Application of Spicatin in the preparation of medicine for preventing or treating non-alcoholic fatty liver disease

Abstract

The present invention discloses the use of Spicatin in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. In an in vitro model, a series of studies have found that Spicatin may downregulate the expression of key lipid synthesis enzymes ACC1 and FASN based on the FXR-SREBP-1c pathway, inhibit the de novo synthesis of fat, and provide candidate compounds for the research of new drugs for the treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of Spicatin in preparing medicines for preventing or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, usually accompanied by other metabolic diseases such as obesity, hypertension, dyslipidemia and type 2 diabetes. It is characterized by significant abnormal liver lipid accumulation. It has become a focus of public health attention because it can develop into fatty liver hepatitis (NASH) accompanied by hepatocyte fibrosis, and even deteriorate into cirrhosis and hepatocellular carcinoma (HCC). Globally, the prevalence of NAFLD is as high as 30.05%, and with people's unhealthy eating habits of high sugar and high fat, the prevalence continues to grow. The occurrence and development of NAFLD is the result of the combined action of multiple molecular mechanisms, including environmental, genetic and metabolic factors, among which abnormal liver lipid accumulation is a key pathological factor. Hepatic lipid accumulation stems from the imbalance between lipid production and clearance. When the production of liver lipids cannot be offset by fatty acid oxidation or the increase in the secretion rate of triglyceride (TG)-rich lipoproteins, excessive lipids accumulate in hepatocytes, leading to fatty degeneration of the liver. Fatty acids can be used as substrates for the production of lipotoxic substances, leading to the generation of toxic lipids, including free fatty acids, TG, free cholesterol (TC), ceramides, etc., which trigger oxidative stress, inflammatory damage and death of hepatocytes and promote the development of NAFLD.

[0003] NAFLD is a common chronic liver disease, and there is currently no officially approved effective drug treatment. The main treatment for NAFLD patients is to reduce metabolic risk factors, such as exercise to lose weight and change diet, but these measures are difficult to implement in daily life and the effect is poor. In addition, most of the drugs under development have significant toxic side effects. It is urgent to find new drugs with low toxicity and effectiveness for the treatment of NAFLD. The FXR agonist obeticholic acid (OCA), as a drug with significant efficacy in phase 3 clinical trials, can effectively regulate lipid and glucose metabolism and reduce liver fibrosis and inflammation, suggesting that FXR agonists have good potential in the treatment of NAFLD.

[0004] Cholesterol regulatory element binding proteins (SREBPs) are important transcription factors that control cholesterol and lipid synthesis. There are three subtypes: SREBP-1c, SREBP-1a, and SREBP-2. The SREBP-1c subtype is mainly present in adult liver and is responsible for regulating the transcription of lipid synthesis-related genes, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase 1 (SCD1). Overexpression of SREBP-1c leads to aggravated fatty degeneration of the liver. Inhibiting the expression of SREBP-1c is a favorable preventive measure that can effectively reduce lipid synthesis in the liver. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor highly expressed in the liver and adipose tissue, which stimulates the transcription of genes related to mitochondrial uptake of fatty acids and fatty acid β-oxidation, thereby promoting lipolysis. FXR is a ligand-activated transcription factor, which is also called bile acid receptor because its transcriptional activity can be activated by bile acid (BA) and its metabolites. FXR is the main regulatory factor controlling bile acid synthesis and enterohepatic circulation. After FXR in the liver is activated by ligand, it can inhibit the expression of SREBP-1c, thereby inhibiting the accumulation of triglycerides in the liver. FXR is similar in structure to PPARα, and after being activated, it can competitively bind to the nucleic acid sequence of the other's reaction element, activate the transcription of genes related to fatty acid β-oxidation, and thus promote fatty acid β-oxidation.

[0005] Swietenia macrophylla King is a fruit of the Meliaceae family. It is cool, antipyretic, astringent, and has great medicinal value. Studies have shown that Swietenia macrophylla King has biological activities such as hypoglycemic, lipid-lowering, anti-oxidant, anti-inflammatory, analgesic, antihypertensive, antibacterial, stomach-protecting, and antidiarrheal. In the early stage of the present invention, more than 30 chemical components including Spicatin were separated from the Swietenia macrophylla King extract. Studies have shown that Spicatin can effectively inhibit the proliferation of esophageal cancer cells. However, there is no literature report on the use of Spicatin in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide the use of Spicatin in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, and to provide candidate compounds for the research of new drugs for treating non-alcoholic fatty liver disease.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides the use of Spicatin in preparing a medicine for preventing or treating non-alcoholic fatty liver disease.

