Application of benzimidazole amine derivative in medicine for preventing / treating non-alcoholic fatty liver disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of a benzimidazole amine derivative in a medicine for preventing / treating non-alcoholic fatty liver disease, and the benzimidazole amine derivative has a structural formula as shown in a formula (1) or a salt thereof. It is proved for the first time that the active molecule improves the antioxidant level, inhibits oxidative stress and reduces lipid accumulation by activating an Nrf2-ARE signal channel, remarkably improves the non-alcoholic fatty liver disease, is expected to serve as an alternative medicine for preventing / treating the disease, and has potential application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of benzimidazole amine derivatives as potential drugs for preventing / treating nonalcoholic fatty liver disease (NAFLD). Background Art

[0002] NAFLD is one of the most common chronic liver diseases in the world. It is closely related to human metabolism. Its main feature is excessive fat deposition in liver cells, which can easily progress to non-alcoholic fatty liver disease, liver fibrosis, cirrhosis and even liver cancer. As the incidence of obesity and type 2 diabetes increases year by year, the prevalence of NAFLD related to them is also growing rapidly. The current treatment strategy for this disease is mainly lifestyle changes, such as adjusting diet and increasing exercise.

[0003] Based on this, it is necessary to develop small molecule drugs for this disease. Summary of the invention

[0004] The object of the present invention is to provide a use of a benzimidazole amine derivative in a drug for preventing / treating NAFLD.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides an application of a benzimidazole amine derivative in a drug for preventing / treating non-alcoholic fatty liver disease.

[0007] Furthermore, the benzimidazole amine derivative is of the structural formula (1) or a salt thereof:

[0008]

[0009] R1=H,Br;

[0010] R2 is one of the following structural formulas:

[0011]

[0012] Furthermore, the benzimidazole amine derivative is specifically one of the following structural formulas:

[0013]

[0014] Furthermore, the benzimidazole amine derivatives are used to protect HepG2 cell damage induced by free mixed acid.

[0015] Furthermore, the benzimidazole amine derivatives are used to activate the Nrf2-ARE pathway to promote the increase of Nrf2 protein levels, induce Nrf2 to enter the cell nucleus, and increase the protein expression levels of downstream antioxidant and detoxification molecules, including quinone oxidoreductase 1 and glutamate-cysteine ​​ligase catalytic subunit; wherein, the Nrf2 refers to nuclear factor NF-E2-related factor 2, and the ARE refers to antioxidant response element.

[0016] Furthermore, the benzimidazole amine derivatives are used to reduce intracellular lipid accumulation and lipid peroxidation, inhibit reactive oxygen species aggregation, and enhance the cell's resistance to damage in a free mixed acid-induced non-alcoholic fatty liver disease cell model.

[0017] Furthermore, the benzimidazole derivatives are used as active ingredients and a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0019] (1) It is the first time to confirm that benzimidazole amine derivatives have a significant improvement effect on NAFLD. The benzimidazole amine derivatives provided by the present invention are used in the treatment or prevention of NAFLD and have a significant improvement effect on the disease.

[0020] (2) Further mechanistic studies have shown that the benzimidazole amine derivative A5 can activate the Nrf2-ARE pathway, increase the protein expression levels of downstream antioxidant and detoxification molecules, improve lipid metabolism, etc., and can subsequently be used as a reserve drug for the treatment of NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effects of benzimidazole amine derivatives on the survival rate of HepG2 cells and their protective effects on HepG2 cell damage induced by free fatty acid (FFA);

[0022] Figure 2 The effect of benzimidazole amine derivative A5 on fat accumulation in HepG2 cells induced by FFA;

[0023] Figure 3 The effect of benzimidazole amine derivative A5 on lipid peroxidation in HepG2 cells induced by FFA;

[0024] Figure 4 The effect of benzimidazole amine derivative A5 on ROS level in HepG2 cells induced by FFA;

[0025] Figure 5The effect of benzimidazole amine derivative A5 on the expression of NQO1, GCLC and other proteins in HepG2 cells;

[0026] Figure 6 This is the effect of benzimidazole amine derivative A5 on the protein expression level and distribution of Nrf2 in HepG2 cells.

