Use of luteolin and / or trans-UCA in the preparation of a drug for removing bis(2-ethylhexyl) phthalate

Abstract

The present invention discloses the use of luteolin and / or trans-UCA in the preparation of a drug for removing bis(2-ethylhexyl) phthalate. The present invention introduces an innovative method, that is, using a visual DEHP tracer system and a compound library as the basis for screening potential drugs; the emphasis is on directly starting the screening process from DEHP itself and its specific targets; it is first discovered that luteolin and its regulated endogenous trans-UCA are identified as potential functional drugs and metabolites for effectively eliminating DEHP in the liver; the target molecule Uroc1 of luteolin is the main target for the body to effectively eliminate DEHP and its related pollutants in the liver.

Description

Technical Field

[0001] The present invention relates to the use of luteolin and / or trans-UCA in the preparation of a drug for removing bis(2-ethylhexyl) phthalate, and belongs to the field of drug applications. Background Art

[0002] At present, phthalate esters (PAEs) account for approximately 65% of the global plasticizer market, and bis(2-ethylhexyl) phthalate (DEHP) is the most prominent PAE compound. Since there is no chemical bond connection with the polymer, DEHP has a significant tendency to escape, that is, it can easily release from plastic materials and then penetrate into the atmosphere, edible substances or liquids. Therefore, this chemical substance is commonly detected in food packaging paper, disposable food containers, children's toys and cosmetics. As an exogenous compound, the accumulation of DEHP exposure is particularly obvious in the liver, exceeding other organs such as the gastrointestinal tract and kidneys. Therefore, the liver is the main target organ for DEHP accumulation. Epidemiological evidence reveals an inherent connection between DEHP exposure and lipid metabolism disorders, liver injury and non-alcoholic fatty liver disease, which poses a great threat to the overall stability of public health and social economy.

[0003] As a non-persistent pollutant, DEHP rapidly spreads to the entire peripheral tissue before being cleared from the body. Notably, when it reaches the liver, DEHP is metabolized in the body into more harmful compounds and disrupts normal metabolic pathways by increasing oxidative stress and triggering inflammation, and the damage mechanism to the body is very complex. Many monomeric drugs derived from traditional Chinese medicine, such as lycopene and icariin, which are known for their antioxidant or anti-inflammatory properties, can reduce the damaging consequences of DEHP in the liver and other peripheral tissues. However, these drugs can only relieve symptoms and do not solve the fundamental problem of DEHP retention in human tissues. Therefore, reducing the accumulation of DEHP in the liver and shortening its retention time may provide a new treatment strategy for eliminating the negative impact of DEHP on human health.

[0004] In China, traditional Chinese medicine (TCM) classical prescriptions are hailed as traditional empirical prescriptions in ancient medical books and have been accumulated by ancient physicians through hundreds of years of clinical practice. These prescriptions have the advantages of high safety and fine formulation. In addition, a large number of records and corresponding treatment methods for detoxification are contained in ancient TCM literature. For example, the "Three-bean Drink" composed of mung beans, adzuki beans, black beans, and liquorice in "Compendium of Materia Medica" has remarkable properties in dissipating heat, detoxifying, nourishing the liver, and promoting lung hydration. With the development of network pharmacology, various chemical monomers have been proven to be the dominant and functional components for treating various diseases. For example, Qingfei Decoction, an herbal formula commonly used to treat acute lung injury, still has an unclear mechanism of action. Some studies have successfully identified the active compounds and their potential targets by using network pharmacology and molecular docking systems. These studies not only reveal the therapeutic effects of individual chemical monomers but also help to better understand their pharmacological mechanisms, thus accelerating the clinical transformation process of TCM. Therefore, a comprehensive network pharmacological analysis of TCM detoxification prescriptions will help to identify effective drugs, more effectively eliminate redundant DEHP in the human liver, and clarify their potential molecular targets.

[0005] However, the difficult-to-track nature of DEHP poses great difficulties for its detection, resulting in the lack of high-throughput screening technology and limited drug options for specifically targeting and promoting the elimination of DEHP. To solve this problem, autoradiography technology has been used, using 3 H-labeled DEHP to trace its distribution in rat testes. However, this method requires long-term development of the test parts, the use of specialized instruments or equipment, and the implementation of additional safety precautions to protect the experimenters. In addition, due to the lack of a carboxyl group (-COOH) in DEHP, it is difficult to form a cross-link with the amino group (-NH2) present on the fluorescent probe. Therefore, it is crucial to establish an innovative visual detection and tracking system for DEHP. At the same time, due to the lack of a visual drug screening platform, drugs for eliminating DEHP have not been studied and their specific mechanisms are not clear. Summary of the Invention

[0006] Object of the Invention: The technical problem to be solved by the present invention is to provide a method for tracing the elimination of di-(2-ethylhexyl) phthalate by luteolin using a visual DEHP tracing system.

