Construction method of hepatocyte lipid accumulation model and application of hepatocyte lipid accumulation model in screening of lipid-lowering functional substances

Abstract

The invention discloses a construction method of a hepatocyte lipid accumulation model and application of the hepatocyte lipid accumulation model in screening of lipid-lowering functional substances, and belongs to the technical field of detection. The method comprises the following steps: culturing AML12 cells by using a DMEM / F-12 culture medium containing 10% of FBS, 1 * ITS and 1% of P / S, carrying out combined intervention for 24-72 hours by using 2%-3% of ethanol and 50-100 [mu] M of palmitic acid for molding, and quantifying the intracellular lipid accumulation degree through BODIPY fluorescence detection. The model simulates pathological mechanisms of alcoholism and high fat diet, AML12 cells retain key enzymes of alcohol metabolism, the cell viability is maintained at 60%-80% after modeling, and the lipid fluorescence intensity is remarkably improved compared with that of a blank group. The model can efficiently screen lipid-lowering and liver-protecting functional substances for relieving alcoholic fatty liver, is sensitive and quantifiable in detection, and provides a reliable tool for screening related medicines and extracts.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and specifically proposes a method for constructing a hepatocyte lipid accumulation model and its application in screening lipid-lowering substances. Background Technology

[0002] With changes in residents' lifestyles, the combination of long-term heavy drinking and a high-fat diet has led to a year-on-year increase in the incidence of alcoholic liver disease (ALD). The pathological progression of alcoholic liver disease begins with alcoholic fatty liver disease (AFLD). Its core pathological mechanism is as follows: after alcohol intake, it is mainly metabolized in the liver. Ethanol is metabolized into acetaldehyde via alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) pathways. This process is accompanied by the production of a large amount of reduced nicotinamide adenine dinucleotide (NADH), which not only inhibits fatty acid β-oxidation in hepatocytes but also promotes fat synthesis. At the same time, the free fatty acids (FFA) provided by a high-fat diet further increase the metabolic burden on liver mitochondria. Together, these factors trigger the large-scale generation of reactive oxygen species (ROS), leading to lipid peroxidation damage, destroying cell membrane structure and organelle function, and ultimately causing abnormal lipid accumulation in hepatocytes. If not intervened in time, it can further develop into alcoholic hepatitis (AH) and alcoholic cirrhosis (ALC), seriously threatening human health.

[0003] Currently, research and screening of active ingredients for alcoholic fatty liver disease heavily rely on in vitro cell models. However, existing models have significant limitations and cannot meet the needs of clinical simulation and efficient screening. Specific problems are as follows: The modeling mechanism is disconnected from clinical pathology: Existing models mostly use single-factor induction. For example, the "sodium palmitate-induced 3T3-L1 adipocyte model" disclosed in CN115851585A only simulates free fatty acid overload with palmitic acid and is used for non-alcoholic fatty liver disease (NAFLD) related research, without involving the effect of alcohol metabolism on lipid accumulation. The "alcohol-stimulated AML12 cell model" disclosed in CN117147815A, although it focuses on the damage of alcohol to cells, only detects indicators such as ethanol metabolism enzyme activity and reactive oxygen species, without designing detection and evaluation links for hepatocyte steatosis (lipid accumulation), a core feature of alcoholic fatty liver disease. The "oleic acid + palmitic acid-induced non-alcoholic fatty liver cell model" disclosed in CN109929794A also lacks alcohol intervention and cannot simulate the dual pathological inducing factors of "alcohol abuse + high-fat diet" in clinical practice, resulting in insufficient targeting of the model for alcoholic fatty liver disease.

