Assay for screening of specific hepatotoxic components of epimedium and psoralea

Abstract

The application provides a kind of assay method for screening specific liver injury components of Epimedium and Psoralea, comprising the steps of preparing mother liquor, preparing diluent, measuring after cell administration, animal experiment verification and metabolomics analysis.The application excavates the components of Epimedium and Psoralea medicinal materials, and finds that immune stress induced by TNF-alpha, immune promoting components in Epimedium and Psoralea and susceptible components of liver injury synergistically cause liver injury, which provides potential biological target for early warning, diagnosis and treatment of liver injury caused by compatibility of Epimedium and Psoralea, and ensures safe and reasonable use of clinical drugs.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine component detection technology. Specifically, this application relates to a detection method for screening specific liver injury components of Epimedium and Psoralea corylifolia. Background Technology

[0002] Epimedium and Psoralea corylifolia are commonly used tonic herbs in traditional Chinese medicine. Epimedium tonifies kidney yang, strengthens tendons and bones, and dispels wind and dampness, while Psoralea corylifolia warms the kidneys and assists yang, promotes qi circulation and relieves asthma, warms the spleen and stops diarrhea, and can be used externally to dispel wind and remove blemishes. Both are pungent and warm in nature, and can also tonify kidney yang. They are commonly used in combination with other tonifying herbs in prescriptions for treating kidney yang deficiency, liver and kidney deficiency, lung and kidney qi deficiency, or spleen and kidney yang deficiency. In recent years, there have been reports of drug-induced liver injury caused by traditional Chinese medicine preparations such as Zhuanggu Guanjie Wan and Xianling Gubao capsules. The National Medical Products Administration has also issued corresponding notices, which may be related to the hepatotoxicity of Epimedium and Psoralea corylifolia in the formulas. Studies have shown that both Epimedium and Psoralea corylifolia can induce liver injury under immune stress, and the damage is more severe when used together than when used alone.

[0003] In recent years, reports of liver damage caused by traditionally "non-toxic" Chinese herbal medicines have been frequent, raising widespread concern about the safety of Chinese medicine and sparking controversy within the industry. For a long time, research on liver damage caused by Chinese medicine has mainly focused on direct hepatotoxicity, with little attention paid to idiosyncratic liver injury from the perspective of individual susceptibility. TNF-α is a key immunological factor in evaluating drug-induced idiosyncratic liver injury. Studies on the immunological mechanisms of idiosyncratic liver injury have shown that TNF-α synergistically activates the TNF-α-mediated MAPK / NF-κB signaling pathway, leading to the secretion of large amounts of inflammatory cytokines, causing an imbalance in the liver's immune microenvironment, inducing hepatocyte apoptosis, and resulting in liver damage.

[0004] Because the toxic chemicals in Epimedium and Psoralea corylifolia that induce liver injury are unclear, a method for screening specific liver-injury components of Epimedium and Psoralea corylifolia is needed. This study aims to elucidate the role of TNF-α-induced immune stress, immune-promoting components, and liver-injury-susceptibility components in Epimedium and Psoralea corylifolia in causing liver injury. The goal is to provide potential biological targets for early warning, diagnosis, and treatment of liver injury caused by the combination of Epimedium and Psoralea corylifolia, ensuring their safe and rational clinical use. Summary of the Invention

[0005] The purpose of this application is to provide a method for screening specific liver injury components of Epimedium and Psoralea corylifolia. The specific implementation steps include:

[0006] a. Preparation of stock solutions containing monomers of TNF-α, Epimedium, and Psoralea corylifolia

[0007] TNF-α stock solution: Prepare a 0.1 mg / mL stock solution from TNF-α powder;

[0008] Preparation of mother liquor of chemical components of Epimedium and Psoralea: 10 Epimedium monomer components and 11 Psoralea monomer components were selected, and appropriate amounts of each component were accurately weighed and placed in 1.5 mL sterile EP tubes. The components were dissolved in cell-grade DMSO to prepare mother liquor, which was then stored at -80℃ for later use.

