regulation, that is, pathways. There is also directional regulation between each pathway. KEGG displays and analyzes protein and expression information in the overall network. Through KEGG analysis of differentially expressed proteins between groups, a total of 63 pathways were enriched, and the top 15 were selected to draw a dotplot diagram, as shown in the following figure: Figure 5 It is mainly enriched in pathways such as metabolism, RNA transport, spliceosome, ribosome, endoplasmic reticulum protein processing, and DNA replication, suggesting that specific proteins directly binding to evodiamine play a certain role in the processing and modification of DNA, RNA, and protein molecules.

[0087] In the exploration of omics mechanisms, it has been found that tight junctions play a certain role in evodiamine-induced hepatotoxicity. In the enrichment pathway of pullIdown target proteins, the tight junction pathway is also enriched, and the key protein ZO-1 of this pathway is caught.

[0088] 3. Changes in the toxic response of L02 cells to evodiamine after Z0-1 knockdown.

[0089] 4. Verification of Z0-1 siRNA transfection results.

[0090] Z0-1 siRNA was labeled with FlTC fluorescent label during synthesis. When siRNA was transfected into cells, green fluorescence could be observed in the cells. The cells were placed under a fluorescence microscope 24 hours after transfection, and the transfection of cells was observed through the green fluorescence channel. The results are as follows: Figure 6 As shown, green fluorescence can be seen distributed in the cells, indicating that ZO-1 siRNA has been transfected into the cells and successfully expressed under this transfection condition.

[0091] Western blot results Figure 7 As shown, each group had a clear specific band at 195 kDa. There was no significant difference in protein expression between the L02-siNC group and the normal control group, indicating that the transfection process did not affect the expression of Z0-1 protein. The band in the L02-si-Z0-1 transfection group was significantly weakened, and the protein expression content was significantly lower than that in the L02-siNc group (P < 0.05), indicating that si-Z0-1 was successfully transfected into the cells and interfered with the normal expression of Z0-1.

[0092] Figure 7 n=3, compared with the normal control group, P<0.05.

[0093] 5. The toxic effect of evodiamine on the proliferation of Z0-1 knockdown L02 cells.

[0094] The cell survival rate of each group after evodiamine treatment was as follows Figure 8 As shown, the survival rate of normal L02 cells after administration was 76.30±0.76%, and the survival rate of cells in the L02-siNC+EVO group was 69.42±3.33%, with no significant difference between the two groups; the survival rate of cells in the L02-siZ0-1+EVO group was 70.39±7.18%, which was significantly increased compared with the L02-siNC+EVO group (P<0.05). The above results suggest that knocking down ZO-1 in L02 cells can significantly improve the toxic effects of evodiamine on cell survival and proliferation.

[0095] Figure 8 n = 3, P < 0.01 compared with group L02;

[0096] Compared with the L02+EV0 group, ns means no significant difference; compared with the L02-siNC+EV0 group, P<0.01).

[0097] 6. Effect of evodiamine on apoptosis of ZO-1 knockdown L02 cells.

[0098] Western blot test results Fig. 9 As shown, each group had a clear band at 32 kDa. After the administration of evodiamine, although there was no significant difference in protein expression between the L02-siZO-1+EVO group and the L02-siNC+EVO group, the overall level was reduced, suggesting that evodiamine may reduce the expression of apoptotic proteins after ZO-1 knockdown.

[0099] Fig. 9 n=3, ns indicates no significant difference compared with the idle control group.

[0100] 7. Changes in the toxic response of L02 cells to evodiamine after ZO-1 overexpression

[0101] 8. Verification of ZO-1 siRNA transfection results

[0102] Western blot test results Fig.10 As shown, each group had a clear specific band at 195 kDa. There was no significant difference in the protein expression content between the L02-OE-NC group and the normal control group, indicating that the transfection process and the blank plasmid did not affect the expression of ZO-1 protein; the band of the L02-OE-ZO-1 transfection group was significantly enhanced, and the protein expression content was significantly increased compared with the L02-OE-NC group (P < 0.05), indicating that the OE-ZO-1 plasmid was successfully transfected into the cells and achieved high expression.