[0009] The present invention uses a targeted cell screening model of SREBP-1c and PEPCK, which are key genes for lipid synthesis and gluconeogenesis, and finds that Spicatin can dose-dependently inhibit the activity of the promoter of SREBP-1c, a key regulatory gene for lipid synthesis; the present invention uses a HepG2 / NCTC 1469 hepatocyte lipid accumulation model induced by 0.5 mM oleic acid (OA) and 0.25 mM palmitic acid (PA), and finds that Spicatin can significantly reduce the triglyceride (TG) content of HepG2 cells; the present invention uses a RAW 264.7 cell inflammation model induced by 100 ng / mL lipopolysaccharide (LPS), and finds that Spicatin can significantly reduce the release of NO in RAW 264.7 cells, and significantly inhibit the expression of proinflammatory cytokines IL-1β and IL-6.

[0010] The present invention studies the lipid-lowering molecular mechanism of Spicatin. RT-qPCR results show that Spicatin can downregulate the mRNA expression levels of SREBP-1c, ACC1, and FASN induced by the lipid synthesis agonist TO901317, and increase the mRNA expression levels of FXR and PPARα. Western Blot results show that Spicatin significantly increases the protein expression level of FXR, and significantly reduces the protein expression levels of SREBP-1c and FASN. This study further transfected FXR-siRNA into primary mouse hepatocytes to knock down the expression of FXR. After the action of Spicatin, the expression level of FXR mRNA was significantly increased, and the mRNA expression level of SREBP-1c was significantly reduced. In summary, Spicatin may alleviate the lipid accumulation of hepatocytes through the FXR-SREBP-1c pathway.

[0011] Furthermore, the drug comprises an effective amount of Spicatin and a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient of the present invention includes a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant, a pH regulator, etc., and the amount thereof is the conventional amount in the art.

[0012] Preferably, the drug is in the form of tablets, capsules, granules, oral liquid preparations, or intravenous or intramuscular injections.

[0013] The pharmaceutical dosage form of the present invention can be prepared according to conventional preparation methods in the art.

[0014] The present invention provides a new use of Spicatin in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. In an in vitro model, a series of studies have found that Spicatin may downregulate the expression of key lipid synthesis enzymes ACC1 and FASN based on the FXR-SREBP-1c pathway, inhibit the de novo synthesis of fat, and provide a candidate compound for the research of new drugs for treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of Spicatin on the promoter activity of SREBP-1c and PEPCK;

[0016] Figure 2 The effect of Spicatin on the viability of HepG2 / NCTC 1469 cells;

[0017] Figure 3 The effect of Spicatin on total triglyceride in HepG2 / NCTC 1469 cells;

[0018] Figure 4 Effects of Spicatin on the number of lipid droplets in HepG2 cells (A: blank group; B: OA+PA model group; C: positive control group, obeticholic acid concentration was 10 μM; D, E and F: experimental group Spicatin, concentrations were 5, 10 and 20 μM);

[0019] Figure 5 Effects of Spicatin on the expression of lipid metabolism-related genes FXR, SREBP-1c, ACC1, FASN, and PPARα in NCTC 1469 cells;

[0020] Figure 6 Effects of Spicatin on the expression of lipid metabolism-related proteins FXR, SREBP-1c, FASN and PPARα in NCTC 1469 cells;

[0021] Figure 7 The effect of Spicatin on the expression of lipid metabolism-related genes FXR and SREBP-1c in primary mouse hepatocytes transfected with FXR-siRNA;

[0022] Figure 8 The effect of Spicatin on the survival rate of RAW 264.7 cells;

[0023] Fig. 9 The effect of Spicatin on NO release induced by LPS in RAW 264.7 cells;

[0024] Fig.10The effect of Spicatin on LPS-induced inflammatory factors IL-1β and IL-6 in RAW 264.7 cells. Implementation

[0025] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0026] 1.1 Main experimental materials and sources

[0027] (1) Cells: HepG2 cells (human hepatoma cells) were donated by the Institute of Virology, School of Life Sciences, Wuhan University and are maintained by our laboratory.

[0028] (2) Bacteria strain: E. coli DH5α was purchased from Shanghai Ang Yu Biotechnology Co., Ltd. and used for plasmid transformation and amplification.

[0029] (3) Experimental drugs: Spicatin was isolated from the extract of the fruit of the Chinese angelica dahurica, which was obtained from Anhui University of Traditional Chinese Medicine.

[0030] (4) Main experimental reagents: DMEM high-glucose medium and fetal bovine serum were purchased from Gibco Biotechnology Co., Ltd.; DNA transfection reagent was purchased from CodeGene Technology Co., Ltd.; Dual-luciferase reporter gene detection system kit was purchased from Promega (Beijing) Biotechnology Co., Ltd.