[0027] Statistical analysis: Graphpad Prism9 statistical software was used to analyze the data, and One-way ANOVA was used for significance analysis. * Compared with the control group, P < 0.05. ** Compared with the control group, P < 0.01. *** Compared with the control group, P < 0.001. **** represents P < 0.0001 compared with the control group; & Compared with the model group, P < 0.05. && The difference was significant when P<0.01 compared with the model group. The difference was significant when P<0.05. The results were expressed as mean±standard error (Mean±SE). DETAILED DESCRIPTION

[0028] The pathogenesis of NAFLD is relatively complex. In recent years, the "multiple parallel strikes" theory has become more popular. This theory is considered to be a more accurate description of the pathogenesis of NAFLD. In this theory, multiple factors such as oxidative stress, abnormal lipid metabolism, insulin resistance, inflammatory response, mitochondrial dysfunction, immune disorders and intestinal flora imbalance contribute to the development of NAFLD. Oxidative stress is caused by the imbalance between the production and elimination of reactive oxygen species (ROS) in the body. Excessive ROS can cause damage to proteins, lipids and nucleic acids. Studies have shown that inhibiting oxidative stress can effectively improve lipid metabolism, alleviate oxidative damage and delay the development of NAFLD.

[0029] Nrf2 is the main transcription factor that regulates the expression of various antioxidant proteins. Activating the Keap1-Nrf2-ARE pathway can induce the increase of antioxidant and detoxification enzyme levels in the body, thereby protecting cells from oxidative damage and playing an important role in the antioxidant defense system. Under physiological conditions, Nrf2 binds to its inhibitory partner Keap1 protein, which mediates the ubiquitination and degradation of Nrf2 through the E3 ubiquitin ligase complex. When attacked by electrophilic reagents or in a state of oxidative stress, Nrf2 is released from the Keap1 protein anchor, enters the cell nucleus, forms heterodimers with the small Maf protein, and binds to the antioxidant response element (ARE) in the promoter region of downstream target genes, thereby activating the expression of downstream antioxidant molecules (quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1) and glutamate-cysteine ​​ligase catalytic subunit (GCLC)). Small molecule activators targeting Nrf2 can effectively activate the Keap1-Nrf2-ARE pathway and may be used as potential drug molecules for NAFLD. Benzimidazole amine derivatives have multiple functions, and the present invention provides their mechanisms of action as Nrf2 activators and in NAFLD.

[0030] The present invention is further described below in conjunction with the accompanying drawings. The following examples are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0031] This example explores the mechanism of protection of HepG2 cells by benzimidazole amine derivatives. The preparation method of the benzimidazole amine derivatives is as follows:

[0032] Compound A1-A4: dissolve the acid and the activator in a solvent, add different types of amines under ice bath, then add the acid binder and the amide coupling agent and continue stirring to room temperature to obtain the benzimidazole amine derivatives; wherein the solvent is N,N-dimethylformamide, the activator is 1-hydroxy-7-azabenzotriazole (HOAt), the acid binder is N-methylmorpholine (NMM), and the amide coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0033] The preparation method specifically comprises: dissolving 1 eq of substituted benzoic acid and 1.2 eq of HOAt in DMF, stirring for 10 min, and then adding benzimidazole amine; then, adding 0.7 eq of NMM at 0°C, stirring for 10 min, adding 1.2 eq of EDCI, maintaining stirring at 0°C for 1.5 h, and reacting at room temperature for 12 h; spin-drying the DMF, extracting, and column-passing to obtain the benzimidazole amine derivative.

[0034] Compound A5: dissolve benzimidazole amine in a solvent to obtain a reaction system; add benzaldehyde to the reaction system, and drop a small amount of glacial acetic acid, react for 6 hours under a nitrogen environment to obtain a reaction solution; then place the reaction at 0°C, slowly add a reducing agent to the reaction solution, return to room temperature and react for 12 hours to obtain the benzimidazole amine derivative; wherein the solvent is methanol and the reducing agent is sodium cyanoborohydride.

