Small molecule composition that negatively regulates the Nrf2 signaling pathway and its application methods

Abstract

The present invention belongs to the fields of pharmaceutical technology and health food, and specifically provides a low-molecular composition containing licocalcone A, licocalcone C, licocalcone D, licocalcone E, homonnetin, glabrol, and licoflavone C, which negatively regulates the Nrf2 signaling pathway and is used for the prevention or treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the fields of pharmaceutical technology and health food, and specifically provides a low-molecular composition that negatively regulates the Nrf2 signaling pathway and is used for the prevention or treatment of negative regulation of the Nrf2 signaling pathway (information, signal) transmission pathway or non-alcoholic fatty liver disease and the like.

Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or metabolic dysfunction-associated fatty liver disease (MASH) is one of the common chronic liver diseases, and its onset process and progression process are very complex and usually include the following multiple stages. Glucolipid metabolism disorder - fat accumulation in hepatocytes, oxidation of fat in hepatocytes - liver inflammation, liver fibrosis accompanied by inflammation - liver cirrhosis, and then hepatocyte carcinogenesis - the emergence of liver cancer. In the research of the present inventors, it has been found that the above process is related to multiple cell signaling pathways. Among them, the activity and suppression of the signal or information transmission pathway of the main mediator (KEAP1-NRF2) that controls the body's defense mechanism against oxidative stress and electrophilic substances have an extremely great influence on the onset and progression of the disease. The transcription regulatory factor (NRF2) gene with a basic leucine zipper structure activates a region (gene cluster) where hundreds of downstream genes and almost the same genes are present in multiple copies on genomic DNA, and produces main functions such as antioxidant, protection against cell damage, and suppression of inflammation. Therefore, transcription regulatory factor (NRF2) agonists are usually considered effective for the treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0003] Licorice is widely applied in crude drugs and is used with the highest frequency in traditional prescriptions of crude drugs, so it is called the king of crude drugs. However, licorice in traditional Chinese medicine prescriptions is usually used as a decoction. From the perspective of the pharmaceutical view of the material basis, most of the medicinal components are water-soluble molecules.

[0004] Licochalcone A is the main non - water - soluble chalcone molecule in licorice and is an agent that acts on the above - mentioned main mediator (KEAP1 - NRF2) pathway similar to most licochalcones or other chalcone molecules, and should positively regulate the Nrf2 signaling pathway. However, the inventors of the present invention have discovered during research that one of the licorice extracts rich in licochalcone A actually exerts a negative regulatory effect on the expression of the Nrf2 gene during the onset and progression of non - alcoholic fatty liver disease, that is, it becomes an Nrf2 antagonist. This discovery was originally something to be rejected, but as a result of continuous detailed research based on years of research experience, it finally led to obtaining a drug or health food with sufficient beneficial effects for the treatment of non - alcoholic fatty liver disease, which no one could have imagined.

Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new low - molecular - weight composition that negatively regulates the Nrf2 signaling pathway but effectively prevents or treats non - alcoholic fatty liver disease. In addition, the present invention provides a pharmaceutical preparation or health food containing the low - molecular - weight composition and an application method, etc.

[0006] Specifically, the first object of the present invention is to provide a low - molecular - weight composition that contains licochalcone A, licochalcone C, licochalcone D, licochalcone E, formononetin, glabrol, and licoflavone C and negatively regulates the Nrf2 signaling pathway. The composition exerts a negative regulatory effect on the expression of the Nrf2 gene during the onset and progression of non - alcoholic fatty liver disease.

[0007] In the present invention, licochalcone A, licochalcone C, licochalcone D, licochalcone E, formononetin, glabrol, and / or licoflavone C may be extracted from plants or artificially synthesized. Ideally, a suitable plant raw material is Glycyrrhiza inflata Batalin.

[0008] In the first objective composition of the present invention, the weight ratio of licocalcon A, licocalcon C, licocalcon D, licocalcon E, hormononetin, glabrol and licoflavone C is preferably 60-75:2-6:1-4:5-10:1-5:1-5:1-5.

[0009] Moreover, in the first objective composition of the present invention: the weight ratio of licocalcon A, licocalcon C, licocalcon D, licocalcon E, hormononetin, glabrol and licoflavone C is preferably 65-75:3-5:1-3:6-8:2-3:2-3:1-2.

[0010] The second objective of the present invention is to provide a preparation comprising the first objective composition of the present invention and a pharmaceutically or food acceptable additive. The preparation may be a pharmaceutical preparation or a health food.

[0011] The term "pharmaceutically acceptable additive" in this specification includes carriers, excipients, diluents, etc. that are compatible with the drug active ingredient and are pharmaceutically acceptable. The preparation of pharmaceutical preparations using pharmaceutically acceptable additives is a technique well known to those skilled in the art of the present invention. The pharmaceutical preparations used in the present invention contain nicotinamide mononucleotide, mogroside and erythritol as active ingredients, and the active ingredients are combined with pharmaceutically acceptable adjuvants (carriers, excipients, diluents, etc. well known to those skilled in the art of the present invention) to prepare various preparations. The preferred choices are dosage forms such as tablets, pills, capsules (including sustained release or delayed release dosage forms), powders, suspensions, granules, syrups, emulsions, suspensions, etc. and various solid and liquid preparations of sustained release dosage forms, and the oral administration form is preferred.