[0031] 1.2 Experimental methods

[0032] Dual luciferase reporter gene assay

[0033] (1) Transformation: Take out the competent E. coli cells (DH5α) stored at -80 ℃, gently place them on ice to thaw, pipette 1 μL of the target plasmid into 14 μL of DH5α, gently flick the bottom of the EP tube with your fingers to mix, place the EP tube in an ice bath for 30 min, place the EP tube in a 42 ℃ water bath, heat shock for 80 s, then quickly return to the ice bath for 3 min. Do not shake the tube during the process.

[0034] (2) Amplification: Add 300 μL of liquid LB medium without ampicillin that has been restored to room temperature to the treated competent cell mixture, and incubate in a shaker set at 37°C and 150 rpm for 1 h. Dilute the bacterial solution to an appropriate multiple, apply it to the LB solid medium containing 0.1% ampicillin with a spreading rod, and incubate it in a 37°C constant temperature incubator overnight after the liquid is absorbed. Pick a single colony and inoculate it into the LB liquid medium containing 0.1% ampicillin, and shake it at 37°C overnight. Take the bacterial solution in the logarithmic growth phase and mix it with 50% sterilized glycerol in a ratio of 1:1, and store the strain at -20°C for a short period of time for subsequent experiments.

[0035] (3) Extract plasmid: Collect the bacteria by centrifugation at 4000 rpm and extract the plasmid according to the instructions of the kit. Take 2 μL of the extracted plasmid and measure the concentration and purity on an ultra-microbiological detector. Perform agarose gel electrophoresis to check the purity of the plasmid. Store the remaining plasmid at -20 ℃.

[0036] (4) Transfection: HepG2 cells were inoculated into 24-well plates and cultured overnight. When the cell density reached 70%, new complete culture medium was replaced 2 h in advance for transfection. According to the calculated ratio, the plasmid was diluted with pure culture medium and mixed evenly. An equal proportion of transfection reagent was added to the dilution and gently mixed. After standing for 25 min, the incubated transfection complex was gently dripped into the cell culture medium and gently mixed. After 24 h of culture, the complete culture medium containing different concentrations of Spicatin was replaced.

[0037] (5) Detection: Continue to culture for 24 h. According to the instructions of the kit, dilute the lysate with ultrapure water, add it to the cell plate and place it on a shaker for 20 min. Pipette the supernatant and place it in an ice box. Detect the chemiluminescence value of the enzyme in the lysate after the reaction with the working solution substrate on the microplate reader to detect the effect of different concentrations of Spicatin on the promoter activity.

[0038] 1.3 Experimental Results

[0039] The effect of Spicatin on the promoter activity of SREBP-1c and PEPCK, key genes for lipid synthesis and gluconeogenesis, was detected by dual luciferase reporter gene assay. Figure 1 (* p <0.05,** p <0.01 vs Control; n=3), Spicatin can dose-dependently inhibit the activity of the promoter of SREBP-1c, a key regulatory gene for lipid synthesis ( p <0.01), and inhibited the promoter activity of the key gluconeogenesis gene PEPCK to a certain extent.

[0040] The above results indicate that Spicatin can significantly inhibit the activity of the promoter of SREBP-1c, a key regulatory gene for lipid synthesis, and has the potential to inhibit de novo lipogenesis.

[0041] 2.1 Main experimental materials and sources

[0042] (1) Cells: HepG2 cells were donated by the Institute of Virology, School of Life Sciences, Wuhan University; NCTC 1469 cells (normal mouse liver cells) were purchased from Hangzhou Haisheng Biotechnology Co., Ltd. and are preserved in this laboratory.

[0043] (2) Main experimental reagents: DMEM high glucose medium and fetal bovine serum were purchased from Gibco Biotechnology Company;

[0044] CCK-8 reagent was purchased from DOJINDO; sodium oleate, sodium palmitate and Oil Red O powder were purchased from SIGMA Biotechnology, USA; TG detection kit was purchased from Beijing Pulilai Biotechnology Co., Ltd.

[0045] 2.2 Experimental methods

[0046] (1) Cell viability assay

[0047] In a clean bench, HepG2 / NCTC 1469 cells were seeded into 96-well plates at an appropriate density with 100 μL per well. After 24 h, the culture medium was replaced with medium containing different concentrations of Spicatin. After culturing for another 24 h, the original culture medium was discarded and 100 μL of culture medium containing 10% CCK-8 reagent was added to each well. CO 2 The cells were incubated in the incubator for half an hour, and the optical density was detected at a wavelength of 450 nm using an ELISA reader. The cell survival rate of the blank group was used as a control to calculate the effect of each concentration of Spicatin on the cell survival rate.

