And theaflavin-3apos; application of gallate and medicine prepared from gallate

Abstract

The invention belongs to the technical field of pharmacotherapeutics, and particularly relates to application of theaflavin-3 '-gallate and a medicine prepared from theaflavin-3'-gallate. The theaflavin-3 '-gallate is used as a KEAP1-NRF2 inhibitor and is competitively combined with the KEAP1 to destroy the inhibition effect of the KEAP1 on the NRF2, so that the content of the NRF2 is increased, and the expression of downstream NQO1 and GCLC is improved. Besides, the theaflavin-3 '-gallate protects the liver function by inhibiting the increase of the contents of AST and ALT, and reduces the oxidative stress and lipid accumulation of the liver, so that the theaflavin-3'-gallate has the potential to become a medicine for treating the ethanol-induced liver injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmacotherapeutics, and specifically relates to the application of theaflavin-3'-gallate as a KEAP1-NRF2 inhibitor in the preparation of a drug for preventing and treating alcoholic liver injury and the prepared drug. Background Art

[0002] Alcoholic Liver Disease (ALD) is one of the main causes of chronic liver diseases worldwide. Chronic and excessive alcohol consumption can cause extensive liver lesions, such as alcoholic steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Compared with the latest progress in viral hepatitis, the pharmacological treatment of ALD patients has made little progress.

[0003] The liver plays a key role in ethanol metabolism. In hepatocytes, three main oxidative pathways metabolize ethanol. Ethanol is metabolized by ADHs, CYP2E1, and catalase into the intermediate acetaldehyde. Subsequently, acetaldehyde is further metabolized into acetic acid under the catalysis of aldehyde dehydrogenase (ALDH). Subsequently, acetic acid is converted into acetyl-CoA by acetyl-CoA synthase and then enters the tricarboxylic acid cycle. However, the conversion of acetaldehyde to acetic acid is the key rate-limiting step in the alcohol oxidation metabolism pathway, which is mediated by human ALDH1A1, ALDH1B1, and ALDH2. This easily leads to the accumulation of acetaldehyde in the body, resulting in an increase in (ROS) in the liver. ROS can interact with proteins, DNA, and lipids in hepatocytes, increasing the level of oxidative stress, thereby leading to lipid peroxidation and an increase in the level of malondialdehyde (MDA), while consuming a large amount of antioxidant substances, such as superoxide dismutase (SOD) and glutathione (GSH), thus weakening the antioxidant defense ability of the body, especially the liver. This acetaldehyde-induced oxidative stress plays a crucial role in the development of alcoholic liver disease (ALD).

[0004] At present, there is no specific drug for the treatment of alcoholic liver disease. The most effective treatment method to alleviate the ALD process is still alcohol abstinence plus nutritional treatment. It is very necessary to develop new drugs for the treatment of ALD. Summary of the Invention

[0005] Aiming at the treatment problem of ALD, one of the purposes of the present invention is to provide the application of theaflavin-3'-gallate as a KEAP1-NRF2 inhibitor.

[0006] Theaflavin-3'-gallate competitively binds with KEAP1, disrupts the binding of KEAP1 to NRF2, that is, disrupts the inhibitory effect of KEAP1 on NRF2, increases the content of NRF2, and increases the expression of downstream NQO1 and GCLC.

[0007] Furthermore, the theaflavin-3'-gallate is used for preparing a drug for preventing and / or treating alcoholic liver disease.

[0008] A second object of the present invention is to provide a drug for treating alcoholic liver disease, which contains a pharmaceutically effective dose of theaflavin-3'-gallate.

[0009] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0010] Preferably, the pharmaceutically acceptable carrier includes excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents, and other adjuvants with one or several functions; such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.

[0011] Preferably, the drug is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.

[0012] The beneficial effects of the present invention are as follows:

[0013] The Kelch-like ECH-associated protein 1-(Keap1-)-nuclear factor-erythroid 2-related factor 2 (Nrf2) system can be used to monitor oxidative stress and control the transcription of multiple antioxidant enzymes. When not stressed, Nrf2 is captured in the cytoplasm by Keap1, preventing its translocation to the nucleus. Keap1, as an E3 ubiquitin ligase, promotes the ubiquitination and proteasomal degradation of Nrf2, thus maintaining a low level of Nrf2. When cells are subjected to oxidative stress (such as increased ROS production) or electrophilic attacks, certain cysteine residues of Keap1 are modified, leading to a conformational change. This modification blocks the ubiquitination of Nrf2, allowing Nrf2 to dissociate from Keap1 and activate the expression of a series of antioxidant enzymes and compatible proteins, such as superoxide dismutase (SOD), glutathione S-transferases (GSTs), and heme oxygenase-1 (HO-1). Some studies have shown that inhibiting KEAP1-NRF2 plays an important role in treating oxidative stress and lipid metabolism in ALD.

