Cell-based new drug screening method for inhibiting lipid accumulation

Abstract

The invention discloses a cell-based new drug screening method for inhibiting lipid accumulation. The method comprises the following steps: culturing a hepatocyte line, inoculating the hepatocyte line into a culture plate, and adhering the hepatocyte line to the wall for overnight; adding free fatty acid and to-be-detected compounds with different concentrations or an equal amount of solvent into the culture plate for incubation; the method comprises the following steps of: firstly, incubating cells with D-PBS (D-Phosphate Buffer Solution), fixing the cells with paraformaldehyde, washing the cells with D-PBS, incubating the cells with D-PBS containing BODIPY 493 / 503 and DAPI in a dark place, washing the cells with D-PBS, and finally measuring the fluorescence intensity of BODIPY 493 / 503 and the fluorescence intensity of DAPI by using a microplate reader; calculating the ratio of the BODIPY 493 / 503 fluorescence intensity to the DAPI fluorescence intensity, and evaluating the lipid content according to the ratio; and comparing the lipid content of the experimental group with the lipid content of the control group to screen out the drug for inhibiting lipid accumulation. The method is simple and easy to implement, does not depend on expensive instruments and equipment, and can be widely applied to different laboratories and under different conditions.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a cell-based new drug screening method for inhibiting lipid accumulation. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD), defined as steatosis in which intrahepatic lipid content exceeds 5% of liver wet weight in the absence of heavy alcohol consumption, viral hepatitis, or other causes of chronic liver disease (CLD), has become one of the most common causes of CLD worldwide. Unfortunately, NAFLD is a multifactorial disease with a steadily increasing prevalence worldwide and an unmet medical need, as no specific surgical interventions and drugs have been approved as treatments for NAFLD to date. Worse, many of the drugs currently in use are known to induce steatosis, which may lead to interruptions in clinical trials or withdrawal of drugs from the market.

[0003] At present, several cell-based detection methods have been reported that use cultured primary hepatocytes or hepatoma cells to evaluate drug-induced steatosis by detecting intracellular lipid content or identifying early protein markers or analyzing the expression profiles of transcription factors or genes related to lipid metabolism. Since fatty liver is a complex disease with multiple pathogenic factors, it is more direct and reliable to screen drugs that inhibit steatosis by lipid droplet quantification. At present, several screening methods based on lipid quantification have been reported. They mainly use Oil Red O, Nile Red or BODIPY 493 / 503, combined with flow cytometry or automated cell image analysis. These methods are either cumbersome to operate or rely on expensive instruments, which is not conducive to widespread promotion. Some studies have also used Nile Red, AdipoRed or BODIPY558 / 568C 12 Combined with fluorescence measurement to determine lipid content. These methods are faster, but also have some limitations. For example, Nile red can not only stain neutral lipids, but also phospholipids. BODIPY 493 / 503 is a lipid droplet-specific fluorescent dye that is easy to detect and not interfered by other lipids. It is commonly used in fluorescence microscopy and flow cytometry. CN116297369A discloses a method for quantitatively analyzing intracellular lipid content based on Bodipy 493 / 503 fluorescent probe, which uses Bodipy 493 / 503 fluorescent probe and NucBlue TMThe nuclear stain is used to stain the intracellular lipids and the cell nucleus respectively, and finally the cells are observed under a fluorescence microscope. After the cells are extracted with isopropanol, the fluorescence intensity of the extract is measured using a full-wavelength multifunctional microplate reader. Although the document uses a microplate reader to measure the fluorescence intensity of BODIPY 493 / 503, it is aimed at the extract obtained by organic solvent extraction, and the direct fluorescence detection of cells still relies on a fluorescence microscope. Using BODIPY 493 / 503 to stain lipid droplets, (1) lipid quantification by fluorescence microscopy or (2) lipid quantification by flow cytometry after digestion of adherent cells or (3) extracting with organic solvents and measuring the fluorescence intensity of the extract by fluorescence measurement to analyze lipid content, these three methods have the disadvantages of expensive detection instruments and / or cumbersome operations when screening new drugs that inhibit lipid accumulation.

[0004] Among the reported methods for evaluating drug-induced steatosis, PI, WST-1, MTT, or LDH assays are used to detect cell viability to evaluate drug toxicity, but CCK-8 has not been reported. PI needs to be fixed, and MTT needs to be dissolved in an organic solvent, and neither can maintain cell activity; although the LDH assay can maintain cell activity, it requires supernatant collection, which is time-consuming and laborious; WST-1 and CCK-8 have no obvious toxicity to cells, and do not require supernatant collection, cell washing, cell collection, or additional dissolution steps, making them significantly superior to PI, MTT, and LDH assays, and CCK-8 is more stable, more sensitive, more soluble, and easier to preserve than WST-1. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a cell-based method for screening new drugs for inhibiting lipid accumulation, so as to solve the problem of lack of effective therapeutic drugs for non-alcoholic fatty liver disease (NAFLD). The screening method is simple, does not rely on expensive instruments, and is easy to promote.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] A cell-based method for screening new drugs for inhibiting lipid accumulation, comprising the following steps:

[0008] Cell culture: Hepatocyte cell lines were cultured and seeded into culture plates and allowed to adhere overnight;

[0009] Cell treatment: Add free fatty acids and different concentrations of the test compound or an equal amount of solvent to the culture plate and incubate together;

[0010] Fluorescence measurement: Wash the cells with D-PBS, then fix the cells with paraformaldehyde, wash them with D-PBS again, then incubate them with D-PBS containing BODIPY 493 / 503 and DAPI in the dark, wash the cells with D-PBS, and finally use a microplate reader to measure the fluorescence intensity of BODIPY 493 / 503 and the fluorescence intensity of DAPI;

[0011] Data analysis: The ratio of BODIPY 493 / 503 fluorescence intensity to DAPI fluorescence intensity was calculated and used to evaluate lipid content. Drugs that can inhibit lipid accumulation were screened out by comparing the lipid content of the experimental group and the control group.

[0012] Preferably, in the above technical solution, in the cell culture step, the liver cell line is a human liver cancer cell line or a mouse liver cell line, the human liver cancer cell line is Huh7 and HepG2, and the mouse cell line is a mouse liver cancer cell line Hepa1-6 and a normal mouse liver cell line AML12.