[0004] Inappropriate cell line selection affects model fidelity: Commonly used cell lines for in vitro models have metabolic defects or reliability issues: Human hepatocellular carcinoma cell lines (such as HepG2), although highly proliferating, have extremely low CYP2E1 (a key enzyme in alcohol metabolism) expression and are difficult to induce with alcohol, making it impossible to accurately reproduce the metabolic process of alcohol in hepatocytes; the human normal hepatocyte cell line L02 is not included in authoritative depository institutions such as the US ATCC and the European ECACC, and short tandem repeat (STR) sequence analysis shows that L02 cells in most laboratories are cross-contaminated, resulting in the loss of hispatocyte-specific lipid metabolism and alcohol metabolism functions, casting doubt on the reliability and reproducibility of experimental results; adipocyte cell lines such as 3T3-L1 differ significantly from hepatocytes in physiological function and metabolic pathways, and cannot reflect the liver-specific alcohol-lipid interaction damage mechanism.

[0005] The detection methods lack precision and have low screening efficiency: Existing models for detecting lipid accumulation mostly use Oil Red O staining (such as CN115851585A), which can only qualitatively or semi-quantitatively observe lipid droplet distribution and cannot accurately quantify the degree of lipid accumulation; moreover, some models have not established clear "model effectiveness judgment criteria", such as not limiting the range of cell viability (excessive damage will lead to a large number of cell deaths, which cannot reflect lipid accumulation, while insufficient damage is difficult to simulate pathological conditions), resulting in poor model stability and making it difficult to use for batch and efficient screening of functional substances.

[0006] Furthermore, while traditional animal models can simulate alcoholic liver injury to some extent, they suffer from drawbacks such as long dosing cycles (usually more than 4 weeks), complex procedures, high costs, and significant individual variability. Moreover, they require invasive methods for evaluating efficacy, such as liver tissue triglyceride (TG) detection and Oil Red O staining, failing to meet the demands for "rapid, low-cost, and high-throughput" methods in the initial screening stage of active ingredients. Therefore, developing an alcohol-induced hepatocyte lipid accumulation model that accurately simulates clinical pathological mechanisms, has reliable cell lines, and allows for quantifiable detection is crucial to overcoming the bottleneck in screening active ingredients for alcoholic fatty liver disease. Summary of the Invention

[0007] In view of this, the present invention proposes a method for constructing a model of lipid accumulation in hepatocytes induced by alcohol combined with palmitic acid, and the application of this model in screening substances that can alleviate alcohol-induced lipid accumulation in hepatocytes, so as to achieve efficient and accurate in vitro screening of substances related to alcoholic fatty liver.

[0008] The technical solution of this invention is achieved as follows: This invention provides a method for constructing a hepatocyte lipid accumulation model. This model uses normal mouse hepatocytes AML12 as cell carriers and is constructed through combined intervention with alcohol and palmitic acid. Specific steps include: (1) Cell culture: AML12 cells were cultured in DMEM / F-12 complete medium containing 10% fetal bovine serum (FBS), 1× insulin-transferrin-selenium (ITS), and 1% penicillin-streptomycin (P / S) at 37°C in a 5% CO2 incubator until the cell confluence reached 80%-90%; (2) Cell seeding: After digesting the AML12 cells cultured in step (1) with trypsin, the cell density was adjusted to 1×10⁻⁶. 4 / ml-1×10 5 / ml, evenly seeded into 96-well cell culture plates; (3) Combined modeling: After the AML12 cells to be inoculated adhered and grew for 12-24 hours (at which time the cells were in the logarithmic growth phase), anhydrous ethanol and palmitic acid were added to the culture medium for combined intervention, wherein the intervention concentration of anhydrous ethanol was 2%-3% (volume percentage) and the intervention concentration of palmitic acid was 50-100μM. (4) Model validation: After 24-72 hours of combined intervention, cell viability and lipid accumulation were detected. If cell viability decreased to 60%-80% of the unmodeled control group, and the fluorescence intensity of lipid accumulation detected by BODIPY fluorescent dye showed a significant difference compared to the control group (p<0.05), then the hepatocyte lipid accumulation model was obtained. The effectiveness of the model was verified by two indicators: ① Cell viability detection: The cell viability of the model group decreased to 60%-80% of the unmodeled control group (this range ensures that the hepatocytes are in a pathological damage state and avoids the large number of cell deaths affecting the lipid accumulation detection); ② Lipid accumulation detection: The intracellular lipids were stained with BODIPY fluorescent dye. The fluorescence intensity of lipid accumulation in the model group showed a significant difference compared to the control group (p<0.05), indicating that the model was successfully constructed.

