Application of glaucocalyxin A in preparation of medicine for preventing / treating non-alcoholic fatty liver disease

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of glaucocalyxin A in preparation of medicine for preventing / treating non-alcoholic fatty liver disease. It is proved for the first time that GLA reduces lipid accumulation and resists oxidative stress through an Nrf2-ARE endogenous antioxidant pathway, has an obvious improvement effect on NAFLD, is expected to serve as an alternative medicine for treating NAFLD subsequently, and has potential application value in clinical treatment of the NAFLD.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the use of Glaucocalyxin A (GLA) in the preparation of a medicine for preventing / treating nonalcoholic fatty liver disease (NAFLD). Background Art

[0002] As one of the important organs of the human body, the liver has multiple functions such as detoxification, metabolism, secretion, and biotransformation. It participates in nutrient metabolism and energy balance, and regulates multiple physiological processes such as iron metabolism, bilirubin metabolism, intestinal pathogen clearance, and immune regulation. However, the liver is also stimulated by various pathological factors, especially oxidative stress, electrophilic substances, and toxic substances, which cause structural and functional disorders and produce corresponding diseases. NAFLD is a chronic progressive liver disease related to metabolism, caused by multiple factors, and characterized by excessive fat deposition in hepatocytes, including simple fatty liver (SFL) and its evolution of non-alcoholic steatohepatitis (NASH), fatty liver fibrosis, and irreversible cirrhosis and even hepatocellular carcinoma (HCC). In recent years, with the increase in the prevalence of type 2 diabetes and obesity, the prevalence of NAFLD has also increased rapidly.

[0003] Therefore, it is very necessary to provide a solution that helps prevent or treat NAFLD. Summary of the invention

[0004] The object of the present invention is to provide an application of GLA in preparing a medicine for preventing / treating NAFLD.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides application of cyanocalyxin A in preparing medicine for preventing / treating non-alcoholic fatty liver disease.

[0007] Further, the structural formula of the bluecalyx acetone is as follows:

[0008]

[0009] Furthermore, the blue calyx factor A is used to activate the Nrf2-ARE pathway; wherein the Nrf2 refers to nuclear factor NF-E2-related factor 2, and the ARE refers to the antioxidant response element.

[0010] Furthermore, the bluecalyx factor A has a protective effect on HepG2 cell damage induced by free fatty acids.

[0011] Furthermore, the bluecalyx acetone is used to upregulate the expression of heme oxygenase, quinone oxidoreductase 1 and glutamate-cysteine ​​ligase catalytic subunit in HepG2 cells at the protein level to enhance the capacity of the endogenous antioxidant defense system in HepG2 cells.

[0012] Furthermore, the blue calyx acetone is used to reduce lipid accumulation in hepatocytes caused by free fatty acids and enhance the anti-damage ability of hepatocytes in non-alcoholic fatty liver disease induced by free fatty acids.

[0013] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0014] (1) It was confirmed for the first time that GLA has a significant improvement effect on NAFLD with minimal toxic side effects.

[0015] (2) The present invention can subsequently be used as a reserve drug for the treatment of NAFLD and has potential application value in the clinical treatment and prevention of NAFLD.

[0016] In the present invention, GLA has a significant improvement effect on NAFLD by activating the Nrf2-ARE pathway. Among them, Nrf2 refers to nuclear factor NF-E2 related factor 2, and ARE refers to antioxidant response element. First, GLA has a protective effect on HepG2 cell damage induced by free fatty acid (FFA). Secondly, GLA can regulate the expression of heme oxygenase (HEME oxygenase-1, HO-1), quinone oxidoreductase 1 (NAD(P)H:quinoneoxidoreductase 1, NQO1) and glutamate-cysteine ​​ligase catalytic subunit (glutamate-cysteineligase catalytic, GCLC) in HepG2 cells at the protein level, and enhance the ability of the endogenous antioxidant defense system in HepG2 cells. Finally, GLA has a preventive and therapeutic effect on FFA-induced NAFLD, can reduce the lipid accumulation of hepatocytes caused by free fatty acids, and enhance the anti-damage ability of hepatocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of GLA on the activity of HepG2 cells;