[0014] Theaflavin-3'-gallate (TF2B) is a natural polyphenolic compound, mainly derived from the fermentation process of black tea. During the production of black tea, the polyphenols in fresh leaves undergo oxidative polymerization under the action of polyphenol oxidase, thereby forming theaflavin substances, among which TF2B is an important component of theaflavin substances. Structurally, TF2B is composed of two catechin molecules connected by a C-C bond, and its chemical structure contains multiple phenolic hydroxyl groups and ester groups. TF2B has not been clinically applied yet, but as a component of dietary supplements or functional foods, TF2B has certain application potential in cardiovascular diseases, diabetes, neurodegenerative diseases, as well as cancer and antiviral infections. However, the preventive and therapeutic effects of TF2B in alcoholic liver disease have not been reported.

[0015] Based on the relationship between the KEAP1-NRF2 pathway and ALD, this application discovered a novel KEAP1-NRF2 inhibitor - TF2B. TF2B has a high affinity for KEAP1 protein. Through experiments, it was verified that TF2B can decrease the protein level of KEAP1 in vivo and increase the protein expression of NRF2 and its downstream targets NQO1 and GCLC. This invention also demonstrated that TF2B protects liver function by inhibiting the increase in AST and ALT contents, reduces liver oxidative stress and lipid accumulation. TF2B has the potential to become a drug for treating ethanol-induced liver injury. Brief Description of the Drawings

[0016] Figure 1 For the results of the cell viability experiment in Example 1, where A is the toxicity experiment of the compound on AML-12 cells, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the normal group; B is the effect of the compound on the inhibition of cell viability induced by ethanol, P < 0.001 compared with the control group, ***P < 0.001 compared with the model group.

[0017] Figure 2 For the results of immunofluorescence assay of NRF2 with 10 Μm TF2B in cells in Example 2.

[0018] Figure 3 For the liver tissue sections of mice in Example 3, where A is the HE staining result and B is the Oil Red O staining result.

[0019] Figure 4 Shows the effects of TF2B on the levels of ALT, AST, T-CHO, and TG in ALD model mice in Example 3, P < 0.001 compared with the control group, ***P < 0.001 compared with the model group.

[0020] Figure 5Effect of TF2B on SOD, MDA, and GSH levels in ALD model mice in Example 3, ##P < 0.001 compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model group.

[0021] Figure 6 Protein blotting analysis of the inhibitory effect of TF2B on KEAP1-NRF2 in ALD model mice in Example 3, and determination of the protein expression of NRF2 downstream targets NQO1 and GCLC. Detailed implementation manners

[0022] The technical solutions of the present invention will be described in more detail below with reference to the examples.

[0023] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0024] TF2B used in the experiment was purchased through the official website of TargetMol (PubChem ID: 459630106).

[0025] Example 1

[0026] CCK-8 experiment for detecting cytotoxicity

[0027] Mouse hepatocytes AML-12 were seeded into 96-well plates. When the cells reached 50% confluence, the culture medium was replaced with fresh medium (10% fetal bovine serum, 1% 100× penicillin-streptomycin), and 10 μM of the natural compound was added simultaneously to determine the toxicity of the compound. 200 mM ethanol was added simultaneously with 10 μM of the natural compound to determine the change in cell viability. After culturing in a 37°C incubator for 24 h, 10 μL of CCK-8 was added to each well and incubated for 1 - 2 h, and the absorbance was measured at 450 nm using a microplate reader. The cell proliferation ratio of the drug-treated group relative to the control group was calculated. The blank group was the medium without cells, and the control group was the cell group without drugs.

[0028] Cell survival rate = (OD of experimental group 450 - OD of blank group 450 ) / (OD of control group 450 - OD of blank group 450 ).

[0029] Then, SPSS software was used to calculate IC 50 . The results were expressed as the mean ± SD of three experiments. The results are shown in Figure 1 as follows.

[0030] Figure 1 Panel A in [reference] shows the toxicity of the compound to AML-12 cells at a concentration of 10 μM. Figure 1Figure B shows the effect of 10 μM natural compound on the inhibition of cell viability induced by ethanol. After treatment with ethanol, the cell viability of AML-12 cells decreased significantly.

