Application of Acsl1 as alcoholic liver disease treatment target
By using Triacsin C to inhibit ACSL1 enzyme activity in cell and animal models, the problem of lipid metabolism disorder in alcoholic liver disease was solved, achieving protection and relief of hepatocyte damage, and providing a simple and economical treatment option.
Patent Information
- Application Number
- CN202511076132.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-01
- Publication Date
- 2025-12-12
AI Technical Summary
In the current technology, lipid metabolism disorders in alcoholic liver disease have not been effectively intervened, leading to aggravated hepatocyte damage. The role of ACSL1 has not been systematically elucidated, and the effects of existing inhibitors are limited.
Triacsin C was used as an inhibitor of ACSL1. By intervening in its expression or enzyme activity in cell and animal models, the conversion of fatty acids to acyl-CoA was inhibited, thereby intervening in lipid accumulation and damage in alcoholic hepatocytes.
It effectively reduces lipid accumulation in hepatocytes, lowers liver damage markers, and protects the liver from alcohol-induced damage. The procedure is simple and low-cost.
Smart Images
Figure CN121109542A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically involving the application of Acsl1 as a therapeutic target for alcoholic liver disease. Background Technology
[0002] Alcoholic liver disease (ALD) is a chronic liver disease caused by long-term or excessive alcohol consumption. Its main characteristics include fatty liver, hepatitis, and liver fibrosis, which may eventually progress to cirrhosis and liver cancer. Studies have shown that alcohol intake can significantly interfere with hepatic lipid metabolism, leading to abnormal lipid accumulation in hepatocytes. Among the abnormal regulatory mechanisms of lipid metabolism, members of the acyl-CoA synthetase long-chain family (ACSL) are key enzymes in fatty acid metabolism, responsible for converting free fatty acids into acyl-CoA, the first step in fatty acid biosynthesis or degradation. The ACSL family includes members such as ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6. Among them, ACSL1 is highly expressed in the liver and is one of the key enzymes regulating hepatic fatty acid metabolism and energy balance. ACSL1 mainly participates in the activation of saturated fatty acids in hepatocytes, and its activation products can further enter the triglyceride synthesis pathway or the mitochondrial β-oxidation pathway. Under metabolic stress or disease conditions, ACSL1 expression is upregulated, promoting lipid synthesis or accumulation. For example, in models of diabetes and non-alcoholic fatty liver disease (NAFLD), high ACSL1 expression is closely related to hepatic lipid deposition. In alcoholic liver disease, the role of ACSL1 has not been systematically elucidated. Existing literature suggests that alcohol can activate ACSL1 transcriptional expression through oxidative stress and inflammatory signaling pathways, potentially promoting abnormal lipid deposition and exacerbating hepatocyte damage. Inhibiting ACSL1 may help to address alcohol-induced lipid metabolism disorders at their source by reducing fatty acid activation and lipid synthesis, thereby alleviating the progression of ALD.
[0003] Triacsin C, a natural product originally isolated from Streptomyces, selectively inhibits ACSL activity, blocking the conversion of fatty acids to acyl-CoA, thereby inhibiting lipid synthesis and accumulation. Therefore, Triacsin C was selected as an inhibitor of Acsl1 in this invention to verify its potential therapeutic value in alleviating lipid accumulation in alcoholic hepatocytes. Summary of the Invention
[0004] In order to overcome the problems in the prior art, this invention intervenes in the expression or enzyme activity of Acsl1 at the cellular level and in animal models to observe its alleviating effect on alcohol-induced lipid accumulation in hepatocytes and liver damage, thereby verifying its feasibility and effectiveness as a therapeutic target, and proposes a technical solution for the application of Acsl1 as a therapeutic target for alcoholic liver disease.
[0005] The present invention is implemented using the following technical solutions:
[0006] In a first aspect, the present invention provides an application of Acsl1 as a therapeutic target for alcoholic liver disease.
[0007] Secondly, this invention provides the use of Acsl1 in the preparation of drugs for the prevention or treatment of alcoholic liver disease.
[0008] Furthermore, the drug for preventing or treating alcoholic liver disease is a reagent that inhibits the activity of the Acsl1 enzyme.
[0009] Thirdly, the present invention provides the use of reagents that inhibit Acsl1 enzyme activity in the preparation of drugs for the prevention or treatment of alcoholic liver disease.
[0010] Furthermore, the reagent used to inhibit Acsl1 enzyme activity is Triacsin C.
[0011] Fourthly, the present invention provides a drug for the prevention or treatment of alcoholic liver disease, wherein the reagent for inhibiting Acsl1 enzyme activity is Triacsin C.