[0103] Fig.10 n=3, compared with the normal control group, P<0.05.

[0104] 9. The toxic effect of evodiamine on the proliferation of ZO-1 overexpressing L02 cells.

[0105] The cell survival rate of each group after evodiamine treatment was as follows Fig.11 As shown in the results, the survival rate of normal L02 cells after administration was 78.04±5.48%, and the survival rate of cells in the L02-OE-NC+EV0 group was 72.41±5.55%, with no significant difference between the two groups; the survival rate of cells in the L02-OE-ZO-1+EVO group was 59.46±4.50%, which was significantly lower than that in the L02-OE-NC+EVO group. The above results suggest that overexpression of ZO-1 in L02 cells can significantly aggravate the toxic effects of evodiamine on cell survival and proliferation (P<0.05).

[0106] Fig.11 n=3, compared with the L02 group, P<0.01; compared with the L02+EVO group, ns means no significant difference; compared with the L02-OE-NC+EVO group, P<0.01.

[0107] Western blot test results Fig.12 As shown, each group had a clear band at 32 kDa. After evodiamine administration, the protein expression level in the L02-OE-ZO-1+EVO group was significantly increased compared with that in the L02-OE-NC+EVO group (P < 0.05), indicating that ZO-1 overexpression aggravated the cell apoptosis induced by evodiamine.

[0108] Fig.12 n=3, compared with the idle control group, P<0.05.

[0109] 4. Summary and Discussion

[0110] The present invention screened evodiamine-specific binding target proteins by pull-down combined with high-throughput mass spectrometry, and found that evodiamine could interact with a variety of proteins. Combined with the results of multi-omics research on the toxicity mechanism of evodiamine, it was suggested that the destruction of TJ protein might be an important pathway for evodiamine to affect the hepatotoxicity of bile acid homeostasis in L02 cells. Therefore, ZO-1, which was fished into TJ, was selected, and the relationship between its expression level and evodiamine-induced hepatotoxicity was explored by targeted knockdown and overexpression of ZO-1 through transfection technology.

[0111] ZO proteins include ZO-1, ZO-2 and ZO-3, which are peripheral proteins located at the junction site and can recruit various types of proteins to the cytoplasmic scaffold proteins on the surface of the junction. The three can interact with each other, and can also interact with several transmembrane proteins (OCLN, CLDN, JAM-A) and actin. They can also directly bind to gap junctions (GJ), indicating that ZO proteins have crosstalk with other cell adhesion structures and have the function of cell-cell adhesion and communication. It has been reported that the knockout of ZO-1 in epithelial cells does not affect the formation of tight junctions. The loss of ZO-1 causes tight junctions to recruit ZO-2 from the cytoplasm, resulting in a biased distribution of ZO-2. Mice with ZO-1 knockout still maintain intact tight junction structures and bile acid levels.

[0112] In addition, Z0 protein not only interacts with transmembrane junction proteins to form TJ structural organization, but also interacts with some other cytoplasmic proteins to produce high-order molecular structures at the junction site and recruit proteins involved in signal transduction and transcriptional regulation. These proteins are involved in cell proliferation and differentiation, giving Z0 functions far beyond its role in regulating paracellular permeability. In recent years, increasing evidence has shown that Z0 protein is not only associated with structural barrier mechanisms, but also involved in signal transduction and transcriptional regulation. ZO-1 can affect cell proliferation by binding to transcription factors, which are localized in the nucleus and regulate gene expression. Among them, ZO-1-related nucleic acid binding protein Y box transcription factor (ZONAB) is a dual resident protein located in TJ and / or the nucleus, and its expression depends on the proliferation state of the cell and the expression of ZO-1 at the contact site between cells. ZONAB binds to the promoter region of cyclin D1 (CycD1) and proliferating cell nuclear antigen (PCNA), initiates transcription and promotes cell proliferation, while ZO-1 reduces the localization of ZONAB in the nucleus by forming a junction site with ZONAB. If ZO-1 is missing in the binding site, ZONAB shuttles into the nucleus to initiate cell proliferation, and overexpression of ZO-1 causes ZONAB to redistribute from the nucleus to the cytoplasm, inhibiting cell proliferation. Therefore, evodiamine may interact with ZO-1 in the cytoplasm to interfere with the normal cell proliferation transcription process, which is also a possible direction for its induction of toxic response.

