Application of timosaponin BII in preparation of medicine for preventing and treating alcoholic liver disease

Abstract

The invention belongs to the technical field of pharmacotherapeutics, and particularly relates to application of timosaponin BII in preparation of a medicine for preventing and treating alcoholic liver injury. A novel KEAP1-NRF2 inhibitor-TBII is found through a virtual screening method, TBII and KEAP1 protein have high affinity, and experiments verify that TBII can reduce the protein level of KEAP1 in vivo and improve protein expression of NRF2 and downstream targets NQO1 and GCLC of NRF2. It is proved that TBII can protect the liver function by inhibiting increase of AST and ALT content, oxidative stress and lipid accumulation of the liver are reduced, and TBII has the potential to become a medicine for treating ethanol-induced liver injury.

Description

Technical Field

[0001] The invention belongs to the technical field of drug therapy, and specifically relates to the application of timosaponin BII in the preparation of drugs for preventing and treating alcoholic liver disease. Background Art

[0002] Alcoholic liver disease (ALD) is one of the leading causes of chronic liver disease worldwide. Chronic and excessive drinking can cause a wide range of liver lesions, such as alcoholic steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Compared with the latest advances in viral hepatitis, there has been little progress in the pharmacological treatment of ALD patients.

[0003] Oxidative stress, endoplasmic reticulum stress, impaired autophagy, and mitochondrial dysfunction are all involved in the development of ALD, among which oxidative stress is considered to be an important contributing factor to the progression of ALD. Oxidative stress manifests as an imbalance between oxidants and antioxidants. When the capacity of the body's antioxidant system decreases, the level of inactivated reactive oxygen species (ROS) increases, the redox state reaches a dangerous level, and the adverse effects of oxidants appear. ROS are small amounts of highly reactive molecules naturally produced during the body's metabolic reactions, responsible for the performance of cellular functions, from signal transduction pathways, defense against invading microorganisms, gene expression to promoting growth or death, and can react with complex cellular molecules (such as lipids, proteins, or DNA) and destroy them. Acute and chronic ethanol treatment increases the production of ROS, reduces cellular antioxidant levels, and enhances oxidative stress in many livers. Excessive ROS can damage cellular proteins, lipids, and DNA, impairing the liver's antioxidant defense system, including reducing GSH levels and SOD activity, leading to fatal cell damage.

[0004] At present, there is no specific drug for the treatment of alcoholic liver disease. The most effective treatment to alleviate the progression of ALD is still abstinence from alcohol plus nutritional therapy. It is very necessary to explore the pathogenesis of ALD patients and develop new therapeutic drugs. Summary of the invention

[0005] In view of the difficulty in treating ALD, one of the objectives of the present invention is to provide the use of Anemarrhena saponin BII in the preparation of drugs for preventing and treating alcoholic liver disease.

[0006] The second object of the present invention is to provide a medicine for treating alcoholic liver disease, which contains a pharmaceutically effective dose of Anemarrhena saponin BII.

[0007] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0008] Preferably, the pharmaceutically acceptable carrier includes one or more functional auxiliary materials selected from the group consisting of excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents; such as starch, lipids, wax, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and the like.

[0009] Preferably, the drug is any one of an injection, tablet, pill, capsule, suspension or emulsion.

[0010] The beneficial effects of the present invention are:

[0011] Nuclear factor erythroid 2-related factor 2 (NRF2) is the main regulator of the adaptive antioxidant response to oxidative stress in cells, which helps control redox homeostasis and prevents oxidative damage by detoxifying oxidants and electrophiles. In the basal state, NRF2 binds to Kelch-like ECH-associated protein 1 (KEAP1) and exists in the cytoplasm. This binding paves the way for NRF2 ubiquitination and degradation by the proteasome, keeping NRF2 at a low level. When attacked by electrophiles or ROS, the sensory cysteine ​​of KEAP1 is modified, and the released NRF2 is not inhibited and ubiquitinated by KEAP1, translocates to the nucleus and successively binds to small MAF proteins and antioxidant response elements ARE, activating the transcription of downstream detoxification enzymes and antioxidant enzyme genes, including GCLC, NQO1, and HO-1, to maintain intracellular redox balance. Studies have shown that inhibiting KEAP1-NRF2 plays an important role in the treatment of oxidative stress and lipid metabolism caused by ALD.

