CN112675156A - Application of erythritol in preparation of medicine for treating non-alcoholic fatty liver disease - Google Patents
Application of erythritol in preparation of medicine for treating non-alcoholic fatty liver disease Download PDFInfo
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- CN112675156A CN112675156A CN202110076155.3A CN202110076155A CN112675156A CN 112675156 A CN112675156 A CN 112675156A CN 202110076155 A CN202110076155 A CN 202110076155A CN 112675156 A CN112675156 A CN 112675156A
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Abstract
The invention discloses application of erythritol in preparation of a medicine for treating non-alcoholic fatty liver disease. The pharmacological experiment shows that: erythritol has good therapeutic effect on non-alcoholic fatty liver disease mouse model and liver cell lipid accumulation model. The experimental result proves that the erythritol can effectively reduce the lipid content in the liver cell by regulating the expression of acetyl coenzyme A carboxylase (ACC) and cholesterol regulatory element binding protein 1c (SREBP-1c) of the liver cell; in non-alcoholic fatty liver mice, erythritol significantly reduces lipid accumulation in the liver and can significantly inhibit the levels of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase. In conclusion, erythritol can exert a regulating effect on blood lipids by inhibiting the expression of SREBP-1 and ACC.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of erythritol in preparation of a medicine for treating non-alcoholic fatty liver disease.
Background
Non-alcoholic fatty liver disease (NAFLD) is a syndrome caused by degeneration and accumulation of fat in liver parenchymal cells due to non-alcoholic action, and is classified into simple fatty liver, fatty hepatitis and liver cirrhosis and hepatocellular carcinoma related thereto according to whether diseased tissues are accompanied by inflammatory reaction and fibrosis. The non-alcoholic fatty liver disease is divided into primary and secondary, the primary is related to insulin resistance and heredity, and the secondary is related to obesity, diabetes, high blood fat disease, environment and the like.
The currently available drugs for treating NAFLD mainly include: (ii) insulin potentiating agent: rosiglitazone and pioglitazone, which are thiazolidinediones, can relieve the occurrence of NAFLD by improving hepatic steatosis and insulin sensitivity, but have no obvious effect on relieving hepatic fibrosis; ② the hypolipidemic drugs: fibrates, statins or Robucco, which can alleviate the occurrence of NAFLD by reducing blood lipid; ③ the medicine for treating liver diseases, vitamin E and polyene phosphatidylcholine can play a role in assisting the treatment of liver diseases through antioxidation, anti-inflammation and anti-fibrosis, thereby relieving the occurrence of NAFLD.
However, the long-term taking of the medicines can bring certain toxic and side effects to the liver. Therefore, the problem to be solved is to develop a medicine with small harm to human body and quick action and curative effect. Natural small molecule compounds have been studied extensively as components of Chinese medicine. It has good therapeutic effect on diseases, low adverse side effect, and high safety. Currently, the use of natural small molecule compounds for the treatment of NAFLD has received much attention from researchers.
Erythritol (Erythritol) is prepared by performing enzymolysis and fermentation on starch such as wheat and corn, separating, purifying and drying. Erythritol has little influence on blood sugar, has high tolerance of organism, and has antioxidant activity. However, the therapeutic effect of erythritol on nonalcoholic fatty liver disease has not been studied and reported.
Disclosure of Invention
Technical problem to be solved
In view of the defects of the prior art, the invention provides the application of erythritol in medicaments for preventing and treating non-alcoholic fatty liver disease, and explains the action mechanism of erythritol for reducing fat and protecting liver. In the invention, erythritol is firstly proposed to reduce the lipid level of liver cells and the lipid level of mouse liver, and the contents of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in the mouse liver are obviously reduced, so that the effects of reducing fat and protecting liver are exerted.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: application of erythritol in preparing medicine for treating non-alcoholic fatty liver disease is provided.
Further, the erythritol has the effect of reducing the lipid level in the liver cells.
Further, the non-alcoholic fatty liver disease model of the present invention is mouse non-alcoholic fatty liver disease induced by tyloxapol.
Furthermore, the erythritol has the function of inhibiting the expression of the cholesterol production protein.
Furthermore, the erythritol has the function of inhibiting the expression of fat synthesis protein.
Furthermore, the erythritol can reduce the concentration of glutamic-pyruvic transaminase in mouse liver.
Further, the structural formula of the erythritol is as follows:
further, the molecular weight of erythritol is as follows: 122.120.
further, the erythritol of the invention has a purity of 99% and is purchased from Shanghai-source leaf science and technology Co., Ltd, CAS No.: 149-32-6, goods number: s11140-100 g.
(III) advantageous effects
The application of the erythritol in preparing the medicine for treating the non-alcoholic fatty liver disease has the following beneficial effects:
the invention firstly illustrates the lipid-lowering and liver-protecting effects of erythritol: erythritol (250mg/ml) significantly reduced free fatty acid-induced lipid accumulation in AML12 cells; in addition, erythritol can reduce the lipid content of HEPG2 cells by regulating the expression of acetyl-coa carboxylase (ACC) and cholesterol regulatory element binding protein 1c (SREBP-1 c); in non-alcoholic fatty liver mice, erythritol (200mg/kg) significantly reduced lipid accumulation in the liver and significantly inhibited the levels of aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT). In conclusion, erythritol can exert a regulatory effect on lipids by inhibiting the expression of SREBP-1 and ACC.