[0009] b. Prepare diluted solutions of TNF-α, epimedium, and psoralen monomer components.

[0010] Take out the TNF-α stock solution, place it at room temperature in the dark for 2 hours, and then dilute it with pure DMEM medium for later use.

[0011] Take the stock solution of each monomer component and dilute it with pure DMEM medium to prepare monomer component dilutions of different concentrations for later use;

[0012] c. Cell drug delivery and assay

[0013] Hepatocytes were seeded into 96-well cell culture plates and cultured in an incubator. When the drug was administered, the cell culture medium was aspirated, and an appropriate amount of TNF-α diluent or its DMSO carrier was added to each well in advance. After a certain period of time, an appropriate amount of the corresponding concentration of Epimedium and / or Psoralea monomer component diluent or its DMSO carrier was added to each well, and the wells were placed in an incubator for culture. Afterward, the supernatant was aspirated and the amount of lactate dehydrogenase released was measured, which was used as an indicator to evaluate cytotoxicity.

[0014] d. Animal experiments to verify

[0015] A TNF-α-induced immune-specific liver injury model was established in mice: mice were first given TNF-α solution or its carrier, and after a period of time, they were given different concentrations of icariin and / or psoralen or its carrier. Blood was collected from the eyes of mice at the set time of administration, and the blood was collected in vitro using anticoagulant tubes containing heparin sodium. At the same time, mouse liver tissue was collected. The levels of alanine aminotransferase and lactate dehydrogenase in mouse plasma were measured. ALT and LDH were used as preliminary indicators to verify in vivo that immune-promoting components and liver injury susceptibility components synergistically induce liver injury.

[0016] e. Metabolomics analysis

[0017] After processing, liver tissue samples were analyzed using ultra-high performance liquid chromatography and tandem mass spectrometry, and the results were further analyzed using metabolomics.

[0018] Preferably, in step a:

[0019] The 10 selected epimedium monomer components are epimedin, epimedin, epimedin I, epimedin II, epimedin A, epimedin A1, epimedin B, epimedin C, dehydrated epimedin and epimedin A;

[0020] The 11 selected psoralen monomer components are psoralenol, psoralen A, psoralen glycoside, isopsoralen glycoside, psoralen, isopsoralen, isopsoralen dihydroflavonoid, neopsoralen isoflavone, psoralen dihydroflavonoid methyl ether, psoralen methyl and psoralen B.

[0021] The concentrations of the mother liquor of both Epimedium monomer and Psoralea monomer were 50 mg / mL;

[0022] The mother liquor solution is stored at -80℃.

[0023] Preferably, in step b:

[0024] During the assay, the concentration of the TNF-α diluent was 10 ng / mL;

[0025] During the determination, the concentration of the monomer component diluent was 8.33-66.67 μM;

[0026] Prepare the diluent immediately before use.

[0027] Preferably, step c includes:

[0028] Culture: Cells are cultured in an incubator for 24 hours before being administered the drug;

[0029] Pre-stimulation: Aspirate the culture medium, add 100 μL of TNF-α dilution or its DMSO carrier DMEM to each well, incubate in an incubator for 2 hours, and then administer the drug.

[0030] Administration method: Add 50 μL of the corresponding concentration of Epimedium and / or Psoralea monomer components or their DMSO carrier directly to each well;

[0031] Drug concentration: Preferably, when administered in combination, the concentration of psoralen is 33.33 μM, and the concentration range of the other drugs is 6.25-66.67 μM;

[0032] Measurement: After administration, the sample was placed in an incubator and incubated for 24 hours.

[0033] Indicator: The amount of lactate dehydrogenase (LDH) released is measured and used as an indicator to evaluate cytotoxicity.

[0034] Preferably, in step d:

[0035] A heterogeneous liver injury model was established using 18-20g BALB / c mice.