[0009] Meanwhile, this invention also provides a method for applying the above-mentioned model in screening effective substances. The method involves using the constructed hepatocyte lipid accumulation model to evaluate or screen substances that have the effect of alleviating alcohol-induced hepatocyte lipid accumulation. The application steps include: While constructing the model (i.e., while adding anhydrous ethanol and palmitic acid in step (3)), different concentrations of the substances to be screened (such as extracts, monomeric components, etc.) were added to the culture system of the 96-well plate. After 24-72 hours of combined intervention modeling, the intracellular lipid accumulation fluorescence intensity of the substance to be screened group was detected by referring to the lipid accumulation detection method (BODIPY fluorescence staining) in the model validation. If the lipid accumulation fluorescence intensity of the substance to be screened group was significantly lower than that of the model group without the substance to be screened (p<0.05), it was determined that the substance to be screened had the effect of alleviating the lipid accumulation of hepatocytes induced by alcohol combined with palmitic acid, that is, it had the potential lipid-lowering and liver-protecting effects.

[0010] The present invention has the following advantages over the prior art: This invention is the first to use a combined model of "2%-3% alcohol + 50-100μM palmitic acid" to accurately simulate the dual pathological factors of alcohol metabolism inhibiting fatty acid β-oxidation and high-fat diet providing free fatty acids in clinical practice. It reproduces the core pathological features of alcoholic fatty liver (lipid accumulation), solves the problem of existing single-factor models being disconnected from clinical pathology, and provides a more reliable in vitro simulation environment for screening effective substances.

[0011] The use of BODIPY fluorescent dye to detect lipid accumulation allows for direct quantification of lipid accumulation levels through fluorescence intensity. This method is more sensitive and accurate than traditional Oil Red O qualitative staining and is compatible with high-throughput operations in 96-well plates. Furthermore, it establishes clear criteria for model effectiveness, including "cell viability of 60%-80%" and "significant difference in fluorescence intensity," avoiding issues of poor model stability. This significantly shortens the screening cycle for active ingredients (only 24-72 hours), reduces screening costs, and demonstrates a significant improvement in efficiency compared to animal models (over 4 weeks).

[0012] This model is specifically designed for alcohol-induced lipid accumulation in hepatocytes and can be directly used for the initial screening of substances with lipid-lowering and liver-protecting effects related to alcoholic fatty liver. It covers a variety of substance types, including extracts from food and medicine, chemical drug monomers, and natural product extracts (such as the kudzu root, wolfberry, and mulberry leaf extracts verified in the examples). It provides key technical support for drug development and health food development for alcoholic liver disease and has broad application prospects. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a graph showing the effect of ethanol concentration on AML12 cell viability in Example 2 of the present invention; Figure 2 This is a graph showing the effect of palmitic acid concentration on AML12 cell viability in Example 2 of the present invention; Figure 3 This is a comparison of the fluorescence intensity of BODIPY staining in detecting lipid accumulation in hepatocytes in Examples 1 and 2 of the present invention. Figure 4 This is a comparative diagram showing the effects of the combined ethanol and palmitic acid modeling on AML12 cell viability and lipid accumulation in Example 2 of the present invention. Figure 5This is a comparison of the fluorescence intensity of lipid accumulation in AML12 cells after intervention with different medicinal and edible extracts in Example 1 of the present invention. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 Experimental steps Step 1: Cell Preparation Mouse normal hepatocytes AML12 were cultured in DMEM / F-12 complete medium containing 10% fetal bovine serum (FBS), 1× insulin-transferrin-selenium (ITS), and 1% penicillin-streptomycin (P / S) in a 37°C, 5% CO2 incubator. When the cells reached 80% confluence, 0.25% trypsin was added for 2.5 min of digestion. After digestion, the cells were collected by centrifugation, resuspended in the same complete medium, and the cell density was adjusted to 5×10⁶ cells / year. 4 / ml, evenly seeded into 96-well cell culture plates, with an seeding volume of 100μl per well.