[0013] Preferably, in the above technical solution, the free fatty acid in the cell treatment step is a 0.15 mM free fatty acid mixture, and the mixture is 0.1 mM oleic acid (OA) and 0.05 mM palmitic acid (PA).

[0014] Preferably, in the above technical solution, the cell treatment step is specifically: adding fresh culture medium containing free fatty acids and the test compound or the same amount of solvent to the culture plate at the same time, and incubating for 24 hours.

[0015] Preferably, in the above technical solution, the concentration of BODIPY 493 / 503 is 2 μM, and the concentration of DAPI is 5 μg / ml.

[0016] Preferably, in the above technical solution, the microplate reader is a light absorption filter type microplate reader, a full wavelength microplate reader, a fluorescence / chemiluminescence microplate reader, or a multifunctional microplate reader.

[0017] Preferably, in the above technical scheme, after the cell treatment step and before the fluorescence measurement step, a cytotoxicity detection step is also included, wherein the toxicity detection step adopts the CCK-8 method to detect cytotoxicity, and the relative cell viability is determined using the following formula: relative cell viability = (A450 / A630 of the experimental group) / (A450 / A630 of the control group), and the relative viability value is used to evaluate the cytotoxicity of the drug, and screen out low-toxic drugs that can inhibit lipid accumulation; wherein, the cytotoxicity detection and fluorescence measurement are performed first and then in the same culture plate, without the need for parallel plate processing.

[0018] Preferably, in the above technical solution, the cell-based new drug screening method for inhibiting lipid accumulation comprises the following steps:

[0019] (1) Cell culture: Hepatocytes were cultured and seeded into 96-well culture plates and allowed to adhere overnight.

[0020] (2) Cell treatment: free fatty acids and different concentrations of the test compound or an equal amount of solvent were added to the culture plate and incubated for 24 hours;

[0021] (3) Cytotoxicity detection: CCK-8 method was used to detect cytotoxicity, that is, 1 hour before the end of incubation, CCK-8 solution of 10% of the total volume of the culture medium was added to each well, gently mixed, and returned to the cell culture incubator for continued incubation. After 1 hour, the absorbance at 450 nm was measured using a microplate reader, and 630 nm was used as the reference wavelength to evaluate the relative viability of the cells;

[0022] (4) Fluorescence measurement: After the above step (3) is completed, the cells are immediately washed with D-PBS, and then fixed with 4% paraformaldehyde for 10 minutes, washed again with D-PBS, and then incubated in D-PBS containing BODIPY 493 / 503 and DAPI at 37°C in the dark for 30 minutes. The cells are washed three times with D-PBS, and finally the fluorescence intensity of BODIPY 493 / 503 and the fluorescence intensity of DAPI are measured using a multifunctional microplate reader. The excitation wavelength for the fluorescence intensity of BODIPY 493 / 503 is 485 nm, and the emission wavelength is 520 nm; the excitation wavelength for the fluorescence intensity of DAPI is 350 nm, and the emission wavelength is 450 nm.

[0023] (5) Data analysis: The relative cell viability was determined using the following formula: relative cell viability = (A450 / A630 of the experimental group) / (A450 / A630 of the control group), and the relative viability value was used to evaluate drug cytotoxicity. The ratio of BODIPY 493 / 503 fluorescence intensity to DAPI fluorescence intensity was calculated, and the lipid content was evaluated using this ratio. By comparing the cytotoxicity and lipid content of the experimental group and the control group, potential low-toxicity drugs that can inhibit lipid accumulation were screened out.

[0024] A drug for inhibiting lipid accumulation in non-alcoholic fatty liver disease (NAFLD), the drug being obtained by a screening method.

[0025] A pharmaceutical composition for treating non-alcoholic fatty liver disease (NAFLD), comprising a drug and at least one pharmaceutically acceptable carrier.

[0026] A method for evaluating the therapeutic effect of a drug on non-alcoholic fatty liver disease (NAFLD), the method comprising:

[0027] (1) Using screening methods to determine the inhibitory effect of drugs on lipid accumulation;

[0028] (2) Evaluate the therapeutic effect of the drug on NAFLD based on the inhibitory effect.

[0029] A screening kit for potential drugs for inhibiting lipid accumulation, comprising: a liver cell line, a 0.15mM free fatty acid mixture, a 10% CCK-8 solution, 2μM BODIPY 493 / 503 and a 5μg / ml DAPI solution, wherein the free fatty acid mixture is 0.1mM oleic acid and 0.05mM palmitic acid, and the liver cancer cell line is a human liver cancer cell line.

[0030] The above technical solution of the present invention has the following beneficial effects:

[0031] (1) This method is simple and easy to use, does not rely on expensive instruments and equipment, and can be widely used in different laboratories and under different conditions.

[0032] (2) By integrating cytotoxicity and lipid content detection on the same 96-well plate, the screening efficiency is significantly improved, saving cost and time.

[0033] (3) The lipid droplets were stained with BODIPY 493 / 503 fluorescent dyes and the fluorescence intensity of the adherent cells was directly measured by fluorescence measurement to quantify the lipids. DAPI was also stained to calibrate the cell number. The results were accurate, reliable, and highly reproducible. There was no need to digest the adherent cells or use organic solvents for extraction. The operation was simple and did not rely on expensive instruments and equipment, which significantly improved the screening efficiency.

[0034] (4) CCK-8 was used to evaluate drug cytotoxicity. CCK-8 has no obvious toxicity to cells and does not require supernatant collection, cell washing, cell collection or additional lysis steps. It does not affect the subsequent relative quantification results of lipids using BODIPY 493 / 503 staining, so parallel plate processing is not required.

[0035] (5) Using BODIPY 493 / 503 fluorescent dyes and CCK8 method, this method can accurately and sensitively quantify lipid accumulation and assess cell viability, ensuring the reliability and accuracy of the results.

[0036] (6) This method also has lower intra-group variability, which enhances the repeatability and stability of the experiment.