[0012] In a specific embodiment of the present invention, the first objective composition according to the present invention is diluted with a 0.5% CMC-Na solution to form a liquid preparation. The effective dosage for prevention or treatment is estimated based on the dosage of experimental animals as the first objective composition of the present invention.

[0013] As used herein, the term "food-acceptable additive" includes carriers, excipients, diluents, flavoring agents, coloring agents, flavoring agents, etc. that are compatible with food health-active ingredients and acceptable for food. A composition containing nicotinamide mononucleotide, mogroside and erythritol may directly constitute a food or food ingredient or be added to a food or food ingredient. For example, it may be applied to the surface of other foods or mixed with other foods.

[0014] It is a third object of the present invention to provide an application method of the composition of the first aspect of the present invention in the preparation of a reagent for negatively regulating the Nrf2 signaling pathway. Further, it is a fourth object of the present invention to provide a method for negatively regulating the Nrf2 signaling pathway, including the use of the composition of the first object of the present invention.

[0015] It is a fifth object of the present invention to provide an application method of the composition of the first object of the present invention in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease (NAFLD). Furthermore, it is a sixth object of the present invention to provide a method for preventing or treating non-alcoholic fatty liver disease, including the usage of the composition of the first object of the present invention.

[0016] As used herein, the subject of use may be a human or an experimental animal, but a human is preferentially selected.

[0017] Non-alcoholic fatty liver disease is preferentially non-alcoholic steatohepatitis (NASH).

[0018] The preferentially selected non-alcoholic fatty liver disease is also a glycolipid metabolism disorder disease, liver fibrosis, liver cirrhosis or liver cancer.

[0019] To facilitate the understanding of the present invention, specific examples and drawings will be described in detail below for the embodiments of the present invention. It should be particularly pointed out that the following is only an exemplary description and not a limiting matter that restricts the description of the scope of the claims of the present application. Based on the description in this specification, it is obvious that those skilled in the art can make various changes and modifications to the technical scope of the present invention.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0021] The content of the present invention will be described below with reference to examples. Unless otherwise specified, the technical means used in the examples are ordinary means and commercially available ordinary equipment and reagents for those skilled in the art, and the instructions of the manufacturers representing the corresponding equipment and reagents can be referred to separately.

[0022] Example 1 Preparation of Licorice Extract The roots and rhizomes of Glycyrrhiza inflata Batalin were taken, crushed, and extracted by warm soaking with water (60 °C), and the extract was treated separately. The water content of the medicinal residues was dried to (15 ± 5)%, and reflux extraction was carried out using (85 ± 5)% ethanol. The extracts were combined, ethanol was recovered and concentrated to an appropriate amount, added to a treated porous adsorption resin separation tube, and eluted successively with ethanol of different concentrations. The corresponding eluates were collected, ethanol was recovered and concentrated to an appropriate amount, added to a treated polyamide resin separation tube, and eluted successively with ethanol of different concentrations. The corresponding eluates were collected, ethanol was recovered, and concentrated to a high-viscosity paste (paste) state, followed by vacuum drying and pulverization. Among them, for the component (extract) numbered NR218 that showed interesting characteristics in the preliminary test, further in-depth research was carried out.

[0023] Example 2 Confirmation Test of NR218 Extract As a result of commissioning the confirmation test, the chromatogram showing the characteristics of the NR218 extract was as shown in Figure 1. Among them, the chemical structures of the analytical results of the seven main compounds: licocalcone A, licocalcone C, licocalcone D, licocalcone E, formononetin, glabrol, and licoflavone C are shown in Figure 2, and the proportions of the seven compounds are shown in Table 1. The results of HPLC-MS analysis are shown in Table 2 for the low-content components found in addition to the above main components.

[0024]

Table 1

[0025]

Table 2

[0026] Example 3 Preparation of NR218 Composition 7.2 g of licocalcone A, 0.38 g of licocalcone C, 0.22 g of licocalcone D, 0.68 g of licocalcone E, 0.25 g of formononetin, 0.28 g of glabrol, and 0.12 g of licoflavone C were taken and uniformly mixed to obtain the NR218 composition of the stabilized product, which was further used in the in-depth test.