[0018] Figure 2 The effect of different concentrations of FFA on the activity of HepG2 cells;

[0019] Figure 3 GLA protects HepG2 cells from FFA-induced damage;

[0020] Figure 4The effect of GLA on lipid accumulation in HepG2 cells induced by FFA;

[0021] Figure 5 The effect of GLA on ROS levels in HepG2 cells induced by FFA;

[0022] Figure 6 The effect of GLA on lipid peroxidation in HepG2 cells induced by FFA;

[0023] Figure 7 The effect of GLA on the expression of HO-1, NQO1 and GCLC proteins;

[0024] Figure 8 This is the effect of GLA on the expression and distribution of Nrf2 in HepG2 cells.

[0025] 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. **** Compared with the control group, P < 0.0001. & 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±SEM). DETAILED DESCRIPTION

[0026] The “multiple hits” theory involves multiple factors that may act simultaneously, providing a more appropriate description of the pathogenesis of NAFLD. Oxidative stress is one of the many factors that lead to “multiple hits” and is considered to be the main factor in liver damage and disease progression in NAFLD. Oxidative stress refers to a harmful process caused by the imbalance between the excessive formation of reactive oxygen species (ROS) and limited antioxidant defense. If the ability of mitochondria to control this balance is destroyed under continuous oxidative stress, the system will collapse. ROS not only consumes a large amount of antioxidant substances in cells, leading to an imbalance between oxidation and antioxidant activity, causing an increase in NO and lipid peroxidation products MDA, but also greatly reduces GSH-Px and SOD that scavenge oxygen free radicals, ultimately causing nuclear damage to reducing cofactors, oxidation, protein denaturation, lipid peroxidation, inflammation, and fibrosis. In short, oxidative stress is a key factor in inducing non-alcoholic liver damage. Failure to remove excessive ROS in a timely manner will cause serious consequences of liver damage.

[0027] The inventors have found that the Keap1-Nrf2-ARE pathway plays a leading role in the main defense mechanism of cells against oxidative stress and exogenous toxic substances. Its role is to prevent internal and external oxidation and chemical stimulation from damaging cells. This pathway has a positive significance for promoting the stability of the intracellular environment. Under normal and healthy physiological conditions, nuclear factor NF-E2-related factor 2 (Nrf2) can react with Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and localize in the cytoplasm. Keap1 protein binds to an Nrf2 protein in the form of a dimer and further mediates its ubiquitination degradation. When cells are damaged from the outside, oxidative stress is induced due to the generation of ROS on the mitochondrial membrane. In order to cope with internal and external stress conditions such as free radicals and chemical stimulation, the conformation of Keap1 is changed, causing the dissociation and activation of Nrf2 and Keap1. Once activated, Nrf2 enters the cell nucleus and binds to the antioxidant response element (ARE). In response to internal and external harmful stimuli, it triggers the transcription and expression of ARE downstream genes, including proteasomes / molecular chaperones, phase II detoxification enzymes and antioxidant proteins, such as catalase (CAT), quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1), glutamate-cysteine ​​ligase catalytic subunit (GCLC), glutamate-cysteine ​​ligase (GCLM), etc. Free radical-mediated oxidative stress is widely involved in the occurrence and development of NAFLD. The Keap1-Nrf2-ARE pathway is the most important endogenous antioxidant stress pathway that regulates the transcriptional expression of multiple antioxidant enzymes. Compared with previous exogenous antioxidant strategies, its moderate and controllable regulation will open up new ways to prevent and treat NAFLD from in vivo antioxidants.

[0028] Lamiaceae is widely distributed in my country and is widely used by people because of its various biological activities. Studies have found that a variety of active ingredients can be isolated from Lamiaceae, among which GLA is the main one. Past studies have found that GLA has a variety of biological activities, including anti-tumor, anti-coagulant, anti-thrombotic, immunosuppressive, anti-neuroinflammatory, antibacterial and antioxidant activities.