[0007] Technical Solution: To solve the above technical problem, the present invention provides the use of luteolin and / or trans-UCA in the preparation of a drug for reducing or eliminating di-(2-ethylhexyl) phthalate in the body.

[0008] The present invention also provides the use of luteolin and / or trans-UCA in the preparation of a drug for promoting the excretion of di-(2-ethylhexyl) phthalate.

[0009] The present invention also provides the use of luteolin and / or trans-UCA in the preparation of a drug for reducing the content of bis(2-ethylhexyl) phthalate in the liver, serum or urine.

[0010] The present invention also provides the use of luteolin and / or trans-UCA in the preparation of a drug for improving or treating mild liver function injury caused by bis(2-ethylhexyl) phthalate.

[0011] The present invention also provides the use of luteolin and / or trans-UCA in the preparation of a drug for reducing the levels of aspartate aminotransferase and / or alanine aminotransferase.

[0012] Among them, the concentration of luteolin is 1-10 μM.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The present invention introduces an innovative method, that is, using the visualization DEHP tracer system and compound library as the basis for screening potential drugs; the emphasis is on directly starting the screening process from DEHP itself and its specific targets; 2. The present invention discovers for the first time that luteolin and its regulated endogenous trans-UCA are identified as potential functional drugs and metabolites for effectively eliminating DEHP in the liver; 3. The target molecule Uroc1 of luteolin is the main target for the body to effectively eliminate DEHP and its related pollutants in the liver. Description of the Drawings

[0014] Figure 1 It is a schematic flow chart for aptamer screening and csa-DEHP complex synthesis;

[0015] Figure 2 It is the fluorescence spectra of Cy5, aptamer and Aptamer-Cy5;

[0016] Figure 3 It is the schematic diagram of the 1H NMR spectrum of DEHP;

[0017] Figure 4 It is the schematic diagram of the 1H NMR spectrum of the aptamer;

[0018] Figure 5 It is the schematic diagram of the 1H NMR spectrum of the binding of DEHP and the aptamer;

[0019] Figure 6 It is the cytotoxicity detection of csa-DEHP in primary hepatocytes, where N.S. represents no significant difference;

[0020] Figure 7 It is the situation of csa-DEHP in primary mouse hepatocytes at different retention times: a is the representative fluorescence image; b is the relative fluorescence intensity;

[0021] Figure 8 Schematic diagram of the localization of csa-DEHP in organelles;

[0022] Figure 9 Effect of endocytosis inhibitors of different pathways on the cellular uptake of csa-DEHP: a is the representative fluorescence image; b is the effect on the relative fluorescence intensity of cells;

[0023] Figure 10 Effect of different temperatures (changes in the lipid bilayer structure) on the cellular uptake of csa-DEHP: a is the representative fluorescence image; b is the relative fluorescence intensity of cells;

[0024] Figure 11 Situation of primary mouse hepatocytes under the treatment of free Cy5 probe, aptamer-Cy5, and csa-DEHP: a is the representative fluorescence image; b is the effect on the relative fluorescence intensity of cells;

[0025] Figure 12 Schematic diagram of the screening process of drugs promoting the excretion of redundant DEHP in the liver;

[0026] Figure 13 Summary chart of the number of bioactive ingredients;

[0027] Figure 14 Distribution map of repeated active ingredients of 21 traditional Chinese medicines and 37 effective monomers;

[0028] Figure 15 Cytotoxicity analysis of candidate small molecule compounds;

[0029] Figure 16 Situation of primary mouse hepatocytes after incubation with candidate small molecule compounds: a is the fluorescence result chart; b is the relative fluorescence intensity;

[0030] Figure 17 LC-MS analysis for screening small molecule compounds from traditional Chinese medicines that promote the excretion of DEHP in the liver;

[0031] Figure 18 Luteolin promotes the excretion of DEHP in the form of time gradient and concentration gradient: a is the fluorescence result chart; b is the relative fluorescence statistical chart of luteolin promoting the excretion of DEHP in the form of time gradient; c is the relative fluorescence statistical chart of luteolin promoting the excretion of DEHP in the form of concentration gradient;

[0032] Figure 19 Comparison chart of the promoting excretion effects of luteolin on aptamer and csa-DEHP: a is the fluorescence result chart; b is the relative fluorescence statistical chart;

[0033] Figure 20To detect the levels of DEHP in the livers, sera and urine of mice by HPLC-MS;

[0034] Figure 21 For the serum AST and ALT levels of mice in the luteolin treatment group and the control group;

[0035] Figure 22 For the heat map of differentially expressed proteins in the proteomics of primary hepatocytes of mice in the luteolin treatment group and the control group;

