Use of β-hydroxybutyric acid or its salt for preparing a medicament for treating liver fibrosis

Abstract

The present invention discloses the use of β-hydroxybutyric acid or its salt for preparing a medicament for treating liver fibrosis, and claims the use of β-hydroxybutyric acid or its pharmaceutically acceptable salt for preparing a medicament for treating liver fibrosis. The present invention proves through various experiments that β-hydroxybutyric acid or its pharmaceutically acceptable salt can significantly inhibit liver fibrosis with excellent effects. This indicates that β-hydroxybutyric acid or its pharmaceutically acceptable salt has the prospect of being developed and prepared into a medicament for treating liver fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of β-hydroxybutyric acid or its salts for preparing a medicament for treating hepatic fibrosis. Background Art

[0002] Hepatic fibrosis (HF) is the result of abnormal deposition of various extracellular matrices (ECMs) caused by an excessive repair response induced by various chronic liver diseases such as genetically related diseases, chronic viral hepatitis, alcoholic hepatitis, cholestasis, and drug-induced liver injury. The occurrence and development mechanisms of hepatic fibrosis are related to hepatic stellate cells (HSCs), hepatic macrophages, endoplasmic reticulum stress (ERs), and autophagy. Hepatic fibrosis is a dynamic process that will continue to develop into liver cirrhosis and even hepatocellular carcinoma without intervention. Therefore, delaying or reversing the occurrence and development of hepatic fibrosis is of great significance. Summary of the Invention

[0003] The object of the present invention is to provide the use of β-hydroxybutyric acid or its salts for preparing a medicament for treating hepatic fibrosis.

[0004] The above object of the present invention is achieved by the following technical solutions:

[0005] The use of β-hydroxybutyric acid or its pharmaceutically acceptable salts for preparing a medicament for treating hepatic fibrosis.

[0006] Preferably, the medicament uses β-hydroxybutyric acid or its pharmaceutically acceptable salts as the active ingredient and is formulated into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

[0007] More preferably, the carrier is in solid, liquid or semi-solid form.

[0008] More preferably, the dosage form includes injections, tablets, capsules, and drops.

[0009] Advantageous Effects:

[0010] The present invention proves through various experiments that β-hydroxybutyric acid or its pharmaceutically acceptable salts can significantly inhibit hepatic fibrosis with excellent effects. This indicates that β-hydroxybutyric acid or its salts have the prospect of being developed into a medicament for treating hepatic fibrosis. Brief Description of the Drawings

[0011] Figure 1: BHB inhibits TGF-β-induced activation of LX-2. qRT-PCR analysis of the mRNA of fibrosis genes (1A); Western blot analysis of the protein expression levels of α-SMA and COL1A1 (1B).

[0012] Figure 2 : BHB inhibits the activation of primary mouse hepatic stellate cells. qRT-PCR analysis of the mRNA expression levels of fibrosis genes (2A); Immunofluorescence staining analysis of the positive expression level of α-SMA (2B).

[0013] Figure 3 : BHB ameliorates BDL-induced inflammatory liver injury and liver fibrosis in mice. Determination of the contents of ALT and AST in mouse serum (3A, 3B); H&E staining and Sirius red staining of the liver (3C); Changes in the transcriptional levels of fibrosis-related genes Acta2, Col1a1, Col3a1, Ctgf, and Tgfb1 in the mouse liver (3D).

[0014] Figure 4 : BHB ameliorates CCl4-induced chronic liver injury and fibrosis development in mice. Determination of the contents of ALT and AST in mouse serum (4A, 4B); H&E staining and Sirius red staining of the liver (4C); Changes in the transcriptional levels of fibrosis-related genes Acta2, Col1a1, Col3a1, Ctgf, and Tgfb1 in the mouse liver (4D).

[0015] Figure 5 : Sodium BHB ameliorates BDL-induced inflammatory liver injury and liver fibrosis in mice. Determination of the contents of ALT and AST in mouse serum (5A, 5B); H&E staining and Sirius red staining of the liver (5C).

[0016] Figure 6 : Sodium BHB ameliorates CCl4-induced chronic liver injury and fibrosis development in mice. Determination of the contents of ALT and AST in mouse serum (6A, 6B); H&E staining and Sirius red staining of the liver (6C). Detailed implementation manners

[0017] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention thereby.