[0012] Timosaponin belongs to the saponin class of compounds. It exists in the dried rhizomes of Anemarrhena asphodeloides Bge., a plant of the Liliaceae family, and is one of the main active ingredients of Anemarrhena asphodeloides. Timosaponin BⅡ (TBⅡ) is one of the main components of Anemarrhena asphodeloides. In clinical applications, Timosaponin BⅡ is mainly used to treat Alzheimer's disease, depression, diabetes and its complications, osteoporosis, platelet aggregation and other problems. In addition, it has also been used to improve the symptoms of cerebral ischemia, showing good therapeutic effects.

[0013] The paper (Liu Fen. Study on the mechanism of timosaponin improving rat insulin resistance through Nrf2 signaling pathway [D]. Guangzhou University of Chinese Medicine, 2020.) reported that in rats with insulin resistance induced by long-term high-fat diet feeding, timosaponin can improve the body's oxidative stress response through the Nrf2 signaling pathway, alleviate lipid metabolism disorders in the body, and thus protect liver tissue. Patent CN112168833A discloses the medical uses of timosaponin enzymatic hydrolysis products and its main component timosaponin AⅢ, and discloses the use of timosaponin AⅢ in drugs for the prevention and treatment of non-alcoholic fatty liver disease. However, there is no report on the preventive and therapeutic effect of timosaponin BII in alcoholic liver disease.

[0014] Based on the relationship between the KEAP1-NRF2 pathway and ALD, the present invention discovered a new type of KEAP1-NRF2 inhibitor, TBII, for the first time through a virtual screening method. TBII has a high affinity for the KEAP1 protein. Experimental verification shows that TBII can reduce the protein level of KEAP1 in the body and increase the protein expression of NRF2 and its downstream targets NQO1 and GCLC. The present invention also proves that TBII protects liver function by inhibiting the increase of AST and ALT levels, reducing oxidative stress and lipid accumulation in the liver. TBII has the potential to become a drug for the treatment of ethanol-induced liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the cell viability experiment in Example 2, wherein A is the toxicity of 37 compounds to AML-12 cells; and B is the effect of the compounds on the inhibition of cell viability induced by ethanol.

[0016] Figure 2 This is the result of the total antioxidant capacity of cells in Example 3.

[0017] Figure 3 These are the results of the cell NRF2 immunofluorescence assay in Example 4.

[0018] Figure 4 These are mouse liver tissue sections in Example 5, wherein A is the HE staining result, and B is the Oil Red O staining result.

[0019] Figure 5 These are the test results of AST (A in the figure), ALT (B in the figure), T-CHO (C in the figure) and TG (D in the figure) in the mouse serum in Example 5.

[0020] Figure 6 The levels of SOD, MDA and GSH in the mouse serum (A in the figure) and liver tissue homogenate (B in the figure) samples in Example 5.

[0021] Figure 7 The results of Western blotting experiments in Example 5. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in more detail below in conjunction with the embodiments.

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

[0024] The TBII used in the experiment was purchased from the official website of Shanghai Taoshu (PubChem ID: 44575945).

[0025] Example 1

[0026] Receptor structure-based virtual screening:

[0027] The crystal structure of KEAP1 (PDB code: 4L7B) was obtained from the official website of Protein Data Bank (PDB, https: / / www.rcsb.org / ). Using Discovery Studio, the active pocket of the receptor protein was identified and simulated docking was performed with a natural product compound library containing 4533 small molecules. Finally, the top 50 compounds with the highest docking result scores were selected. The compounds are referred to Table 1.