[0012] Fifthly, the present invention provides a method for detecting the effect of inhibiting Acsl1 activity on alcohol-induced hepatocyte damage, comprising the following steps:
[0013] (S.1) Treat AML12 cells with Triacsin C at a concentration of 8 μM-12 μM for 23-25 h;
[0014] (S.2) Add ethanol at a concentration / volume ratio of 1% and continue treatment for 23-25 hours;
[0015] (S.3) Oil Red or Nile Red fluorescent staining is used to assess the degree of intracellular lipid accumulation.
[0016] Sixthly, the present invention provides a method for evaluating the effect of Acsl1 inhibitors in alleviating alcohol-induced liver injury in vivo, comprising the following steps:
[0017] (N.1) An acute alcoholic liver injury model was established using male C57BL / 6J mice;
[0018] (N.2) The concentration of Triacsin C administered to the model animals was 9-11 mg / kg; the frequency of administration of Triacsin C was once every two days for one week;
[0019] (N.3) Blood and liver tissue were collected, serum AST, ALT and TG levels were measured, and lipid accumulation in liver tissue was analyzed.
[0020] The present invention has the following beneficial effects:
[0021] (1) This invention observes the effect of Acsl1 on alleviating alcohol-induced lipid accumulation and liver damage by intervening in the expression or enzyme activity of Acsl1 at the cellular level and in animal models, thereby verifying its feasibility and effectiveness as a therapeutic target.
[0022] (2) The present invention is simple and convenient to operate, has low cost, and is conducive to widespread promotion in production. Attached Figure Description
[0023] Figure 1 The diagram shows the effect of Acsl1 inhibitor treatment on ethanol treatment in the AML12 cell line provided by this invention; Note: a: Representative Oil Red staining results of the control group, ethanol treatment group and ethanol and Triacsin C treatment group; b: Quantitative analysis of the positive staining area in a, the data are expressed as mean ± standard deviation (n=5); c: Flow cytometry results of Nile Red staining.
[0024] Figure 2 Figure e.g., Effect of Acsl1 inhibitor treatment in an acute gavage model of mice; Note: a: Schematic diagram of establishing an acute alcohol gavage mouse model and Triacsin C treatment; b: Schematic diagram of Triacsin C's function in protecting the liver from lipid accumulation; c: Oil Red staining image of mouse liver; d: Statistical analysis of lipid accumulation in liver tissue sections of mice treated with or without Triacsin C after Oil Red staining, data are expressed as mean ± standard deviation (n = 6 mice); Figure eg: Comparison of serum AST, ALT, and TG levels in a mouse model of alcoholic liver injury treated with or without Triacsin C, data are expressed as mean ± standard deviation (n = 6 mice), all statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test. Detailed Implementation
[0025] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.
[0026] Example 1: Method for detecting the effect of Acsl1 inhibition on alcohol-induced hepatocellular damage
[0027] 1. Experimental Methods:
[0028] (S.1) Three groups of AML12 cells were set up: the first group was the negative control group treated with PBS, the second group was the positive control group treated with ethanol at a final concentration of 1% and the third group was the experimental group treated with Triacsin C at a final concentration of 10 μM and ethanol at a final concentration of 1%
[0029] (S.2) Continue treatment with ethanol for a period of time: AML12 normal mouse hepatocytes were seeded in 6-well plates using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. Once the cells reached 50% confluence, Triacsin C was added to a final concentration of 10 μM, and incubation was continued for 24 hours. After treatment, anhydrous ethanol was added to a final concentration of 1% (v / v), and treatment continued for another 24 hours. To minimize the impact of ethanol evaporation, the culture plates were sealed with sealing film during the ethanol treatment process.
[0030] (S.3) Staining assessment of intracellular lipid accumulation: AML12 cells treated with ethanol were gently washed twice with PBS, followed by the following two detection methods:
[0031] (1) Oil Red Staining: A modified Oil Red staining kit was used, following the instructions. Cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and then stained with Oil Red O staining solution for 15 minutes. The cells were then gently rinsed with 75% ethanol to remove background staining, and the nuclei were counterstained with hematoxylin. Finally, the cells were washed with PBS and mounted. The distribution of lipid droplets was observed under a microscope and photographed. Under the microscope, lipid droplets in the tissue cells appeared bright red, the nuclei appeared blue, and the stroma was colorless. Figure 1 a) Five random views were photographed, and ImageJ (version 1.52) was used to perform statistical analysis of the red area. Figure 1 b).
[0032] (2) Nile Red fluorescence staining and flow cytometry: Nile Red working solution to a final concentration of 1 μg / mL was added to the cells and incubated at room temperature in the dark for 30 minutes. After incubation, the cells were washed with PBS, collected, and their fluorescence intensity was detected by flow cytometry using the FITC channel (Ex 488nm / Em 575nm). Data analysis was performed using FlowJo V10 software to compare the differences in lipid droplet accumulation among different treatment groups. Figure 1 c).