[0029] At present, there are no relevant reports on the role of GLA in NAFLD, so it is of great significance to further study GLA and its possible mechanism for improving 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] 1.1 Reagents: DMEM medium (Gibco); fetal bovine serum FBS (Gibco); penicillin-streptomycin solution, 0.25% trypsin-EDTA, 4% paraformaldehyde (Biyuntian); Oil Red O stain (Nanjing Jiancheng); oleic acid, palmitic acid (Aladdin); DMSO, anhydrous methanol, anhydrous ethanol (Shanghai Lingfeng); GLA (Pus Biotechnology); Bodipy dye (MCE); DCFH-DA probe, bovine serum albumin (BSA) (Biyuntian); MTT (Biyuntian); Nrf2 antibody, Lamin B antibody (Nature Biosciences); β-Actin antibody, Keap1 antibody, HO-1 antibody, NQO1 antibody, GCLC antibody, goat anti-rabbit secondary antibody (Biyuntian); RIPA cell lysate, cytoplasmic-nuclear protein extraction kit, Bradford protein concentration determination kit (Biyuntian); EC chemiluminescence detection kit (Affinity Biotechnology); 0.45μm PVDF membrane (Millipore).

[0032] 1.2 Instruments: microplate reader (Thermo); inverted fluorescence microscope (Olympus); constant temperature CO2 cell culture incubator (Thermo); oven (Jinghong); electronic balance (Mettler Toledo); refrigerated centrifuge (Xiangyi); high pressure sterilizer (Shanghai Shen'an); constant temperature water bath (Shanghai Jinghong); constant temperature metal bath (Dalong); gel imaging system (Bio-Rad); electrophoresis apparatus, vertical electrophoresis tank (Junyi).

[0033] Example 1: HepG2 cell culture

[0034] The oxidative damage model established by human liver cancer cell HepG2 cells is an ideal cell model widely used in the study of the molecular mechanism of natural antioxidants. HepG2 cells are a highly differentiated transformed cell line that is easy to culture, has good characteristics, and relatively intactly retains the main functions of liver parenchymal cells such as lipoprotein secretion; in addition, the steady-state function of its antioxidant defense system is relatively higher than that of hepatocytes and other non-transformed cells, and the changes in response to different conditions are easier to detect, making it a reliable model. HepG2 cells were cultured in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2 incubator, and cells in the logarithmic growth phase were used for experiments.

[0035] Example 2: MTT assay to detect cytotoxic activity

[0036] HepG2 cells in the logarithmic growth phase were taken and 5×10 3The density of cells was inoculated in a 96-well plate and incubated in an incubator for 24 hours. After the cells adhered to the wall, a certain concentration gradient of GLA (0.5μM, 1μM, 2μM, 5μM, 10μM, 20μM) was set to treat HepG2 cells for 24 hours, and the OD value at a wavelength of 570nm was detected by MTT method, and the cell survival rate was calculated according to the formula. Cell survival rate = (As / Ac) × 100%, As: experimental well (with cells, MTT and drugs); Ac: control well (with cells and MTT).

[0037] Figure 1 To investigate the effect of GLA on the activity of HepG2 cells, compared with the control group, 0.5 μM and 1 μM GLA had almost no toxicity to HepG2 cells. Therefore, subsequent experiments were performed using 0.5 and 1 μM GLA to exclude the effects of cytotoxicity.

[0038] Example 3: Determining the FFA modeling concentration and establishing a NAFLD cell injury model

[0039] The cytotoxicity of FFA to HepG2 cells was detected. The FFA ratio was 2:1 (the final concentration of 20mM oleic acid and the final concentration of 10mM palmitic acid were mixed at a molar ratio of 1:1, and the FFA concentration was based on the oleic acid concentration). HepG2 was treated with 0.25mM, 0.5mM, 1.5mM, and 2mM FFA for 24h, and the cell survival rate was detected by MTT method.