[0027] Example 4 Effects and Therapeutic Actions of NR218 Composition on the NRF2 Pathway in a Rat Non-Alcoholic Steatohepatitis Model Induced by a High-Fat Diet [Test Materials] Feed for model preparation: High-fat feed (88% basal feed + 10% lard + 2% cholesterol), purchased from Sprague Dawley (Beijing) Biotechnology Co., Ltd. License number SCXK (Beijing) 2019-0010, certificate number 1103242000022188. Animals: SPF male Wistar rats, 160 ± 10 g, purchased from Sprague Dawley (Beijing) Biotechnology Co., Ltd. License number SCX (Beijing) 2019-0010. Drugs: NR218 Composition, Bisicyclol Tablets, Beijing Union Pharmaceutical Factory, Specification 50 mg / tablet, Lot Number 191116. Rosuvastatin Capsules, Yangtze River Pharmaceutical Group Co., Ltd., Specification 20 mg / capsule, Lot Number 19030461. Obeticholic Acid, Hubei Jiuzhou Kangda Biotechnology Co., Ltd., Specification 100 g / bag, Lot Number 20200210. Equipment: Multifunction Microplate Reader, Guangzhou Darui Biotechnology Co., Ltd., Model Number H1M. Multi-Sample Tissue Grinder, Shanghai Jingxin Industrial Development Co., Ltd., Model Number Tissuelyser-24. Cooling Centrifuge, Model Number Micro 21R. Centrifuge, Thermo Fisher, Model Number LR58495. Electronic Balance, Shanghai Yueping Electronic Balance, Model Number YP10001. Analytical Balance, METTLER TOLEDO, Model Number PL602-L. Vertical Pressure Steam Sterilizer, Chongqing Yamato Technology Co., Ltd., Model Number SN510C. Electrophoresis Device Power Supply, Beijing Liuyi Instrument Factory, Model Number DYY-6C. Protein Electrophoresis and Transfer System, BIO-RAD, Model Number Mini-PROTEAN. Materials: High-density lipoprotein cholesterol (HDL-C) test kit, Nanjing Jiancheng Bioengineering Institute, product number A112-1-1, lot number 20201012. Low-density lipoprotein cholesterol (LDL-C) test kit, Nanjing Jiancheng Bioengineering Institute, product number A113-1-1, lot number 20201012. Non-esterified fatty acid (NEFA) reagent kit, Nanjing Jiancheng Bioengineering Institute, product number A042-2-1, lot number 20201012. Aspartate aminotransferase (AST / GOT) reagent kit, Nanjing Jiancheng Bioengineering Institute, product number C010-2-1, lot number 20201012. Alanine aminotransferase (ALT / GPT) reagent kit, Nanjing Jiancheng Bioengineering Institute, product number C009-2-1, lot number 20201012. Total cholesterol (T-CHO) test kit, Nanjing Jiancheng Bioengineering Institute, product number A111-1-1, lot number 20200815. Triglyceride (TG) test kit, Nanjing Jiancheng Bioengineering Institute, product number A110-1-1, lot number 20200814. Uric acid (UA) test kit, Nanjing Jiancheng Bioengineering Institute, product number C012-2-1, lot number 20201012. Glucose quantification reagent kit, Jinclon Biotechnology Co., Ltd., product number SH152W, lot number 20201012. Rat fasting insulin (FINS) enzyme-linked immunosorbent assay (ELISA) reagent kit, Jinclon Biotechnology Co., Ltd., product number SEKR-0160, lot number 20201012. High-efficiency RIPA tissue rapid lysate, Solarbio Science & Technology Co., Ltd., product number R0010, production lot number 20200926. BCA reagent kit, Solarbio Science & Technology Co., Ltd., product number PC0020, production lot number 20201010. SIRT1 antibody, Cell Signaling technology, product number 9475S. AMPK α1 antibody, Proteinteck, product number 10929-2-AP. NF-κB p65 antibody, Proteinteck, product number 10745-1-AP. Nrf2 antibody, Proteinteck, product number 16396-1-AP. GAPDH antibody, Proteinteck, product number 10494-1-AP.

[0028] [Test method] (1) Establishment of a Rat Model of Non-Alcoholic Steatohepatitis Induced by a High-Fat Diet and Drug Administration One hundred and twenty Wistar rats were randomly divided into 8 groups of 15 rats each: a normal control group, a model group, a bicyclol positive control group, a rosuvastatin positive control group, an obeticholic acid positive control group, a high-dose NR218 group (90 mg / kg), a medium-dose group (30 mg / kg), and a low-dose group (10 mg / kg). In the normal control group, the rats were fed a normal diet and bred for 12 weeks. In each of the other groups, the rats were fed a high-fat diet (88% basal diet + 10% lard + 2% cholesterol) and bred for 12 weeks. After model establishment, the corresponding drug (10 ml / kg) was administered to each of the dosing groups, and an equal volume of 0.5% CMC-Na solution was administered to the normal control group and the model group. Gastric administration was performed once a day for 8 consecutive weeks. During the test period, the research animals were allowed free access to water and food, the animal room was kept quiet, natural light was introduced, the temperature was maintained at 25 ± 0.5 °C, and the humidity was maintained at 55 ± 5%. The body weight of the rats was measured once a week, and the body weight data were recorded.

[0029] (2) Sample Collection and Test Measurement (i) Consideration of the General Condition of the Animals The body weight was measured once a week, and the change in the body weight of the rats in each group was considered.

[0030] (ii) Test Measurement of Serum Biochemical Indicators Related to Non-Alcoholic Steatohepatitis On the last day of the 8th week of dosing, all rats after the final dosing were placed under fasting but with free access to water for 16 h. After anesthesia was induced by intraperitoneal injection of 20% ethyl carbamate solution (10 ml / kg), blood was collected from the abdominal aorta. The blood was allowed to stand for 1 h and then centrifuged at 4 °C at 3000 revolutions per minute for 15 min. The upper-layer serum was collected and stored at -80 °C. According to the instructions of the biochemical indicator test measurement reagent kit, the levels of HDL-C, LDL-C, NEFA, AST, ALT, UA, TC, TG, FPG, and FINS in rat serum were measured, and the rat insulin resistance index [(HOMA-IR) = FPG (mmol / L) × FINS (mIU / L) / 22.5] was considered.