[0018] Example 1: β-Hydroxybutyric acid for the treatment of liver fibrosis

[0019] I. Experimental materials

[0020] 1. Experimental animals

[0021] C57BL / 6 male mice, 6-8 weeks old, were housed in separate cages at a constant temperature (23-25°C) and purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0022] 2. Instruments and equipment

[0023] FRESCO17 high-speed refrigerated centrifuge (ThermoFisher Scientific, USA); JXFSTPRP-24 tissue grinder (Shanghai Jingxin Company); biological safety cabinet (ThermoFisher Scientific, USA); CCL-170B-8 cell culture incubator (ThermoFisher Scientific, USA); FV3000 laser confocal microscope (Olympus, Japan); Synergy 2 multifunctional microplate reader (BioTek, USA); Thermomixer comfort metal bath heater (eppendorf, Germany); Orbital Shaker TS-10 decolorization shaker (Kylin-Bell Lab Instraments Co., Ltd., Haimen City); XI Cell vertical electrophoresis tank (BIO-RAD, USA); PowerPac Basic electrophoresis instrument (BIO-RAD, USA); Tanon-520 gel imaging system (Shanghai Tianneng Technology Co., Ltd.); QuantStudio3 real-time fluorescence quantitative PCR instrument (ThermoFisher Scientific, USA).

[0024] 3. Experimental reagents and drugs

[0025] Carbon tetrachloride (CCl4) (Shanghai McLean Biochemical Technology Co., Ltd.); Corn Oil (Corn Oil) (Shanghai Yuanye Biotechnology Co., Ltd.); β-hydroxybutyric acid (BHB) (Sigma-Aldrich, Germany); β-hydroxybutyric acid sodium salt (BHB sodium salt) (Santa Cruz, USA); RPMI.1640 culture medium (including double antibody) (Nanjing Keygen Biotechnology Co., Ltd.); Percoll (Sigma-Aldrich, USA); DNase I (Invitrogen, USA); Nycodenz (Sigma-Aldrich, USA); TGF-β (MedChemExpress, USA); Triton TMX-100 (Sigma-Aldrich, USA); FITC-conjugated goat anti-rabbit IgG (Yeasen Biotech Co., Ltd., Shanghai); anti-fluorescence quenching mounting medium (strong) (Beyotime Biotechnology Co., Ltd., Shanghai); DAPI staining solution (Beyotime Biotechnology Co., Ltd., Shanghai); RIPA strong lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai); 30% (w / v) acrylamide / methylene bisacrylamide solution (Sangon Biotech, Shanghai); 1 M pH = 6.8 Tris-HCl buffer solution and 1.5 M pH = 8.8 Tris-HCl buffer solution (Beyotime Biotechnology Co., Ltd., Shanghai); protein loading marker (ThermoFisher Scientific, USA); RNAisolater Total RNA Extraction Reagent (Yeasen Biotech Co., Ltd., Shanghai); qPCR SYBR Green Master Mix (antibody method, Low Rox) (Yeasen Biotech Co., Ltd., Shanghai); RT-gDNA digestion SuperMix (Yeasen Biotech Co., Ltd., Shanghai); Glutamic-pyruvic transaminase (GPT / ALT) activity detection kit (Nanjing Jiancheng Bioengineering Institute); Glutamic-oxaloacetic transaminase (GOT / AST) activity detection kit (Nanjing Jiancheng Bioengineering Institute).

[0026] II. Experimental methods

[0027] 1. Activation induction and grouping of hepatic stellate cells (cell model)

[0028] 1.1 Activation induction and grouping of human hepatic stellate cell line LX2

[0029] Divided into control group, model group, and model drug administration group. LX2 was starved for 4 hours using RPMI-1640 medium containing double antibiotics without fetal bovine serum. After starvation, the model group was replaced with RPMI-1640 medium containing double antibiotics with 1% FBS and TGF-β was added to the medium at a concentration of 10 ng / mL for maintenance growth and activation induction. The model drug administration group was replaced with a drug-containing medium (10 ng / mL TGF-β + 0.05 mM / 0.5 mM / 5 mM BHB), and the control group was cultured only with 1% RPMI-1640 medium. Subsequent experiments were carried out after 24 hours of culture.