[0036] Figure 23 For the MOE analysis of the binding potential between luteolin and differentially expressed proteins;

[0037] Figure 24 For the changes in the transcriptional and protein levels of Uroc1 in cells after DEHP treatment: a is the statistical chart of Uroc1 transcription; b is the change in Uroc1 protein level;

[0038] Figure 25 For the analysis of the changes in the transcriptional and protein levels of Uroc1 in vivo and in vitro in the luteolin treatment group and the DEHP group: a is the; Uroc1 transcription in primary hepatocytes treated with DEHP and Luteolin; b is the Uroc1 protein expression in primary hepatocytes treated with DEHP and Luteolin; c is the Uroc1 protein expression in the livers of mice after intragastric administration of DEHP and Luteolin;

[0039] Figure 26 For the CHX chase map of luteolin accelerating the degradation of Uroc1 protein: a is the; Uroc1 protein expression after CHX treatment; b is the statistical analysis of the Uroc1 protein half-life;

[0040] Figure 27 For the schematic diagram of luteolin-dependent ubiquitination pathway for degrading Uroc1: a is the protein result diagram; b is the protein statistical chart;

[0041] Figure 28 For the result diagram of the molecular docking analysis between Uroc1 and luteolin;

[0042] Figure 29 For the effect of predicting the influence of binding site mutations on the secondary structure of Uroc1 protein by the SOPMA algorithm;

[0043] Figure 30 For the Western blot analysis of the ubiquitination level of Uroc1 protein in primary hepatocytes of mice;

[0044] Figure 31 For the effect of mutating the Uroc1 A270 and V272 sites on the role of luteolin in promoting the excretion of DEHP: a is the fluorescence schematic diagram; b is the relative fluorescence statistical chart;

[0045] Figure 32 Schematic diagram of the effect of interfering with Uroc1 expression to promote the excretion of DEHP in primary mouse hepatocytes: a is the fluorescence schematic diagram; b is the relative fluorescence statistical chart;

[0046] Figure 33 DEHP levels in the liver, serum and urine of mice overexpressing Uroc1;

[0047] Figure 34 Serum AST and ALT levels in Uroc1-overexpressing mice;

[0048] Figure 35 Schematic diagram of the role of Uroc1 in histidine metabolism in the body;

[0049] Figure 36 Cytotoxicity detection of Uroc1 upstream metabolites and DEHP co-stimulation: a is the cytotoxicity detection of L-His in primary hepatocytes; b is the cytotoxicity detection of trans-UCA in primary hepatocytes; c is the cytotoxicity detection of DEHP and L-His co-stimulating primary hepatocytes; d is the cytotoxicity detection of DEHP and trans-UCA co-stimulating primary hepatocytes;

[0050] Figure 37 Effect of Uroc1 upstream metabolites on the promotion of csa-DEHP excretion: a is the fluorescence schematic diagram; b is the relative fluorescence statistical chart;

[0051] Figure 38 HPLC-MS detection of the effect of Uroc1 upstream metabolites on the promotion of csa-DEHP excretion;

[0052] Figure 39 Situation of trans-UCA promoting the excretion of DEHP in the liver in the form of time gradient and concentration gradient: a is the fluorescence result chart; b is the relative fluorescence statistical chart at different times; c is the relative fluorescence statistical chart at different concentrations;

[0053] Figure 40 DEHP levels in the liver, serum and urine of trans-UCA treatment group and control group mice;

[0054] Figure 41 Serum AST and ALT levels of trans-UCA treatment group and control group mice;

[0055] Figure 42 Heat map of differentially expressed proteins in proteomics of primary hepatocytes of trans-UCA treatment group and control group mice;

[0056] Figure 43Gene GO analysis of proteomics results;

[0057] Figure 44 Phosphorylation level of ERK1 / 2 protein after DEHP treatment: a is the phosphorylation level of ERK1 / 2 protein in primary hepatocytes treated with DEHP; b is the corresponding protein statistics;

[0058] Figure 45 Effect of inhibiting lysosome-related pathways on the promotion of redundant DEHP clearance in mouse liver by luteolin / trans-UCA: a is the fluorescence result map; b is the relative fluorescence intensity;

[0059] Figure 46 Schematic diagram of the effect of LC-MS analysis on the effect of inhibiting lysosome-related pathways on the action of luteolin / trans-UCA. Detailed implementation manners

[0060] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0061] Example 1 Synthesis and characterization of csa-DEHP