[0113] The results of the present invention show that the toxic response of L02 cells knocked down ZO-1 to evodiamine is weakened, but the level of apoptotic proteins does not change significantly: after the overexpression of ZO-1 protein, the toxic effect of evodiamine on cell survival and proliferation is aggravated, and the level of apoptotic proteins increases significantly. This suggests that the toxic effect of evodiamine is correlated with the expression level of ZO-1, which may be due to the fact that evodiamine acts on ZO-1, destroying the TJ structure, affecting the polarity of hepatocytes, blocking the efflux of bile acids, and accumulating in cells, causing hepatocyte damage.

[0114] Example 2

[0115] refer to Fig.13 , a method for evaluating the hepatotoxicity of evodiamine:

[0116] Liver damage caused by evodiamine in normal animals Liver damage evaluation: C57 mice were adaptively raised for one week, during which the weight, water intake, appetite, mental state, etc. of the mice were observed to ensure that the experimental mice were normal and without abnormalities.

[0117] 1. Experimental plan:

[0118] 1. Grouping scheme: blank group, 4% DDS treatment group, evodiamine treatment group (800 mg / kg), DDS treatment group + evodiamine treatment group (800 mg / kg)

[0119] 4% Dextran sulfate sodium (DSS): Take 4g DSS in a 250mL conical flask, add 100mL distilled water, stir thoroughly until all the powder is completely dissolved, and the 4% DSS solution is obtained after it is evenly distributed. Store at 4℃ away from light.

[0120] 0.5% sodium carboxymethyl cellulose (CMC-Na): Weigh 0.5g of CMC-Na and put it into a 200mL beaker. Add about 80mL of ultrapure water and place it on a magnetic stirrer. Heat and stir until it becomes clear. After cooling, adjust the volume to 100mL to prepare 0.5% CMC-Na.

[0121] 20 (40) mg / mL evodiamine: Weigh 20 (40) mg of evodiamine into a mortar, add 1 mL of 0.5% CMC-Na, vortex mix to prepare a uniform suspension, and store in a refrigerator at 4°C until use.

[0122] 2. Drug treatment: The blank group was not treated in any way, and only provided with normal drinking water and food; the model group was given 4% DSS aqueous solution every day for seven consecutive days. After the model was successfully established, the drug-treated group was given intragastric administration once a day for one week, during which the weight, appetite, water intake, mental state, etc. of the mice were observed and recorded. The model group was given the same amount of CMC-Na solution.

[0123] 3. At the end of the experiment, after fasting for 24 hours, samples of serum, liver, intestine and other tissues were collected for subsequent experiments.

[0124] 4. The liver and intestinal tissues of mice in different treatment groups were fixed with 4% paraformaldehyde, dehydrated and transparent, embedded in wax, sliced ​​and mounted, dewaxed and HE stained, dehydrated and transparent, and sealed. The slices were placed under a slice scanner for observation, photography and analysis.

[0125] 5. Collect blood from mice in different treatment groups and measure liver enzymes according to the instructions of the kit. Repeat the experiment three times for each sample.

[0126] 6. Collect liver and intestinal tissues and use western blot technology to determine the effect of evodiamine on Z0-1 protein. 2. Experimental results

[0127] 1. Compared with the normal group, the body weight of mice in other treatment groups showed a significant downward trend, among which the body weight of mice in the IBD model group was the lowest. Compared with the model group, the body weight of mice in the evodiamine treatment group increased significantly.

[0128] 2. HE staining results showed that compared with the normal group, the liver tissue treated with evodiamine was significantly damaged, with fatty degeneration, shrinkage of the nucleus, unclear cell boundaries, and liver lesions with inflammatory cell infiltration. Compared with the normal group, the intestinal tissue of the model group was significantly damaged, and the intestine of the IBD model group was severely damaged, with irregular crypt surfaces, and even disappearance of crypt structure and abnormal inflammatory infiltration. Compared with the model group, evodiamine treatment had a significant improvement effect on IBD model damage and inflammation. Most importantly, compared with normal animals treated with evodiamine, evodiamine significantly improved the liver damage effect of IBD model mice.