[0031] (iii) Consideration of liver index After blood was collected from the abdominal aorta of rats, liver tissue was separated, thoroughly washed with physiological saline, blotted with filter paper, weighed, and the liver index was calculated. Liver index = wet liver weight / body weight × 100%.

[0032] (iv) Pathological examination of rat liver tissue HE staining and Oil Red staining: After blood was collected from the abdominal aorta of rats, liver tissue was separated, fixed with 10% neutral formalin, and processed according to the normal specimen preparation procedure, including embedding, sectioning, deparaffinization, clearing with xylene, staining, and mounting. The pathological changes in the liver tissue of rats in each group were observed under a microscope.

[0033] (v) Detection and measurement of non-alcoholic steatohepatitis-related proteins Immunohistochemical examination: Paraffin-embedded liver tissue sections with a thickness of 5 μm were cultured with the target antibody for 90 minutes, and then cultured with a peroxidase-conjugated secondary antibody for 30 minutes. Subsequently, they were cultured with "streptavidin-peroxidase-biotin complex" at room temperature for 20 minutes. After color development, observation and analysis were performed under a microscope.

[0034] Western blotting method: An appropriate amount of liver tissue was taken, lysed using a high-efficiency RIPA tissue rapid lysate and PMSF to extract proteins. The BCA Protein Assay Kit was used to quantitatively measure the extracted liver tissue proteins. According to the Western blotting method, GAPDH was used as an internal reference substance to measure the changes in the expression of AMPK, SIRT1, NF-κB, and Nrf2 proteins. Quantitative analysis was performed using ImageJ1.48 software.

[0035] (3) Statistical analysis The test results were expressed as mean ± standard deviation (X±SD), and one-way analysis of variance was performed using SPSS software version 17.0. P<0.05 indicated the presence of a significant difference, and P<0.01 indicated the presence of a highly significant difference. All results were visualized using GraphPad Prism software (version 6.02).

[0036] [Test Results] (1) Consideration of the body weight changes of rats in each group The analysis results of the body weight shown in Table 3 indicated an increasing trend in rat body weight during the 12 weeks before model establishment. After dosing in each group, the body weight decreased to a certain extent, but no significant difference was observed.

[0037]

Table 3

[0038] (2) Effect of NR218 on the liver index of non-alcoholic steatohepatitis rats After the test, the liver index of rats in each group was considered. As shown in Table 4, compared with the normal control group, the liver index of the model group was significantly increased (P<0.01). Compared with the model group, the liver indexes of the bicyclol, lovastatin, obeticholic acid positive control group, and the high, medium, and low dose groups of NR218 rats were all significantly decreased (P<0.01 or P<0.05), and the effects of the medium and high doses of NR218 showed superiority over the positive control drug.

[0039]

Table 4

[0040] (3) Effect of NR218 on the liver tissue pathology of non-alcoholic steatohepatitis rats Figure 3 shows the results of HE. The hepatocytes in the normal control group were arranged neatly, the structure of the hepatic lobule was clear, no fatty degeneration was observed in the cells, and no infiltration of inflammatory cells was observed. The hepatocytes of the model group rats were swollen, and fat droplet vacuoles of uneven size were observed in the cells. The cell nucleus migrated to the edge, showing globular degeneration and infiltration of inflammatory cells was observed. Compared with the model group, the fat degeneration situation in each dosing group was reduced to a certain extent, and the inflammatory cells decreased. This was most significant in the high-dose group of NR218, and the shape of the hepatocytes was basically normal.

[0041] Figure 4 shows the Oil Red O staining results. The fat content in the liver tissue of the model group was significantly increased, the arrangement of hepatocytes was irregular, and the loss of cytoskeleton was observed. Many of the intracellular lipid droplets in the model group were multilocular and large, and many of the lipid droplets were distributed in flakes. After specimen embedding, aggregation of lipid droplets on the section surface was often confirmed. In the hepatocytes of rats in the NR218 administration group, a small number of minute lipid droplets were scattered, and obvious improvements were observed in both the level of fatty degeneration and the content of lipid droplets compared with the model group.

[0042] (4) Effect of NR218 on serum biochemical indexes of non-alcoholic steatohepatitis rats According to the instructions of the biochemical index test measurement reagent kit, the serum ALT, AST, TC, TG, HDL-C, LDL-C, UA, NEFA, FPG, and FINS levels of rats were tested and measured. The test results are as shown in Table 5. Compared with the normal control group, the serum ALT, AST, TC, TG, LDL-C, UA, and NEFA levels of rats in the model group were all significantly increased (P<0.01 or P<0.05), and the HDL-C level was significantly decreased (P<0.01). Compared with the model group, NR218 significantly decreased the serum AST, ALT, TC, TG, LDL-C, UA, and NEFA levels of rats, and at the same time increased the content of beneficial lipoprotein HDL-C (P<0.01 or P<0.05), improving the liver function and lipid metabolism of non-alcoholic steatohepatitis. As a result of considering the insulin resistance index (HOMA-IR) of rats in each group, compared with the normal control group, the HOMA-IR of rats in the model group was significantly increased (P<0.01), and compared with the model group, NR218 significantly decreased the insulin resistance of non-alcoholic steatohepatitis rats. From the above results, it was shown that NR218 can significantly improve the liver function of non-alcoholic steatohepatitis rats, decrease the blood lipid content, and improve insulin resistance.