[0040] Figure 2 The effect of different concentrations of FFA on the activity of HepG2 cells. Compared with the control group, 0.5 mM FFA significantly reduced cell viability, but the cells still survived, enough to continue subsequent experiments, and this concentration was used as the experimental concentration.

[0041] Example 4: MTT assay for the protective effect of GLA on FFA-induced HepG2 cell damage

[0042] In order to detect the protective effect of GLA against FFA-induced oxidative damage within a non-toxic concentration range, HepG2 cells were first treated with different concentrations of GLA (0.5 μM, 1 μM) for 24 h, and then treated with 0.5 mM FFA for 24 h.

[0043] Figure 3 Compared with the control group, the cell survival rate of the model group treated with FFA alone was significantly reduced, while pretreatment with non-toxic concentrations of GLA could significantly reduce the cytotoxicity caused by FFA, and with the increase of GLA concentration, the survival rate of HepG2 cells also increased accordingly. Therefore, it is shown that GLA can effectively inhibit the decrease in HepG2 cell viability caused by FFA and has a cell protective effect.

[0044] Example 5: Identification of lipid deposition by Oil Red O staining

[0045] How to effectively inhibit the increase of cellular lipid droplets induced by free fatty acids has become the key to in vitro research on anti-NAFLD. Oil Red O is a fat-soluble azo dye with strong fat solubility and lipid staining properties, and can specifically stain neutral lipids such as triglycerides in cells or tissues. Therefore, it is particularly important to use Oil Red O staining to identify the degree of lipid deposition and find the concentration of potential anti-NAFLD drugs.

[0046] Take HepG2 cells in the logarithmic growth phase and adjust the concentration to 5×10 4 / well, 1 mL per well was inoculated in a 12-well plate and cultured for 16h-18h. Different concentrations of GLA solution (0.5μM, 1μM) were prepared to replace the original culture medium, and a control group (normal cells without any treatment), a model group (only FFA treatment) and a sample group (GLA and FFA treatment, only GLA treatment) were set up, and placed in an incubator for continued pre-protection for 24h. The culture medium was replaced, and 0.5 mL of FFA with a final concentration of 0.5mM was added, and cultured at 37℃ for 24h.

[0047] Staining related experimental steps: (1) Aspirate the cell culture medium, add PBS to wash 1-2 times, add 500μL 4% paraformaldehyde to each well, and place in the incubator to fix the cells for 30 minutes. (2) Prepare the staining solution at a fixed time. Note that it must be prepared under light-proof conditions. Preparation of Oil Red O staining solution: Dilute the Oil Red O stock solution: diluent in a ratio of 5:2 and filter. The staining solution should not be stored for more than 2 hours and should be prepared before use. (3) After the cells are fixed, aspirate the paraformaldehyde, add 500μL staining solution to each well, and stain in a 37℃ incubator for 15 minutes. (4) After staining, discard the staining solution, add 60% isopropanol to each well, remove excess dye, and keep the background clean. (5) Add PBS to wash 2-3 times, and place under an inverted fluorescence microscope for observation and photography.

[0048] Figure 4 In the experimental example, the effect of GLA on lipid accumulation of HepG2 cells induced by FFA was shown. It can be found that the cells in the control group had clear outlines and no lipid droplet accumulation; the cells in the group treated with GLA alone also had no lipid droplet accumulation; a large number of red lipid droplets appeared in the cytoplasm of HepG2 cells incubated with FFA alone; compared with the model group, GLA pre-protected lipid droplet accumulation in a dose-dependent manner. Therefore, GLA can resist the fatty degeneration of HepG2 cells and reduce the lipid accumulation of cells caused by FFA, which provides an important basis and reference for subsequent studies on the inhibition of fat accumulation.