[0048] (2) TG content determination

[0049] TG induction solution: Accurately weigh 50 mg sodium oleate (OA) and dissolve it in 1 mL methanol. Ultrasonicate to fully dissolve it to obtain 164.24 mM OA mother solution, which is stored in a refrigerator and away from light. Accurately weigh 28 mg sodium palmitate (PA) and dissolve it in 1 mL sterile water, and dissolve it in a metal bath at 80°C to obtain 100.57 mM PA mother solution, which is stored in a refrigerator and away from light. When preparing TG induction solution, dilute the OA mother solution to 0.5 mM and the PA mother solution to 0.25 mM with complete culture medium containing 10% BSA.

[0050] HepG2 cells were cultured until they were in good condition and seeded in 6-well plates. When the cell density reached 40%, all groups except the blank group were replaced with TG induction solution. After 24 h, the experimental group was replaced with complete culture medium containing different concentrations of Spicatin, the positive control group was replaced with complete culture medium containing 10 μM obeticholic acid (OCA), and the blank group and model group were replaced with complete culture medium. After 24 h, the cells were washed three times with PBS, 150 μL of lysis solution was added to each well, and the lysis was performed for 30 min. The supernatant was aspirated into a 1.5 mL EP tube, and a portion of the supernatant was diluted tenfold to measure the protein concentration. The EP tube was placed in a 70 ° C water bath for 10 min, centrifuged at 2000 rpm at room temperature for 5 min, and the supernatant was added with working solution and reacted at 37 ° C for 20 min. The OD value was detected at a wavelength of 550 nm, and the TG level was calculated by dividing it by the corresponding protein concentration. The TG content of the model group was set as 100%, and the relative TG content of other groups was calculated.

[0051] (3) Oil Red O staining

[0052] Oil Red O dye solution: accurately weigh the solid Oil Red O and dissolve it in isopropanol as much as possible, shake it evenly after completely dissolving it under ultrasound in the dark, filter the insoluble impurities with two layers of filter paper under pressure, and store it in the dark and refrigerate. When using, mix the mother liquor with ultrapure water in a ratio of 3:2, and after it is completely dissolved, filter it under pressure with three layers of filter paper to remove the insoluble matter, and use it immediately after preparation.

[0053] HepG2 cells were cultured until they were in good condition and seeded in 6-well plates. When the cell density reached 40%, all groups except the blank group were replaced with TG induction solution. After 24 hours, the experimental group was replaced with complete culture medium containing different concentrations of Spicatin, and the positive control group was replaced with complete culture medium containing 10 μM OCA. After 24 hours, the cells were washed twice with PBS, soaked in 60% isopropanol for 45 seconds, and then the isopropanol was discarded. The diluted and filtered Oil Red O staining solution was added and stained for 30 minutes in the dark, followed by rinsing with 60% isopropanol and washing with PBS twice to remove excess staining solution. Oil Red O staining solution is a fat-soluble dye that can dye the lipid droplets of cells orange-red. The fat content of the cells can be clearly observed by observing the staining of the cells under a microscope.

[0054] 2.3 Experimental Results

[0055] (1) This study first evaluated the safety of Spicatin using the CCK-8 method. Figure 2As shown in the figure (*p<0.05, **p<0.01 vs Control; n=3), starting from 25 μM, the activity of HepG2 was significantly decreased (p<0.05), and at 100 μM, Spicatin had obvious toxicity to NCTC 1469 cells (p<0.01). Combined with the results of promoter activity, this study selected 5, 10 and 20 μM as the experimental concentrations of Spicatin to further explore its effect on cellular lipid accumulation.

[0056] (2) Free fatty acids have strong lipotoxicity and the ability to induce lipid production, so they are often used as inducers for the hepatocyte lipid accumulation model. In this study, a hepatocyte lipid accumulation model was established by inducing HepG2 / NCTC1469 cells with 0.5 mM oleic acid (OA) and 0.25 mM palmitic acid (PA). The TG content results showed that Figure 3 As shown ( # p <0.05, ## p <0.01 vs blank group; * p <0.05,** p <0.01 vs OA+PA model group; n=3), compared with the blank group, the TG content in the OA+PA model group increased significantly ( p <0.01), Spicatin can dose-dependently reduce the TG content of HepG2 cells (p<0.01).

[0057] (3) Oil Red O staining results are as follows Figure 4 As shown, after OA and PA induction, the number of red lipid droplets in the model group increased significantly, and after Spicatin treatment, the number of lipid droplets decreased to varying degrees.

[0058] The above results indicate that Spicatin can significantly inhibit the free fatty acid-induced TG production in hepatocytes and alleviate hepatic fat accumulation.