[0043]

Table 5

[0044] (5) Effect of NR218 on the expression of related proteins in the liver tissue of non-alcoholic steatohepatitis rats The expression status of AMPK, NF-κB, Nrf2 and SIRT1 proteins in the liver tissue of non-alcoholic steatohepatitis rats was examined by immunohistochemistry. As shown in the results of Figure 5, high, medium and low doses of NR218 significantly decreased the expression levels of NF-κB and Nrf2 proteins in the liver tissue of non-alcoholic steatohepatitis rats to varying degrees, and significantly increased the expression levels of AMPK and SIRT1 proteins in the liver tissue. In addition, compared with the normal control group shown in Table 6, the expression of AMPK and SIRT1 proteins in the model group was significantly decreased (P<0.01), and the expression of NF-κB and Nrf2 was significantly increased (P<0.01). Compared with the model group, high, medium and low doses of NR218 all significantly increased the expression levels of AMPK and SIRT1 proteins (P<0.01 or P<0.05), inhibited the expression levels of NF-κB and Nrf2 proteins (P<0.01 or P<0.05), and the best results were obtained in the high-dose range. The above results suggest the possibility of treating non-alcoholic steatohepatitis with NR218 by regulating inflammation and oxidative stress through the AMPK / SIRT1 / NF-κB signaling axis.

[0045]

Table 6

[0046] [Conclusion of the test] In this experiment, a rat non-alcoholic steatohepatitis model established by feeding a high-fat diet was used to confirm that NR218 can reduce the level of hepatic steatosis in non-alcoholic steatohepatitis rats, decrease the liver index, improve liver function and lipid metabolism, and reduce blood lipids and insulin resistance index. At the protein level, NR218 significantly increased the expression levels of liver AMPK and SIRT1 proteins and inhibited the expression levels of NF-κB and Nrf2 proteins. The above results suggest that NR218 has a significant effect on improving non-alcoholic steatohepatitis induced by a high-fat diet, improves inflammation and antioxidant stress through the AMPK / SIRT1 / NF-κB signaling axis, and indicates the possibility of realizing this effect.

[0047] Example 5 High-fat diet + CCL 4 Inhibitory and therapeutic effects of NR218 on the NRF2 pathway in a rat non-alcoholic steatohepatitis model induced by combination [Test materials] Diet for model establishment: High-fat diet (88% basal diet + 10% lard + 2% cholesterol), purchased from Beijing Keao Xieli Feed Co., Ltd. Feed permit number SCXK (Beijing) 2019-0003, feed certificate number 1112622000028380. Carbon tetrachloride (CCL 4 ), Shanghai Macklin Biochemical Co., Ltd., lot number C10921057, olive oil, Sinopharm Chemical Reagent Co., Ltd., lot number 0191008.

[0048] Animals: SPF male Wistar rats, 160 ± 10 g, purchased from Spf (Beijing) Biotechnology Co., Ltd. Permit number SCX (Beijing) 2019-0010.

[0049] Drugs: NR218 composition, bicyclol tablets, Beijing Union Pharmaceutical Factory, specification 50 mg / tablet, lot number 191116. Rosuvastatin capsules, Yangtze River Pharmaceutical Group Co., Ltd., specification 20 mg / capsule, lot number 19030461. Obeticholic acid, Hubei Jiuzhou Kangda Biotechnology Co., Ltd., specification 100 g / bag, lot number 20200210.

[0050] Equipment: Multifunction microplate reader, US BioTek, model Synergy H1. Multi-sample tissue grinder, Shanghai Jingxin Industrial Development Co., Ltd., model Tissuelyser-24. Centrifuge, Thermo Fisher Scientific, model PICO 17. Electronic balance, Shanghai Yueping Electronic Balance, model YP10001. Analytical balance, METTLER TOLEDO, model AL204. Vertical autoclave, Chongqing Yamato Scientific Co., Ltd., model SN510C. Electrophoresis apparatus, Beijing Liuyi Instrument Factory, model DYY-6C. Low-temperature centrifuge, Thermo Fisher Scientific, model FRESCO21.