Drawings
FIG. 1 is a graph of the oil-Red-O staining of intracellular lipid droplets of AML12 after treatment in example 1;
FIG. 2 is a graph of SREBP-1c and ACC expression in HEPG2 cells after treatment in example 2;
FIG. 3 is a graph showing the measurement of the TG content in the liver of mice after the treatment of example 3;
FIG. 4 is a graph of mouse liver oil red O staining after treatment in example 4;
FIG. 5 is a graph showing the AST content in the liver of mice after the treatment of example 5;
FIG. 6 is a graph of the determination of the ALT content in the liver of mice after the treatment of example 5;
indicates that as compared to the blank group,*p<0.05，**p<0.01; # denotes that in comparison to the model set,#p<0.05，##p<0.01; abbreviations: TG: triglycerides, ACC: acetyl-CoA carboxylase, SREBP-1 c: cholesterol regulatory element binding protein 1c, ALT: glutamic-pyruvic transaminase, AST: glutamic-oxalacetic transaminase.
Detailed Description
The detailed operation method of this example is as follows:
material
Erythritol is provided by Shanghai Yuanye science and technology Co., Ltd, and has a commodity number of S11140-100 g; SPF male C57BL/6 mice, 20-22 g, available from Liaoning Biotechnology Ltd; TG, AST and ALT measurement kits were purchased from Nanjing construction Co., Ltd.
EXAMPLE 1AML12 cell oil red O staining experiment
The experimental method comprises the following steps:
(1) AML12 cells were cultured and seeded in six-well plates. The cell culture medium formulation was 4.5g/L glucose F12 medium, 10% fetal bovine serum and 1% streptomycin and 1% ITS, 40ng/ml dexamethasone (Sigma, D4902-100 mg). Cells were cultured in 5% CO2And a cell culture chamber at 37 ℃ for 12 hours.
(2) The original culture solution was removed and the cells were cultured with F12 containing only 4.5g/L glucose as the culture solution, and the AML12 cells were starved for 3 hours.
(3) Co-incubating AML12 cells with 250mg/ml erythritol and 1000 μ M free fatty acid (oleic acid: palmitic acid ═ 2:1) for 24 hours; among them, the group was divided into a blank group (F12 medium), a model group (1000. mu.M free fatty acid), a control group (250mg/ml erythritol), and an experimental group (250mg/ml erythritol + 1000. mu.M free fatty acid).
(4) The cell culture medium was discarded, and the cells were washed 2 times with PBS and then fixed with 4% paraformaldehyde (ORO Fixative Fixative) for 20-30 minutes.
(5) The fixative was discarded, the cells were washed 2 times with distilled water and rinsed for 5 minutes with 60% isopropanol.
(6) 60% isopropanol is discarded, and 0.5% oil red O staining solution is added for staining for 10-20 minutes.
(7) The staining solution was discarded and the cells were washed 3-5 times with distilled water until there was no excess staining. Adding hematoxylin staining solution, and counterstaining the cell nucleus for 1-2 minutes.
(8) The dye solution is discarded, washed with water for 2-5 times, and the lipid droplets are observed by an optical microscope with the observation multiple of 200 times.
(9) The experimental results are shown in figure 1.
Figure 1AML12 cell oil red O staining results show that the model group can increase the generation of lipid droplets in AML12 cells, and the experimental group erythritol can significantly reduce the lipid droplet increase induced by 1000 μ M free fatty acid.
Experimental example 2Western blot detection of lipid regulatory protein expression in HEPG2 cells
Experimental methods
(1) HEPG2 cells were cultured and plated in six-well plates at a density of 1X 10 per well6And (4) cells. The cell culture medium formula comprises a DMEM culture medium containing 4.5g/L glucose, 10% fetal calf serum and 1% streptomycin. Cells were cultured in 5% CO2And a cell culture chamber at 37 ℃ for 12 hours.
(2) The original culture was removed and replaced with DMEM containing only 4.5g/L glucose as the culture medium, and the cells were starved for 3 hours using HEPG 2.
(3) Co-incubating HEPG2 cells with 250mg/ml erythritol and 1000 μ M free fatty acid (oleic acid: palmitic acid ═ 2:1) for 24 hours; among them, the group was divided into a blank group (DMEM medium), a model group (1000. mu.M free fatty acid), a control group (250mg/ml erythritol), and an experimental group (250mg/ml erythritol + 1000. mu.M free fatty acid).
(4) The medium was aspirated, cells were washed with PBS, scraped, collected in a centrifuge tube, centrifuged at 1000 rpm for 5 minutes. After washing, the cells were collected and pelleted in a clean EP tube.
(5) The experiment was operated at low temperature. Mixing lysate, phosphatase inhibitor and 1% PMSF to obtain protein lysate, adding into EP tube, shaking, and extracting total protein from HEPG2 cell.