[0030] 1.2 Activation induction and grouping of primary mouse hepatic stellate cells

[0031] Primary hepatic stellate cells can be spontaneously activated after 7 days of culture, and the culture medium is changed every 24 hours during this period. After activation, they are divided into a model control group and model drug administration groups with 0.05 mM, 0.5 mM, and 5 mM BHB.

[0032] Extraction of primary mouse hepatic stellate cells: Mice were anesthetized with sodium pentobarbital and fixed on the operating table. The abdomen was incised to expose the liver. The liver was gently lifted with a cotton swab moistened with normal saline to expose the portal vein and inferior vena cava. The front-end needle of the peristaltic pump tubing was inserted into the portal vein and fixed with an arterial clamp. The peristaltic pump was turned on at a perfusion rate of 3 mL / min. If the catheterization was successful, the liver should turn white rapidly. Once the catheterization was confirmed successful, the inferior vena cava should be immediately cut to release the pressure of the system (portal vein - liver - inferior vena cava), allowing the perfusion fluid to flow out from the inferior vena cava. After the perfusion, the liver was completely removed and placed in pre-cooled incomplete DMEM high-glucose medium containing double antibiotics, and transferred to the laminar flow hood for operation. The gallbladder was removed with forceps, and the liver was torn to allow the cells to flow out fully. The obtained cell suspension was passed through a 70-μm cell strainer into a 50-mL centrifuge tube pre-added with DNase at a volume ratio of 1%. It was shaken in a 37°C water bath for 20 min to allow sufficient digestion reaction. Differential centrifugation was used to remove the liver parenchymal cells, and the non-parenchymal cell precipitate was collected. 2 mL of Nycodenz solution was added to the precipitate, and it was pipetted and resuspended. The cell resuspension was transferred to a 15-mL centrifuge tube, and 1 mL of PBS was slowly layered on top of the Nycodenz solution. It was centrifuged horizontally at 1350 rcf at 20°C for 16 min. A distinct white ring of cells could be seen at the stratified layer, and it was carefully aspirated into a pre-prepared DMEM high-glucose medium containing double antibiotics with more than 10 times the amount of 10% fetal bovine serum. It was centrifuged at 700 rcf at room temperature for 5 min to obtain primary hepatic stellate cells.

[0033] 2. Establishment and grouping of mouse liver fibrosis models (animal models)

[0034] 2.1 Establishment and grouping of mouse bile duct ligation (BDL) models

[0035] They were divided into 4 groups: sham operation group (Sham), sham operation drug administration group (Oil + BHB), BDL model group (BDL), and model drug administration group (BDL + BHB). Mice were anesthetized with sodium pentobarbital and fixed on the operating table. The abdominal epidermis was incised, the common bile duct was dissected free, ligated with 5-0 suture, and the abdominal cavity was sutured. Starting from the third day after surgery, BHB (100 mg / kg) was injected intraperitoneally every other day, and the mice were sacrificed after 14 days.

[0036] 2.2 Establishment and grouping of mouse CCl4 models

[0037] It was divided into 4 groups: blank control group (Oil), blank drug administration group (Oil + BHB), model group (CCl4), and model drug administration group (CCl4 + BHB). According to 20% CCl4 / corn oil, 2 mL / kg was intraperitoneally injected. The control group was injected with the same dose of corn oil twice a week. The experimental period was 4 weeks. BHB (100 mg / kg) was intraperitoneally injected every 48 h. During this period, the status and weight changes of the mice were recorded.

[0038] 3. Detection of Fibrosis Gene Expression Levels

[0039] 3.1 Detection of Fibrosis Gene Expression Levels in Animal Tissues

[0040] (1) Total RNA extraction: The tissues stored in an ultra-low temperature refrigerator at -80 °C were ground into a homogeneous powder in liquid nitrogen. Take 20 mg of the ground powder and add 1 mL of Trizol lysis solution, and homogenize it with a tissue homogenizer at 60 Hz for 60 s. Add 200 μL of chloroform to the tissue homogenate, shake it vigorously up and down for 20 s, and transfer the tissue homogenate to ice and let it stand for 5 min. Centrifuge at 12,000 g at 4 °C for 10 min, carefully aspirate 400 μL of the upper aqueous phase into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, gently invert it up and down to mix evenly, and let it stand at 4 °C for 10 min. Centrifuge at 12,000 g at 4 °C for 10 min, carefully discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water, gently flick the bottom of the tube to suspend the white precipitate for sufficient washing, and let it stand at 4 °C for 5 min. Centrifuge at 12,000 g at 4 °C for 5 min, and discard the supernatant. Invert the centrifuge tube open-end down on a flat piece of paper to aspirate the excess ethanol, dry it for 3 - 5 min, add an appropriate amount of DEPC water to dissolve the precipitate, and gently pipette to dissolve the precipitate.