[0062] Using an ssDNA aptamer containing a Cy5 probe and highly specific for targeting DEHP to achieve visual tracing of DEHP ( Figure 1 ). Pipette 50 μL of 0.2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 μL of 0.2 mM N-hydroxysuccinimide (NHS) into a 2 mL EP tube, and then add 15 μL of 10 mM ssDNA aptamer. Vortex thoroughly for 10 min. A cross-linking reaction occurs between the ssDNA aptamer and EDC / NHS molecules, and an amide bond is formed between the carboxylic acid group of the ssDNA aptamer and the amino group of Cy5. Dialyze the reaction mixture with deionized water for 3 days using a dialysis membrane (molecular cut-off value: 4000 MW) and freeze-dry to obtain the aptamer conjugated with Cy5, Aptamer-Cy5 (the sequence is from the publicly disclosed patent (application number: 202210683778.1, SEQ ID No. 10 of this patent)). Fluorescence spectroscopy analysis shows ( Figure 2 ) that the aptamer-Cy5 complex (Aptamer-Cy5) is at 650

[0063] Absorption peaks appear at both nm and 670 nm, overlapping with the absorption spectra of the ssDNA aptamer and Cy5 itself, indicating the successful grafting of Cy5 to the aptamer. Subsequently, taking advantage of the strong affinity and specificity of the aptamer, the freeze-dried aptamer-Cy5 complex after dialysis was dissolved in 1 mL of ddH2O, then 100 μL of 10 μM DEHP was added, and gently stirred overnight in the dark at 4 °C. The dialysis technique was used to remove the unbound free DEHP, and finally, a ternary complex csa-DEHP with stable binding of DEHP, aptamer, and Cy5 was obtained. In the 1 1H NMR spectrum of DEHP, the hydrogen signal peaks of the benzene ring are at 7.25 ppm and 7.22 ppm, while the signal peaks of the methylene groups on the carbon chain are at 4.2 ppm and 1.6 ppm. In the 1H NMR spectrum of the aptamer, the methylene signal peak is at 3.4 ppm, and the amino signal is at 1.9 ppm. As Figures 3 - 5 shown, in the 1H NMR spectrum of csa-DEHP, the hydrogen signal peak of the benzene ring in DEHP and the signal peak of the amino group of the aptamer were detected, and the signal peak of the amino group of the aptamer underwent a chemical shift to 2.3 ppm, indicating that DEHP binds to the Cy5-labeled aptamer through hydrogen bonds.

[0064] To verify the stability of the system and its effect on cell viability, the present invention used the CCK-8 method (the kit was purchased from Nanjing Jiancheng Bioengineering Institute) to analyze the potential cytotoxicity of the system. As Figure 6 shown, different concentrations of csa-DEHP had no effect on the survival rate of primary hepatocytes of mice (6-week-old healthy male C57 / BJ6 mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.). Moreover, as Figure 7 and Figure 8As shown, csa-DEHP remained stable in the cytoplasm after 48 h and was enriched in mitochondria (Mito) and lysosomes (Lyso). To further explore the cellular uptake mechanism of csa-DEHP, the present invention utilized a variety of endocytosis inhibitors, including Filipin (1 μg / mL) to block caveolin-mediated endocytosis, Chlorpromazine (CPZ, 10 μg / mL) to block clathrin-mediated endocytosis, and 5-(N-Ethyl-N-isopropyl) Amiloride (EIPA, 10 μg / mL) to block macropinocytosis. To illustrate the cellular uptake mechanism of csa-DEHP, primary mouse hepatocytes were pre-incubated with serum-free medium containing the above inhibitors for 30 min. Then, the cells were incubated for an additional 12 h with a medium containing both 150 nM csa-DEHP and different inhibitors. Samples were then collected, washed three times with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, stained with DAPI for nuclei, washed three times with PBS, and 500 μL of PBS was re-added. The intracellular fluorescence distribution and intensity were observed under a laser scanning confocal microscope (LSCM). As Figure 9 shown, after adding the inhibitors, the uptake rate of csa-DEHP by hepatocytes decreased significantly; among them, Figure 9 CTL in Figure 9 a and csa-DEHP in Figure 10 b were both treated with csa-DEHP alone for 12 h without adding inhibitors. Then, primary mouse hepatocytes were treated with csa-DEHP at 4 °C and 37 °C for 12 h, respectively. The results were as Figure 11 shown, the uptake rate of csa-DEHP by hepatocytes decreased significantly at 4 °C. These results indicate that endocytosis and macropinocytosis are the main pathways for cellular uptake of csa-DEHP; and temperature also plays a role in the uptake process. This is because low temperature can change the lipid bilayer structure, and the uptake of csa-DEHP is inhibited at 4 °C, suggesting that csa-DEHP still retains its lipophilic characteristics. In addition, primary mouse hepatocytes were treated with free Cy5 probe (Cy5), aptamer-Cy5 (Amtamer-Cy5), and csa-DEHP for 12 h, respectively. The results were as Figure 11 shown, compared with the treatment with free Cy5 probe and aptamer-Cy5, csa-DEHP had a longer retention time in primary mouse hepatocytes, indicating that DEHP is a key factor for the effectiveness of the csa-DEHP visualization system.

