Application of RCTR1 in preparation of medicine for preventing or treating fatty liver disease related to metabolic dysfunction

Abstract

The invention provides application of RCTR1 in preparation of a medicine for preventing or treating fatty liver diseases related to metabolic dysfunction. Animal experiments show that the RCTR1 can alleviate diet-induced liver injury, reduce the activity of ALT and AST in serum, and alleviate fat vacuoles, ballooning, hepatocyte necrosis and inflammatory cell infiltration existing in liver tissues; meanwhile, diet-induced liver fat change is reduced, the content of TG in the liver is reduced, and the fatty degeneration degree in liver tissue is reduced; and diet-induced liver fibrosis is inhibited. The RCTR1 has the potential of being developed into drugs for preventing or treating fatty liver diseases related to metabolic dysfunction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the treatment of fatty liver disease associated with metabolic dysfunction, and specifically to the use of RCTR1 in the preparation of drugs for preventing or treating fatty liver disease associated with metabolic dysfunction. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is the most common chronic liver disease in the world and the main cause of elevated serum transaminases in healthy people undergoing physical examinations. The global prevalence of metabolic dysfunction-associated fatty liver disease is estimated to be 32.4%, with a higher prevalence in men than in women, and the prevalence is increasing year by year.

[0003] The causes of metabolic dysfunction-associated fatty liver disease mainly include obesity, genetics, insulin resistance, and intestinal microbial disorders. Metabolic dysfunction-associated fatty liver disease (MASH) is an important link in the development of MAFLD from simple steatosis (NAFL) to liver fibrosis. The main pathological characteristics of MASH are interlobular inflammation, accompanied by varying degrees of steatosis, insulin resistance, hepatocellular damage, and fibrosis, which can lead to diabetes and liver cancer in severe cases. However, there is still no effective treatment for MASH. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides the use of RCTR1 in preparing a drug for preventing or treating fatty liver disease associated with metabolic dysfunction.

[0005] Unless otherwise specified, the parts described in the present invention are all parts by weight and the percentages described are all mass percentages.

[0006] The present invention adopts the following technical solution:

[0007] Use of RCTR1 in the preparation of a drug for preventing or treating fatty liver disease associated with metabolic dysfunction, the structural formula of RCTR1 is as follows:

[0008]

[0009] In one embodiment of the present invention, the metabolic dysfunction-associated fatty liver disease is metabolic dysfunction-associated steatohepatitis.

[0010] In one embodiment of the present invention, the use is to reduce the activities of ALT and AST in serum.

[0011] In one embodiment of the present invention, the application is to reduce the degree of fat vacuoles, ballooning, hepatocyte necrosis, and inflammatory cell infiltration in liver tissue.

[0012] In one embodiment of the invention, the use is to reduce triglyceride content in the liver.

[0013] In one embodiment of the present invention, the use is to reduce the degree of fatty degeneration in liver tissue.

[0014] In one embodiment of the invention, the use is to inhibit diet-induced liver fibrosis.

[0015] Beneficial effects:

[0016] The present invention provides the use of RCTR1 in the preparation of a drug for preventing or treating fatty liver disease associated with metabolic dysfunction. Animal experiments show that RCTR1 can reduce diet-induced liver damage, reduce diet-induced serum ALT and AST activities, reduce fat vacuoles, ballooning, hepatocyte necrosis, and inflammatory cell infiltration in liver tissue; it also reduces diet-induced liver steatosis, reduces the content of TG in the liver, reduces the degree of fatty degeneration in liver tissue; and inhibits diet-induced liver fibrosis. It can be seen that RCTR1 has the potential to be developed into a drug for preventing or treating fatty liver disease associated with metabolic dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the chemical structure diagram of RCTR1;

[0018] Figure 2 This is a graph showing the results of serum ALT / AST activity testing;

[0019] Figure 3 This is the result of HE staining of liver tissue;

[0020] Figure 4 This is the result of the liver tissue TG content test;

[0021] Figure 5 This is the result of Oil Red O staining of liver tissue;

[0022] Figure 6 This is the result of Sirius red staining of liver tissue;

[0023] Figure 7 This is the result of aSMA immunohistochemical staining of liver tissue. DETAILED DESCRIPTION