[0051] Materials: High-density lipoprotein cholesterol (HDL-C) test kit, Nanjing Jiancheng Bioengineering Institute, product number A112-1-1, lot number 20201010. Low-density lipoprotein cholesterol (LDL-C) test kit, Nanjing Jiancheng Bioengineering Institute, product number A113-1-1, lot number 20201010. Alanine aminotransferase (ALT / GPT) test kit, Nanjing Jiancheng Bioengineering Institute, product number C009-2-1, lot number 20200912. Aspartate aminotransferase (AST / GOT) test kit, Nanjing Jiancheng Bioengineering Institute, product number C010-2-1, lot number 20200929. Uric acid (UA) test kit, Nanjing Jiancheng Bioengineering Institute, product number C012-2, lot number 20200907. Free fatty acid (NEFA) measurement reagent kit, Nanjing Jiancheng Bioengineering Institute, product number A042-2-1, lot number 20201010. Fasting plasma glucose (FPG) content measurement reagent kit, GeneCopoeia (Beijing) Biotechnology Co., Ltd., product number SH152W, lot number 20201016. Total cholesterol (TC) test measurement reagent kit, Nanjing Jiancheng Bioengineering Institute, product number A111-1-1, lot number 20200825. Triglyceride (TG) test measurement reagent kit, Nanjing Jiancheng Bioengineering Institute, product number A110-1-1, lot number 20200825. Rat fasting insulin (FINS) enzyme-linked immunosorbent assay (ELISA) reagent kit, Shanghai Enzyme-linked Biotechnology Co., Ltd., product number ml302840. Rat type III procollagen (PC-III) test measurement reagent kit, Shanghai Enzyme-linked Biotechnology Co., Ltd., product number ml038007. Rat laminin (LN) test measurement reagent kit, Shanghai Enzyme-linked Biotechnology Co., Ltd., product number ml823654. Rat type IV collagen (IV-C) test measurement reagent kit, Shanghai Enzyme-linked Biotechnology Co., Ltd., product number ml038234. Rat hyaluronic acid (HA) test measurement reagent kit, Shanghai Enzyme-linked Biotechnology Co., Ltd., product number ml852321. SIRT1 antibody, Cell Signaling technology, product number 9475S. AMPK α1 antibody, Proteinteck, product number 10929-2-AP. NF-κB p65 antibody, Proteinteck, product number 10745-1-AP.Nrf2 antibody, Proteintech, product number 16396-1-AP. GAPDH antibody, Proteintech, product number 10494-1-AP.

[0052] [Test method] (1) Replication and dosing of a rat non-alcoholic steatohepatitis model induced by combination of high-fat diet and CCL 4 4 120 male Wistar rats were randomly divided into 8 groups of 15 rats each according to the random number table method: normal control group, model group, bicyclol positive control group, rosuvastatin positive control group, obeticholic acid positive control group, NR218 high-dose group (90 mg / kg), medium-dose group (30 mg / kg), and low-dose group (10 mg / kg). In the normal control group, rats were fed a normal diet. In the other rats, for the first 6 weeks, only a high-fat diet was fed, and from the 7th week, 30% CCL 4 olive oil solution was subcutaneously injected from the back at a dose of 0.2 ml / 100 g, twice a week for a total of 4 times. After successful replication of the model, the corresponding drugs (10 ml / kg) were administered intragastrically to each dosing group, and an equal volume of 0.5% CMC-Na solution was continuously administered to the normal control group and the model group once a day for 10 weeks. During the test period, the experimental animals had free access to drinking water and food, the animal room was kept quiet, natural light was introduced, the temperature was 25 ± 0.5 °C, and the humidity was 55 ± 5%. The body weight of the rats was measured once a week and the body weight data were recorded.

[0053] (2) Sample collection and test measurement (i) Observation of the general condition of rats The body weight was measured once a week, and after the test, the body weight change of rats in each group was compared.

[0054] (ii) Measurement of liver index After blood was collected from the abdominal aorta of the rats, the liver tissue of the rats was separated, washed thoroughly with physiological saline, blotted with filter paper, and then weighed to calculate the liver index. Liver index = wet liver weight / body weight × 100%

[0055] (iii) Measurement of serum biochemical indicators of non-alcoholic steatohepatitis At the end of the 10th week of dosing, all rats after the last dose were placed under fasting for 16 hours with free access to water. After anesthesia was induced by intraperitoneal injection of 20% ethyl carbamate solution (10 ml / kg), blood was collected from the abdominal aorta, allowed to stand for 1 hour, centrifuged at 3000 rpm for 15 minutes at 4°C, and the upper-layer serum was taken and stored at -80°C. In accordance with the instructions of the biochemical indicator test kit, the levels of HDL-C, LDL-C, NEFA, AST, ALT, UA, TC, TG, PCIII, IV-C, LN, and HA in rat serum were measured.

[0056] (iv) Calculation of the HOMA-IR index In accordance with the instructions of the reagent kit, the FPG content in serum was measured by colorimetry, and the FINS concentration in serum was measured by radioimmunoassay. The HOMA-IR index was calculated according to the formula HOMA-IR = FPG (mmol / L) × FINS (mIU / L) / 22.5.

[0057] (v) Histopathological observation of tissues After blood was collected from the rat abdominal aorta, liver tissues were separated, fixed with 10% neutral formalin, and processed through embedding, sectioning, deparaffinization, clearing with xylene, staining, and mounting according to the normal specimen preparation procedure. Subsequently, HE staining, Masson staining, and oil red staining were performed respectively, and the pathological changes of the liver tissues of rats in each group were observed under a microscope.

[0058] (vi) Measurement of non-alcoholic steatohepatitis-related proteins Immunohistochemical examination: Paraffin-embedded liver tissue sections with a thickness of 5 μm were cultured with the target antibody for 90 minutes and then cultured with peroxidase-conjugated secondary antibody for 30 minutes. Subsequently, they were cultured with "streptavidin-peroxidase-biotin complex" for 20 minutes at room temperature. After color development, observation and analysis were performed under a microscope.