[0129] 3. Compared with the normal group, the liver function level of the evodiamine-treated group increased significantly. Compared with the normal mice treated with evodiamine, the liver function level of the IBD model mice treated with evodiamine was significantly alleviated.

[0130] 4. Western blot results showed that compared with the blank group, Z0-1 in the liver tissue of the evodiamine-treated group was significantly increased. Compared with the blank group, the levels of Z0-1 in the liver and intestine of IBD model mice were significantly reduced; compared with the lBD model, Z0-1 in the liver and intestine of the evodiamine-treated group was significantly increased.

[0131] 3. Summary

[0132] High doses of evodiamine can cause liver toxicity in mice, but under the IBD model, the hepatotoxicity of evodiamine is significantly reduced. This may be due to the fact that the Z0-1 protein in the liver-gut axis is significantly reduced under the IBD model. This indicates that the hepatotoxicity and therapeutic activity of evodiamine in IBD are related to its promotion of Z0-1 protein levels.

[0133] In summary, the specific activation mechanism between evodiamine and tight junction proteins needs further research. The association between liver-injury drugs and tight junction proteins also provides a new treatment direction for bile acid accumulation caused by drug-induced liver injury.

[0134] From the above, we can know that:

[0135] The present invention is directed to the technical problem: However, the current studies on the toxicity characteristics of evodiamine all rely on traditional toxicity evaluation models, using single cells or normal animals to evaluate the effects of evodiamine on in vitro hepatocytes and in vivo animal liver tissue cell structure and biochemical indicators, and then confirm the hepatotoxic effect of evodiamine. The evaluation conditions of the existing evaluation model are high-dose treatment, which is not combined with the actual clinical use environment of Evodiamine, and cannot systematically and accurately reflect the hepatotoxic nature of evodiamine. In addition, the direct hepatotoxic target of evodiamine has not yet been confirmed, and the hepatotoxic mechanism of evodiamine cannot be fully explained.

[0136] The present invention provides precise targets for understanding the toxicity mechanism of EV0, which will help to achieve precise regulation in future drug development and toxicity prevention. At the same time, the efficacy and toxicity mechanism of EV0 in different states of the body are compared from the perspective of Z0-1 protein expression in the liver-gut axis, providing direct evidence for the theory of toxic Chinese medicine using poison to fight poison.

[0137] By adopting the technical solutions of the above embodiments and through the above settings, the present application can certainly solve the above technical problems and achieve the following technical effects:

[0138] Through biotin labeling technology and pull-down, mass spectrometry and other methods, we successfully confirmed that the direct target of evodiamine in the liver is zo-1. This discovery provides a precise molecular target for understanding the toxic mechanism of evodiamine, which will help to more accurately regulate in future drug development and toxicity prevention.

[0139] Based on the idea of ​​toxicity-effect conversion, a hepatotoxicity evaluation model of evodiamine was established. This model not only takes into account the dose factor, but also the impact of the animal's body state on drug safety. This will help to more comprehensively evaluate the safety of evodiamine in drug development and clinical applications, and reduce potential drug risks.

[0140] Mechanistic studies have found that evodiamine-induced liver injury is associated with upregulated zo-1 protein expression levels in the liver. However, compared with healthy animals, the Z0-1 protein in the liver-gut axis is downregulated overall under inflammatory bowel disease conditions. Therefore, the upregulation of Z0-1 in the liver-gut axis by evodiamine may only manifest as a therapeutic effect on inflammatory bowel disease under inflammatory bowel disease conditions, and will no longer cause liver injury. This discovery provides a new perspective for understanding the mechanism of action of evodiamine in the liver-gut axis, and can also provide a deeper understanding of the mechanism of action of evodiamine under different physiological and pathological conditions, thereby providing a scientific basis for personalized drug treatment. By regulating specific targets, the efficacy and safety of drugs can be achieved more accurately.

[0141] Evodiamine is toxic to the mouse liver under normal conditions, but under the IBD model, its liver toxicity is significantly reduced and it shows an improving effect on intestinal inflammation; this study provides important basis for the potential application of evodiamine in the treatment of IBD and suggests that it may have different pharmacological effects under different pathological conditions.