[0037] In conclusion, the screening method of the present invention provides a practical and efficient tool for NAFLD drug development, and is expected to accelerate the process of new drug discovery and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0039] Figure 1 To select the optimal concentration of BODIPY 493 / 503 and DAPI in fluorescence measurement. Wherein: A, Schematic diagram of Huh7 cells treated with free fatty acids (FFAs) or equal doses of solvent. Huh7 cells were treated with 0.3mM FFAs (0.2mM oleic acid / 0.1mM palmitic acid (0.2OA / 0.1PA)) or equal amounts of solvent for 24 hours (hour, h). Con: solvent-treated cells; 0.2O: 0.2mM OA; 0.1P: 0.1mM PA; 0.2O / 0.1P: a mixture of 0.2mM OA and 0.1mM PA. B, Absolute fluorescence intensity of BODIPY 493 / 503 in the FFAs-treated and control groups. n=4. Data are expressed as mean ± SEM. C, Relative fluorescence intensity of BODIPY 493 / 503 in the FFAs-treated and control groups. The lipid droplet content is expressed as the relative fluorescence intensity of BODIPY 493 / 503, that is, the fluorescence intensity of BODIPY 493 / 503 in each well was first divided by the protein concentration of the well for calibration, and then normalized to the control value. n = 4. Data are expressed as mean ± SEM. D, Absolute fluorescence intensity of DAPI in the FFAs-treated and control groups. n = 4. Data are expressed as mean ± SEM. E, Relative fluorescence intensity of BODIPY 493 / 503 in the FFAs-treated and control groups. The lipid droplet content is expressed as the relative fluorescence intensity of BODIPY 493 / 503, that is, the fluorescence intensity of BODIPY493 / 503 in each well was first divided by the DAPI fluorescence intensity of the well for calibration, and then normalized to the control value. n = 4. Data are expressed as mean ± SEM.

[0040] Figure 2 Figure 3. Detection of lipid droplets by fluorometry using BODIPY 493 / 503 was affected by fixation but not by CCK-8. A, Schematic diagram of treatment of Huh7 cells with FFAs or an equal dose of solvent to evaluate the effect of fixation on our fluorometry. B, BODIPY 493 / 503 treated and control groups.

[0041] The lipid droplet content was expressed as the relative fluorescence intensity of BODIPY 493 / 503, as Figure 1As described in the legend to figure E. n = 6. Data are presented as mean ± SEM. C, Schematic diagram of Huh7 cells treated with FFAs or an equal dose of solvent to evaluate the effect of CCK-8 on our fluorometric assay. D, Relative fluorescence intensity of BODIPY 493 / 503 in the FFAs-treated and control groups. Lipid droplet content is expressed as the relative fluorescence intensity of BODIPY 493 / 503, as Figure 1 As described in the legend of Figure E. n = 6. Data are presented as mean ± SEM.

[0042] Figure 3 The quantitative results of lipid droplet detection by fluorimetry using BODIPY 493 / 503 are comparable to those of fluorescence microscopy but with higher accuracy. Wherein: A, Schematic diagram of Huh7 cells treated with FFAs or an equal dose of solvent to test the accuracy of our fluorimetry. B, Relative fluorescence intensity of BODIPY 493 / 503 in the FFAs-treated and control groups. The lipid droplet content is expressed as the relative fluorescence intensity of BODIPY 493 / 503, as Figure 1 As described in the legend to Figure E. n = 30. Data are presented as mean ± SD.

[0043] ****P<0.0001vs.Con group. SD: standard deviation; CV: coefficient of variation.

[0044] CD, Huh7 cells were treated with 0.1OA / 0.05PA or an equal amount of solvent for 24 h.

[0045] 493 / 503 stains intracellular lipid droplets, and then lipids are quantified by fluorescence microscopy. Representative images are shown in (C), and lipid quantification data are shown in (D). Green represents BODIPY 493 / 503-stained lipid droplets, while blue represents DAPI-stained cell nuclei. n=8. Data are presented as mean ± SD.

[0046] ****P<0.0001vs.Con group.

[0047] Figure 4Figure 2 shows the cytotoxicity and lipid overload of free fatty acids at different concentrations on hepatocytes. A is a schematic diagram of treating Huh7 and HepG2 cells with FFAs or an equal dose of solvent to determine the appropriate concentration of FFAs required for drug screening. Huh7 and HepG2 cells were treated with different concentrations of FFAs (0.042O / 0.021P, 0.1O / 0.05P, 0.2O / 0.1P, 0.4O / 0.2P) or an equal dose of solvent control for 24 h. The cytotoxicity was then evaluated by the CCK-8 method, and the lipid overload was evaluated by fluorimetry using BODIPY 493 / 503. BC, the cytotoxicity of the FFAs-treated group and the control group is shown in (B), and the lipid overload is shown in (C). n=4. Data are expressed as mean ± SEM. *P<0.05,

[0048] ****P<0.0001vs.Con group.

[0049] Figure 5 The concentration-dependent effects of steatosis-inducing compounds on lipid accumulation in Huh7 cells. Wherein: AJ, Huh7 cells were treated as described in Example 5, and lipid accumulation was assessed by fluorimetry using BODIPY 493 / 503. The effects of amiodarone hydrochloride (A), doxycycline hydrochloride (B), tetracycline hydrochloride (C), clofibrate (D), sodium valproate (E), cyclosporine A (F), fenofibrate (G), tamoxifen (H), tianeptine sodium salt (I) and ticlopidine (J) on lipid accumulation are shown as the relative fluorescence intensity of BODIPY 493 / 503, as shown in Figure 1 As described in the legend of Figure E. Compared with the control group, the lipid droplet contents of the 10 positive drug treatment groups increased in a dose-dependent manner. n = 4. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Con group.

[0050] Figure 6.Concentration-dependent effects of non-steatosis-inducing compounds on lipid accumulation in Huh7 cells. Wherein: AE, Huh7 cells were treated as described in the Example, and lipid accumulation was assessed by fluorimetry using BODIPY 493 / 503. The effects of sodium citrate (A), amikacin (B), colchicine (C), rotenone (D), and tert-butylhydroperoxide (E) on lipid accumulation are shown as the relative fluorescence intensity of BODIPY 493 / 503, as shown in Figure 2. Figure 1 As described in the legend of Figure E. Compared with the control group, the five negative drugs had no significant effect on lipid droplet content. n = 4. Data are expressed as mean ± SEM.