[0028] Table 1 Top 50 natural small molecule compounds

[0029]

[0030]

[0031] After excluding compounds that were unavailable or reported in the literature to be related to the KEAP1-NRF2 pathway and had obvious toxicity and adverse reactions, the remaining 37 compounds were purchased for subsequent experiments.

[0032] Example 2

[0033] CCK-8 assay for cytotoxicity

[0034] Mouse liver cells AML-12 were inoculated into 96-well plates. When the cells grew to 50%, new culture medium (10% fetal bovine serum, 1% 100× penicillin-streptomycin) was replaced, and 10μM natural compounds were given at the same time to measure the toxicity of the compounds. 200mM ethanol was given and 10μM natural compounds were added to measure the changes in cell viability. After culturing in a 37°C incubator for 24h, 10μL CCK-8 was added to each well, incubated for 1-2h, and the absorbance was detected at 450nm using an enzyme reader. The cell proliferation ratio of the drug group relative to the control group was calculated. The blank group was a group with no cells but only culture medium, and the control group was a group with no drug cells.

[0035] Cell survival rate = (OD 450 -Blank group OD 450 ) / (control group OD 450 -Blank group OD 450 ).

[0036] Then, IC was calculated using SPSS software. 50 The results are presented as mean ± SD of three experiments. Figure 1 shown.

[0037] Figure 1 A in the figure shows the toxicity of the remaining 37 compounds to AML-12 cells at a concentration of 10 μM. It can be seen that only 6 compounds (1a, 1b, 1e, 1j, 4c, 4f) have obvious cytotoxicity. Figure 1 B in the middle shows the effect of 10 μM natural compounds on the inhibition of cell viability induced by ethanol. After AML-12 cells were treated with ethanol, the cell viability was significantly reduced. Among the compounds without obvious cytotoxicity, 7 compounds (2a, 2d, 2e, 3c, 3g, 4d, 4i) can largely reverse this trend. These 7 compounds were further selected for further experiments.

[0038] Example 3

[0039] Determination of total cellular antioxidant capacity

[0040] The total antioxidant capacity of the seven small molecule compounds screened in Example 2 was determined. AML-12 cells were incubated with various compounds at different concentrations (2 μM, 5 μM, 10 μM) for 2 h, and then stimulated with 200 mM ethanol for 24 h, and compared with the normal group and the model group.

[0041] 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) is a colorimetric agent that is oxidized to green ABTS+ under the action of an appropriate oxidant. About 1 million cells were collected and placed in 200 μL of ice-cold PBS solution. Ultrasound was used to fully disrupt the cells and release the antioxidants therein. The cells were centrifuged at about 12,000 g at 4°C for 5 minutes, and the supernatant of each group was taken. The cell supernatant and the standard were added to the prepared ABTS working solution, incubated at room temperature for 2-6 minutes, and the absorbance was detected at 745 nm using an enzyme reader. The results are shown in Figure 2. Figure 2 shown.

[0042] Figure 2It was shown that in the ethanol-induced cell model, the total antioxidant capacity of the cells was significantly lower than that of the untreated normal group cells. The total antioxidant capacity of the cells treated with compounds 2a, 2d, 3c, and 3g were improved to varying degrees, and the antioxidant capacity of 2d-Timosaponin BII (TBII) was more advantageous than other compounds and showed dose dependence. Therefore, TBII was selected for subsequent experiments.

[0043] Example 4

[0044] NRF2 immunofluorescence assay

[0045] AML-12 cells were grown on coverslips and treated with 10 μM TBII when they grew to 30%. After 24 hours, they were fixed with 4% paraformaldehyde and blocked with 3% BSA solution for 40 minutes. After that, NRF2 primary antibody (1:200) was spread on the coverslips and placed in a culture dish in a 4°C refrigerator overnight. The next day, after incubation with fluorescent secondary antibody (rabbit antibody) for 1 hour, the cell nuclei were stained with DAPI for 10 minutes. Images were taken using a fluorescent inverted microscope. The results are shown in Figure 2. Figure 3 shown.