[0142] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, they can be fixedly connected, detachably connected, or integrated; they can be mechanically connected, electrically connected, or able to communicate with each other; they can be directly connected, or indirectly connected through an intermediate medium, and they can be internally connected between two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0143] Obviously, the embodiments described above are only some embodiments of the present invention, rather than all embodiments. The preferred embodiments of the present invention are given in the accompanying drawings, but they do not limit the patent scope of the present invention. The present invention can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Although the present invention has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific embodiments, or to perform equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of the present invention, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present invention.

Claims

1. A method for confirming the toxicity target of evodiamine, characterized in that: The following steps are involved: s1: Quantification of tissue protein. After 24 hours of cell administration, the culture medium was discarded, the cells were washed twice with pre-cooled PBS, and then lysed at 4°C with lysis buffer containing 1× cocktail protease inhibitor. The cells were vortexed once every half an hour for 2 hours. The protein extract obtained by lysis was centrifuged at 4°C and 13000rpm for 15 minutes. The supernatant was transferred to a new 1.5mL EP tube and centrifuged again under the same conditions to collect the supernatant. After partial dilution, the concentration was determined by BCA method. S2: Biotin-labeled evodiamine, labeling biotin at the appropriate site of evodiamine to obtain Biotin-evodiamine; S3: Balance the fixed avidin gel, resuspend the gel solution upside down, and pipette 50 μL of the suspension into the labeled SpinColumn tube; S4: fixation of biotin and biotin-evodiamine, followed by blocking of biotin and capture of biotin-evodiamine, and then cleaning and electrophoresis of the spin column tube; S5: Protein pretreatment and sample loading, followed by protein identification and bioinformatics analysis. During the protein identification process, the mass spectrometry data obtained are searched through the Proteome Discoverer2.4 software to identify the protein type.

2. The method for confirming the toxicity target of evodiamine according to claim 1, characterized in that: During the equilibration and fixation of the avidin gel, the gel solution was inverted and resuspended, 50 μL of the suspension was aspirated into a marked Spin Column tube, the bottom stopper was plugged and placed in a collection tube, 250 μL of TBS was added, the top cover was tightened, mixed, the top cover and the bottom stopper were removed, the Spin Column tube was centrifuged at 1250 g for 50 s, TBS was discarded, and the tube was reinserted into the collection tube, 250 μL of TBS was added, and the subsequent operation was repeated twice, and the bottom stopper was plugged at the bottom of the Spin Column tube.

3. The method for confirming the toxicity target of evodiamine according to claim 1, characterized in that: During the fixation of biotin and Biotin-evodiamine, 300 μL of 5 mM biotin and Biotin-evodiamine were added to the Spin Column tube, the top cover and bottom plug were tightened, and the tube was gently shaken and incubated at 4° C. on a rotating platform for 60 minutes. After the incubation, the top cover and bottom plug of the Spin Column tube were removed, placed in a collection tube, centrifuged at 1250 g for 60 seconds, and placed back in the collection tube.

4. The method for confirming the toxicity target of evodiamine according to claim 1, characterized in that: The biotin blocking and biotin-evodiamine capture process is as follows: 250 μL of biotin blocking solution is added to the Spin Column tube, mixed by inverting, and incubated at room temperature for 5 minutes. After the incubation, the top cover of the Spin Column tube is removed, and the tube is placed in a collection tube, centrifuged at 1250 g for 50 seconds, and the above operation is repeated once. TBS 250 μL is added to the Spin Column tube, the top cover is tightened, mixed by inverting, the top cover is removed, and the tube is placed in a collection tube, centrifuged at 1250 g for 50 seconds, and the above operation is repeated once, and the tube is placed in the collection tube again; Add 300 μL of captured protein sample solution to the Spin Column tube, tighten the top cover, and incubate overnight at 4°C on a rotating platform. After the incubation, remove the top cover and bottom plug of the spin column tube, place it in a collection tube, centrifuge at 1250g for 60s, remove the spin column tube from the collection tube, cover the collection tube, place it on ice, and place the spin column tube in a new collection tube for elution.