[0051] Figure 7 The newly established fluorescence assay correctly identified the inhibitory effect of ML261 on lipid accumulation. Wherein: AB, AML12 cells and Hepa1-6 cells were treated as described in Example 5 and Example 6. The effect of ML261 on lipid accumulation in AML12 cells (A) and Hepa1-6 cells (B) is shown as the relative fluorescence intensity of BODIPY 493 / 503, as shown in Figure 1 As described in the legend of Figure E. ML261 can dose-dependently reduce the lipid droplet content in AML12 cells and Hepa1-6 cells. n = 6. Data are expressed as mean ± SEM. ****P < 0.0001 vs. Con group.

[0052] Figure 8 The concentration-dependent effects of steatosis-inducing compounds on the cytotoxicity of Huh7 cells. Among them: AJ, Huh7 cells were treated as described in Example 5, and the cytotoxicity was evaluated by CCK-8 method. The effects of amiodaronehydrochloride (A), doxycycline hydrochloride (B), tetracycline hydrochloride (C), clofibrate (D), sodium valproate (E), cyclosporine A (F), fenofibrate (G), tamoxifen (H), tianeptine sodium salt (I) and ticlopidine (J) on cytotoxicity were expressed as relative cell viability. Compared with the control group, these 10 positive drugs showed different degrees of cytotoxicity within the selected working concentration range. n = 4. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Con group.

[0053] Fig. 9 The concentration-dependent effects of non-steatosis-inducing compounds on the cytotoxicity of Huh7 cells. Among them: AE, Huh7 cells were treated as described in Example 5, and the cytotoxicity was evaluated by CCK-8 method. The effects of sodium citrate (A), amikacin (B), colchicine (C), rotenone (D), and tert-butyl hydroperoxide (E) on cytotoxicity were expressed as relative cell viability. n = 4. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Con group.

[0054] Fig.10 The cytotoxic effects of ML261 on AML12 cells and Hepa1-6 cells. Wherein: AB, AML12 cells and Hepa1-6 cells were treated as described in Example 5 and Example 6. The effects of ML261 on the cytotoxicity of AML12 cells (A) and Hepa1-6 cells (B) are expressed as relative cell viability. ML261 has no significant cytotoxicity to AML12 cells and Hepa1-6 cells within the working concentration range of 0.01-10 μM. n=6. Data are expressed as mean ± SEM.

[0055] Fig.11 Fluorescence microscopy was used to detect the inhibitory effect of ML261 on lipid accumulation. Wherein: AB, Hepa1-6 cells were treated as described in the example. Intracellular lipid droplets were stained with BODIPY 493 / 503, and then lipids were quantified by fluorescence microscopy. Representative images are shown in (A), and lipid quantification data are shown in (B). Green represents BODIPY 493 / 503-stained lipid droplets, while blue represents DAPI-stained cell nuclei. n=4. Data are expressed as mean ± SEM. **P<0.01, ****P<0.0001vs.Con group. DETAILED DESCRIPTION

[0056] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0057] The present application aims to develop a simple, cell-based, expensive instrument-independent, and easily scalable 96-well method for screening new low-toxic drugs that inhibit lipid accumulation.

[0058] The method first uses CCK-8 to detect cytotoxicity, and then uses BODIPY493 / 503 to detect lipid content by fluorimetry. First, to determine whether BODIPY493 / 503 can be used for fluorimetry, Huh7 cells were treated with free fatty acids (FFAs) or an equal amount of solvent, and lipid droplets were quantified by fluorimetry and fluorescence microscopy using BODIPY 493 / 503, respectively, and the inter-group relative quantification results and intra-group variability of lipid droplets by the two methods were compared. Second, Huh7 cells were treated with FFAs or an equal amount of solvent, and it was evaluated whether CCK-8 affected the subsequent relative quantification of lipid droplets by BODIPY 493 / 503. Finally, we used 10 known steatosis inducers and 5 negative controls as well as ML261, a known inhibitor of hepatic lipid droplet formation, to verify the reliability of this newly developed method.

[0059] The technical solution of the present application is described in detail below in conjunction with the examples. The reagents or instruments used in the present application can be obtained from the market or other commercial channels unless otherwise specified.

[0060] Example 1 Cell culture and reagents

[0061] Cell culture: Select appropriate cell lines (such as Huh7, AML12, Hepa1-6 cells) for culture to simulate the liver environment. Specifically:

[0062] Human liver cancer cell lines Huh7 and HepG2 and mouse liver cancer cell line Hepa1-6 were cultured in high glucose DMEM medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA), 100 units / ml penicillin and 100 mg / ml streptomycin (Thermo Fisher Scientific). Normal mouse liver cell line AML12 was cultured in DMEM / F-12 medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA), 5 μg / ml ITS (Insulin-Transferrin-Selenium) (Life Technologies), 40 ng / ml dexamethasone, 100 U / ml penicillin, 100 μg / ml streptomycin and 0.25 g / L glutamine. All cells were cultured in a 37°C, 5% CO2 incubator.

[0063] Ten known steatosis-inducing agents (amiodarone hydrochloride, doxycycline hydrochloride, tetracycline hydrochloride, clofibrate, sodium valproate, cyclosporine A, fenofibrate, tamoxifen, tianeptine sodium salt, ticlopidine) and five negative controls (sodium citrate, amikacin, colchicine, rotenone, tert-butyl hydroperoxide) were purchased from GlpBio (CA, USA). ML261 (catalog number HY-111179) was purchased from Med Chem Express (NJ, USA). BODIPY 493 / 503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-boronic acid-3a,4a-diaza-s-indene, Catalog No. D3922) was purchased from Invitrogen (Madrid, Spain). DAPI (Catalog No. D9542) was purchased from Sigma.

[0064] Example 2 Detection of lipid droplets and calibration of cell number

[0065] (1) Detection of lipid droplets by fluorescence microscopy

[0066] Cells were seeded into 8-well chambers and allowed to adhere overnight. After the incubation, the cells were washed once with D-PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed once with D-PBS, and then incubated in D-PBS containing 2μM BODIPY 493 / 503 and 5μg / ml DAPI at 37°C for 30 minutes in the dark. Next, the cells were washed three times with D-PBS. Finally, the cells were immediately photographed using a fluorescence microscope.