[0046] Depend on Figure 3 It can be seen that compared with the normal group, treatment with 10 μM TBII can significantly increase the level of intracellular NRF2.

[0047] Example 5

[0048] Mouse model of alcoholic fatty liver disease

[0049] 1) Experimental animals and groups:

[0050] Wild-type C57BL / 6 male mice weighing more than 20g aged 10-12 weeks (purchased from Jiangsu Jicui Yaokang Biological) were used and given ordinary feed and tap water. The experiment was conducted after one week of free drinking and eating to adapt to the environment. 48 mice were randomly assigned to the normal group, model group, drug-treated groups including TBII low-dose group (25 mg / kg), TBII medium-dose group (50 mg / kg), TBII high-dose group (100 mg / kg), and positive drug silymarin (SLY) group (50 mg / kg).

[0051] 2) Modeling and drug administration

[0052] 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 with 30 mL of liquid feed for each mouse; on the 3rd day, the normal group was fed with control liquid feed, and the model group and the drug-treated group were fed with a mixture of 1 / 3 alcohol liquid feed and 2 / 3 control liquid feed, and the feed was prepared with 30 mL for each mouse; on the 4th day, the normal group was fed with control liquid feed, and the model group and the drug-treated group were fed with a mixture of 2 / 3 alcohol liquid feed and 1 / 3 control liquid feed, and the feed was prepared with 30 mL for each mouse; from the 5th to the 15th day, the normal group was fed with control liquid feed, and the model group and the drug-treated group were fed with alcohol liquid feed, and the feed was prepared with 30 mL for each mouse, and at the same time, the drug-treated group was gavaged with corresponding doses of drugs, and the normal group and the model group were gavaged with the same amount of tap water; on the 16th day, the normal group was gavaged with 0.45 g / ml (9 g maltose dissolved in water with a final volume of 20 ml) dextrin at a dose of 10 ml / kg. The model group and the drug-treated group were gavaged with 31.5% alcohol solution (v / v) at a dose of 10 ml / kg. Nine hours after gavage, blood was collected from the eyeballs of the mice using tissue forceps, and the liver tissue was dissected. The large lobe of the liver was used for Oil Red O and HE staining, and part of the liver was placed in paraformaldehyde fixative for tissue protein extraction and subsequent experiments.

[0053] 3) Histological evaluation

[0054] After taking the mouse liver tissue, part of the liver tissue was fixed with formalin for about 24 hours, and the rest 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 pathology section scanner. The results are as follows Figure 4 shown.

[0055] Figure 4 Middle A shows that compared with the normal group, the model group mice had obvious fat vacuoles in the liver and a large number of inflammatory cells infiltrated in the liver, while the mice treated with TBII significantly alleviated this change. Figure 4 Figure B shows that compared with the normal group, the model group mice had severe fat accumulation and obvious fatty degeneration in the liver, while the mice treated with TBII showed significant improvement. In conclusion, HE and Oil Red O staining showed that TBII can significantly improve liver fat metabolism in mice in the ALD model.

[0056] 4) Determination of ALT, AST, TG, T-CHO in vivo

[0057] Collect whole blood from each group of mice, place the collected blood samples at room temperature for about 30 minutes, then put the blood samples into a centrifuge, set the appropriate speed (3000rpm, centrifuge for 10-15 minutes), and take 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 triglycerides (TG) in the serum. The results are as follows Figure 5 shown.