[0065] Example 2 Network Pharmacology Analysis and Drug Screening of New Drugs with Hepatic Redundant DEHP Capacity

[0066] As Figure 12 shown, in order to screen for drugs that can promote the liver to clear redundant DEHP, the present invention analyzed 128 traditional Chinese medicine detoxification prescriptions from traditional Chinese medicine classical formulas and traditional Chinese medicine chemical composition libraries (https: / / organchem.csdb.cn / scdb / main / TCM_introduction.ASP), and a total of 21 traditional Chinese medicines with a frequency of occurrence ≥ 10 times were found (Table 1).

[0067] Table 1 Active ingredients of 21 traditional Chinese medicines

[0068]

[0069]

[0070] Subsequently, in-depth analysis was carried out using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (http: / / LSP.nwu.edu.cn / TCPMSP.PHP). Drug components were screened according to the criteria of oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18, and half-life ≥ 4 h, and a total of 387 effective small molecule compounds were obtained. As Figure 13 and Figure 14 shown, in order to identify the effective monomer components, Venn and topological analyses (using Cytoscape software to calculate the Degree value) were performed on the above 387 effective small molecule compound results, and 37 effective monomers were screened out, among which 15 monomers with Degree ≥ 3 were obtained (Table 2).

[0071] Table 2 Topological calculations of effective small molecule compounds

[0072]

[0073] On the other hand, the present invention also performed CCK-8 analysis to evaluate the potential toxicity of these drugs to primary mouse hepatocytes. Eight of the above-mentioned candidate small molecule compounds were selected for cytotoxicity analysis, and the results are as Figure 15 shown. Kaempferol and Paeoniflorin showed cytotoxicity. Finally, the present invention obtained 6 important monomer compounds (Luteolin, Wogonin, Isorhamnetin, Quercetin, Stigmasterol, and Baicalin) with the potential to promote the liver to excrete redundant DEHP.

[0074] Example 3 Screening and efficacy confirmation of drugs for promoting the excretion of redundant DEHP in the liver

[0075] To verify the effect of the above six monomeric drugs in promoting the excretion of redundant DEHP from the livers of mice, in the present invention, primary mouse hepatocytes were exposed to csa-DEHP for 12 hours (h) to simulate environmental exposure to DEHP, and then co-incubated with the above drugs for 24 h. As Figure 16 shown, the results of confocal laser scanning microscopy analysis indicated that luteolin had the strongest ability to promote the excretion of DEHP from the liver.

[0076] LC-MS analysis further confirmed this result ( Figure 17 ).

[0077] Steps: (1) Sample pretreatment:

[0078] Tissue: 50 mg of liver was placed into a 1.5 mL EP tube, 2 mL of methanol was added, homogenized with magnetic beads, 10 ng / mL of internal standard DEHP-D4 (solvent: methanol, prepared in advance), 100 μL was added, sonicated for 10 min, vortexed for 5 min, centrifuged at 14000 rpm at 4 °C for 10 min. 1.5 mL of the supernatant was taken and transferred to a new EP tube, dried under nitrogen, re-dissolved in 200 μL of acetonitrile, and vortexed for 5 min. Then, centrifuged at 14000 rpm at 4 °C for 10 min, 180 μL of the supernatant was taken and transferred to an insert tube (the insert tube is a device for small volumes in liquid phase detection, and the insert tube is placed inside the injection vial), the bottle cap was tightened (to prevent volatilization of organic solvents), and then loaded for detection.

[0079] Cells: After sample collection, PBS was added for washing, repeated 3 times to remove residual culture medium. After washing, 1 mL of PBS was aspirated and added to a 3.5 cm sample cell dish, and the cells were collected in a 1.5 mL EP tube. The cells were centrifuged at 14000 rpm for 5 min to precipitate the cells. The upper layer of liquid was aspirated and discarded, leaving 100 μL at the bottom of the tube. 100 μL of internal standard DEHP-D4 (10 ng / mL) and 400 μL of methanol were added, vortexed for 5 s, sonicated 3 times (at 4 °C, intensity 60%, working for 10 s, pausing for 10 s, total duration 3 min), vortexed for 5 min, centrifuged at 14000 rpm for 10 min, then 80% of the supernatant, i.e., 480 μL, was aspirated for nitrogen drying. After nitrogen drying, re-dissolved in acetonitrile. Vortexed for 5 min, sonicated for 10 min, then centrifuged at 14000 rpm for 10 min, and the supernatant was taken. Loaded into the insert tube of the injection vial and loaded for analysis.

[0080] (2) LC-MS conditions:

[0081] a. Mobile phase: A: pure water, B: 90% methanol, 5.0 mM ammonium acetate (ammonium acetate was completely dissolved in water and then methanol was added). The gradient elution program is shown in Table 3-1.