[0059] 3.1 Main experimental materials and sources

[0060] (1) Cells: NCTC 1469 cells were purchased from Hangzhou Hisun Biotechnology Co., Ltd. and maintained in this laboratory.

[0061] (2) Main experimental reagents: DMEM high-glucose medium, RPMI 1640 medium and fetal bovine serum were purchased from Gibco Biotechnology Co., Ltd.; PCR primers were purchased from Beijing Qingke Biotechnology Co., Ltd.; TO901317 was purchased from APExBIO, USA; FXR and FASN antibodies were purchased from Cell Signaling Technology, USA; SREBP-1c and PPARa antibodies were purchased from Wanlei Biotechnology Co., Ltd.; InvitroRNA TM Cell transfection reagents were purchased from Invivo Biotechnology Co., Ltd.

[0062] 3.2 Experimental methods

[0063] (1) Isolation and culture of primary mouse hepatocytes

[0064] Solution C: Contains 480 mM potassium chloride, 120 mM potassium phosphate dibasic, and 120 mM magnesium sulfate.

[0065] Calcium chloride solution: contains 1 M anhydrous calcium chloride, 1 M CaCl 2 •2H 2 O.

[0066] Krebs Ringer solution: 1 L contains 7 g sodium chloride, 2 g sodium bicarbonate, 3.6 g glucose, 5 mL 1M HEPES (pH = 7.45) solution, 10 mL solution C, flush with oxygen for 2 h before use, and store in a sealed container at 4°C.

[0067] Perfusion solution I: Add 60 μL of 50 mM EGTA to 30 mL of Krebs Ringer solution and mix well before use.

[0068] Perfusion solution II: Add 42 μL 2 M CaCl to 30 mL Krebs Ringer solution before use. 2 Mix well and set aside for later use. Add 15 mg of collagenase and mix well before perfusion.

[0069] Primary hepatocyte culture medium: 160 mL RPMI 1640 medium containing 10% FBS, double antibody, 3 nM insulin, and 1 nM dexamethasone.

[0070] ① Prepare the cell culture well plate in the clean bench in advance. After 30 minutes of UV, spread 2 mL of collagen solution in each well of the well plate to moisten each bottom surface, and blow dry on the bench. Before isolating hepatocytes, prepare a 50 mL centrifuge tube, a 5 mL pipette, a 1 mL pipette gun, a 70-mesh cell sieve, and a 1 mL syringe in the clean bench and sterilize with UV for 30 minutes.

[0071] ②Prepare cannula, syringe (10 mL, 1 mL), 320 U / mL heparin, foam board, 75 cm 2 Use large dishes, 50mL centrifuge tubes, collagenase, etc. to prepare heparin sodium solution (2 mg / mL), 1% pentobarbital sodium solution, and perfusion fluids I and II.

[0072] ③ After anesthetizing the mouse, put the perfusion fluids I and II into a 37 ℃ constant temperature water bath, wipe the mouse skin with 75% alcohol, cut the mouse abdominal skin with a U-shaped cutter, separate the mouse inferior vena cava, and tie a false knot around the inferior vena cava with surgical thread. Insert the catheter cannula into the inferior vena cava below the false knot, withdraw the needle to see if there is blood return. If the blood return quickly fixes the false knot, tie another knot to double fix it, fix the catheter by hand, and carefully withdraw the cannula. Immediately inject 300 μL of heparin, connect the catheter of the inferior vena cava to the 10 mL syringe containing perfusion fluid I, cut the portal vein, and push the syringe so that the perfusion time of perfusion fluid I does not exceed 2 min.

[0073] ④ After the perfusion of fluid I is completed, perfusion of fluid II is injected, and the perfusion speed is controlled so that the perfusion time is controlled within 5 min, until cracks or blisters appear on the liver.

[0074] ⑤ Stop perfusion, open the mouse chest, clamp the superior vena cava near the liver with hemostatic forceps, separate the liver and place it in a culture dish with ice-cold PBS, transfer it to the clean bench, place the cell sieve in the mouth of a 50 mL centrifuge tube, tear the liver capsule with tweezers, add ice-cold 1640 culture medium, and use a 1 mL syringe to push the head to assist the cells to pass through the sieve. After standing for 5 minutes, aspirate the supernatant with a pipette, gently add ice-cold 1640 culture medium to suspend and wash the cells, centrifuge at 4 ° C, 50 g for 2 minutes, discard the upper liquid, and repeat twice.

[0075] ⑥ Add 20 mL of primary hepatocyte culture medium to suspend the cells and inoculate the cells in CO 2 Culture in incubator.