[0035] The preparation method specifically comprises: dissolving 1eq of benzimidazole amine in methanol to obtain a reaction system; adding 1eq of substituted benzaldehyde to the reaction system, and dripping 0.1eq of glacial acetic acid, reacting for 6h under a nitrogen environment to obtain a reaction solution; then placing the reaction at 0°C, slowly adding 2.5eq of sodium cyanoborohydride to the reaction solution, and returning to room temperature to react for 12h. After the reaction is completed, water is added to quench, methanol is spin-dried, extracted, and the obtained product is subjected to column chromatography to obtain the benzimidazole amine derivative. The five benzimidazole amine derivatives specifically involved in the present invention have a specific preparation method as follows:

[0036] General method for preparing 1H-benzimidazole-2-amine amide derivatives A1-A4:

[0037] Dissolve substituted benzoic acid or phenylacetic acid (1.50mmol) in DMF (4mL), add HOAt (1.95mmol) to the system and stir for 10min, then add 2-aminobenzimidazole (1.50mmol) to the above system, add N-methylmorpholine (1.05mmol) to it under ice bath and stir for 10min, finally add EDCI (1.95mmol) to the system, keep 0℃ for 1h, return to room temperature for 12h. TLC monitors the completion of the reaction, concentrate the reaction solution under vacuum, extract the obtained oily liquid with ethyl acetate (15mL*3), wash the organic phase with saturated brine, collect the organic phase, add anhydrous sodium sulfate to dry, and concentrate in vacuo. The obtained product is subjected to column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain the target compound.

[0038] N-(6-Bromo-1H-benzimidazol-2-yl)benzamide (A1) was obtained as a white solid in a yield of 63%. 1 HNMR (400MHz, DMSO-d6): δ12.39 (s, 2H), 8.12 (d, J = 7.9Hz, 2H), 7.66-7.61 (m, 2H), 7.55 (t, J=7.6Hz, 2H), 7.43 (d, J=8.4Hz, 1H), 7.27 (dd, J=8.5, 2.1Hz, 1H).

[0039] N-(6-Bromo-1H-benzoimidazol-2-yl)-o-methylbenzeneacetamide (A2) was obtained as a white solid with a yield of 66%.1 HNMR (400MHz, DMSO-d6): δ12.19(s,1H),11.88(s,1H),7.59(s,1H),7.38(d,J =8.4Hz,1H),7.30-7.27(m,1H),7.23-7.15(m,4H),3.82(s,2H),2.32(s,3H).

[0040] N-(6-Bromo-1H-benzoimidazol-2-yl)-3-bromobenzamide (A3) was obtained as a white solid in a yield of 66%. 1 HNMR (400MHz, DMSO-d6): δ12.44(s,2H),8.30(d,J=2.1Hz,1H),8.10(d,J=7.8Hz,1H),7.81(d,J=7.9Hz, 1H), 7.63 (s, 1H), 7.50 (td, J=7.9, 1.6Hz, 1H), 7.42 (dd, J=8.4, 1.6Hz, 1H), 7.29 (dt, J=8.6, 1.8Hz, 1H).

[0041] Benzamide-1H-benzimidazol-2-amine (A4) was obtained as a white solid in a yield of 64%. 1 HNMR (400MHz, DMSO-d6): δ12.28(s,2H),8.16-8.12(m,2H),7.63-7.58(m,1H), 7.53(t,J=7.4Hz,2H), 7.46(dd,J=5.9,3.2Hz,2H), 7.13(dq,J=6.7,3.5Hz,2H). 13 C NMR(101MHz, DMSO-d6): δ168.3,148.9,134.3,132.0,128.4,128.3,121.5,113.4.HRMS(ESI,m / z):Calcd.for C 14 H 11 N3O[M+H] + 238.09748,found:238.0982.