[0082] Table 3 Gradient elution table

[0083]

[0084] b. Chromatographic column: Agilent ZORBAX RRHD Eclipse Plus C18, 2.1×100 mm, 1.8 μm (Part No. 959758 - 902). Column temperature: 50 °C, injection volume: 10 μL, autosampler temperature: 4 °C. The instrument settings for LC - MS are shown in Table 4.

[0085] Table 4 Triple quadrupole LC - MS conditions

[0086]

[0087]

[0088] After treating primary hepatocytes with csa - DEHP for 12 h and then removing csa - DEHP, the primary hepatocytes were treated with different concentrations or for different times of Luteolin and then sampled. The treatment times of Luteolin were set as 0, 6, 12, 24 h, and the concentration gradient of Luteolin treatment was set as 0, 1, 5, 10 μM. It was found that Luteolin could promote the excretion of redundant DEHP from primary mouse hepatocytes in a time - and concentration - gradient - dependent manner ( Figure 18 ). Then, after treating with Cy5, Aptamer - Cy5 or csa - DEHP for 12 h respectively, the primary hepatocytes were treated with 10 μM Luteolin for 24 h to analyze the promoting effect of Luteolin on the excretion of aptamer and csa - DEHP. Figure 19 It was shown that Luteolin did not affect the intracellular content of free Cy5 or Cy5 - modified aptamer. Among them, Figure 19 CTL in it represents the promoting effect of aptamer and csa - DEHP after treating primary hepatocytes with 0.1% DMSO for 24 h.

[0089] To further confirm the promoting effect of Luteolin on excretion, mice were given DEHP by gavage at a dose of 10 mg / kg / d for 28 days, and in the last 14 days, Luteolin was given by gavage at a dose of 10 mg / kg / d (using corn oil as the control, CTL) for treatment. The experimental animals selected were 6 - week - old healthy male C57 / BJ6 mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.). The mice were housed in a standard laboratory environment with a temperature of 24 ± 2 °C, a relative humidity of 50% - 60%, free access to food and water, illuminated by ordinary fluorescent lamps, and the light cycle was controlled by an automatic time - control switch at 12L:12D. Methanol was used to extract and separate DEHP from mouse liver tissue, serum and urine, and after nitrogen blowing and re - dissolution, HPLC / MS analysis was performed using the isotope internal standard method (same conditions and treatment steps as above). As Figure 20As shown in the results, luteolin reduced the DEHP content in the liver, serum and urine of mice. In addition, the serum AST and ALT levels of mice in the luteolin treatment group and the control group were measured using the CCK-8 method. Figure 21 As shown in the results, the mild liver function damage caused by DEHP was improved, which was specifically manifested by a significant decrease in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of mice gavaged with DEHP.

[0090] Example 4 Uroc1 is the molecular target of luteolin in promoting DEHP excretion

[0091] In order to identify the molecular targets of luteolin in promoting DEHP efficacy, the present invention conducted label-free quantitative proteomic analysis on primary mouse hepatocytes treated with luteolin. The cells were initially treated with 10 μM DEHP for 12 h, and then after removing DEHP, they were treated with 10 μM luteolin (with 0.1% DMSO as a control) for another 12 h. After standard protein digestion analysis (sequencing by Shanghai Zhongke New Life Biotechnology Co., Ltd.) and LC / MS / MS analysis (conditions and processing steps were the same as in Example 3), a detailed proteomic analysis was performed (performed by Shanghai Zhongke New Life Biotechnology Co., Ltd.). The obtained MS raw data of each sample were combined, and MaxQuant software (version 1.6.14) was used for identification and quantitative analysis. As Figure 22 As shown, the expression levels of 21 proteins changed after luteolin treatment.

[0092] Subsequently, the present invention performed molecular docking analysis. Molecular docking was used to simulate the binding efficacy of luteolin to differentially expressed proteins, and the 3D structure of the mouse protein of the candidate target was obtained from the PDB database (http: / / www.rcsb.org / ) in PDB format. The 3D conformers of the candidate compound were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) in SDF format. Subsequently, the structure was imported into MOE to obtain the docking score and perform statistics. It was finally determined that Uroc1 had the highest binding affinity potential (such as Figure 23 shown).