[0076] ⑦ After 6 hours, the medium was changed for experimental treatment. According to the experimental plan, InvitroRNA TM FXR-siRNA or NC-siRNA (40 nM) was transfected into cells using cell transfection reagent. After 24 h of culture, the primary hepatocyte culture medium containing different concentrations of Spicatin was replaced. After 24 h of culture, the cell RNA was extracted.

[0077] (2) Total RNA extraction and RT-qPCR detection

[0078] ① Extraction of total RNA

[0079] Prepare sterile, enzyme-free 1.5 mL and 2 mL EP tubes, PCR tubes, 1 mL, 200 μL and 20 μL pipette tips, and chloroform, isopropanol, and anhydrous ethanol. Irradiate with UV light in a clean bench for 30 min.

[0080] NCTC 1469 cells were inoculated in a 6-well plate. When the cell density reached 40%, the model was established for 24 h. After 24 h, the drug-treated group was replaced with complete medium containing different concentrations of Spicatin, and the blank group and model group were replaced with complete medium. After 24 h of drug administration, the medium was discarded in the clean bench, washed twice with PBS, and 1 mL of Trizol was added and allowed to stand for 5 min. The cells were blown and beaten for more than ten times until Trizol was no longer sticky, and then aspirated into a sterile, enzyme-free 1.5 mL EP tube, 200 μL of chloroform was added and vigorously mixed for 15 S, and then allowed to stand at room temperature for 15 min, and then centrifuged at 12,000 rpm in a centrifuge precooled at 4°C for 15 min. Avoid touching the middle and lower layers, carefully aspirate 400 μL of the upper clear liquid into a new sterile enzyme-free 1.5 mL EP tube and place on ice, add an equal amount of isopropanol and gently invert it to mix, then place it on ice for 10 min, centrifuge it at 12000 rpm in a 4℃ centrifuge for 10 min, and you can see a layer of white precipitate at the bottom. Pour out the supernatant in the clean bench, add 1 mL of pre-cooled 75% ethanol prepared with DEPC water, gently flick up the precipitate at the bottom of the EP tube, invert the EP tube to wash the precipitate, centrifuge it at 7600 rpm for 5 min, pour out the supernatant, centrifuge it again for 1 min, then aspirate and discard the supernatant, dry it next to an alcohol lamp for 3 min to evaporate the ethanol, add 15~30 μL of DEPC water according to the amount of precipitate, and repeatedly blow the precipitate with a pipette to mix it.

[0081] 4 μL of RNA solution was taken into a PCR tube, and 2 μL was used to detect the content and purity of RNA using an ultra-microbiological detector; 1% agarose gel was prepared with 1xTAE, and heated in a microwave oven. After it was no longer hot, 10% EB was added and shaken evenly, and then poured into the gel plate and the comb was inserted in time. After solidification for 20 min, the comb was removed, and the gel plate was placed in the electrophoresis tank, filled with 1xTAE solution, and 2 μL of RNA solution was taken with a 10 μL pipette and mixed evenly with 0.5 μL of loading buffer, and then spotted in the comb hole of the agarose gel. After 20 min of electrophoresis, the gel was taken out and placed in a gel imager for scanning to determine the integrity of the RNA.

[0082] ② Synthesis of cDNA

[0083] 1) Calculate the volume of RNA solution required for 4 μg RNA, and prepare the reverse transcription system on ice according to the instructions (see Table 2.1). Place the PCR tube in the instrument and reverse the RNA to cDNA according to the reaction conditions of 37℃ for 15 min, 98℃ for 5 min, and 4℃ for 30 min. Store the obtained cDNA at -20℃ for subsequent reactions.

[0084] Table 1 Reverse transcription reaction system

[0085]

[0086] ③ RT-qPCR detection

[0087] SYBR Green fluorescence staining was used to detect the mRNA expression levels of related genes.

[0088] Table 2 Gene primer sequences

[0089]

[0090] (3) Total protein extraction and target protein detection

[0091] ①Total protein extraction

[0092] NCTC 1469 cells were inoculated in 6-well plates. When the cell density reached 40%, the model group and the experimental group were modeled with 1 μM lipid synthesis agonist TO901317 for 24 h. After 24 h, the experimental group was replaced with complete culture medium containing different concentrations of Spicatin, and the blank group and the model group were replaced with complete culture medium. After 24 h of administration, the cells were washed twice with PBS, and 150 μL of the pre-prepared RIPA lysis buffer was added to each well, and lysed in an ice bath shaker for 30 min. After the end, the lysate was collected in a 1.5 mL EP tube, centrifuged in a pre-cooled centrifuge for 10 min, and the supernatant was aspirated into a clean EP tube.