[0042] Preparation method of N-benzyl-1H-benzimidazol-2-amine (A5):

[0043] 2-aminobenzimidazole (1.88mmol) was dissolved in methanol, and then benzaldehyde (1.88mmol) was added to the system and a small amount of glacial acetic acid was added dropwise thereto. The reaction was allowed to react for 6h under a nitrogen environment, and then the reaction was placed at 0°C, sodium cyanoborohydride (4.69mmol) was slowly added to the reaction solution, and the reaction was restored to room temperature for 12h. The reaction was monitored by TLC. After the reaction was completed, water was added to quench the reaction solution, and the reaction solution was concentrated under vacuum, extracted with ethyl acetate (15mL*3), and the organic phase was washed with saturated brine, the organic phase was collected, dried with anhydrous sodium sulfate, and concentrated under vacuum. The obtained product was subjected to column chromatography (petroleum ether: ethyl acetate = 1:4) to obtain a white solid with a yield of 44%. 1 HNMR (400MHz, DMSO-d6): δ7.31(t,J=7.3Hz,2H),7.24(d,J=7.0Hz,1H),7.19(d,J=7.1Hz,2H),7.14(d,J= 7.8Hz,1H),7.04(d,J=7.7Hz,1H),6.92(t,J=7.5Hz,1H),6.81(t,J=7.5Hz,1H),6.59(s,2H),5.26(s,2H). 13 C NMR(101MHz, DMSO-d6): δ155.1,143.0,137.3,134.2,128.5,127.3,127.0,120.5,118.1,114.8,107.9,44.7.HRMS(ESI,m / z):Calcd.for C 14 H 13 N3[M+H] + 224.1181, found: 224.1187. Next, the biological activities of the five synthesized compounds were tested.

[0044] Reagents: DMEM culture medium (Gibco); fetal bovine serum FBS (Gibco); DMSO (Shanghai Lingfeng); Oil Red O staining kit (Jiancheng Biotechnology); 4% paraformaldehyde (Biyuntian); oleic acid and palmitic acid (Aladdin); Bodipy 581 / 591C11 probe (MCE); DCFH-DA (Aladdin); bovine serum albumin (BSA) (Biyuntian); MTT (Biyuntian); Nrf2 antibody (Nature Biosciences); β-Actin antibody, Lamin B antibody, NQO1 antibody, GCLC antibody, goat anti-rabbit secondary antibody (Biyuntian); RIPA cell lysis buffer (Biyuntian); cytoplasmic-nuclear protein extraction kit (Biyuntian); ECL chemiluminescence detection kit (Novozyme); 0.45 mm PVDF membrane (Millipore).

[0045] Instruments: constant temperature CO2 cell culture incubator (Thermo); microplate reader (Thermo); high pressure sterilizer (Shanghai Shen'an); electronic balance (Mettler Toledo); inverted fluorescence microscope (Olympus); constant temperature metal bath (Dalong); refrigerated centrifuge (Xiangyi); constant temperature water bath (Shanghai Jinghong); gel imaging system (Bio-Rad); electrophoresis instrument and electrophoresis tank (Junyi).

[0046] Example 1: MTT assay to detect the toxicity of benzimidazole amine derivatives to HepG2 cells

[0047] All compounds were diluted with DMSO solution to prepare 10 mM stock solution and stored at -20°C for use. HepG2 cells were cultured at 5×10 3 / well were inoculated in a 96-well plate and incubated in an incubator. After 1 day, all compound stock solutions were diluted with fresh culture medium and treated with HepG2 cells at a concentration of 10 μM for 24 hours. Among them, the drug-treated group was the sample group, the group treated with an equal volume of DMSO alone was the control group, and the group without cells was the blank group. Next, 50 μL of MTT solution containing a concentration of 0.5 mg / mL was added to each culture well, and the OD value at a wavelength of 570 nm was detected by the MTT method. The cell survival rate was calculated according to the following formula: Cell survival rate % = (OD 样品组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100.