[0024] In order to further illustrate the present invention and its advantages, the technical solution of the present invention is further illustrated by specific implementation methods below. It should be understood that these embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0025] The raw materials and reagents used in the present invention are all commercially available products, among which RCTR1 was purchased from Cayman Company, USA (RCTR1 structural formula is as follows Figure 1As shown in the figure, hematoxylin and eosin dyes were purchased from Amresco, USA, Oil Red O dye was purchased from Sigma-Aldrich, USA, Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TG) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute, and Sirius staining kit was purchased from Beijing Soleborg Technology Co., Ltd. Common (CD) and Western diet (WD) feeds were from Nantong Trophy Feed Technology Co., Ltd., immunohistochemistry kits and DAB staining kits were from CST, USA, and aSMA goat anti-rabbit antibody was from Wuhan Proteintech.

[0026] Example 1

[0027] 1. Model and grouping: Male C57BL / 6 mice aged 6–8 weeks (Chongqing Medical University Experimental Animal Center) were divided into 3 groups (n=6): a normal diet (CD) feed group, a Western diet (WD) feed model group, and a RCTR1+Western diet (WD) feed group. The normal diet (CD) feed group was fed with a normal diet for 16 weeks; the Western diet (WD) feed model group was fed with WD for 16 weeks to induce a metabolic dysfunction-associated fatty liver disease (MASLD) model; the RCTR1+Western diet (WD) feed group was fed with WD for 8 weeks and then given 5ug / kg of RCTR1 intraperitoneally 3 times a week for 8 weeks. After 16 weeks, the mice were anesthetized by intraperitoneal injection of pentobarbital. After removing the mouse eyeballs with forceps, the whole blood of the mice was collected in an EP tube pre-treated with heparin. The mice were then dissected to fully expose the liver and subcutaneous fat and photographed, and the liver was irrigated from the left ventricle through the systemic circulation using pre-cooled PBS buffer. After the blood in the liver was irrigated, the liver, spleen, fat, pancreas and other tissues of the mouse were removed and placed in pre-cooled saline. The whole blood of the mouse was left to stand at 4°C for 2 hours and then centrifuged (4°C, 3600rpm, 10min). The supernatant was stored in a -80°C ultra-low temperature refrigerator for the detection of ALT, AST, TC and TG in the mouse serum. The middle lobe of the liver was taken and divided into 6 portions of about 0.5cm 3 Tissue blocks. Two of the liver tissues were embedded in OCT and quickly placed in liquid nitrogen for quick freezing and then stored in a -80°C ultra-low temperature refrigerator for Oil Red O staining and DHE staining; another two were fixed in 4% PFA for 48 hours and used for H&E staining and immunohistochemistry detection; the last two were fixed in 4% PFA for 6 hours, and then dehydrated with 15% and 30% sucrose for 12 hours respectively, and then the dehydrated liver tissues were embedded in OCT and stored in a -80°C ultra-low temperature refrigerator for immunofluorescence staining experiments. Another 0.2g of liver tissue was taken in an enzyme-free cryotube, placed in dry ice and mailed for single-cell sequencing. Take 1mm from the remaining middle lobe of the liver 3The liver tissue of different sizes was stored in glutaraldehyde fixative for electron microscopy. Finally, the remaining liver tissue was stored in an ultra-low temperature refrigerator.

[0028] 2. Serum ALT and AST activity determination

[0029] (1) Take the serum out of the -80°C freezer and rewarm it at 4°C for later use;

[0030] (2) Prepare a 96-well plate and add 200 μL of reagent 1 in the kit to each well using a pipette;

[0031] (3) Sample loading: prepare three replicate wells for each test sample, with 10 μL in each replicate well;

[0032] (4) Set the parameters of the microplate reader (temperature 37°C, oscillation during the cycle, total kinetic cycle time 3 min, interval 60 s, absorbance wavelength 340 nm);

[0033] (5) Use a pipette to add 50 μL of reagent 2 in the kit, then immediately place it in the microplate reader and detect ALT and AST activities according to the kit instructions.

[0034] 3. Determination of liver TG content

[0035] (1) Take 0.1 g of liver tissue, add 900 μl of anhydrous ethanol, and ultrasonically grind in an ice-water bath to make a 10% liver tissue homogenate;

[0036] (2) Centrifuge at 2500 rpm for 10 minutes and collect the supernatant;

[0037] (3) Take 2.5 μl of double distilled water, standard and tissue homogenate respectively and add them to 250 μl of working solution. Detect the absorbance of the sample at 510 nm using an enzyme marker, and then calculate the TG content in the liver tissue (mmol / kg fresh weight) using the formula.