[0059] Western blotting method: 80 mg of liver tissue from each rat was taken under aseptic conditions, dissolved using a high-efficiency RIPA tissue rapid lysate and PMSF, and liver tissue proteins were extracted. The extracted liver tissue proteins were quantitatively measured using a Protein Assay BCA Kit (BCA Protein Assay Kit), and the specific operation method was carried out according to the instructions. According to the Western blotting method, the changes in the expression of AMPK, SIRT1, NF-κB, Nrf2, and GAPDH proteins were measured. Quantitative analysis was performed using ImageJ 1.48 software.

[0060] (3) Statistical analysis The test results were expressed as mean ± standard deviation (X±SD). One-way analysis of variance was performed using SPSS 17.0 statistical software, and then the LSD test was adopted for comparison between the two groups. P<0.05 indicates the presence of a significant difference, and P<0.01 indicates the presence of a very significant difference. All results were visualized using GraphPad Prism software (version 6.02).

[0061] [Test results] (1) Consideration of the change in body weight of rats in each group According to the results shown in Table 7 of the statistical analysis of the body weight of rats per week, the body weight of rats in each group before dosing continuously increased with the passage of breeding time. After dosing, the rate of increase in rat body weight clearly decelerated. When compared with the model group, the body weights of rats in the bicyclol, lovastatin, and obeticholic acid positive control groups and the low, medium, and high dose groups of NR218 all showed a decreasing trend, but no significant difference was observed.

[0062] [Table 7] JPEG2025516078000013.jpg228164

[0063] (2) The effect of NR218 on the liver index of rats with non-alcoholic steatohepatitis induced by the combination of high-fat diet + CCL 4 As a result of examining the liver indices of each group of rats, in the comparison shown in Table 8 with the normal control group, the liver index of the model group rats was significantly increased (P<0.01). In comparison with the model group, the liver index of the bicyclol positive control group rats was significantly decreased. It was confirmed that at low, medium, and high doses of NR218, all could significantly decrease the rat liver index (P<0.01). From the above results, NR218 can effectively suppress the liver hypertrophy of rats with non-alcoholic steatohepatitis induced by the combination of high-fat diet + CCL 4 and it was confirmed that all the effects were better than those of the positive control drug.

[0064]

Table 8

[0065] (3) High-fat diet + CCL 4 Effect of NR218 on serum indices of rats with non-alcoholic steatohepatitis induced by the combination of high-fat diet + CCL In accordance with the instructions regarding the test measurement of the reagent kit, the levels of AST, ALT, HDL-C, LDL-C, UA, NEFA, TC, TG, FPG, FINS, PCIII, IV-C, LN, and HA in the serum of rats were tested and measured. In comparison with the normal control group of the test results shown in Table 9 and Table 10, the expressions of serum ALT, AST, TC, TG, LDL-C, UA, PCIII, IV-C, LN, and HA in the model group rats were all significantly increased (P<0.01), and the expression of HDL-C was significantly decreased (P<0.01). In comparison with the model group, high, medium, and low doses of NR218 decreased the levels of serum AST, ALT, LDL-C, UA, NEFA, TC, TG, PCIII, IV-C, LN, and HA in rats to varying degrees (P<0.01 or P<0.05), significantly increased the content of beneficial lipoprotein HDL-C (P<0.01 or P<0.05), and significantly improved the liver function, blood lipid levels, and liver fibrosis levels of rats with non-alcoholic steatohepatitis.

[0066]

Table 9

[0067]

Table 10

[0068] (4) High-fat diet + CCL 4 Effect of NR218 on liver tissue pathology of rats with non-alcoholic steatohepatitis induced by combination use (i) In the results of HE staining shown in Figure 6, the hepatocytes of rats in the normal control group were arranged neatly, the structure of the hepatic lobule was clear, no fatty degeneration was observed in the cells, and no infiltration of inflammatory cells was observed. The hepatocytes of rats in the model group were swollen, relatively large lipid droplets were observed in the cells, the cell nucleus moved to the edge, showing globular degeneration, the hepatic sinusoids were clearly dilated, obvious infiltration of inflammatory cells was observed, and the liver collagen content was clearly increased, suggesting the success of model establishment. Compared with the model group, the high, medium, and low dose groups of NR218 all decreased the number of lipid particles in hepatocytes and the collagen content in liver tissue to varying degrees, significantly reduced the infiltration level of inflammatory cells and the level of hepatocyte necrosis, and the effect was more significant than that of the positive control drugs obeticholic acid, rosuvastatin, and bicyclol.

[0069] (ii) In the results of the Oil Red O staining test shown in Fig. 7, the hepatocyte nuclei of rats in the normal control group were lightly stained, and there were no fat droplets stained red. The hepatocytes of rats in the model group were swollen, and a large number of large vesicular red fat droplets were observed in the cytoplasm. Some of them fused into large fat droplets, and most of the fat droplets were distributed in flakes. The arrangement of hepatocytes was irregular, and the loss of the cytoskeleton was observed. Compared with the model group, in the hepatocytes of rats in the bicyclol, lovastatin, obeticholic acid positive control group and the high, medium, and low dose groups of NR218, a small number of tiny small fat droplets were scattered, and large fat droplets were rarely observed. However, both the level of fatty degeneration and the content of fat droplets were clearly improved, and the dose-effect of NR218 was more significant than that of the positive control drugs bicyclol, lovastatin, and obeticholic acid.