[0048] Figure 1 A is the effect of five benzimidazole amine derivatives on the viability of HepG2 cells. Compared with the control group, at a concentration of 10 μM, A1-A4 were all significantly toxic to HepG2 cells, while A5 showed no significant toxicity. In subsequent experiments, A5 with less toxicity was selected to exclude the influence of cytotoxicity.

[0049] Example 2: MTT assay to detect the protective effect of benzimidazole amine derivatives on FFA-induced HepG2 cell damage

[0050] In order to detect the protective effect of A5 on FFA-induced oxidative damage within the non-toxic concentration range, HepG2 cells were first treated with 10 μM A5 for 24 h, and then damaged with free mixed acid (FFA) for 24 h. FFA used the following formula: 20 mM oleic acid and 10 mM palmitic acid were mixed in equal volumes, and the FFA concentration was based on the oleic acid concentration. The MTT method was then used to detect cell viability.

[0051] like Figure 1As shown in B, after FFA treatment alone, the survival rate of HepG2 cells was significantly reduced to about 50% of the control group. However, in the group treated with 10μM A5 for 24h before FFA injury, the survival rate of cells increased significantly. The results show that A5 can effectively inhibit the decrease in the survival rate of HepG2 cells caused by FFA and protect HepG2 cells from injury.

[0052] Example 3: Identification of lipid deposition in HepG2 cells by Oil Red O staining

[0053] The Oil Red O staining method was used to detect whether the active molecules could effectively inhibit the increase of cellular lipid droplets induced by FFA, which is the key to the in vitro study of the compound's anti-NAFLD effect. Oil Red O is a fat-soluble azo dye that can specifically dye neutral lipids such as triglycerides in cells or tissues red, which is simple and intuitive.

[0054] HepG2 cells were cultured at 5×10 4 / well were inoculated in a 12-well plate. After 1 day of culture, the A5 stock solution was diluted with fresh culture medium and HepG2 cells were treated with a concentration of 10 μM for 24 hours. Next, the old culture medium was discarded and 0.5 mL of new culture medium containing 0.5 mM FFA was added to each well. Among them, the control group was only added with an equal volume of DMSO, the model group was only added with FFA, and the sample group was only added with A5 and A5+FFA. Next, the experiment was carried out according to the operating instructions in the Oil Red O staining kit, including paraformaldehyde fixation, staining solution preparation, staining, washing, taking pictures, etc.

[0055] Figure 2 The effect of A5 on lipid accumulation in HepG2 cells induced by FFA. The results showed that there was no lipid droplet accumulation in the control group and the group treated with A5 alone, a large number of red lipid droplets appeared in the cells treated with FFA alone, and in the group treated with FFA after A5 pretreatment, lipid droplet accumulation decreased significantly. This shows that A5 can effectively reduce intracellular lipid accumulation caused by FFA, providing an important basis for subsequent research.

[0056] Example 4: Detection of lipid peroxidation by Bodipy 581 / 591C11 staining

[0057] Bodipy 581 / 591C11 can be used to detect intracellular lipid peroxidation. HepG2 cells were cultured at 5×10 4 / well were inoculated in a 12-well plate, and 1 day later, HepG2 cells were treated with 5μM and 10μM A5 for 24h. The old culture medium was discarded, and 0.5mL of new culture medium containing 0.5mM FFA was added to each well for continued culture. Among them, the control group was only added with an equal volume of DMSO, the model group was only treated with FFA, and the A5+FFA group was the sample group. Bodipy 581 / 591C11 dye mother solution was diluted with serum-free culture medium to a final concentration of 10μM, and the old culture medium was replaced and cultured for 30min. The culture medium was discarded, washed 3 times with PBS, and the well plate was placed under an inverted fluorescence microscope to observe the staining and take pictures.