[0038] 4. HE staining of liver tissue sections

[0039] (1) Place the paraffin sections of liver tissue in a 70°C oven for 3 h, then quickly remove the sections from the oven, dewax them in xylene, and shake overnight;

[0040] (2) Soak the tissue sections in 100%, 95%, 80%, 75%, and 50% ethanol for 5 min respectively to fully hydrate the tissues;

[0041] (3) After staining with hematoxylin for 9 min, reverse blueing was performed in saturated lithium carbonate for 30 s;

[0042] (4) Then place the tissue section in eosin for staining for 2 minutes;

[0043] (5) Dehydrate the tissue sections using ethanol of different concentrations and seal the sections with neutral resin.

[0044] 5. Oil Red O Staining of Liver Tissue

[0045] (1) Cut the quick-frozen liver tissue into 8-10 μm tissue slices and fix them in 75% ethanol for 10 min;

[0046] (2) Soak tissue sections in double distilled water three times for 5 min each time to wash away the OCT embedding agent in the tissue;

[0047] (3) Soak the tissue sections in 60% isopropanol for 3 min and then stain them in Oil Red O working solution for 10 min;

[0048] (4) Adjust the color under the microscope with 60% isopropanol to wash away the floating color on the sections;

[0049] (5) After washing the tissue sections with double distilled water, stain with hematoxylin for 5 min and then reverse blue in lithium carbonate for 30 s;

[0050] (6) Seal the sections with glycerol-gelatin sealing medium and observe the results under a microscope.

[0051] 6. Sirius Staining of Liver Tissue Sections

[0052] (1) Cut liver tissue paraffin sections into 5 μm thick sections, place them in a 65°C oven for 2 h, remove the tissue sections from the oven, quickly place them in xylene for dewaxing, and shake them overnight;

[0053] (2) Soak the tissue sections in anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and 50% ethanol for 5 min respectively to hydrate the liver tissue;

[0054] (3) After adding Sirius stain solution for 8 minutes, the Sirius stain solution was washed away with double distilled water;

[0055] (4) Soak in 95% ethanol and anhydrous ethanol for 10 seconds respectively, and then dehydrate in xylene for 10 minutes;

[0056] (5) Add 20 μl of neutral resin, cover with a coverslip and remove all bubbles, and observe the red fiber results under an electron microscope.

[0057] 7. Immunohistochemistry of Liver Sections

[0058] (1) Dewaxing: Place the paraffin sections in a 60°C oven, wait for the surface paraffin to melt, and then wash in xylene for 30 min;

[0059] (2) Hydration: The sections were placed in 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and water in sequence for 5 min each time for a total of 25 min for gradient hydration treatment;

[0060] (3) Antigen repair: Place an appropriate amount of citric acid repair solution in a 500 mL beaker and heat it in a microwave oven until it is slightly boiling. Then place the slices on a slice rack and put them in the beaker. Cover the beaker with a plastic wrap with a small hole. Heat on low heat for 30 minutes to keep the liquid slightly boiling. Take out the beaker, place it at room temperature, and let it cool naturally.

[0061] (4) Wash the sections three times with 1× PBS buffer, 5 min each time;

[0062] (5) Quenching: Place the sections in endogenous peroxidase quenching solution for 30 min;

[0063] (6) Wash the sections three times with 1× PBS buffer, 5 min each time;

[0064] (7) Blocking: Add an appropriate amount of 10% animal serum to the tissue spot, place it in a wet box, and incubate it in a 37°C incubator for 1 h;

[0065] (8) Primary antibody incubation: Gently wipe off the excess liquid on the surface of the slice, add the primary antibody solution (1:100) to the tissue point, and incubate in a 4°C refrigerator for 14-18 h;

[0066] (9) Remove the slices and rewarm at room temperature for 20 min;

[0067] (10) Wash the sections three times with 1× PBS buffer, 5 min each time;

[0068] (11) Secondary antibody incubation: Gently wipe off the excess liquid on the surface of the slice, add biotin-labeled secondary antibody solution to the tissue point, and incubate in a 37°C incubator for 1 h;

[0069] (12) Wash the sections three times with 1× PBS buffer, 5 min each time;

[0070] (13) Add an appropriate amount of horseradish peroxidase to the slices and incubate at 37°C for 30 min;

[0071] (14) Wash the sections three times with 1× PBS buffer, 5 min each time;

[0072] (15) Prepare DAB or AEC staining solution according to the instructions, stain, and observe the staining effect under an upright white light microscope. After staining, wash the sections in 1× PBS buffer;

[0073] (16) Hematoxylin staining and blueing: The sections were stained in hematoxylin for 6 min, then washed with slowly running water and blued in saturated lithium carbonate solution for 1 min.