[0036] TNF-α is administered via tail vein injection;

[0037] The TNF-α dosage is 10 μg / kg;

[0038] Mice were given the corresponding drug 2 hours after TNF-α injection;

[0039] Icariin II and psoralen were prepared using 0.5% CMC-Na solution;

[0040] Preferably, the concentration of icariin II is 25 mg / kg, and the dosage of psoralen is 50 mg / kg;

[0041] The administration method was by gavage;

[0042] Mice were sacrificed 16 hours after administration;

[0043] The levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in mouse plasma were used as the measurement indicators.

[0044] Preferably, the hepatocytes in step c are human normal hepatocyte L02 cell line or human hepatocellular carcinoma HepG2 cell line, preferably human hepatocellular carcinoma HepG2 cell line.

[0045] Preferably, the cell seeding concentration in step c is 5 × 10⁻⁶. 5 ~10×10 5 Cells / mL, preferably, the cell seeding concentration is 9 × 10⁶. 5 per mL.

[0046] Preferably, the substance that induces cellular or animal immune stress in step ad is TNF-α.

[0047] Preferably, the cell culture medium in step c is prepared from a mixture of DMEM medium, fetal bovine serum and 10,000 U / ml penicillin and streptomycin. Preferably, the volume percentages of DMEM medium, fetal bovine serum and 10,000 U / ml penicillin and streptomycin in the cell culture medium are 89%, 10% and 1%, respectively.

[0048] This invention mines the components of Epimedium and Psoralea corylifolia, discovering that TNF-α-induced immune stress, along with immune-boosting and liver-damage-susceptibility components in Epimedium and Psoralea corylifolia, synergistically lead to liver damage. This provides potential biological targets for early warning, diagnosis, and treatment of liver damage caused by the combination of Epimedium and Psoralea corylifolia, ensuring their safe and rational clinical use. Attached Figure Description

[0049] Figure 1 shows the cytotoxicity detection results of various components in Epimedium; A and J represent icariin, epimedin, epimedin I, epimedin II, icariin A, epimedin A1, epimedin B, epimedin C, dehydrated epimedin, and epimedin A, respectively.

[0050] Figure 2 shows the cytotoxicity test results of various components in Psoralea corylifolia; AK represents psoralen, psoralen A, psoralen glycoside, isopsoralen glycoside, psoralen, isopsoralen, isopsoralen dihydroflavonoid, neopsoralen isoflavone, psoralen dihydroflavonoid methyl ether, psoralen methyl ether, and psoralen B, respectively.

[0051] Figure 3 shows the cytotoxicity test results of psoralen and different epimedium monomers in combination; AJ represents the combination of different components and dosages.

[0052] Figure 4 shows the cytotoxicity test results of psoralen and different epimedium monomers in combination; AH represents the combination of different components and dosages.

[0053] Figure 5 shows the cytotoxicity test results of psoralen A and different psoralen monomers in combination; AJ represents the combination of different components and dosages.

[0054] Figure 6 shows the cytotoxicity test results of psoralen A and different psoralen monomers in combination; AJ represents the combination of different components and dosages.

[0055] Figure 7 The results of cytotoxicity assays for the combined administration of psoralen and icariin II;

[0056] Figure 8 shows the results of plasma biomarker detection in mice after administration of TNF-α, psoralen, and icariin II (A is ALT and B is LDH).

[0057] Figure 9 shows the results of liver tissue metabolomics analysis; AC: OPLS-DA score; DF: goodness of fit and predictive ability; GI metabolite differences;

[0058] Figure 10 shows the Venn diagram analysis results (A) of multiple combinations of TNF-α, psoralen, and icariin II, as well as the analysis results of the KEGG pathway. Figure 10B ). Detailed Implementation

[0059] Example 1: Main Methods and Materials

[0060] Cell culture

[0061] HepG2 human hepatocellular carcinoma cells were selected because they are insensitive to the harmful effects of cytokines but simultaneously express TNF-α and IFN-γ receptors. Furthermore, in terms of cytotoxic interactions between specific liver-injury drugs and cytokines, HepG2 cells responded similarly to primary mouse and human hepatocytes. HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified incubator containing 5% carbon dioxide. Cells from passages 3-7 were used in this experiment, and only cells with a viability greater than 90% were employed.