[0093] In addition, total RNA was extracted from liver tissue and primary hepatocytes using TRIzol reagent, and cDNA was synthesized using total mRNA as a template using a reverse transcription kit, and gene expression was detected using qPCR and SYBR green kits. Figure 24 and Figure 25As shown, although the mRNA expression level of Uroc1 in mouse liver did not change, luteolin treatment significantly reduced the protein content of Uroc1 in the DEHP-treated group. Then, the half-life of Uroc1 protein was determined by cycloheximide (CHX) chase assay. Primary mouse hepatocytes were treated with 10 μM DEHP for 12 h and luteolin for 12 h. After 24 h, DMEM (Sigma-Aldrich, St. Louis, MO, USA) containing 10 μg / mL CHX was added. Cells were harvested at 0, 12, 24, 36, 48, and 72 h for western blot analysis. The results of the CHX chase experiment showed that luteolin could significantly accelerate the protein degradation of Uroc1, resulting in the shortening of the half-life of this protein to 31.7 h (as Figure 26 shown).

[0094] Example 5 Ala270 and Val272 of Uroc1 protein are the key sites for luteolin to promote its ubiquitination and degradation

[0095] To further clarify the mechanism by which luteolin reduces the Uroc1 protein level, the present invention used MG132 (a protein ubiquitination inhibitor, Sigma-Aldrich, St. Louis, MO, USA) and bafilomycin A1 (Baf A1) (an inhibitor of autophagosome-lysosome-mediated protein degradation, MCE, New Jersey, USA) to inhibit the protein degradation pathway. Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h and 10 μM luteolin for a single time for 6 h. Subsequently, these cells were further treated with a combination of 10 μM luteolin + 10 μM MG132 (MG132 + Luteolin) or 10 μM luteolin + 20 nM bafilomycin A1 (Baf A1) (Baf A1 + Luteolin) for 6 h. Figure 27It was shown that MG132 treatment partially inhibited luteolin-induced degradation of Uroc1 protein, suggesting that the ubiquitin-proteasome is deeply involved in this degradation process. Among them, CTL+DEHP: Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h and treated with 0.1% DMSO for 12 h; MG132+DEHP: Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h, treated with 0.1% DMSO for 6 h, and treated with 10 μM MG132 for 6 h; Baf A1+DEHP: Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h, treated with 0.1% DMSO for 6 h, and treated with 20 nM Baf A1 for 6 h; CTL+DEHP+Luteolin: Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h and stimulated with 10 μM luteolin once for 12 h; MG132+DEHP+Luteolin: Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h, stimulated with 10 μM luteolin once for 6 h, and co-stimulated with 10 μM luteolin and 10 μM MG132 for 6 h; Baf A1+DEHP+Luteolin: Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h, stimulated with 10 μM luteolin once for 6 h, and co-stimulated with 10 μM luteolin and 20 nM Baf A1 for 6 h.

[0096] In addition, to clarify the regulatory site of luteolin on the degradation of Uroc1 protein, the present invention performed molecular docking analysis (the steps were the same as in Example 4). The results showed that luteolin potentially binds to the Ala270 and Val272 sites of the Uroc1 protein (as Figure 28 shown). Then, the present invention intended to mutate the above sites in order to confirm the important roles of the Ala270 and Val272 sites. As Figure 29 shown, the present invention used the SOPMA algorithm (NPS@: SOPMA secondary structure prediction (ibcp.fr)) to analyze the secondary structure of the Uroc1 protein after amino acid mutation of Ala270 and Val272. The results showed that the site mutation did not affect its natural secondary structure composition. As Figure 30 shown, when the Ala270 or Val272 site was mutated alone, the decrease in the level of luteolin-induced Uroc1 protein was partially reversed. When both sites were mutated simultaneously, the degradation effect of luteolin on the Uroc1 protein was completely reversed. Functionally, overexpression of Uroc1 significantly reversed the promoting effect of luteolin on the excretion of redundant DEHP in primary mouse hepatocytes. In contrast, when Uroc1 was double-mutated at the Ala270 and Val272 sites, this phenomenon disappeared (as Figure 31Shown). Consistent with these results, the fluorescence signal of csa-DEHP decreased after knockout of Uroc1, and knockout of Uroc1 promoted the excretion of DEHP from primary mouse hepatocytes (as Figure 32 Shown). Conversely, AAV8-mediated liver-specific overexpression of Uroc1 significantly reversed the function of luteolin in promoting the excretion of DEHP from the mouse liver. Specifically, compared with the luteolin-treated group, after liver-specific overexpression of Uroc1, the levels of DEHP in the mouse liver, serum, and urine were significantly increased. At the same time, the levels of serum AST and ALT also showed a reversal (as Figure 33 , Figure 34 Shown). The above results further emphasized the conclusion of the present invention, that is, Uroc1 is a specific target protein of luteolin and can mediate the beneficial effect of luteolin on the excretion of DEHP from the liver.