[0093] After mixing, take a portion of the protein supernatant and dilute it 10 times with ultrapure water. Use the BCA protein quantification kit to measure the protein concentration of each group. Dispense the protein stock solution according to the 30 μg mass, add 5X protein loading buffer (protein to buffer volume ratio of 4:1), mix well, and denature in a 100℃ metal bath for 10 min. After denaturation, store in a -80℃ refrigerator.

[0094] ② SDS-PAGE polyacrylamide gel electrophoresis

[0095] Before the experiment, clean and assemble the glass plate and gel rack, and invert them to dry.

[0096] Prepare 10% or 6% lower gel (separation gel) according to the molecular weight of the target protein. Add 6 mL of lower gel solution in the middle of the glass plate and then add 1 mL of isopropanol to isolate the air. Place it horizontally at 37 °C for about 30 min to promote gelation. Prepare 5% upper gel (concentration gel). Add 2 mL in the middle of the glass plate and gently insert a comb that has been washed and dried in advance on top to avoid bubbles under the comb. Place it horizontally at 37 °C for 30 min. The specific ingredients and ratios are shown in Table 3.

[0097] Table 3 SDS-PAGE gel formula

[0098]

[0099] Assemble the gelled plate and put it into the electrophoresis box with the electrophoresis solution, carefully pull out the comb and check and adjust the comb holes. Take out the protein sample and thaw it, centrifuge it and gently blow it to mix it, slowly and evenly add it to the loading well, and add the protein maker according to the molecular weight of the protein; run the gel at a voltage of 80 V, and when the red band of the maker appears, adjust the voltage to 120 V and continue the electrophoresis until the end.

[0100] ③ Transfer

[0101] Prepare the clips, sponge pads and filter paper for transfer in advance. After the electrophoresis, according to the molecular weight of the target protein and the indication of the maker band, carefully cut a certain width of gel on the opened glass plate, cut out a PVDF membrane of appropriate size according to the size of the gel and activate it in methanol for 1 min, arrange the black bottom of the clip, sponge pad, 3 layers of freshly cut filter paper, gel, PVDF membrane, 3 layers of filter paper, sponge pad, and white upper layer of the clip in this order from bottom to top, and fix the clip, then transfer the membrane into the electrotransfer box filled with ice-cold electrotransfer solution.

[0102] ④ Closed

[0103] Prepare TBST cleaning solution and blocking solution in advance, take out the electro-transferred PVDF membrane from the clamp and mark it, place it in TBST and wash it on a shaker, place it in the blocking solution after 2 minutes so that it is completely immersed, and block it on a shaker for 60 minutes.

[0104] ⑤ Incubate with primary antibody

[0105] The blocked PVDF membrane was washed in 1×TBST for 2 min, the primary antibody diluent and the required antibody were aspirated to prepare the primary antibody for incubation, the washed PVDF membrane was placed in the corresponding primary antibody, and incubated overnight on a rotator in a chromatography cabinet at 4°C.

[0106] ⑥ Incubation with secondary antibody

[0107] The PVDF membrane incubated overnight was removed and washed three times in TBST, each time for 10 min. The secondary antibody used for incubation was prepared with blocking solution, and the washed PVDF membrane was placed in the corresponding secondary antibody and incubated on a shaker at room temperature for 60 min.

[0108] ⑦ Exposure

[0109] The incubated PVDF membrane was removed and washed three times in TBST, each time for 10 min. Prepare the luminescent solution in a dark place, pour a certain amount of ECL luminescent solution on the washed PVDF membrane, and place the membrane on the exposure plate for exposure in a chemiluminescence imager. The protein bands were analyzed with Image J software, and the relative expression of the target protein was calculated using the internal reference protein as a control.

[0110] (4) Data processing and statistical analysis

[0111] The experimental results are presented as mean ± standard deviation (Mean ± SD), and the data (3 or more) were analyzed using GraphPad statistical software. All analyses were performed using Unpaired student's t Test or One-way ANOVA test, p A difference < 0.05 was considered statistically significant.

[0112] 3.3 Experimental Results

[0113] (1) Based on the above results, Spicatin has a significant lipid-lowering activity. In this study, 1 μM of the lipid synthesis agonist TO901317 was used on NCTC 1469 cells for 24 h to establish a lipid accumulation model in vitro. Figure 5 As shown in the figure, after NCTC 1469 cells were treated with 1 μM TO901317, the expression of FXR mRNA in the model group was significantly downregulated ( p <0.01), SREBP-1c and ACC1 mRNA expressions were significantly upregulated ( p <0.01), FASN mRNA expression was upregulated to a certain extent. After the action of Spicatin, the expression of FXR mRNA was significantly increased at 10 and 20 μM ( p <0.01), and significantly reduced the expression of SREBP-1c, FASN and ACC1 mRNA starting from 5 μM ( p <0.01). At 20 μM, the lipid oxidation gene PPARα mRNA was significantly increased ( pIn summary, Spicatin can upregulate the expression of FXR and PPARα mRNA and inhibit the expression of lipogenesis genes SREBP-1c, FASN and ACC1 mRNA.