[0033] 2. Experimental Results:
[0034] (1) The level of intracellular lipid accumulation was shown by Oil Red and Nile Red staining. The results showed that the level of intracellular lipid accumulation was significantly reduced in the Acsl1 inhibitor Triacsin C treatment group compared with the ethanol-only treatment group. Figure 1 a).
[0035] Example 2: Effects of Acsl1 inhibitors on acute alcoholic liver injury in mice
[0036] 1. Model animals:
[0037] Eight-week-old male C57BL / 6J mice, weighing 20–22g, were selected and acclimatized for one week before the experiment. They were then divided into three groups of six mice each. All animal experiments were conducted in accordance with the institution's animal ethics guidelines.
[0038] 2. Experimental Methods:
[0039] (N.1) Establishment of alcoholic liver injury model: The experimental group mice were given 4 g / kg body weight of anhydrous ethanol (diluted to 20% v / v) by gavage to simulate an acute alcoholic liver injury model in a single gavage; the control group was given an equal volume of water.
[0040] (N.2) Acsl1 inhibitor intervention: Eight hours after ethanol gavage, mice in the experimental group were administered Triacsin C (10 mg / kg) dissolved in 3% DMSO via gavage, while the control group received an equal volume of the solvent. The administration frequency was once every two days for one week. Figure 1 ab).
[0041] (N.3) Blood and Tissue Sample Collection and Testing: After fasting for 8 hours following the last administration, mice were anesthetized, and whole blood was collected and incubated overnight at 4°C. Serum was then separated by centrifugation at 1000×g for 15 minutes. Serum samples were used to determine the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglycerides (TG) using a biochemical analyzer. Figure 2e.g., mouse livers were simultaneously collected, fixed with 4% paraformaldehyde, and stained with Oil Red O, following the same staining procedure as in cell experiments. The stained tissue sections were observed under a microscope and images were taken. ImageJ software (version 1.52) was used to analyze the proportion of Oil Red O positive areas to assess the degree of lipid accumulation in the liver tissue. Figure 2 cd).
[0042] 3. Experimental Results:
[0043] like Figure 1 In the AML12 cell line, ethanol treatment induced lipid accumulation, while pretreatment with Triacsin C reversed this effect. In an acute mouse gavage model, treatment with 10 mg / kg Triacsin C showed no significant hepatotoxicity and did not affect blood lipids. Figure 2 g), and can also resist lipid accumulation in hepatocytes caused by ethanol ( Figure 2 cd), serum liver injury markers showed a significant decrease ( Figure 2 e) reduces the damage of ethanol to liver cells to some extent.
[0044] The above experimental results can be used to develop pharmaceutical compositions for the treatment or prevention of alcoholic hepatocellular damage, wherein the active ingredient and pharmaceutical carrier of the drug include Triacsin C, wherein Triacsin C exerts its effect by inhibiting Acsl1 activity.
Claims
1. Application of Acsl1 as a therapeutic target for alcoholic liver disease.
2. Application of Acsl1 in the preparation of drugs for the prevention or treatment of alcoholic liver disease.
3. The application as described in claim 2, characterized in that, The drug for preventing or treating alcoholic liver disease is a reagent that inhibits the activity of the Acsl1 enzyme.
4. Application of reagents that inhibit Acsl1 enzyme activity in the preparation of drugs for the prevention or treatment of alcoholic liver disease.
5. The application as described in claim 4, characterized in that, The reagent used to inhibit Acsl1 enzyme activity is Triacsin C.
6. A drug for the prevention or treatment of alcoholic liver disease, characterized in that, It contains reagents that inhibit the activity of Acsl1 enzyme.
7. The medicament for preventing or treating alcoholic liver disease as described in claim 6, characterized in that, The reagent used to inhibit Acsl1 enzyme activity is Triacsin C.
8. A method for detecting the effect of inhibiting Acsl1 activity on alcohol-induced hepatocyte damage, characterized in that, Includes the following steps: (S.1) Treat AML12 cells with Triacsin C at a concentration of 8 μM-12 μM for 23-25 h; (S.2) Add ethanol at a concentration / volume ratio of 1% and continue treatment for 23-25 hours; (S.3) Oil Red or Nile Red fluorescent staining is used to assess the degree of intracellular lipid accumulation.
9. A method for evaluating the effect of Acsl1 inhibitors in alleviating alcohol-induced liver injury in vivo, characterized in that, Includes the following steps: (N.1) An acute alcoholic liver injury model was established using male C57BL / 6J mice; (N.2) The concentration of Triacsin C administered to the model animals was 9-11 mg / kg; the frequency of administration of Triacsin C was once every two days for one week; (N.3) Blood and liver tissue were collected, serum AST, ALT and TG levels were measured, and lipid accumulation in liver tissue was analyzed.