5. The method for confirming the toxicity target of evodiamine according to claim 4, characterized in that: Add 250 μL Wash Buffer to each Spin Column tube, gently invert to mix, rinse, incubate at room temperature for 1 min, remove the top cover and bottom plug of the Spin Column tube after incubation, place it in a collection tube, centrifuge at 1250 g for 50 s, repeat the above steps three times, for the last rinse, add Wash Buffer, transfer the liquid in the tube and the beads to a 1.5 mL centrifuge tube, discard the supernatant after centrifugation, add 50 uL 2× Loading buffer to resuspend the beads, shake and mix, place in a boiling water bath for 5 min, cool on ice, then load the sample, perform electrophoresis at 80 V with 10% separation gel, increase the voltage to 120 V when the marker bands are clearly separated, perform Coomassie Brilliant Blue staining and gel cutting at the end of electrophoresis.

6. The method for confirming the toxicity target of evodiamine according to claim 5, characterized in that: The cut strips were decolorized, reduced and alkylated, enzymatically hydrolyzed, and enzymatically hydrolyzed polypeptides were extracted. The samples were desalted on a Ziptip C18 column and then dried in vacuo. 0.1% FA was added to the dried samples for redissolution. 1 to 2 ug of the samples were loaded onto the machine. The mobile phases A and B were 0.1% formic acid-water and 0.1% formic acid-acetonitrile, respectively. The elution gradient was 0 to 3 min, 3 to 23 min, 23 to 42 min, 42 to 47 min, 47 to 48 min, and 48 to 60 min. The flow rate was 200 nL / min. The tandem mass spectrometry detection adopted a data-dependent scanning mode, a full scan resolution of 60,000, a mass-to-charge ratio range of m / z 350-1800, an HCD fragmentation mode, and a collision energy of 30 eV.

7. The method for confirming the toxicity target of evodiamine according to claim 1, characterized in that: During the bioinformatics analysis, the differential proteins of the experimental group and the control group were compared according to the grouping information. It was considered that the differential proteins of the evodiamine group excluding the intersection of the two groups were evodiamine-specific binding proteins. The results were displayed using a Venn diagram, and KEGG enrichment analysis was performed on the evodiamine-specific binding proteins.

8. The method for confirming the toxicity target of evodiamine according to claim 7, characterized in that: After the bioinformatics analysis, the mRNA sequence of Z0-1 was obtained from the NCBI database, purified by HPLC, and FITC-labeled Z0-1 siRNA was synthesized by conventional chemical synthesis. A negative control siRNA was designed and synthesized by a random shuffle method.

9. The method for confirming the toxicity target of evodiamine according to claim 8, characterized in that: After the Z0-1 siRNA is synthesized, the Z0-1 siRNA is transfected, and the effects of evodiamine on the proliferation toxicity of Z0-1 knockdown L02 cells and the effects of evodiamine on the apoptosis of Z0-1 knockdown L02 cells are verified through experiments.

10. A method for evaluating the hepatotoxicity of evodiamine, applicable to the method for confirming the toxicity target of evodiamine according to any one of claims 1 to 9, characterized in that: The following steps are involved: S1: C57 mice were adaptively raised for one week, during which the weight, water intake, appetite, and mental state of the mice were observed to ensure that the experimental mice were normal; S2: C57 mice were divided into blank group, 4% DDS treatment group, evodiamine treatment group, and DDS treatment group + evodiamine treatment group; S3: Drug treatment: The blank group was not treated with any treatment, and only provided with normal drinking water and food; the model group was given 4% DSS aqueous solution every day for seven consecutive days; after the model was successfully established, the drug-treated group was given intragastric administration once a day for one week, during which the weight, appetite, water intake, and mental state of the mice were observed and recorded; and the model group was given the same amount of CMC-Na solution; S4: After the experiment, serum, liver, and intestinal tissues were sampled; The liver and intestinal tissues of mice in different treatment groups were then placed in 4% paraformaldehyde for treatment, and then the slices were placed under a slice scanner for observation, photography, and analysis; Liver and intestinal tissues were collected, and western blot technology was used to determine the effect of evodiamine on Z0-1 protein.

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