[0062] Drug preparation

[0063] An immune stress model was constructed using TNF-α. 0.1 mg / mL stock solution was prepared from TNF-α powder tubes according to the manufacturer's instructions, aliquoted, and stored at -80℃ for later use. Ten icariin monomers were selected for screening: icariin, icariin, icariin I, icariin II, icariin A, icariin A1, icariin B, icariin C, dehydrated icariin, and icariin A. Eleven psoralen monomers were also selected: psoralen, psoralen A, psoralen glycoside, isopsoralen, psoralen, isopsoralen, isopsoralen dihydroflavonoid, neopsoralen isoflavone, psoralen dihydroflavonoid methyl ether, psoralen, and psoralen B. Accurately weigh appropriate amounts of each reference standard, dissolve them in cell-grade DMSO to prepare a stock solution with a concentration of 50 mg / mL, and store it in aliquots at -80℃ for later use.

[0064] Cytotoxicity assay

[0065] HepG2 cells were used at a rate of 9 × 10 4 Cells were seeded at a density of cells / well in 96-well plates, cultured in complete medium for 24 hours, and then drug-induced. TNF-α stock solution was incubated at room temperature in the dark for 2 hours before drug administration, then diluted to the desired concentration with pure DMEM medium. Cells were treated with TNF-α (10 ng / mL) or its carrier (DMEM) for 2 hours, followed by treatment with different concentrations of the test component or its carrier (DMEM). After 24 hours of exposure to the drug and / or TNF-α, cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) into the culture medium using the Promega CytoTox96 Non-Radioactive Cytotoxicity Assay kit.

[0066] Quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons among multiple groups, and t-test was used for comparisons between two groups. p < 0.05 indicated a significant difference, and p < 0.01 indicated a highly significant difference.

[0067] Animal experiments verified

[0068] A TNF-α-induced immune-specific liver injury model was established using 18-20g BALB / c mice. First, mice were injected intravenously with recombinant TNF-α (10 μg / kg). Two hours later, different concentrations of the test components or their carriers (0.5% CMC-Na) were administered by gavage. At predetermined administration times, the mice were enucleated to collect blood, which was then collected in anticoagulant tubes containing heparin sodium. Simultaneously, mouse liver tissue was collected. The levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in mouse plasma were measured using a Shanghai enzyme-linked ELISA kit. ALT and LDH were used as preliminary indicators to verify the synergistic induction of liver injury by immune-boosting components and liver injury susceptibility components. The research group mainly investigated the administration time and dosage to optimize the experimental design. All experiments and procedures involving mice were conducted in accordance with the guidelines of the Animal Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences.

[0069] Quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons among multiple groups, and t-test was used for comparisons between two groups. p < 0.05 indicated a significant difference, and p < 0.01 indicated a highly significant difference.

[0070] liver tissue metabolomics analysis

[0071] Liver tissue sample processing: Remove the sample from the -80℃ freezer and thaw on ice. Weigh 20 mg into the corresponding numbered centrifuge tube, add a steel ball, and homogenize using a ball mill (30 Hz) for 20 s. Then, centrifuge at 3000 rpm for 30 s at 4℃ until the sample reaches the bottom of the tube. Add 1 mL of lipid extraction buffer containing internal standard (methyl tert-butyl ether: methanol = 3:1, V / V), and vortex for 15 min. Add 200 μL of water, vortex for 1 min, and centrifuge at 12000 rpm for 10 min at 4℃. Transfer 200 μL of the supernatant to the corresponding numbered centrifuge tube, concentrate to dryness, add 200 μL of lipid reconstitution solution (acetonitrile: isool = 1:1, V / V), vortex for 3 min, centrifuge at 12000 rpm for 3 min, and transfer the supernatant for LC-MS / MS analysis.