[0017] Step 2: Grouping and Intervention Cells in 96-well plates were incubated for 18 hours. Once the cells had fully adhered to the plates, they were divided into 5 groups, with 3 replicates per group. Control group: Only fresh DMEM / F-12 complete culture medium was replaced, with no other interventions; Model group: Replace with complete culture medium containing 2.5% anhydrous ethanol (volume percentage) + 80 μM palmitic acid; Kudzu root extract group: The complete culture medium containing 2.5% anhydrous ethanol + 80 μM palmitic acid + kudzu root extract was replaced, and the concentrations of kudzu root extract were 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively. Goji berry extract group: Replace with complete culture medium containing 2.5% anhydrous ethanol + 80μM palmitic acid + goji berry extract, with a goji berry extract concentration of 100μg / ml; Mulberry leaf extract group: The culture medium was replaced with a complete medium containing 2.5% anhydrous ethanol + 80 μM palmitic acid + mulberry leaf extract, and the concentration of mulberry leaf extract was 100 μg / ml; All groups were placed in a 37℃, 5% CO2 incubator for 48 hours for intervention.

[0018] Performance verification (1) Cell viability assay (CCK-8 assay) After the intervention, add 10 μl of CCK-8 reagent to each well, gently shake the 96-well plate to mix the reagent evenly, and incubate in an incubator for 2 hours; use an ELISA reader to detect the absorbance value of each well at a wavelength of 450 nm, and calculate the cell viability of each group with the cell viability of the blank group as 100% (cell viability = (absorbance of experimental group - absorbance of blank reagent) / (absorbance of blank group - absorbance of blank reagent) × 100%).

[0019] (2) Lipid accumulation detection (BODIPY fluorescence staining method) After the cell viability assay was completed, the culture medium in the wells was discarded, and the cells were gently washed twice with phosphate-buffered saline (PBS). 100 μl of PBS containing 5 μM ODI PY 493 / 503 fluorescent dye was added to each well, and the cells were incubated in a dark incubator for 30 min. The staining solution was discarded, and the cells were washed twice with PBS to remove unbound dye. The fluorescence intensity of each well was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 515 nm. The ratio of the fluorescence intensity of each group to that of the model group was calculated.

[0020] Experimental results

[0021] The results show: 1. The model constructed in this invention (2.5% ethanol + 80 μM palmitic acid intervention in AML12 cells) meets the efficacy criteria. The cell viability of the model group decreased to 70.2% compared with the blank group (within the 60%-80% effective damage range), and the fluorescence intensity of lipid accumulation was significantly increased compared with the blank group with extremely significant difference (p < 0.001), successfully reproducing the core pathological features of lipid accumulation in hepatocytes of alcoholic fatty liver. 2. This model can efficiently screen for active ingredients: the three medicinal and edible extracts, kudzu root, wolfberry, and mulberry leaf, can all significantly reduce intracellular lipid accumulation. Among them, kudzu root extract showed a concentration-dependent lipid-lowering effect (the lipid accumulation reduction reached 47.0% at 200 μg / ml, p < 0.001), while wolfberry and mulberry leaf extracts also achieved a reduction of about 30% at a concentration of 100 μg / ml (p < 0.01).

[0022] Example 2 Experimental steps Step 1: Cell Preparation Same as in Example 1, AML12 cells were cultured to 80% confluence, then digested and the density adjusted to 5 × 10⁶ cells / year. 4 / ml, seeded in 96-well plates, and cultured for 18 hours until cells adhered.

[0023] Step 2: Concentration gradient design and intervention Anhydrous ethanol concentration gradients of 1%, 2%, 2.5%, 3%, and 4% (volume percentage) and palmitic acid concentration gradients of 30 μM, 50 μM, 80 μM, 100 μM, and 150 μM were set up to form 25 "ethanol + palmitic acid" combined groups (5×5). A blank group (no intervention) was also set up, with 3 replicates for each group. The corresponding concentrations of ethanol and palmitic acid were added to the wells of each combined group, and the blank group was replaced with fresh culture medium. All groups were intervened for 48 h.