[0031] Example 2

[0032] NRF2 immunofluorescence assay

[0033] AML-12 cells were grown on cover slips. When they grew to 30%, they were treated with 10 μM TF2B. After 24 h, they were fixed with 4% paraformaldehyde and blocked with 3% BSA solution for 40 min. Then, the primary antibody against NRF2 (1:200) was applied to the cover slips and incubated overnight in a culture dish at 4°C. The next day, after incubation with the fluorescent secondary antibody (rabbit anti-) for 1 h, the nuclei were stained with DAPI for 10 minutes. Images were taken using a fluorescence inverted microscope. The results are as Figure 2 shown.

[0034] As Figure 2 can be seen, compared with the normal group, the treatment with 10 μM TF2B can significantly increase the level of NRF2 in cells.

[0035] Example 3

[0036] Mouse model of alcoholic fatty liver

[0037] 1) Experimental animals and grouping:

[0038] Wild-type C57BL / 6 male mice aged 10 - 12 weeks with a body weight exceeding 20 g (purchased from Jiangsu Jicui Yakang Biotechnology) were used. After being given normal feed and tap water and allowed to drink and eat freely to adapt to the environment for one week, the experiment was carried out. 48 mice were randomly assigned to the normal group, the model group, and the drug administration groups including the low-dose TF2B group (20 mg / kg), the medium-dose TF2B group (40 mg / kg), the high-dose TF2B group (60 mg / kg), and the positive drug silymarin (SLY) group (50 mg / kg).

[0039] 2) Modeling and drug administration

[0040] On the 1st and 2nd days, both the normal group and the model group were fed with control liquid feed, and the feed was prepared at 30 mL per mouse; on the 3rd day, the normal group was fed with control liquid feed, and the model group and the drug administration group were fed with a mixture of 1 / 3 alcohol liquid feed and 2 / 3 control liquid feed, and the feed was prepared at 30 mL per mouse; on the 4th day, the normal group was fed with control liquid feed, and the model group and the drug administration group were fed with a mixture of 2 / 3 alcohol liquid feed and 1 / 3 control liquid feed, and the feed was prepared at 30 mL per mouse; from the 5th to the 15th day, the normal group was fed with control liquid feed, and the model group and the drug administration group were fed with alcohol liquid feed, and the feed was prepared at 30 mL per mouse. At the same time, the drug administration group was intragastrically administered with the corresponding dose of the drug, and the normal group and the model group were intragastrically administered with an equal amount of tap water; on the 16th day, the normal group was intragastrically administered with dextrin at a concentration of 0.45 g / ml (9 g of maltose dissolved in water to a final volume of 20 ml), and the dose was 10 ml / kg. The model group and the drug administration group were intragastrically administered with a 31.5% alcohol solution (v / v), and the dose was 10 ml / kg. 9 hours after intragastric administration, blood was collected from the mouse eyeballs with tissue forceps, and the liver tissue was dissected. The large lobes of the liver were used for oil red O and HE staining, and part of the liver was placed in paraformaldehyde fixative for extracting tissue proteins and subsequent experiments.

[0041] 3) Histological evaluation

[0042] After taking the mouse liver tissue, part of the liver tissue was fixed with formalin for about 24 h, and the remaining part was stored at -80 °C. The paraffin-embedded tissue sections were 5 μm thick and stained with hematoxylin and eosin (HE) and oil red O. Finally, the images were observed with a pathological section scanner, and the results are as Figure 3 shown.

[0043] Figure 3 As shown in A, compared with the normal group, the fat vacuoles in the liver of the model group mice were obvious, and a large number of inflammatory cell infiltrations appeared in the liver, while the changes in the mice treated with TF2B were significantly reduced. Figure 3 As shown in B, compared with the normal group, the fat accumulation in the liver of the model group mice was severe and the fatty degeneration was obvious, while there was a significant improvement in the mice treated with TF2B. In summary, through HE and oil red O staining, it was shown that TF2B could significantly improve the liver fat metabolism in ALD model mice.

[0044] 4) Determination of the in vivo contents of ALT, AST, TG, and T-CHO

[0045] Collect the whole blood of mice in each group. Place the collected blood samples at room temperature and let them stand for about 30 minutes. Then put the blood samples into a centrifuge, set an appropriate rotation speed (3000 rpm, centrifuge for 10 - 15 minutes), and aspirate the supernatant as the sample to be tested. After diluting the serum in a certain proportion, use a biochemical analyzer to directly detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (T-CHO), and total triglyceride (TG) in the serum. The results are as Figure 4 shown.