[0041] (2) cDNA synthesis (RNA reverse transcription): Reverse transcription: Remove the residual genomic DNA. Prepare a mixture in an RNase free centrifuge tube, and the components are RNase free ddH2O, 5X gDNA digester Mix, and template RNA. Gently pipette and incubate at 42 °C for 2 min.

[0042] Configure the reverse transcription reaction system, mix the reaction solution from the first step with 4X Hifair III SuperMix plus, and set the reverse transcription program as follows:

[0043] Temperature Time 25℃ 5 min 55℃ 15 min 85℃ 5 min

[0044] (3) qRT-PCR Quantification: The reaction system (10 μL) required for qRT-PCR is as follows: primer : cDNA (1×) : luciferase = 1 : 4 : 5. Add them to the corresponding wells of the PCR white plate respectively. After finishing, stick the film on it, scrape it flat, centrifuge briefly for 1 min, and then put it into the real-time fluorescence quantitative PCR instrument to set the program for the experiment. The qRT-PCR program is as follows:

[0045]

[0046] Primer sequences:

[0047]

[0048] 3.2 Detection of the gene expression level of stellate cell fibrosis

[0049] (1) Total RNA extraction: Take out the cells, discard the culture medium, wash once with PBS, add an appropriate amount of Total RNA Extraction Reagent, cover and pipette up and down repeatedly to lyse the cells, and place at room temperature for 5 min to completely dissociate ribosomes. Take out the cells, discard the culture medium, wash once with PBS, add an appropriate amount of Total RNA Extraction Reagent, cover and pipette up and down repeatedly to lyse the cells, and place at room temperature for 5 min to completely dissociate ribosomes.

[0050] The remaining experimental steps are the same as those in 3.1 Detection of the gene expression level of animal tissue fibrosis.

[0051] Primer sequences:

[0052] ① The primary hepatic stellate cells are the same as those in 3.1;

[0053] ② LX2

[0054]

[0055] 4. Detection of the protein expression levels of COL1A1 and α-SMA in hepatic stellate cells

[0056] (1) Total cell protein extraction: Discard the culture medium. Add 1 mL of PBS buffer solution to the adherent cells for washing, pipette the washing solution, add 100 μL of 1× loading buffer to each well, scrape the cells thoroughly with a cell scraper, and transfer them to a 1.5 mL centrifuge tube.