[0072] Chromatographic and mass spectrometry acquisition conditions: The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). The liquid phase conditions mainly include: Column: Thermo Accucore™ C30 column (id 2.1 x 100 mm, 2.6 μm); Mobile phase: Phase A: acetonitrile / water (60 / 40, V / V) (containing 0.% formic acid, 10 mmol / L formic acid), Phase B: acetonitrile / isopropanol (10 / 90, V / V) (containing 0.% formic acid, 10 mmol / L formic acid); Mobile phase gradient: 0 min: A / B (80:20, V / V), 2 min: (70:30, V / V), 4 min: (40:20, V / V). The mass spectrometry parameters were as follows: 60, V / V; 9 min (15:85, V / V); 14 min (10:90, V / V); 15.5 min (5:95, V / V); 17.3 min (5:95, V / V); 17.5 min (80:20, V / V); 20 min (80:20, V / V); flow rate 0.35 mL / min; column temperature 45℃; injection volume 2 μL. The main mass spectrometry conditions included: electrospray ionization (ESI) temperature 500℃; mass spectrometry voltage 5500 V in positive ion mode; mass spectrometry voltage -4500 V in negative ion mode; ion source gas 1 (GS1) 45 psi; gas 2 (GS2) 55 psi; curtain gas (CUR) 35 psi; and collision-activated dissociation (CAD) parameter set to Medium. In the triple quadrupole, each ion pair is scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0073] Analysis results of Example 2

[0074] Using LDH as a detection indicator to screen for components of specific liver injury

[0075] as follows Figure 1A As shown in Figure J, none of the 10 components of Epimedium showed significant toxicity to HepG2 cells within the set concentration range, nor did they show significant toxicity under TNF-α-mediated immune stress (p < 0.05). Therefore, these 10 components may all be good immune-promoting components.

[0076] like Figure 2AAs shown in Figure -K, the cytotoxicity of psoralen and neopsoralen isoflavones, components of Psoralea corylifolia, increased with increasing drug concentration within a specific concentration range, and the cytotoxicity was significantly increased under TNF-α stimulation (p < 0.05). Since psoralen has almost no cytotoxicity when administered alone, but its cytotoxicity is significantly increased under TNF-α stimulation (p < 0.05), we selected psoralen as a susceptible component for further studies. Furthermore, the other monomeric components of Psoralea corylifolia are likely to be beneficial immunomodulatory components.

[0077] like Figure 3A -J and Figure 4A As shown in Figure -H, when psoralen was administered in combination with 10 different icariin monomers, the LDH release was measured. The results showed that at a dose of 66.67 μM, the synergistic toxicity index of psoralen and icariin was the highest, at 1.93; at doses of 33.33 and 16.67 μM, the synergistic toxicity index of psoralen and icariin B was the highest, at 1.33 and 2.22, respectively; and at doses of 16.67 and 8.33 μM, the synergistic toxicity index of psoralen and icariin II was the highest, at 3.18 and 1.71, respectively.

[0078] like Figure 5A -J and Figure 6A As shown in Figure J, when psoralen was administered in combination with 10 different psoralen monomers, the LDH release was measured. The results showed that at a dose of 66.67 μM, the synergistic toxicity index of psoralen and psoralen dihydroflavonoid methyl ether was the highest, at 2.52; at doses of 33.33 and 25 μM, the synergistic toxicity index of psoralen and isopsoralen dihydroflavonoid was the highest, at 3.14 and 3.02, respectively; and at doses of 16.67 and 8.33 μM, the synergistic toxicity index of psoralen and neopsoralen isoflavone was the highest, at 3.23 and 2.72, respectively.

[0079] Preferably, icariin, icariin B, and icariin II from Epimedium, as well as psoralen dihydroflavonoid methyl ether, isopsoralen dihydroflavonoid, and neopsoralen isoflavone from Psoralea corylifolia, are all considered as good immune-boosting components. Since psoralen A and icariin II have relatively high synergistic toxicity indices and are derived from Psoralea corylifolia and Epimedium respectively, they are more representative. Therefore, psoralen A and icariin II were finally selected for subsequent cell and animal experiments.