[0046] Figure 4 In which A is the result of AST and B is the result of ALT. It can be seen that compared with the normal group, the levels of AST and ALT in the model group of mice are significantly increased, suggesting that ethanol exposure can cause liver cell damage and inflammation. However, in the mice treated with TF2B, the levels of AST and ALT are significantly decreased, indicating that TF2B can reduce the liver damage caused by ethanol. Figure 4 In which C is the result of T-CHO and D is the result of TG. It can be seen that compared with the normal group, the levels of T-CHO and TG in the model group of mice are significantly increased, suggesting lipid metabolism disorder in mice. After intervention with TF2B, the T-CHO and TG in the serum of mice are significantly decreased, indicating that TF2B can relieve ethanol-induced liver damage by regulating lipid metabolism.

[0047] 5) Determination of the in vivo contents of SOD, MDA, and GSH

[0048] Collect the whole blood of mice in each group. Place the collected blood samples at room temperature and let them stand for about 30 minutes. Then put the blood samples into a centrifuge, set an appropriate rotation speed (3000 rpm, centrifuge for 10 - 15 minutes), and aspirate the supernatant as the serum sample to be tested. Take 0.03 - 0.05 g of the liver in each group, add an appropriate amount of normal saline, mechanically homogenize it under ice-water bath conditions, centrifuge and filter it through a 0.5 μm filter to obtain the supernatant, which is used as the liver tissue homogenate sample to be tested. Determine the levels of SOD, MDA, and GSH in the serum samples and liver tissue homogenate samples according to the method in the instruction manual. The results are as Figure 5 shown.

[0049] According to Figure 5 the results of A (serum sample) and B (liver tissue homogenate sample) in it, compared with the normal group, the content of MDA in the serum and liver of the model group of mice is increased, suggesting the occurrence of lipid peroxidation, and the contents of SOD and GSH are decreased, suggesting the reduction of the body's antioxidant capacity and the state of oxidative stress. In the mice treated with TF2B, the level of MDA is significantly decreased compared with the model group, and the levels of SOD and GSH are significantly increased, proving that TF2B can enhance the antioxidant capacity of the mouse liver, reduce ethanol-induced lipid peroxidation, and alleviate the occurrence of oxidative stress in mice.

[0050] 6) Detection of the ability of TF2B to inhibit the KEAP1-NRF2 pathway by Western Blot (WB) assay

[0051] Extract proteins from the liver tissues of mice in each group using RIPA lysis buffer containing 1% PMSF. Ultracentrifuge at 12,000 g for 30 min at 4°C, collect the supernatant, add protein loading buffer, and denature at 100°C for 10 min to obtain protein samples. Analyze the inhibitory ability of TF2B on the KEAP1-NRF2 pathway by Western Blot. The results are as Figure 6 shown.

[0052] It can be seen that compared with the normal group, the expression of KEAP1 protein increased in the model group of mice, while the expression of NRF2 and its downstream targets NQO1 and GCLC proteins decreased, suggesting a downregulation of the antioxidant gene level in mice. In mice treated with TF2B (50 mg / kg), compared with the model group, the content of KEAP1 decreased significantly, and the expression of NRF2 downstream targets NQO1 and GCLC proteins increased significantly. The results indicate that TF2B binds to KEAP1, disrupts the binding of KEAP1 to NRF2, increases the content of NRF2, and upregulates the expression of downstream NQO1 and GCLC.

[0053] The above experiments indicate that TF2B can serve as a novel KEAP1-NRF2 inhibitor and has the potential to be developed into a drug for the treatment of alcoholic liver disease.

[0054] The above are only the preferred practical examples of the present invention and are not intended to limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of theaflavin-3'-gallate as a KEAP1-NRF2 inhibitor.

2. The application according to claim 1, characterized in that The theaflavin-3'-gallate is used for preparing a drug for preventing and / or treating alcoholic liver disease.

3. A drug for treating alcoholic liver disease, which contains a pharmaceutically effective dose of theaflavin-3'-gallate.

4. The drug according to claim 3, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.

5. The drug according to claim 4, characterized in that, The pharmaceutically acceptable carrier includes a carrier having one or several functions of excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents.

6. The medicament according to claim 4, wherein The drug is any one of injection, tablets, pills, capsules, suspensions or emulsions.

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