[0067] (2) Fluorescence detection of lipid droplets

[0068] The cells were seeded into 96-well black transparent bottom culture plates (Corning, NY, USA) and allowed to adhere overnight. After the incubation, the cells were washed once with D-PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed once with D-PBS, and then incubated in D-PBS containing 2 μM BODIPY 493 / 503 and 5 μg / ml DAPI at 37°C for 30 minutes in the dark. Next, the cells were washed three times with D-PBS.

[0069] Finally, the fluorescence intensity of BODIPY493 / 503 (ex485nm, em 520nm) was measured using a multifunctional microplate reader eplex (Tecan, Austria), and then the fluorescence intensity of DAPI (ex 350nm, em 450nm). The lipid droplet content was expressed as the relative fluorescence intensity of BODIPY 493 / 503, that is, the ratio of the fluorescence intensity of BODIPY493 / 503 of the sample to the fluorescence intensity of DAPI of the sample was first calculated, and then the ratio of all samples in the control group was averaged, and both groups were divided by the average to obtain the value for plotting.

[0070] Example 3 Cytotoxicity Detection

[0071] The cytotoxicity of the tested compounds was determined by the CCK-8 method (TransGen, Beijing, China).

[0072] In short, 0.5-1 (hour, h) before the end of culture, CCK-8 solution with a final concentration of 10% was added to each well of the 96-well plate, gently and quickly mixed, and then the plate was immediately returned to the cell culture incubator. After 0.5-1h, the absorbance (absorbance, A) at 450nm (with 630nm as the reference wavelength) was measured using an ELISA reader. The relative cell viability was determined using the following formula: relative cell viability = (A450 / A630 of the experimental group) / (A450 / A630 of the control group).

[0073] Example 4 Fluorometry Accuracy Verification

[0074] The accuracy of the newly established lipid droplet detection method, fluorimetry, was validated by measuring the intraplate variability and consistency with the results of lipid determination by fluorescence microscopy.

[0075] Half of the wells in a 96-well plate were treated with 0.15 mM FFA mixture (0.1 mM oleic acid / 0.05 mM palmitic acid (0.1 OA / 0.05 PA)), and the other half were treated with an equal amount of solvent. Lipid droplets were detected by fluorimetry 24 hours later, and the relative value of lipid droplet content between the 0.1 OA / 0.05 P group and the control group was calculated. The mean and coefficient of variation (CV) of the relative value were calculated to evaluate the intra-plate variability.

[0076] In order to evaluate the consistency at the same time, the cells were seeded at the same density in an 8-well chamber and treated exactly the same as in the 96-well plate. Lipid droplets were then detected by fluorescence microscopy. The relative value of lipid droplet content between the 0.1OA / 0.05PA group and the control group was also calculated, and the mean and CV of the relative value were calculated.

[0077] Example 5 Drug treatment and effectiveness verification

[0078] Cells were seeded into 96-well black clear-bottom culture plates. For 10 known steatosis inducers and 5 negative controls, Huh7 cells were pre-incubated with 63 μM OA and PA mixture (2:1 ratio) for 14 h, and then replaced with fresh medium containing different concentrations of test compounds or the same amount of solvent for another 24 h.

[0079] For ML261, AML12 cells and Hepa1-6 cells were incubated with a mixture of 150 μM OA and PA (2:1 ratio) and different concentrations of ML261 or an equal amount of solvent for 24 h. Stock solutions of all compounds were prepared in DMSO or water and diluted in culture medium to obtain the desired final concentration. The final concentration of DMSO in the culture medium did not exceed 0.5% (v / v).

[0080] Example 6 Screening of potential drugs

[0081] Screening process for new drugs that inhibit lipid accumulation:

[0082] Cells were seeded into 96-well black transparent bottom culture plates. After overnight attachment, Huh7 cells were incubated with a mixture of 150 μM OA and PA (2:1 ratio) (0.1O / 0.05P) and different concentrations of test compounds or an equal amount of solvent for 24 h. Cytotoxicity was then detected using the above CCK-8 method, and lipid droplet content was immediately detected using the above fluorometry.

[0083] Example 7 Statistical Processing

[0084] As shown in the figure, all experimental results are expressed as mean ± SEM or mean ± SD. Statistical analysis was performed using GraphPad Prism software version 8.0.1. Student t test and one-way analysis of variance were used to compare two groups and multiple groups, respectively. P < 0.05 was used as the standard for significant difference.

[0085] Experimental results:

[0086] (I) Determining the optimal concentration of BODIPY 493 / 503 and DAPI for fluorescence measurement

[0087] (1) Since BODIPY 493 / 503 has not been reported to be useful in fluorometry, we first determined the optimal concentration of BODIPY 493 / 503 in fluorometry.

[0088] Huh7 cells were treated with 0.3 mM FFA mixture (0.2 mM oleic acid / 0.1 mM palmitic acid (0.2OA / 0.1PA)) or an equal amount of solvent for 24 h ( Figure 1 The lipid droplet content was then measured by fluorimetry using increasing working concentrations of BODIPY 493 / 503 (1, 2, 4, 8, 16 μM) and the cell number was calibrated using protein concentration.

[0089] The results showed that as the concentration of BODIPY 493 / 503 increased, the absolute fluorescence intensity of BODIPY 493 / 503 in the FFA-treated and control groups also increased ( Figure 1 However, compared with the control group, the fold change of BODIPY493 / 503 fluorescence intensity in the FFA-treated group did not change with the increase of BODIPY 493 / 503 working concentration ( Figure 1 Figure C).

[0090] Therefore, this study determined that 2 μM was the optimal concentration of BODIPY 493 / 503, at which a relatively ideal fluorescence intensity (greater than 10,000 but not too high) could be obtained.

[0091] (2) Determine the optimal concentration of DAPI.

[0092] Similarly, Huh7 cells were treated with FFAs (0.2OA / 0.1PA) or an equal dose of solvent for 24 h. Then, lipid droplet content was detected by fluorimetry using BODIPY 493 / 503 at a working concentration of 2 μM, and cell number was calibrated using increasing working concentrations of DAPI (2.5, 5, 10, 20, 40 μg / ml).