[0114] (2) Based on the above results, this study further tested Spicatin as a protein related to lipid metabolism. Figure 6 As shown in the figure, after NCTC 1469 cells were treated with 1 μM TO901317, the expression of FXR and PPARα proteins in the model group was downregulated to a certain extent, and the expression of SREBP-1c protein was significantly upregulated ( p <0.05), the expression of FASN protein was upregulated to a certain extent, and after the action of Spicatin, the expression of FXR protein was significantly increased at 10 and 20 μM ( p <0.05), and significantly down-regulated the expression of SREBP-1c and FASN proteins starting from 5 μM in a dose-dependent manner ( p <0.05), and had no significant effect on the expression of lipid oxidation gene PPARα protein.

[0115] (3) To further verify that Spicatin inhibits SREBP-1c by activating FXR, thereby inhibiting de novo lipid synthesis, this study transfected FXR-siRNA into primary mouse hepatocytes to knock down FXR expression. Figure 7 As shown, after mouse primary hepatocytes were transfected with FXR-siRNA, the mRNA expression level of FXR was significantly reduced ( p <0.01), the mRNA expression level of SREBP-1c was significantly increased ( p <0.05), which also verified that SREBP-1c is an important target gene of FXR. Spicatin significantly promoted the expression level of FXR mRNA ( p <0.01), significantly reduced the mRNA expression level of SREBP-1c ( p <0.01). The above results indicate that Spicatin inhibits the expression of SREBP-1c by activating FXR.

[0116] The above results indicate that Spicatin may reduce de novo fat synthesis by activating the expression of FXR and downregulating the expression of SREBP-1c and its downstream target genes ACC1 and FASN.

[0117] 4.1 Main experimental materials and sources

[0118] (1) Cells: RAW 264.7 cells (mouse mononuclear macrophages) were donated by the Institute of Virology, School of Life Sciences, Wuhan University and maintained by our laboratory.

[0119] (2) Main experimental reagents: DMEM high glucose medium and fetal bovine serum were purchased from Gibco Biotechnology Company;

[0120] Lipopolysaccharide (LPS) was purchased from Biosharp Biotechnology Co., Ltd.; NO detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0121] 4.2 Experimental methods

[0122] (1) Cell viability assay

[0123] Same as Example 2 (1) Cell viability detection.

[0124] (2) Determination of NO release

[0125] RAW 264.7 cells were seeded into 96-well plates at an appropriate density, and replaced with new complete medium containing different concentrations of Spicatin after 24 hours. At the same time, LPS induction solution was added to each well to make the final concentration of 100 ng / mL. After 24 hours of treatment, the NO content in the culture medium was detected according to the instructions of the kit.

[0126] (3) Total RNA extraction and RT-qPCR detection

[0127] The same as Example 3 (3) was used for total RNA extraction and RT-qPCR detection.

[0128] 4.3 Experimental Results

[0129] (1) CCK-8 test results are as follows Figure 8 As shown in Figure 3, Spicatin concentrations of 5, 10, and 20 μM had no significant effect on the survival rate of RAW 264.7 cells, but significantly inhibited the activity of RAW 264.7 cells at 40 μM. p <0.05). Therefore, for RAW 264.7 cells, the highest concentration of Spicatin was determined to be 20 μM.

[0130] (2) LPS was used to induce RAW 264.7 cells to establish a cellular inflammation model. The experimental results are as follows: Fig. 9 As shown in Figure 2, Spicatin significantly reduced the release of NO starting at 10 μM ( p <0.01), and this effect was dose-dependent.

[0131] (3) This study further examined the effect of Spicatin on LPS-induced inflammatory factors in RAW 264.7 cells. The RT-qPCR results are shown in Fig.10 As shown in Figure 2, Spicatin significantly reduced the mRNA levels of IL-1β and IL-6 at concentrations of 5, 10, and 20 μM ( p <0.01).

[0132] The above results indicate that Spicatin can inhibit the production of NO in a dose-dependent manner and significantly inhibit the mRNA expression levels of pro-inflammatory cytokines IL-1β and IL-6 to exert anti-inflammatory effects.

Claims

1. Application of Spicatin in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The medicine comprises effective content of Spicatin and a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 1, characterized in that: The dosage form of the drug is tablets, capsules, granules, oral liquid preparations, and intravenous or intramuscular injections.

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