[0070] (iii) Compared with the normal group in the results of the Masson staining test shown in Fig. 8, the collagen content in the liver tissue of rats in the model group was clearly increased, and most of it was deposited in the central vein area and portal vein area of the liver tissue. Most of the collagen depositions showed a reticular pattern. Compared with the model group, the collagen content in the liver tissue of rats in the high, medium, and low dose groups of NR218 was clearly decreased, the deposition of reticular collagen disappeared, and the effects of each dose of NR218 were more significant than those of the positive control drugs bicyclol, lovastatin, and obeticholic acid.

[0071] (5) Effect of NR218 on the expression of non-alcoholic steatohepatitis-related proteins High-fat diet + CCL 4To further explore the potential biological mechanisms of NR218 in the treatment of non-alcoholic steatohepatitis induced by combination, immunohistochemistry was used to examine the expression status of Smad, TGFβ, α-SMA and NS5ATP9 proteins in the liver tissues of non-alcoholic steatohepatitis rats. As shown in the results of Figure 9, high, medium and low doses of NR218 significantly decreased the expression levels of Smad, TGFβ and α-SMA proteins in the liver tissues of non-alcoholic steatohepatitis rats to varying degrees, and significantly increased the expression level of NS5ATP9 protein in the liver tissues. The results of examining the changes in the expression of AMPK, SIRT1, Nrf2 and NF-κB proteins in rat liver tissues using Western blotting and immunohistochemistry tests are shown in Table 11. Compared with the normal control group, the expression levels of AMPK and SIRT1 proteins in the liver tissues of the model group rats were significantly decreased (P<0.01), and the expression levels of NF-κB and Nrf2 proteins were significantly increased (P<0.01). Compared with the model group, in the high, medium and low dose groups of NR218, the expression levels of AMPK and SIRT1 proteins were significantly increased to varying degrees (P<0.01 or P<0.05), and the expression levels of NF-κB and Nrf2 proteins were significantly decreased (P<0.01), and the improvement effect in the high dose group was the best. The above results suggest that NR218 may improve non-alcoholic steatohepatitis when it regulates inflammation, oxidative stress and liver fibrosis through the AMPK / SIRT1 / NF-κB / Smad pathway.

[0072]

Table 11

[0073] [Conclusions of the test] In this test, high-fat diet + CCL 4Using a rat model of non-alcoholic steatohepatitis induced by combination, the general conditions of rats (body weight and liver mass), serum biochemical indexes, and tissue pathology were investigated. As a result, it was confirmed that NR218 could significantly improve the hepatic steatosis and fibrosis levels in rats with non-alcoholic steatohepatitis, and significantly reduce the liver index, blood lipids, and insulin resistance index of rats. At the protein expression level, NR218 significantly increased the protein expression levels of AMPK, NS5ATP9, and SIRT1 in rat liver tissue, and decreased the protein expression levels of NF-κB, Nrf2, Smad, TGFβ, and α-SMA in liver tissue. The above results indicate that NR218 exerts a significant effect in improving non-alcoholic steatohepatitis induced by combination, suggesting that this effect may regulate inflammation and oxidative stress through the AMPK / SIRT1 / NF-κB pathway and achieve the reduction of liver fibrosis through the Smad signal. 4 It exerted a significant effect in significantly improving non-alcoholic steatohepatitis induced by combination, suggesting the possibility that this effect regulates inflammation and oxidative stress through the AMPK / SIRT1 / NF-κB pathway and achieves the reduction of liver fibrosis through the Smad signal.

Claims

1. A small molecule composition comprising licochalcone A, licochalcone C, licochalcone D, licochalcone E, formononetin, glabrol and licoflavone C, which negatively regulates the Nrf2 signaling pathway.

2. 2. The low molecular weight composition according to claim 1, wherein the weight ratio of licochalcone A, licochalcone C, licochalcone D, licochalcone E, formononetin, glabrol to licoflavone C is 60-75:2-6:1-4:5-10:1-5:1-5:1-5.

3. 2. The low molecular weight composition according to claim 1, wherein the weight ratio of licochalcone A, licochalcone C, licochalcone D, licochalcone E, formononetin, glabrol to licoflavone C is 65-75:3-5:1-3:6-8:2-3:2-3:1-2.

4. The low molecular weight composition according to claim 1, which exerts a negative regulatory effect on the expression of the Nrf2 gene during the onset and progression of non-alcoholic fatty liver disease.

5. 2. The low molecular weight composition according to claim 1, wherein licochalcone A, licochalcone C, licochalcone D, licochalcone E, formononetin, glabrol and / or licoflavone C are extracted from plants or artificially synthesized.

6. A formulation comprising the small molecule composition according to any one of claims 1 to 5 and a pharmaceutically or food-acceptable additive.

7. A method for using the small molecule composition according to any one of claims 1 to 5 to prepare a reagent that negatively regulates the Nrf2 signaling pathway.

8. 10. A method for using the low molecular weight composition according to any one of claims 1 to 5 to prepare a drug for preventing or treating non-alcoholic fatty liver disease (NAFLD).

9. 9. The method of claim 8, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

10. 9. The method according to claim 8, wherein the non-alcoholic fatty liver disease is a glycolipid metabolism disorder, liver fibrosis, liver cirrhosis, or liver cancer.

Images