[0080] 1. Establishment of a idiosyncratic liver injury cell model

[0081] Preferably, we selected psoralen as a susceptible component for liver injury and icariin II as an immune-boosting component to establish a idiosyncratic liver injury cell model. Figure 7As shown, when the administration time was 24 h, the TNF-α dose was 10 ng / mL, the psoralen dose was 33.33 μM, and the icariin II dose was 16.67 μM, the LDH release levels in each group showed significant differences (p<0.0005). Therefore, under TNF-α-induced cellular immune stress, the immune-promoting component icariin II and the liver-damage-susceptibility component psoralen have a synergistic effect on hepatocyte damage, and icariin II can enhance the specific hepatocyte-damaging effect of psoralen.

[0082] 2. Establishment of an animal model of idiosyncratic liver injury

[0083] like Figure 8A As shown in Figure B, preferably, when the TNF-α dosage is 10 μg / kg, the psoralen dosage is 50 mg / kg, and the icariin II dosage is 25 mg / kg, the mice are sacrificed 16 h after TNF-α administration, and the changes in plasma ALT and LDH levels in the mice are measured.

[0084] 3. Liver tissue metabolomics analysis

[0085] Analysis was performed using the supervised pattern recognition method OPLS-DA. OPLS-DA scores showed that the normal group and the TNF-α group, the TNF-α + psoralen group and the TNF-α + psoralen + icariin II group, and the TNF-α + icariin II group and the TNF-α + psoralen + icariin II group were dispersed in two distinct regions. Figure 9A (B and C). The goodness-of-fit and predictive power values ​​indicate that the OPLS-DA model has good fitting and predictive capabilities. Figure 9D (E and F) indicates that the metabolic environment of mice in different physiological states has significant differences. A total of 281 differentially expressed metabolites were identified in the normal group compared to the TNF-α group. Compared to the TNF-α group, the normal group showed increased levels of 205 metabolites and decreased levels of 76 metabolites. A total of 135 differentially expressed metabolites were identified in the TNF-α + psoralen group compared to the TNF-α + psoralen + icariin II group. Compared to the TNF-α + psoralen + icariin II group, the TNF-α + psoralen group showed increased levels of 56 metabolites and decreased levels of 79 metabolites. A total of 125 differentially expressed metabolites were identified in the TNF-α + psoralen + icariin II group compared to the TNF-α + psoralen + icariin II group. Compared to the TNF-α + psoralen + icariin II group, the TNF-α + icariin II group showed increased levels of 71 metabolites and decreased levels of 54 metabolites. Figure 9G H and I).

[0086] The differences between the normal group and the TNF-α group were combined with the differences between the TNF-α + psoralen group and the TNF-α + psoralen + icariin II group, and the differences between the TNF-α + icariin II group and the TNF-α + psoralen + icariin II group. Venn diagram analysis revealed that 32 differences between the TNF-α + psoralen group and the TNF-α + icariin II group and the TNF-α + icariin II group did not overlap with the differences between the normal group and the TNF-α group. Figure 10A This indicates that these differential variables are only affected by the synergistic effect of TNF-α and the drug, and not by the drug or TNF-α alone; therefore, they can be considered as variables related to liver injury. Finally, liver injury-related variables were analyzed using the KEGG pathway, and enriched in metabolic pathways such as sphingolipid metabolism, sphingolipid signaling pathway, necrosis, and apoptosis. Figure 10B ).