[0049] Example 6: DCFH-DA probe detection of ROS generation

[0050] The DCFH-DA probe method was used to detect the level of ROS in HepG2 cells. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to produce DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. ROS in the cell can oxidize non-fluorescent DCFH to produce fluorescent DCF. Detecting the fluorescence of DCF can indicate the level of reactive oxygen free radicals ROS in the cell.

[0051] HepG2 cells were inoculated in 12-well cell culture plates with an inoculation volume of 1 mL per well and an inoculation cell density of 5 × 10 4 / well. The inoculated cell culture plate was returned to the cell culture incubator for 24 hours. After attachment, different concentrations of GLA were added, and a control group (cells without any treatment), a model group (only FFA damage) and a sample group (GLA and FFA treatment) were set up. Different concentrations of GLA were first added to the sample group and incubated for 24 hours. DMEM culture medium was added to the control group, and 500 mL of cell culture medium containing 0.5 mM FFA was added to each well of the model group and the sample group, and the culture was continued for 12 hours to stimulate the production of ROS.

[0052] Dilute the DCFH-DA probe stock solution with serum-free DMEM medium at a ratio of 1:1000 to a final concentration of 10 μM. Wash the cells 1-2 times with PBS, add the probe and incubate for 30 minutes in the dark. After the incubation, rinse with PBS 2-3 times to remove the unloaded probe. Observe and take pictures under an inverted fluorescence microscope.

[0053] Figure 5 The effect of GLA on the level of ROS release in HepG2 cells induced by FFA in the experimental example. Compared with the control group, the level of ROS in the model group cells was significantly increased; compared with the model group, the level of ROS in the GLA-administered group cells was significantly reduced. This result shows that GLA can effectively inhibit the accumulation of ROS induced by mixed acids in cells, and the results are concentration-dependent.

[0054] Example 7: Detection of lipid peroxidation by Bodipy 581 / 591C11 staining

[0055] HepG2 cells in the logarithmic growth phase were taken and 5×10 4 / well density was inoculated in 12-well plates, and different concentrations of GLA were added after the cells adhered to the wall. After 24 hours, FFA was added with a final concentration of 0.5mM and cultured for another 12 hours. Bodipy 581 / 591C11 dye stock solution was diluted with serum-free DMEM medium at a ratio of 1:1000 to a final concentration of 10μM. The cells were washed 1-2 times with PBS, and the dye was added and incubated in the dark for 30 minutes. After the incubation, the cells were rinsed with PBS 2-3 times, and a small amount of PBS was added to observe the staining under an inverted fluorescence microscope and take pictures.

[0056] Figure 6 The effect of GLA on lipid peroxidation in FFA-induced HepG2 cells. The fluorescence of oxidized plasma membrane lipids is green, and the fluorescence of reduced lipids is red. Compared with the control group, the FFA-induced HepG2 cells significantly increased the expression of lipid peroxidation. Compared with the model group, the red fluorescence of the GLA-administered group increased and the green fluorescence decreased, indicating that GLA can alleviate lipid peroxidation.

[0057] Example 8: Western Blot Detection of HO-1, NQO1, and GCLC Protein Expression

[0058] As a regulatory sequence in cellular antioxidant genes, the antioxidant element ARE in the cell nucleus can initiate the transcription of a series of downstream antioxidant genes after binding to the transcription factor Nrf2, and upregulate the expression of cellular antioxidant enzymes, such as HO-1, NQO1 and GCLC, thereby enhancing the cellular antioxidant capacity and exerting a protective effect. Therefore, the Western Blot method was first used to investigate the protein expression of HO-1, NQO1 and GCLC in cells to detect whether the Nrf2-ARE endogenous antioxidant signaling pathway is the main target for GLA to exert indirect antioxidant activity.