[0024] Performance verification Similar to Example 1, cell viability (CCK-8 assay) and lipid accumulation fluorescence intensity (BODIPY staining assay) of each group were detected to determine whether each group met the model validity criteria of "cell viability of 60%-80% and lipid accumulation fluorescence intensity significantly different from the blank group (p<0.05)".

[0025] Experimental results

[0026] The results show that: 1. Only the concentration combination specified in this invention (2%-3% ethanol + 50-100 μM palmitic acid) can simultaneously meet the model validity criteria of "cell viability 60%-80%" and "significant lipid accumulation (p < 0.05)". Among them, 2.5% ethanol + 80 μM palmitic acid is the optimal combination (cell viability 70.2%, lipid accumulation fluorescence intensity is 4.5 times that of the blank group, p < 0.001). 2. Below this concentration range (e.g., 1% ethanol + 80 μM palmitic acid), cell viability is too high (92.5%) and there is no statistically significant difference in lipid accumulation (p > 0.05), making it impossible to simulate pathological damage; above this concentration range (e.g., 4% ethanol + 80 μM palmitic acid, 2.5% ethanol + 150 μM palmitic acid), cell viability is below 60%, and massive cell death disrupts the basis for lipid accumulation detection, making it impossible to construct an effective model.

[0027] Example 3 Experimental steps Step 1: Cell Preparation As in Example 1, AML12 cells were seeded in 96-well plates and cultured for 18 hours until adherence.

[0028] Step 2: Intervention and Vitality Gradient Construction Five intervention protocols were set up, with three replicates in each protocol: Control group: No intervention; Low-damage group: 2% ethanol + 50μM palmitic acid (expected activity 75%-80%); Moderately damaged group: 2.5% ethanol + 80μM palmitic acid (expected activity 65%-75%); High damage group: 3% ethanol + 100μM palmitic acid (expected activity 60%-65%). Excessive damage group: 4% ethanol + 120 μM palmitic acid (expected activity <60%). All groups received intervention for 48 hours.

[0029] Performance verification Similar to Example 1, the fluorescence intensity of cell viability and lipid accumulation in each group was detected, and the correlation between viability range and lipid accumulation was analyzed.

[0030]

[0031] The results show: 1. When cell viability is 60%-80% (low / medium / high damage group), the fluorescence intensity of lipid accumulation is 3.3-4.6 times that of the blank group, and the difference is significant (p<0.01 or p<0.001). This can clearly distinguish the difference in lipid accumulation between the "model group" and the potential efficacy substance group, providing an accurate basis for screening. 2. When cell viability was below 60% (excessive damage group), although there was a difference in lipid accumulation compared with the control group (p < 0.05), the large number of cell deaths made the lipid accumulation detection easily interfered with, making it impossible to accurately determine the lipid-lowering effect of the active ingredient and thus lacking practical screening capability.

[0032] Comparative Example 1 Experimental steps Step 1: Cell Preparation As in Example 1, AML12 cells were seeded in 96-well plates and cultured for 18 hours until adherence.

[0033] Step 2: Grouping and Intervention Divided into 4 groups, with 3 duplicate holes in each group: Control group: No intervention; The combined composition of this invention is: 2.5% ethanol + 80 μM palmitic acid; Alcohol only: 2.5% ethanol (palmitic acid-free); Palmitic acid only group: 80 μM palmitic acid (ethanol-free); All groups received intervention for 48 hours.

[0034] Performance verification Same as in Example 1, cell viability and lipid accumulation fluorescence intensity were detected.

[0035] Comparison results

[0036] The results show that: 1. Neither the alcohol-only group (2.5% ethanol) nor the palmitic acid-only group (80 μM palmitic acid) could induce significant lipid accumulation. The fluorescence intensity of lipid accumulation in both groups was only 1.9-2.2 times that of the blank group, and there was no statistical difference with the blank group (p>0.05). The clinical dual pathological cause of "alcohol metabolism + high-fat diet" could not be reproduced. 2. The combined model group of this invention (2.5% ethanol + 80 μM palmitic acid) achieved a synergistic effect of alcohol inhibiting fatty acid β-oxidation and palmitic acid providing free fatty acids, resulting in a lipid accumulation fluorescence intensity 4.5 times that of the blank group (p < 0.001), successfully simulating the core pathological mechanism of alcoholic fatty liver.