Claims

1. A method for screening specific liver-damaging components of Epimedium and Psoralea corylifolia, the method comprising the following steps: a. Preparation of stock solutions containing TNF-α, Epimedium, and Psoralea corylifolia monomers TNF-α stock solution: Prepare a 0.1 mg / mL stock solution from TNF-α powder; Preparation of mother liquor of chemical components of Epimedium and Psoralea: 10 Epimedium monomer components and 11 Psoralea monomer components were selected, and appropriate amounts of each component were accurately weighed and placed in 1.5 mL sterile EP tubes. The components were dissolved in cell-grade DMSO to prepare mother liquor, which was then stored at -80℃ for later use. b. Prepare diluted solutions of TNF-α, epimedium, and psoralen monomer components. Take out the TNF-α stock solution, place it at room temperature in the dark for 2 h, and then dilute it with pure DMEM medium for later use. Take the stock solution of each monomer component and dilute it with pure DMEM medium to prepare monomer component dilutions of different concentrations for later use; c. Cell drug delivery and assay Culture: The human hepatocellular carcinoma HepG2 cell line was seeded into 96-well cell culture plates and cultured in an incubator for 24 hours before being administered the drug. Pre-stimulation: Aspirate the culture medium, add 100 μL of TNF-α dilution or its DMSO carrier DMEM to each well, incubate in an incubator for 2 h, and then administer the drug. Administration: Add 50 μL of the corresponding concentration of Epimedium and / or Psoralea monomer components or their DMSO carrier directly to each well; when administered in combination, the concentration of Psoralea alpha is 33.33 μM, and the concentration range of the other drugs is 6.25-66.67 μM. Measurement: After administration, the sample was placed in an incubator and incubated for 24 hours. Indicator: The amount of lactate dehydrogenase (LDH) released was measured and used as an indicator to evaluate cytotoxicity. d. Animal experiments to verify A TNF-α-induced immune-specific liver injury model was established in mice: mice were first given TNF-α solution or its carrier, and after a period of time, they were given different concentrations of icariin and / or psoralen or its carrier. Blood was collected from the eyes of mice at the set time of administration, and the blood was collected in vitro using anticoagulant tubes containing heparin sodium. At the same time, mouse liver tissue was collected. The levels of alanine aminotransferase and lactate dehydrogenase in mouse plasma were measured. ALT and LDH were used as preliminary indicators to verify in vivo that immune-promoting components and liver injury susceptibility components synergistically induce liver injury. e. Metabolomics analysis After processing, liver tissue samples were analyzed using ultra-high performance liquid chromatography and tandem mass spectrometry, and the results were further analyzed using metabolomics.

2. The testing method according to claim 1, wherein, In step a: The 10 selected epimedium monomer components are epimedin, epimedin, epimedin I, epimedin II, epimedin A, epimedin A1, epimedin B, epimedin C, dehydrated epimedin and epimedin A; The 11 selected psoralen monomer components are psoralenol, psoralen A, psoralen glycoside, isopsoralen glycoside, psoralen, isopsoralen, isopsoralen dihydroflavonoid, neopsoralen isoflavone, psoralen dihydroflavonoid methyl ether, psoralen methyl and psoralen B. The concentrations of both the epimedium monomer and psoralen monomer components in the mother liquor were 50 mg / mL. The mother liquor solution is stored at -80℃.

3. The testing method according to claim 1, wherein, In step b: During the assay, the concentration of the TNF-α diluent was 10 ng / mL; During the assay, the concentration of the monomer component diluent was 8.33-66.67 μM; Prepare the diluent immediately before use.

4. The testing method according to claim 1, wherein, In step d: A heterogeneous liver injury model was established using 18-20 g BALB / c mice. TNF-α is administered via tail vein injection; The dosage of TNF-α is 10 μg / kg; Mice were given the corresponding drug 2 hours after TNF-α injection; Icariin II and psoralen were prepared using 0.5% CMC-Na solution; The dosage of icariin II was 25 mg / kg, and the dosage of psoralen was 50 mg / kg. The administration method was by gavage; Mice were sacrificed 16 hours after administration; The levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in mouse plasma were used as the measurement indicators.

5. The method for screening specific liver injury components according to any one of claims 1-3, wherein, The cell seeding concentration in step c is 9 × 10⁻⁶. 5 per mL.

6. The testing method according to any one of claims 1-3, wherein, In step c, the cell culture medium is prepared by mixing DMEM medium, fetal bovine serum and 10,000 U / ml penicillin and streptomycin, with the volume percentages of DMEM medium, fetal bovine serum and 10,000 U / ml penicillin and streptomycin in the cell culture medium being 89%, 10% and 1%, respectively.

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