[0074] (17) After gradient dehydration, the slices were placed in xylene and sealed with neutral resin. After the slices were dried, the results were observed under an upright microscope;

[0075] (18) Image J software was used to perform semi-quantitative analysis of the observation results.

[0076] 8. Statistical Analysis Data were evaluated and presented as mean and standard deviation (mean ± SEM). One-way ANOVA was used to analyze significant differences between groups. Unpaired t-test was used to analyze statistical significance between two groups. P values ​​less than 0.05 were considered statistically significant.

[0077] Experimental Results

[0078] 1. RCTR1 can alleviate diet-induced liver damage: Compared with the normal diet (CD) group, the ALT and AST activities in the serum of mice fed with the Western diet were significantly increased (p<0.01). After RCTR1 intervention, the ALT (p<0.05) and AST (p<0.01) activities induced by WD were significantly reduced ( Figure 2 ). Similarly, the results of liver tissue HE staining also found that there were a large number of fat vacuoles in the liver tissue of the WD-fed model group mice, accompanied by typical ballooning, hepatocyte necrosis, inflammatory cell infiltration and fibrosis. After RCTR1 intervention, the above pathological features in the liver of mice were significantly alleviated ( Figure 3 ).

[0079] 2. RCTR1 can reduce diet-induced liver steatosis: Analysis of the TG content in the liver tissue of mice showed that compared with the WD model group, the TG content in the liver of mice treated with RCTR1 was significantly decreased (p<0.01) ( Figure 4 ). Oil Red O dye can specifically adsorb triglycerides (TG) and lipids in liver cells, dyeing fat red. Therefore, Oil Red O staining is used to evaluate the degree of fatty degeneration in liver tissue. The experimental results showed that the red lipid droplets and total percentage area in the liver tissue of mice in the WD feeding model group were larger, while the lipid droplets in the liver tissue of mice in the RCTR1 intervention group were smaller, and the total percentage area was also reduced ( Figure 5 ).

[0080] 3. RCTR1 can inhibit diet-induced liver fibrosis: Liver fibrosis is one of the important signs of late-stage NASH. Its main characteristics are the proliferation of fibrous tissue in the liver, the accumulation of a large amount of extracellular matrix, and the formation of nodules and pseudolobules. Sirius Red staining can dye the fibrous tissue in the liver red to judge the severity of liver fibrosis. Figure 6 Sirius red staining showed that compared with the WD-fed model group, the red fibrous tissue in the liver tissue of the mice in the RCTR1 intervention group was significantly reduced. α-SMA is a marker of hepatic stellate cell activation, and the activation of hepatic stellate cells is directly related to the generation of fibrous tissue. Immunohistochemical staining showed that the activation of hepatic stellate cells in the liver tissue of the mice in the WD-fed model group was significantly enhanced, and RCTR1 intervention could significantly inhibit the activation of hepatic stellate cells induced by the WD diet ( Figure 7 ).

Claims

1. Use of RCTR1 in the preparation of a drug for preventing or treating fatty liver disease associated with metabolic dysfunction. The structural formula of RCTR1 is as follows:

2. The use according to claim 1, characterized in that: The metabolic dysfunction-related fatty liver disease is metabolic dysfunction-related fatty hepatitis.

3. The use according to claim 1 or 2, characterized in that: The application is to reduce the activities of ALT and AST in serum.

4. The use according to claim 1 or 2, characterized in that: The application is to reduce the extent of fat vacuoles, ballooning, hepatocyte necrosis and inflammatory cell infiltration in liver tissue.

5. The use according to claim 1 or 2, characterized in that: The application is to reduce the triglyceride content in the liver.

6. The use according to claim 1 or 2, characterized in that: The application is to reduce the degree of fatty degeneration in liver tissue.

7. The use according to claim 1 or 2, characterized in that: The application is to inhibit diet-induced liver fibrosis.

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