[0093] The results showed that with the increase of DAPI concentration, the absolute fluorescence intensity of DAPI in the FFA-treated group and the control group also increased ( Figure 1 However, compared with the control group, the fold change of BODIPY 493 / 503 fluorescence intensity in the FFA-treated group did not change with increasing DAPI working concentration ( Figure 1 Figure E).

[0094] Therefore, this study determined that 5 μg / ml was the optimal concentration of DAPI, at which a relatively ideal fluorescence intensity (greater than 10,000 but not too high) could be obtained. Therefore, in subsequent experiments, 2 μM BODIPY 493 / 503 and 5 μg / ml DAPI were selected.

[0095] (ii) Fluorometric detection of lipid droplets using BODIPY 493 / 503 was affected by fixation but not by CCK-8

[0096] (1) It is known that BODIPY 493 / 503 can stain both fixed and unfixed cells. Next, the effect of fixation on fluorometry was evaluated. Huh7 cells were treated with different concentrations of FFAs (0.1P, 0.2O / 0.1P, 0.4O / 0.2P) or an equal dose of solvent for 24 h ( Figure 2 Figure A).

[0097] Fluorometry results showed that when cells were fixed, the lipid droplet content of the FFAs-treated group increased in a dose-dependent manner compared with the control group. However, when not fixed, the lipid droplet content of the FFAs-treated group did not change with increasing FFA concentrations compared with the control group ( Figure 2 These results indicate that cells need to be fixed for use of BODIPY 493 / 503 in fluorometry.

[0098] (2) CCK-8 is a safe and non-toxic method for detecting cell viability. In order to save costs and improve efficiency, we planned to first use CCK-8 to detect drug toxicity in the same 96-well plate cells, and then use fluorometry to detect the effects of these drugs on lipid accumulation. Therefore, we further evaluated the effect of CCK-8 on fluorometry. Huh7 cells were treated with different concentrations of FFAs (0.1O / 0.05P, 0.2O / 0.1P, 0.20 / 0.2P, 0.4O / 0.2P) or an equal dose of solvent for 24 h ( Figure 2 Figure C).

[0099] Fluorometric analysis showed that the lipid droplet content in the FFAs-treated group increased in a dose-dependent manner compared with the control group, regardless of whether CCK-8 solution was used or not. Although the absolute fluorescence values ​​of BODIPY 493 / 503 and DAPI changed with CCK-8 solution treatment, the ratio between the two was not affected (data not shown). The relative fluorescence intensity of BODIPY493 / 503 in the FFAs-treated group was also not affected compared with the control group ( Figure 2 These results demonstrate that it is feasible to measure cell viability using CCK-8 prior to detecting lipid droplet content by fluorimetry using BODIPY 493 / 503.

[0100] (III) The quantitative results of lipid droplet detection by fluorescence measurement using BODIPY 493 / 503 are comparable to those by fluorescence microscopy but with higher accuracy

[0101] To test the accuracy of our fluorometric assay, we treated 96-well plates or 8-well chamber Huh7 cells with FFAs (0.1O / 0.05P) or an equivalent amount of solvent for 24 h as described previously ( Figure 3 The lipid droplets were then quantified by fluorimetry or fluorescence microscopy using BODIPY 493 / 503, respectively.

[0102] Fluorometric analysis showed that the lipid droplet content in the FFAs-treated group increased significantly compared with the control group, with an average increase of 1.65 (SD = 0.063, CV = 3.83%) ( Figure 3 Figure B). The results of fluorescence microscopy showed that the lipid droplet content in the FFAs-treated group was significantly increased compared with the control group, with an average increase of 1.66 (SD = 0.17, CV = 10.42%) ( Figure 3 These results indicate that fluorimetry can quantify lipid droplets comparable to, but with greater precision than, fluorescence microscopy.

[0103] 4. Determining the appropriate concentration of FFAs for drug screening

[0104] FFA-induced hepatocyte lipid deposition is an ideal in vitro model for studying steatosis. In order to promote and accelerate the development of lipid accumulation and steatosis-related cell damage, the authors used 0.062mM FFAs (OA / PA, 2:1) for 14h before drug treatment in the reported method for evaluating drug-induced steatosis. When validating the selected 15 known steatosis inducers and negative controls in this application, it is planned to pre-treat with FFAs for 14h in advance; and when evaluating the inhibitory effect of ML261 on lipid deposition, it is planned to incubate with FFAs at the same time. Therefore, the appropriate concentration of FFAs was first determined.

[0105] Huh7 and HepG2 cells were treated with different concentrations of FFAs (0.042O / 0.021P, 0.1O / 0.05P, 0.2O / 0.1P, 0.4O / 0.2P) or the same dose of solvent control for 24 h ( Figure 4 Figure A). The results of the CCK-8 experiment showed that when the FFAs concentration was 0.042O / 0.021P, there was no obvious toxicity to Huh7 and HepG2 cells. Starting from 0.1O / 0.05P, obvious dose-dependent toxicity was observed; at the highest concentration, HepG2 cells showed greater toxicity than Huh7 cells ( Figure 4 Figure B).

[0106] Fluorometric analysis showed that when the FFAs concentration was 0.042O / 0.021P, the lipid content of Huh7 cells did not increase significantly compared with the control group, but it increased significantly in HepG2 cells. Starting from 0.1O / 0.05P, the lipid content of Huh7 cells and HepG2 cells showed a significant dose-dependent increase ( Figure 4 Figure C).

[0107] Based on these results, this application selected 0.042O / 0.021P as the concentration of FFAs pretreatment before drug treatment when evaluating drug-induced steatosis; when evaluating the inhibitory effect of drugs on lipid accumulation, 0.1O / 0.05P was selected for simultaneous incubation, and Huh7 cells were selected for subsequent verification experiments.

[0108] (V) Validation of Fluorometry Using Known Steatosis Inducers and Negative Controls

[0109] Ten known steatosis-inducing drugs and five negative controls were selected for validation.

[0110] The working concentrations of these 15 drugs were selected according to routine procedures. Huh7 cells were treated according to Example 1 above.

[0111] The results of CCK-8 assay showed that all 10 positive drugs showed different degrees of cytotoxicity within the selected working concentration range ( Figure 8 AJ Figure); Fluorescence assay results showed that the lipid droplet content of all 10 positive drugs increased in a dose-dependent manner compared with the control group ( Figure 5 This is consistent with the results reported in existing literature.