(6) The protein samples were separated by SDS-PAGE (polyacrylamide gel) electrophoresis and transferred to a solid phase support-PVDF membrane.
(7) The PVDF membrane was blocked with a 5% skim milk solution at room temperature for 1 hour (or 4 ℃ in a refrigerator for 4 hours).
(8) The PVDF membrane was treated with an antibody (primary antibody) to the target protein, incubated well and left overnight in a refrigerator at 4 ℃.
(9) PVDF membranes were treated with anti-rabbit or anti-mouse secondary antibodies with horseradish peroxidase conjugate for 1 hour at room temperature.
(10) Detecting protein bands by using a hypersensitive light-emitting solution ECL, and calculating the gray value of the protein bands by using ImageJ gel analysis software.
(11) The experimental results are shown in figure 2.
The results in FIG. 2 show that erythritol can inhibit the expression of ACC and SREBP-1c in HEPG2 cells. The above results demonstrate that erythritol can regulate lipid balance.
Example 3 assay of TG in mouse liver
The experimental method comprises the following steps:
(1) the mice are raised in an SPF environment with a temperature of 24 +/-1 ℃ and a relative humidity of 40-80%.
(2) Mice were caged and fasted for 12 hours prior to the experiment, and then intraperitoneally injected with 50mg/kg, 100mg/kg, 200mg/kg erythritol for 1 hour and then 500mg/kg tyloxapol for 12 hours. Mice were randomly divided into seven groups: blank group (physiological saline), model group (500mg/kg tyloxapol), control group (200mg/kg erythritol), drug low dose group (50mg/kg erythritol +500mg/kg tyloxapol), drug medium dose group (100mg/kg erythritol +500mg/kg tyloxapol), drug high dose group (200mg/kg erythritol +500mg/kg tyloxapol), and drug positive control group (100mg/kg fenofibrate +500mg/kg tyloxapol).
(3) The mouse liver was completely removed and cut into pieces according to the purpose of the experiment.
(4) Liver weight was accurately weighed, as per weight (g): adding 9 times volume of normal saline (ml) at a ratio of 1:9, mechanically homogenizing in ice-water bath to obtain 10% homogenate, centrifuging at 2500 rpm for 10 min, collecting supernatant, and diluting with normal saline to appropriate concentration.
(5) The TG content in mouse livers was measured using TG test cassettes and absorbance values for each well were determined using a plate reader according to the instructions.
(6) The experimental results are shown in figure 3.
The results in FIG. 3 show that tyloxapol at a concentration of 500mg/kg significantly increased the TG content in the liver of mice compared to the blank group. Erythritol in the high dose group significantly reduced TG levels in mouse livers compared to the model group (p < 0.05).
Example 4 mouse hepatocyte oil Red O staining
(1) A mouse liver with a proper volume is taken, fixed to the center of a sample holder by an OCT embedding medium, and fixed to a sample stage fixer after the embedding medium turns white after standing.
(2) The distance between the blade and the sample was adjusted and the slide was trimmed (the patch was placed in room temperature with a glass slide).
(3) Taking out the glass slide before dyeing, naturally drying the glass slide, and dyeing the glass slide in oil red O after no water vapor exists on the glass slide.
The experimental result is shown in figure 4, and tyloxapol with concentration of 500mg/kg can obviously increase the lipid accumulation in mouse liver cells. Compared with a model group, the erythritol in the experimental group can obviously relieve the liver lipid accumulation of mice.
EXAMPLE 5 determination of AST and ALT content in mouse liver
(1) - (4) Experimental procedure same as in example 3
(5) According to the instruction, the content of AST and ALT in the mouse liver is detected by using a test box, and the absorbance value of each hole is measured by using a microplate reader.
The results in figure 5 and figure 6 show that tyloxapol at 500mg/kg can significantly increase the ALT and AST content in the liver of mice compared with the blank group. Compared with a model group, the erythritol with different concentrations can obviously reduce the AST and ALT contents in the liver of the mouse.
Claims (8)
1. Application of erythritol in preparing medicine for treating non-alcoholic fatty liver disease is provided.
2. Use according to claim 1, characterized in that: the erythritol has the effect of reducing the lipid level in liver cells.
3. Use according to claim 1, characterized in that: the non-alcoholic fatty liver disease model is mouse non-alcoholic fatty liver disease induced by tyloxapol.
4. Use according to claim 1, characterized in that: the erythritol has the function of inhibiting the expression of a cholesterol-producing protein.
5. Use according to claim 1, characterized in that: the erythritol has the function of inhibiting the expression of fat synthesis protein.
6. Use according to claim 1, characterized in that: the erythritol can reduce the concentration of glutamic-pyruvic transaminase in mouse liver.
7. Use according to claim 1, characterized in that: the structural formula of the erythritol is as follows:
8. use according to claim 1, characterized in that: the molecular weight of the erythritol is as follows: 122.120.
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2021
- 2021-01-20 CN CN202110076155.3A patent/CN112675156A/en active Pending
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Application publication date: 20210420 |