[0058] Figure 5 A is the AST result. Figure 5 B in the figure shows the ALT result. It can be seen that compared with the normal group, the AST and ALT levels of the mice in the model group increased significantly, indicating that ethanol exposure can cause liver cell damage and inflammation, while the AST and ALT levels of the mice treated with TBII decreased significantly, indicating that TBII can alleviate liver damage caused by ethanol. Figure 5 C in the middle is the result of T-CHO. Figure 5 D in the middle is the TG result. It can be seen that compared with the normal group, the T-CHO and TG levels of the mice in the model group increased significantly, indicating that the lipid metabolism of the mice was dysregulated. After TBII intervention, the T-CHO and TG levels of the mouse serum decreased significantly, indicating that TBII can alleviate ethanol-induced liver damage by regulating lipid metabolism.

[0059] 5) Determination of SOD, MDA, GSH content in vivo

[0060] Collect whole blood from each group of mice, place the collected blood samples at room temperature for about 30 minutes, then place the blood samples in a centrifuge, set the appropriate speed (3000rpm, centrifuge for 10-15 minutes), and take the supernatant as the serum test sample. Take 0.03-0.05g of liver from each group, add appropriate amount of physiological saline, mechanically homogenize in an ice water bath, centrifuge and filter with a 0.5μm filter to obtain the supernatant as the liver tissue homogenate test sample. According to the method in the manual, the SOD, MDA, and GSH levels of serum samples and liver tissue homogenate samples were measured. The results are as follows: Figure 6 shown.

[0061] according to Figure 6 The results of A (serum sample) and B (liver tissue homogenate sample) show that compared with the normal group, the MDA content in the serum and liver of the model group mice increased, indicating the occurrence of lipid peroxidation, and the SOD and GSH content decreased, indicating that the body's antioxidant capacity decreased and was in an oxidative stress state. Compared with the model group, the MDA level of mice treated with TBII was significantly reduced, and the SOD and GSH levels were significantly increased, proving that TBII can enhance the antioxidant capacity of mouse liver, reduce ethanol-induced lipid peroxidation, and alleviate the occurrence of oxidative stress in mice.

[0062] 6) Western Blot (WB) assay to detect the ability of TBII to inhibit the KEAP1-NRF2 pathway

[0063] The protein in the liver tissue of each group of mice was extracted with RIPA lysis buffer containing 1% PMSF, and ultracentrifuged at 4°C and 12000g for 30 minutes. The supernatant was extracted and denatured at 100°C for 10 minutes after adding protein loading buffer to obtain protein samples. The inhibitory ability of TBII on the KEAP1-NRF2 pathway was analyzed by Western blotting. The results are as follows Figure 7 shown.

[0064] It can be seen that compared with the normal group, the expression of KEAP1 protein in the model group mice increased, and the expression of NRF2 and downstream targets NQO1 and GCLC proteins decreased, indicating a downregulation of the antioxidant gene level in mice. Compared with the model group mice, the KEAP1 content of mice treated with TBII (50 mg / kg) decreased significantly, and the expression of NRF2 downstream targets NQO1 and GCLC proteins increased significantly. The results show that TBII binds to KEAP1, destroys the binding of KEAP1 and NRF2, increases the NRF2 content and improves the expression of downstream NQO1 and GCLC.

[0065] The above experiments show that TBII can be used as a new KEAP1-NRF2 inhibitor and has the potential to be developed into a drug for the treatment of alcoholic liver disease.

[0066] The above are only preferred practical examples of the present invention and are not intended to limit the invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of Anemarrhena saponin BII in the preparation of drugs for preventing and / or treating alcoholic liver disease.

2. A medicine for treating alcoholic liver disease, comprising a pharmaceutically effective dose of Anemarrhena saponin BII.

3. The drug according to claim 2, characterized in that The drug also contains a pharmaceutically acceptable carrier.

4. The drug according to claim 3, characterized in that The pharmaceutically acceptable carrier includes one or more functional carriers selected from the group consisting of excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

5. The drug according to claim 3, characterized in that The medicine is any one of injection, tablet, pill, capsule, suspension or emulsion.

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