[0112] CCK-8 results showed that within the selected working concentration range, among the five negative drugs, citrate and amikacin had no cytotoxicity, while colchicine and rotenone had obvious cytotoxicity even at the lowest concentration, and tert-butylhydroperoxide had obvious cytotoxicity only at the highest concentration ( Fig. 9 AE graph); Fluorometry results showed that all five negative drugs had no significant effect on lipid droplet content compared with the control group ( Figure 6 AE diagram), which is also consistent with the results reported in the existing literature. These results show that the fluorescence determination method of the present application accurately identifies these 15 drugs.

[0113] (VI) Further validation of the fluorescence assay using a known inhibitor of hepatic lipid droplet formation

[0114] ML261 is an inhibitor of hepatic lipid droplet formation. Studies have shown that ML261 can very effectively inhibit lipid droplet formation in a mouse liver cell line (AML12 cells), but has little effect on human liver cells. Therefore, in order to further verify whether the newly established fluorescence assay in this application can be used to screen drugs that inhibit lipid accumulation, AML12 cells were treated according to the example method.

[0115] The results of CCK-8 experiments showed that ML261 had no obvious cytotoxicity in the working concentration range of 0.01-10 μM ( Fig.10 Fluorescence assay results showed that ML261 could reduce lipid droplet content in a dose-dependent manner compared with the control group ( Figure 7 Consistent results were obtained in another mouse-derived liver cell line, Hepa1-6 cells ( Fig.10 Figure B, Figure 7 In addition, consistent results were obtained in Hepa1-6 cells using fluorescence microscopy after the same treatment ( Fig.11 These results indicate that the drug screening system for steatosis based on fluorescence assay is feasible.

[0116] In order to screen potential new drugs that inhibit lipid accumulation, this application developed a cell-based 96-well lipid droplet assay method in this study, in which Huh7 cells were incubated with FFAs (0.1O / 0.05P) and the compound of interest (or an equal dose of solvent) for 24 hours. CCK-8 reagent was added 1 hour before the end of incubation, and cell viability was detected by CCK-8 method 1 hour later. Then, the fluorescent probe BODIPY 493 / 503 was used to detect lipid content by fluorescence measurement, and DAPI was used to calibrate the cell number at the same time. This study confirmed for the first time that BODIPY 493 / 503 can be used for fluorescence measurement, and for the first time confirmed that the use of CCK-8 does not affect the subsequent relative quantitative results of BODIPY 493 / 503 for lipids.

[0117] BODIPY 493 / 503 has been widely used in fluorescence microscopy or flow cytometry, but there is no report of its direct use in adherent cell fluorescence assay. Compared with fluorescence microscopy and flow cytometry, fluorescence assay is more suitable for high-throughput screening because it can reflect overall lipid deposition, sample preparation is simple, data analysis is simple, and it does not rely on expensive instruments. In this application, we confirmed that in adherent cells, the results of lipid quantification by fluorescence assay using BODIPY 493 / 503 are comparable to those of fluorescence microscopy, and the intra-group differences are smaller, indicating that BODIPY 493 / 503 can be used not only for fluorescence assay, but also with higher accuracy. More importantly, the newly established fluorescence assay in this application correctly identified 15 known positive inducers and negative controls of fatty degeneration, and correctly identified the inhibitory effect of ML261 on lipid accumulation. These data show the accuracy and practicality of the method.

[0118] The present application demonstrates that, in the same 96-well plate, after detecting cell viability with CCK-8, lipid accumulation can still be detected by BODIPY493 / 503 by fluorescence assay, and CCK-8 does not affect the relative quantitative results of lipids by BODIPY 493 / 503. This enables the drug screening system of the present application to simultaneously evaluate the effects of drugs on cytotoxicity and lipid deposition without parallel plate processing, thereby greatly saving time and cost.

[0119] FFAs-induced hepatocyte lipid deposition is an ideal in vitro model for studying fatty degeneration, but FFAs are also toxic to cells, so it is very necessary to choose the right concentration. In this application, a series of FFAs concentrations (0.042O / 0.021P, 0.1O / 0.05P, 0.2O / 0.1P, 0.4O / 0.2P) were tested to evaluate their cytotoxicity and induction of lipid deposition. The results showed that at 0.042O / 0.021P, there was no significant change in the cell viability of Huh7 cells and HepG2 cells compared with the control group; Huh7 cell lipid deposition showed an increase without statistical difference, while HepG2 cell lipid deposition showed a slight statistically significant increase. Compared with the control group, within the dose range of 0.1O / 0.05P to 0.4O / 0.2P, the cell viability of Huh7 cells and HepG2 cells showed a significant dose-dependent decrease, and lipid deposition showed a significant dose-dependent increase. Therefore, in this study, Huh7 cells were selected to verify 15 known positive inducers of steatosis and negative controls, and 0.042O / 0.021P was used for pre-incubation for 14h before drug treatment, because this concentration of FFAs treatment had no obvious cytotoxicity, but could accelerate lipid deposition. Studies have shown that ML261 has a significant effect only on lipid deposition in mouse-derived hepatocytes. Therefore, when verifying the inhibitory effect of ML261 on lipid deposition, AML12 cells and Hepa1-6 cells were selected, and FFAs at a concentration of 0.1O / 0.05P were co-incubated with different concentrations of ML261 (or an equal dose of solvent) for 24h. Because although this concentration of FFA has significant cytotoxicity, their cell survival rate is still greater than 80%, but it can induce sufficient lipid accumulation (fold change value greater than 1.5).

[0120] High throughput is crucial for drug screening. In our newly established method in this application, a cell-based 96-well method was used to measure lipid droplets. When screening unknown compounds, since compounds are usually soluble in water or DMSO, if a single dose is selected for preliminary screening and water or DMSO is used as a solvent control, a 96-well plate can test at least 18 compounds at the same time (three replicates for each different treatment). The method of this application can also use 383-well plates or 1536-well plates, but experimental verification is required. In addition, the newly established method of this application can also be used for lipid quantification in cells other than hepatocytes (such as cardiomyocytes).