[0097] Example 6 Trans-UCA can mimic the effect of luteolin in promoting the excretion of redundant DEHP in the liver

[0098] Luteolin has multiple molecular targets. Considering that the development process of de novo chemical drugs targeting Uroc1 is time-consuming and costly, in order to quickly find effective and safe drugs for promoting excretion, the present invention focused on endogenous metabolites related to Uroc1. Given that Uroc1 itself is an enzyme that converts trans-UCA to 4-imidazolone-5-propionate, a decrease in the Uroc1 protein level will lead to an increase in its substrate trans-UCA and its upstream substrates (as Figure 35 Shown), suggesting that these potential endogenous metabolites may have the effect of promoting the excretion of redundant DEHP from the liver. To test this hypothesis, the present invention first performed a CCK-8 assay to evaluate the safe doses of these two metabolites on primary mouse hepatocytes. The results showed that neither L-histidine (L-his) nor trans-UCA affected cell viability even in the presence of DEHP (as Figure 36 Shown). Further laser confocal analysis based on csa-DEHP showed that trans-UCA, rather than L-his, significantly reduced the fluorescence signal of csa-DEHP (as Figure 37 Shown). LC-MS analysis (steps and conditions are the same as in Example 3) further confirmed the above results (as Figure 38 Shown). Although L-his reduced the DEHP content by 50.22%, trans-UCA showed a similar effect to luteolin by reducing the DEHP level by 84.48%. In addition, the effect of trans-UCA on promoting the excretion of DEHP from primary mouse hepatocytes also showed good time and dose dependence (as Figure 39Mice were treated with DEHP (10 mg / kg / d) by oral gavage for 28 days, and then trans-UCA (10 mg / kg / d) was administered daily for 14 days. Animal studies have shown that trans-UCA can also play a role comparable to luteolin in vivo, reducing DEHP levels in the liver, serum, and urine of mice, while also reversing the DEHP-induced increase in serum AST and ALT (as shown in Figure 2). Figure 40 and Figure 41 shown).

[0099] Example 7: Lysosomal pathway coordinated by ERK1 / 2 signaling mediates the clearance of redundant DEHP in mouse liver by luteolin / trans-UCA

[0100] To investigate the downstream signaling pathways of luteolin and its regulated trans-UCA, we performed a proteomic analysis. We treated primary mouse hepatocytes with DEHP for 12 hours and then with trans-UCA for 12 hours. The results showed that after trans-UCA treatment, the expression of 76 proteins changed, of which 27 were upregulated and 49 were downregulated (e.g. Figure 42 As shown). Figure 43 As shown, gene Go analysis (performed by Shanghai Zhongke New Life Biotechnology Co., Ltd.) showed that these changed genes were clustered in the MAPK pathway, especially the pathway related to ERK1 / 2. Then, the phosphorylation level of ERK1 / 2 protein after DEHP treatment was studied. Mouse primary hepatocytes were treated with 10μM DEHP for 12h, and then treated with 10mM trans-UCA or 10μM Luteolin for another 12h, and the phosphorylation level of ERK1 / 2 was detected by protein samples. Protein level detection showed that DEHP stimulation significantly increased the phosphorylation level of ERK1 / 2 protein. In contrast, both trans-UCA and luteolin significantly reversed DEHP-induced ERK1 / 2 phosphorylation ( Figure 44 ).

[0101] Since the lysosome-related pathway is a key process for the excretion of toxins and foreign substances and is strictly regulated by ERK1 / 2 protein, the lysosomal pathway coordinated by ERK1 / 2 signaling is the key pathway mediating the promotion of DEHP clearance in mouse liver by luteolin / trans-UCA. Primary mouse hepatocytes were stimulated with 10 μM DEHP for 12 h and then with 10 μM luteolin for a single time for 6 h. Subsequently, they were further treated with 0.1% DMSO (CTL), 10 mM trans-UCA, 10 mM trans-UCA + 20 nM Baf A1, 10 μM luteolin, or 10 μM luteolin + 20 nM Baf A1 for 6 h. By inhibiting the lysosomal pathway using Baf A1, the present invention found that the beneficial effects of luteolin and trans-UCA on DEHP hepatic clearance were significantly attenuated (as Figure 45 and Figure 46 shown). Considering that the subcellular distribution of csa-DEHP is partially concentrated in lysosomes, this organelle is a key link in the efflux of DEHP.

Claims

1. Luteolin and / or trans- The application of UCA in the preparation of a drug for reducing or removing bis(2-ethylhexyl) phthalate in vivo, characterized in that The said trans- The structural formula of UCA is as follows: 。 2. Application of luteolin and / or trans- UCA in the preparation of a drug for promoting the excretion of bis(2-ethylhexyl) phthalate, characterized in that The said trans- The structural formula of UCA is as follows: 。 3. Luteolin and / or trans- The application of UCA in the preparation of a drug for reducing the content of bis(2-ethylhexyl) phthalate in the liver and serum, characterized in that The said trans- The structural formula of UCA is as follows: 。 4. The application according to any one of claims 1 to 3, characterized in that The concentration of the luteolin is 1 to 10 μM.

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