[0058] Oxidized lipids fluoresce in green, and reduced lipids fluoresce in red. Figure 3 The results showed that compared with the control group, after FFA induction, the red fluorescence decreased and the green fluorescence increased, indicating that the lipid peroxidation level of HepG2 cells was significantly improved. Compared with the model group, the red fluorescence increased and the green fluorescence decreased in the FFA induction group after A5 pretreatment, indicating that A5 can effectively alleviate the lipid peroxidation caused by FFA.

[0059] Example 5: DCFH-DA probe detection of ROS generation

[0060] DCFH-DA is a classic probe for detecting intracellular ROS levels. It can freely pass through the cell membrane and be hydrolyzed by intracellular esterase to generate DCFH. DCFH itself has no fluorescence, but intracellular ROS can oxidize it into DCF with green fluorescence. Therefore, the fluorescence level of DCF in cells can reflect the level of ROS.

[0061] HepG2 cells were cultured at 5×10 4 / well were inoculated in a 12-well plate for culture. One day later, HepG2 cells were treated with A5 at concentrations of 5μM and 10μM. After 24h, 0.5mL of new culture medium containing 0.5mM FFA was added to each well to replace the old culture medium. The control group was treated with an equal volume of DMSO, the model group was treated with FFA only, and the sample group was A5+FFA. After 12h, the DCFH-DA probe was diluted with serum-free culture medium to a final concentration of 10μM. Next, incubate at 37℃ in the dark for 30min, rinse the cells 2-3 times with PBS, and observe and photograph under an inverted fluorescence microscope.

[0062] Figure 4 The effect of A5 on the ROS level in FFA-induced HepG2 cells in the experimental example. Compared with the control group, the ROS level in the model group cells was significantly increased. Compared with the model group, the ROS level in the A5 pretreatment group cells was significantly reduced. The results show that A5 can effectively inhibit the accumulation of ROS induced by FFA in cells and relieve oxidative stress.

[0063] Example 6: Western Blot method to detect the expression levels of antioxidant molecules such as GCLC and NQO1

[0064] The antioxidant response element ARE exists in the cell nucleus. After binding to the transcription factor Nrf2, it can initiate the transcription and translation of a series of downstream antioxidant genes and upregulate the expression of antioxidant molecules in cells, such as NQO1 and GCLC. This can enhance the antioxidant capacity of cells, fight oxidative stress, and exert a protective effect. Based on this, the Western Blot method was used to detect the protein expression of NQO1 and GCLC in cells.

[0065] HepG2 cells were cultured at 3×10 5 / dish was inoculated in a 60mm culture dish, and HepG2 cells were treated with 5μM and 10μM A5 one day later. After 24h, the culture medium was discarded, washed 2-3 times with PBS, and the cells were collected and lysed with RIPA cell lysis buffer. The supernatant obtained after low-temperature centrifugation contained total cell protein. The protein concentration was adjusted to uniformity using the Bradford method, 5× loading buffer was added to each sample, mixed evenly, heated at 100℃ for 5min using a metal bath, and stored at -80℃. Next, an equal amount of sample (20μg) was taken for electrophoresis using 12% SDS-PAGE. After protein separation, the target protein was transferred to a PVDF membrane using an electrotransfer instrument. Blocked with 5% skimmed milk powder for 1h, then incubated with the corresponding primary and secondary antibodies in turn, and developed on a gel imager according to the operating instructions of the ECL chemiluminescence detection kit. Grayscale values ​​were counted using Image J software, and β-actin was used as a protein internal reference.

[0066] Figure 5 The results showed that after 5μM and 10μM A5 were used to treat HepG2 cells for 24h, the expression levels of GCLC and NQO1 proteins increased significantly with the increase of concentration, and showed a dose-dependent effect ( Figure 5 B and Figure 5 C is correct Figure 5 This indicates that A5 can effectively induce the expression of antioxidant molecules in HepG2 cells and enhance the endogenous antioxidant defense system in cells.