[0121] In summary, this application has established a simple and reliable method for screening potential inhibitors of fatty liver, which does not rely on expensive instruments and is easy to promote. Although it cannot obtain more liver toxicity parameters at the same time, it can quickly and effectively identify the cytotoxicity of drugs and their effects on lipid accumulation in the early stage of drug development, and will become a useful tool for the development of drugs for the treatment of NAFLD.

[0122] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A cell-based method for screening new drugs for inhibiting lipid accumulation, characterized in that: The following steps are involved: Cell culture: Hepatocyte cell lines were cultured and seeded into culture plates and allowed to adhere overnight; Cell treatment: Add free fatty acids and different concentrations of the test compound or an equal amount of solvent to the culture plate and incubate together; Fluorescence measurement: Wash the cells with D-PBS, then fix the cells with paraformaldehyde, wash them with D-PBS again, then incubate them with D-PBS containing BODIPY 493 / 503 and DAPI in the dark, wash the cells with D-PBS, and finally use a microplate reader to measure the fluorescence intensity of BODIPY 493 / 503 and the fluorescence intensity of DAPI; Data analysis: The ratio of BODIPY 493 / 503 fluorescence intensity to DAPI fluorescence intensity was calculated and used to evaluate lipid content. Drugs that can inhibit lipid accumulation were screened out by comparing the lipid content of the experimental group and the control group.

2. The cell-based new drug screening method for inhibiting lipid accumulation according to claim 1, characterized in that: The liver cell line is a human liver cancer cell line or a mouse liver cell line, the human liver cancer cell line is Huh7 and HepG2, and the mouse liver cell line is a mouse liver cancer cell line Hepa1-6 and a normal mouse liver cell line AML12; the cell treatment step is specifically as follows: adding fresh culture medium containing free fatty acids and a test compound or the same amount of solvent to the culture plate at the same time, and incubating for 24 hours; the free fatty acid is a 0.15mM free fatty acid mixture, and the mixture is 0.1mM oleic acid and 0.05mM palmitic acid.

3. The cell-based new drug screening method for inhibiting lipid accumulation according to claim 1, characterized in that: The microplate reader is a light absorption filter type microplate reader, a full wavelength microplate reader, a fluorescence / chemiluminescence microplate reader, and a multifunctional microplate reader.

4. The cell-based new drug screening method for inhibiting lipid accumulation according to claim 1, characterized in that: In the fluorescence measurement step, the concentration of BODIPY 493 / 503 was 2 μM, and the concentration of DAPI was 5 μg / ml.

5. The cell-based new drug screening method for inhibiting lipid accumulation according to claim 1, characterized in that: After the cell treatment step and before the fluorescence measurement step, a cytotoxicity detection step is also included. The cytotoxicity detection step adopts the CCK-8 method to detect the cytotoxicity, and the relative cell viability is determined by the following formula: relative cell viability = (A450 / A630 of the experimental group) / (A450 / A630 of the control group). The relative viability value is used to evaluate the cytotoxicity of the drug and screen out low-toxic drugs that can inhibit lipid accumulation; wherein the cytotoxicity detection and the fluorescence measurement are carried out first and then in the same culture plate, without the need for parallel plate processing.

6. The cell-based new drug screening method for inhibiting lipid accumulation according to claim 1, characterized in that: The following steps are involved: (1) Cell culture: Hepatocytes were cultured and seeded into 96-well culture plates and allowed to adhere overnight. (2) Cell treatment: free fatty acids and different concentrations of the test compound or an equal amount of solvent were added to the culture plate and incubated for 24 hours; (3) Cytotoxicity detection: CCK-8 method was used to detect cytotoxicity, that is, 1 hour before the end of incubation, CCK-8 solution of 10% of the total volume of the culture medium was added to each well, gently mixed, and returned to the cell culture incubator for continued incubation. After 1 hour, the absorbance at 450 nm was measured using a microplate reader, and 630 nm was used as the reference wavelength to evaluate the relative viability of the cells; (4) Fluorescence measurement: After the above step (3) is completed, the cells are immediately washed with D-PBS, and then fixed with 4% paraformaldehyde for 10 minutes, washed again with D-PBS, and then incubated in D-PBS containing BODIPY 493 / 503 and DAPI at 37°C in the dark for 30 minutes. The cells are washed three times with D-PBS, and finally the fluorescence intensity of BODIPY 493 / 503 and the fluorescence intensity of DAPI are measured using a multifunctional microplate reader. The excitation wavelength for the fluorescence intensity of BODIPY 493 / 503 is 485 nm, and the emission wavelength is 520 nm; the excitation wavelength for the fluorescence intensity of DAPI is 350 nm, and the emission wavelength is 450 nm. (5) Data analysis: The relative cell viability was determined using the following formula: relative cell viability = (A450 / A630 of the experimental group) / (A450 / A630 of the control group), and the relative viability value was used to evaluate drug cytotoxicity. The ratio of BODIPY 493 / 503 fluorescence intensity to DAPI fluorescence intensity was calculated, and the lipid content was evaluated using this ratio. By comparing the cytotoxicity and lipid content of the experimental group and the control group, potential low-toxicity drugs that can inhibit lipid accumulation were screened out.

7. The cell-based new drug screening method for inhibiting lipid accumulation according to claim 1, characterized in that: The drug is obtained by the screening method according to any one of claims 1 to 6.

8. A pharmaceutical composition for treating non-alcoholic fatty liver disease, characterized in that The method comprises the drug according to claim 7 and at least one pharmaceutically acceptable carrier.

9. A method for evaluating the therapeutic effect of a drug on non-alcoholic fatty liver disease, the method comprising: (1) Determining the inhibitory effect of a drug on lipid accumulation using the screening method described in any one of claims 1 to 6; (2) Evaluating the therapeutic effect of the drug on non-alcoholic fatty liver disease based on the inhibitory effect.

10. A screening kit for potential drugs for inhibiting lipid accumulation, characterized in that: The kit comprises: a liver cell line, a 0.15 mM free fatty acid mixture, a 10% CCK-8 solution, 2 μM BODIPY 493 / 503 and a 5 μg / ml DAPI solution, wherein the free fatty acid mixture is 0.1 mM oleic acid and 0.05 mM palmitic acid, and the liver cancer cell line is a human liver cancer cell line.

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