[0037] Comparative Example 2 Experimental steps Step 1: Cell Preparation AML12 cells (of this invention), HepG2 cells (human liver cancer cells), and L02 cells (human liver cells, cross-contamination confirmed by STR testing) were cultured separately in their respective complete culture media until 80% confluence, then digested and adjusted to a density of 5 × 10⁻⁶ cells / year. 4 / ml, inoculated into 96-well plates, and cultured for 18 hours until adherent.

[0038] Step 2: Intervention Plan All cells were treated with a combination of 2.5% ethanol and 80 μM palmitic acid. The control group was the no-intervention group for each cell line. Each group had 3 replicates and the intervention lasted for 48 hours.

[0039] Performance verification (1) Detection of key enzymes in alcohol metabolism (CYP2E1 activity): The CYP2E1 activity (U / mg protein) of each group of cells was detected using a CYP2E1 enzyme activity detection kit. (2) Lipid accumulation detection: Same as BODIPY staining method in Example 1.

[0040] Comparison results

[0041] The results show that: 1. Human hepatocellular carcinoma cells HepG2 showed low expression of the key enzyme in alcohol metabolism (CYP2E1) (activity only 0.5 U / mg protein), making it impossible to mimic the metabolic process of alcohol in hepatocytes. The fluorescence intensity of lipid accumulation was only 1.8 times that of the control group (p>0.05), rendering the model invalid. 2. Due to cross-contamination, human hepatocyte L02 cells lost their hepatocyte metabolic characteristics (CYP2E1 activity 1.2 U / mg protein), and the fluorescence intensity of lipid accumulation was only 2.1 times that of the control group (p>0.05), raising doubts about the reliability of the experimental results; 3. Normal mouse hepatocytes AML12 retain the complete alcohol metabolism enzyme system (CYP2E1 activity 2.8 U / mg protein), which can achieve synergistic simulation of alcohol metabolism and lipid accumulation. The fluorescence intensity of lipid accumulation is 4.5 times that of the blank group (p < 0.001), and the model is stable and effective.

[0042] Comparative Example 3 Experimental steps Step 1: Cell Preparation As in Example 1, AML12 cells were seeded in 96-well plates and cultured for 18 hours until adherence.

[0043] Step 2: Grouping and Intervention Divided into 4 groups, with 3 duplicate holes in each group: This invention uses: 2.5% ethanol + 80 μM palmitic acid; Low-concentration ethanol group: 1% ethanol + 80μM palmitic acid; High-concentration ethanol group: 4% ethanol + 80μM palmitic acid; High concentration palmitic acid group: 2.5% ethanol + 150μM palmitic acid; All groups received intervention for 48 hours.

[0044] Performance verification Same as in Example 1, cell viability and lipid accumulation fluorescence intensity were detected.

[0045] Comparison results

[0046] The results show that: 1. The low-concentration ethanol group (1% ethanol + 80 μM palmitic acid) had insufficient alcohol damage, resulting in a cell viability of up to 92.5% and no statistically significant difference in lipid accumulation (p > 0.05), which could not simulate the pathological state. 2. In the high-concentration ethanol group (4% ethanol + 80 μM palmitic acid) or the high-concentration palmitic acid group (2.5% ethanol + 150 μM palmitic acid), the cell viability decreased to 45.1%-58.9% (<60%) due to excessive damage. The massive cell death led to the distortion of lipid accumulation detection, and the model was invalid. 3. Only the concentration group of this invention (2.5% ethanol + 80μM palmitic acid) can balance "pathological damage" and "detection feasibility" and meet the model validity standard.

[0047] Comparative Example 4 Experimental steps Step 1: Cell Preparation and Intervention Same as in Example 1, a blank group, a model group, and a kudzu root extract group (100 μg / ml) were set up, and the intervention lasted for 48 hours.