[0059] HepG2 was cultured in a 60mm dish, and different concentrations of GLA were added when the cell confluence reached 60%-70%. After 24 hours, the DMEM medium containing the sample was removed, and pre-cooled PBS was added to each dish to wash the cells twice. The cells in the dish were scraped off with a cell scraper and transferred to a 1.5mL centrifuge tube. Pre-cooled RIPA cell lysis buffer was added to each tube, and protease inhibitors were added to the cell lysis buffer in advance, and then placed on ice for lysis for 30 minutes. After the lysis was completed, centrifuged at 4°C and 10000rpm for 10 minutes, and the supernatant after centrifugation was taken for use to obtain the total cell protein. The protein concentration was adjusted to a uniform level using the Bradford method. An appropriate amount of lysis buffer and 5× loading buffer were added to the sample, and mixed evenly to make the final protein concentration of the sample consistent. The above samples were placed in a metal bath and heated for 5 minutes for protein denaturation, and stored at -80°C for use.

[0060] The steps of SDS-PAGE electrophoresis are as follows:

[0061] (1) Preparation of SDS-PAGE gel: Prepare 12% separation gel and 4% stacking gel according to the gel recipe instructions.

[0062] (2) Electrophoresis: Add an appropriate amount of sample to each loading well and perform electrophoresis at a constant voltage of 85 V until different bands appear in the marker lane. Then increase the electrophoresis voltage to 120 V and stop the electrophoresis according to the position of bromophenol blue from the bottom and the position of the protein marker.

[0063] (3) Transfer: ① Cut the PVDF membrane of appropriate size and activate it in methanol; ② Soak the sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper and sponge in the transfer buffer in the order of the positive electrode to the negative electrode in a sandwich structure, remove the bubbles during placement, and then place it in the plastic splint for transfer; ③ Place the assembled plastic splint in the electrophoresis tank filled with transfer buffer. Transfer conditions: constant current 200mA, 90min.

[0064] (4) Blocking: Place the transferred PVDF membrane in a TBST solution containing 5% skimmed milk powder and block it on a shaker at room temperature for 1 h.

[0065] (5) Primary antibody incubation: Place the blocked PVDF membrane into an antibody incubation box containing primary antibody dilution solution and incubate at 4°C overnight.

[0066] (6) Secondary antibody incubation: ① Rinse the PVDF membrane after primary antibody incubation with TBST three times, each time for 10 min; ② Place the PVDF membrane in an antibody incubation box containing an appropriate amount of secondary antibody dilution and incubate on a shaker for 60 min; ③ Rinse the PVDF membrane after secondary antibody incubation with TBST three times, each time for 10 min.

[0067] Development and photography: Take out the cleaned PVDF membrane, absorb the excess liquid on the membrane with a clean paper towel, add ECL developer (A solution: B solution = 1:1) on the membrane, and develop it on a gel imager when the PVDF membrane is evenly infiltrated with ECL developer. Take pictures and save them. Use Image J software to calculate the gray value, and β-actin is used as the protein internal reference.

[0068] Figure 7 The results showed that after 24h of treatment of HepG2 cells with 0.5μM and 1μM GLA, the expression levels of the above three proteins increased significantly with the increase of concentration, and the difference was statistically significant compared with the control group, and showed a dose-dependent effect. This indicates that GLA can upregulate the expression of HO-1, NQO1 and GCLC in HepG2 cells at the protein level and enhance the ability of the endogenous antioxidant defense system in HepG2 cells.

[0069] Effect of GLA on the expression and distribution of Nrf2 in HepG2 cells.

[0070] The Nrf2-ARE pathway is one of the important participants in the intracellular antioxidant defense system. Given its central role in the cell defense mechanism, the present invention speculates that GLA plays an active role in it, and may promote the expression and nuclear entry of Nrf2, thereby inducing the activation of the Nrf2-ARE signaling pathway. In order to verify this speculation, the present invention conducted a detailed experimental study.