[0067] Example 7: Western Blot Detection of Nrf2 Expression and Distribution in HepG2 Cells

[0068] The Nrf2-ARE pathway is one of the important participants in the intracellular antioxidant defense system. The expression level of Nrf2 and whether it enters the cell nucleus are the key to the activation of this pathway. We speculate that A5 can promote the expression and nuclear entry of Nrf2, thereby inducing the transcription of downstream antioxidant molecules. In order to verify this speculation, the Western Blot method was used for detailed research.

[0069] HepG2 cells were cultured at 3×10 5 / dish was inoculated in a 60 mm dish, and 1 day later, HepG2 cells were treated with 10 μM A5 for 0 h, 1 h, 3 h, and 6 h. Next, the cells were collected, total protein was extracted using RIPA lysis buffer, and proteins in the cytoplasm and nucleus were extracted using a cytoplasm-nucleoprotein extraction kit, and sample preparation, electrophoresis, electrotransfer, blocking, antibody incubation, development, quantification and other operations were performed (see Example 6 for details). When detecting the total Nrf2 level and the Nrf2 level in the cytoplasm, β-actin was used as a protein internal reference. When detecting the Nrf2 level in the nucleus, Lamin B was used as an internal reference.

[0070] Figure 6 The results showed that after 10 μM A5 was used to treat HepG2 cells, the total Nrf2 expression in the cells increased significantly and reached a peak at 1 h ( Figure 6 A, Figure 6 D is the grayscale quantitative result). Nrf2 entering the cell nucleus is a necessary prerequisite for activating the Nrf2-ARE signaling pathway. Figure 6 The results in B show that as the A5 action time increases, the content of Nrf2 in the cell nucleus increases significantly and reaches a peak at 6 hours ( Figure 6 E is right Figure 6 Grayscale quantitative results of B). At the same time, the level of Nrf2 in the cytoplasm also increased ( Figure 6 C, Figure 6 F is the grayscale quantitative result). In conclusion, the above results indicate that A5 promotes the increase of Nrf2 content in HepG2 cells and induces Nrf2 to enter the cell nucleus, laying the foundation for its activation of downstream antioxidant pathways.

[0071] In summary, the above studies show that the benzimidazole amine derivative A5 has a good preventive / therapeutic effect on FFA-induced HepG2 cell liver injury. A5 can significantly improve HepG2 cell damage caused by FFA, inhibit lipid accumulation and lipid peroxidation, and relieve oxidative stress. Mechanism studies have shown that A5 is an effective Nrf2 activator. In HepG2 cells, A5 promotes the increase in Nrf2 expression levels, induces Nrf2 to enter the cell nucleus, thereby activating the Nrf2-ARE pathway and increasing the expression level of downstream antioxidant molecules, which is suitable for use in drugs for the prevention / treatment of NAFLD.

[0072] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. Application of benzimidazole amine derivatives in the prevention / treatment of non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The benzimidazole amine derivative is a structural formula (1) or a salt thereof: R1=H,Br; R2 is one of the following structural formulas:

3. The use according to claim 2, characterized in that: The benzimidazole amine derivative is specifically one of the following structural formulas:

4. The use according to claim 3, characterized in that: The benzimidazole amine derivatives are used to protect HepG2 cell damage induced by free mixed acid.

5. The use according to claim 3, characterized in that: The benzimidazole amine derivatives are used to activate the Nrf2-ARE pathway to promote the increase of Nrf2 protein levels, induce Nrf2 to enter the cell nucleus, and increase the protein expression levels of downstream antioxidant and detoxification molecules, including quinone oxidoreductase 1 and glutamate-cysteine ​​ligase catalytic subunit; Wherein, the Nrf2 refers to nuclear factor NF-E2 related factor 2, and the ARE refers to antioxidant response element.

6. The use according to claim 3, characterized in that: The benzimidazole amine derivatives are used to reduce intracellular lipid accumulation and lipid peroxidation, inhibit the accumulation of reactive oxygen species, and enhance the anti-damage ability of cells in a free mixed acid-induced non-alcoholic fatty liver disease cell model.

7. The use according to claim 3, characterized in that: The benzimidazole derivatives are used as active ingredients and a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation.

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