[0048] Step 2: Parallel Validation of Two Detection Methods (1) Oil Red O staining method (traditional): Discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 30 min; add Oil Red O staining solution for 15 min, wash three times with PBS to remove excess staining solution; add isopropanol to dissolve the stained lipid droplets, and detect the absorbance at 510 nm wavelength using an ELISA reader to obtain a semi-quantitative lipid droplet content. (2) BODIPY fluorescence detection method (this invention): Same as in Example 1, the fluorescence intensity is detected and quantified.

[0049] Comparison results

[0050] The results show that: 1. Traditional Oil Red O staining can only semi-quantitatively measure lipid droplet content and cannot identify the lipid-lowering effect of kudzu root extract (100 μg / ml). The absorbance of lipid droplets in the extract group was not statistically different from that in the model group (p>0.05), which cannot meet the screening requirements. 2. The BODIPY fluorescence detection method of this invention can accurately quantify the degree of lipid accumulation. The fluorescence intensity of the kudzu root extract group was significantly lower than that of the model group (68.2%, p<0.01), which can clearly distinguish the effects of the active ingredients and is suitable for high-throughput operation of 96-well plates.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a model of lipid accumulation in hepatocytes, characterized by, The method comprises the following steps: (1) using DMEM / F-12 complete culture medium containing 10% fetal bovine serum, 1×insulin-transferrin-selenium, 1% penicillin-streptomycin, culturing mouse normal hepatocytes AML12 to 80%-90% confluence; (2) after trypsin digestion of the AML12 cells cultured in step (1), inoculating into a cell culture plate; (3) after the cells adhere, adding anhydrous ethanol and palmitic acid into the culture medium for combined intervention modeling, wherein the intervention concentration of anhydrous ethanol is 2%-3% by volume percentage, and the intervention concentration of palmitic acid is 50-100 μM; (4) after combined intervention modeling for 24-72 h, obtaining a hepatocyte lipid accumulation model.

2. The construction method of claim 1, wherein, In step (2), the cells are seeded into 96-well cell culture plates at a seeding density of 1 x 10 4 / ml to 1 x 10 5 / ml.

3. The construction method of claim 1, wherein, In step (3), the time for cell adhesion is 12-24 h, and the cells are in the logarithmic growth phase before modeling.

4. The construction method of claim 1, wherein, In step (4), after modeling is completed, the cell viability of the hepatocyte lipid accumulation model is reduced to 60%-80% compared with the cell viability of the blank group without modeling.

5. The construction method of claim 1 wherein, In step (4), after modeling is completed, the intracellular lipids are stained with BODIPY fluorescent dye, and the detection shows that the lipid accumulation fluorescence intensity of the model group has a significant difference from that of the blank group, i.e. p<0.

05.

6. The use of the hepatocyte lipid accumulation model according to any one of claims 1 to 5 for screening of lipid-lowering efficacy substances, characterized in that, The application steps comprise: adding a to-be-screened substance into the culture system of the hepatocyte lipid accumulation model constructed by the method of any one of claims 1-5, detecting the intracellular lipid accumulation degree after culture, and determining that the to-be-screened substance is a lipid-lowering effective substance with the effect of relieving alcohol-induced hepatocyte lipid accumulation if the lipid accumulation degree of the to-be-screened substance group is significantly reduced compared with that of the model group without the to-be-screened substance.

7. Use according to claim 6, wherein The to-be-screened substance is selected from at least one of edible and medicinal extract, chemical drug monomer or natural product extract.

8. The use according to claim 6, wherein The to-be-screened substance is added into the culture system at the same time of constructing the hepatocyte lipid accumulation model.

9. The use according to claim 6, wherein The significant reduction of the lipid accumulation degree of the to-be-screened substance group compared with that of the model group without the to-be-screened substance is that the lipid accumulation fluorescence intensity of the to-be-screened substance group after BODIPY fluorescent staining has a significant difference from that of the model group, i.e. p<0.

05.

10. The use according to claim 7, wherein the compound is ###00003### 8 or ###00004### 9. The edible and medicinal extract is selected from at least one of pueraria extract, medlar extract or mulberry leaf extract.

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