[0071] HepG2 cells were cultured in 60mm dishes. When the cell confluence reached 70%-80%, 1μM GLA was added for treatment. After 0h, 1h, 3h, and 6h, the culture medium in the culture dish was aspirated and the cells were washed twice with pre-cooled PBS in each dish. The cells in the dish were scraped off with a cell scraper and transferred to a 1.5mL centrifuge tube. Pre-cooled RIPA cell lysis buffer was added to each tube, and protease inhibitors were added to the cell lysis buffer in advance, and lysis was performed for 30min. After lysis, centrifuged at 4℃ and 10000rpm for 10min, and the supernatant after centrifugation was taken for use to obtain total cell protein. According to the steps in the instructions of the cytoplasmic-nuclear protein extraction kit, nuclear protein and plasma protein of HepG2 cells were extracted, and the protein concentration was adjusted to uniformity using the Bradford method. An appropriate amount of lysis buffer and 5× loading buffer were added to the sample, and mixed evenly to make the final protein concentration of the sample consistent. The above samples were placed in a metal bath and heated for 5min for protein denaturation, and stored at -80℃ for use.

[0072] The SDS-PAGE electrophoresis method was referred to above, and the gray value was calculated using Image J software. Lamin B was used as a nuclear protein internal reference, and β-actin was used as a cytoplasmic protein internal reference.

[0073] Figure 8The results confirmed that GLA promotes the activation of the Nrf2-ARE pathway. As shown in the figure, with the increase of GLA action time, the total Nrf2 expression in the cell increased significantly, while the expression level of its inhibitory chaperone protein Keap1 decreased. Nrf2 entering the nucleus is a necessary prerequisite for activating the Nrf2-ARE signaling pathway. Next, the present invention analyzes whether GLA can achieve the nuclear entry of Nrf2. After GLA stimulated HepG2 cells for 0h, 1h, 3h, and 6h, the expression levels of Nrf2 protein in the cytoplasm and nucleus were measured respectively. The results in the figure show that with the extension of GLA action time, the content of Nrf2 in the nucleus increased significantly and reached a peak at 6h. At the same time, the level of Nrf2 in the cytoplasm also increased. In short, the above results show that GLA promotes the increase of Nrf2 content and accumulates in the nucleus, which can promote the nuclear entry of Nrf2, providing a basis for its subsequent activation of downstream pathways.

[0074] The above research results show that GLA has a preventive and therapeutic effect on FFA-induced NAFLD, can reduce the lipid accumulation of hepatocytes caused by FFA, and enhance the damage resistance of hepatocytes. Nrf2 is a key nuclear transcription factor that can respond to external stress. It enters the cell nucleus in an activated state and interacts with ARE in the cell antioxidant gene to initiate the expression of a series of cell protection proteins that resist oxidative stress damage. It plays an important role in the body's anti-oxidative damage and regulation of life activities. The research of the present invention can show that GLA can not only promote the nuclear translocation and accumulation of Nrf2 in the nucleus in HepG2 cells, but also activate the Nrf2-ARE pathway, thereby upregulating the expression levels of downstream antioxidant proteins such as HO-1, NQO1 and GCLC, and is expected to be developed as a drug for the treatment of NAFLD.

[0075] 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 bluecalyx acetylcholine in the preparation of drugs for preventing / treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The structural formula of the blue calyx amine is as follows:

3. The use according to claim 1, characterized in that: The blue calyx factor A is used to activate the Nrf2-ARE pathway; wherein the Nrf2 refers to the nuclear factor NF-E2-related factor 2, and the ARE refers to the antioxidant response element.

4. The use according to claim 1, characterized in that: The bluecalyx factor A has a protective effect on HepG2 cell damage induced by free fatty acids.

5. The use according to claim 1, characterized in that: The blue calyx acetone is used for regulating the expression of heme oxygenase, quinone oxidoreductase 1 and glutamate-cysteine ​​ligase catalytic subunit in HepG2 cells at the protein level, so as to enhance the ability of the endogenous antioxidant defense system in the HepG2 cells.

6. The use according to claim 1, characterized in that: The bluecalyx acetone is used for reducing lipid accumulation of hepatocytes caused by free fatty acids and enhancing the anti-damage ability of hepatocytes in non-alcoholic fatty liver disease induced by free fatty acids.

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