[0057] (2) Western blot assay: ① Fix the clean thick and thin glass plates on the gel preparation support. Prepare separating gels with different concentrations according to the molecular weight of the target protein. Use a pipette to evenly add the gels between the two plates and quickly add ddH2O to seal and flatten. After the separating gel solidifies, pour off the upper-layer ddH2O and use filter paper to absorb the excess water. Add the prepared stacking gel on top of the separating gel and quickly insert the sample comb. After the stacking gel solidifies, take it out and store it in a 4°C refrigerator for later use; ② Take out the prepared gel, carefully pull out the sample comb, place the device in the electrophoresis tank and add 1× electrophoresis buffer. Thaw the samples, vortex and centrifuge before use. Add the samples to the comb holes in the order of the loading volume, and add 3 μL and 1 μL of protein marker to the left and right sides of each plate respectively; After turning on the power, set the voltage to 80 V. After about 30 min, the protein samples are flattened into a straight line. Switch the voltage to 120 V and continue electrophoresis until the protein samples run to the bottom of the gel plate and then stop electrophoresis; ③ Prepare 1× wet transfer solution in advance, and the preparation ratio is ddH2O: methanol: 10× wet transfer solution = 7: 2: 1. After electrophoresis is completed, turn off the power. Pre-add an appropriate amount of 1× wet transfer solution to the square tank, soak and lay the filter paper and sponge in the transfer membrane clip, with the side with the white clip facing up. Pry open and separate the thin plate, immerse the thick plate together with the gel in 1× wet transfer solution, carefully scrape off the gel, cut off the stacking gel part, and lay it face up on the transfer membrane clip. Use a special spatula to flatten the gel, remove air bubbles, lay the PVDF membrane and filter paper in turn, and use a roller to remove air bubbles each time. After clamping the transfer membrane clip, place it in the transfer tank, pour in 1× wet transfer solution, put an ice pack in the tank, and cover the tank lid. Move the transfer tank to a basin filled with cold water, and add an ice pack to the basin for refrigeration. Turn on the power, adjust the voltage to the maximum of 300 V, set a constant current of 350 mA for 90 min, and the transfer is completed; ④ Prepare 5% non-fat high-protein milk with 1× TBST solution, and prepare it immediately before use. After the transfer is completed, take out the PVDF membrane, place it in 5% non-fat milk powder, and block it on a shaker for 2 h; ⑤ Dilute the primary antibody with 1× TBST solution according to different antibody dilution ratios for later use. Cut the corresponding bands according to the molecular weight of the target protein, add 2 mL of the diluted primary antibody to each band, and press each band well with a laminator. Put the pressed bands into a rotating cylinder and incubate overnight in a 4°C refrigerator; Recover the primary antibody (generally can be reused 3 - 5 times), place the bands in 1× TBST solution, and wash them on a shaker for 10 min. Repeat the washing 3 times. Dilute the secondary antibody with 1× TBST. After laminating is completed, place the bands at room temperature for incubation for 1 h; ⑥ Take out the bands and place them in 1× TBST solution, and wash them on a shaker for 10 min. Repeat the washing 3 times, and then they can be used for development. Prepare the A and B solutions in the Tanon developer in a 1:1 ratio. Take out the bands from the 1× TBST solution and put them into the imaging instrument. Add 500 μL of developer evenly to each band, expose and take pictures, save the photos, and analyze them with Image J analysis software.

[0058] 5. Immunofluorescence assay

[0059] Extract primary mouse hepatic stellate cells and seed them in 35-mm confocal dishes. Control the cell seeding density at about 50%. After 2 hours, change the medium. Thereafter, change the medium every 24 hours and wait for spontaneous activation. In the intervention group, change the medium containing 0.05, 0.5, and 5 mM BHB every day. Start preparing confocal samples after 7 days. Discard the medium, wash once with PBS, add 500 μL of 4% paraformaldehyde solution, and fix at room temperature for 25 min. Discard the paraformaldehyde, add PBS buffer solution, and place it on a shaker for slow washing for 5 min. Repeat the washing 3 times. Weigh 300 mg of BSA into 10 mL of PBS buffer solution and add 20 μL of Triton X-100 to prepare a BSA solution containing Triton. Add 1 mL of the BSA solution to each dish and incubate at room temperature for 2 hours. Discard the BSA solution, add PBS buffer solution, and place it on a shaker for washing for 5 min. Repeat the washing 3 times. Dilute the α-SMA antibody with the BSA solution containing Triton (dilution ratio 1:200). Add 500 μL of the diluted α-SMA antibody to each dish and incubate overnight at 4 °C in the refrigerator. Discard the primary antibody, add PBS buffer solution, and place it on a shaker for washing for 5 min. Repeat the washing 3 times. Weigh 3 mg of BSA into 10 mL of PBS to prepare a 3% BSA solution for diluting FITC-labeled goat anti-rabbit IgG (dilution ratio 1:200). Add 500 μL of the diluted secondary antibody to each dish and incubate in the dark at 37 °C in an incubator for 1 hour. Discard the secondary antibody, add PBS buffer solution, and place it on a shaker for washing for 5 min. Repeat the washing 3 times. Add 500 μL of DAPI staining solution to each dish and incubate in the dark at 37 °C in an incubator for 10 min. Recover the DAPI staining solution, add PBS buffer solution, and place it on a shaker for washing for 5 min. Repeat the washing 3 times. Dilute the anti-fluorescence quenching mounting medium with PBS buffer solution at a ratio of 1:9. Add 500 μL to each dish and observe and take pictures under a laser confocal microscope.

[0060] 6. Determination of alanine aminotransferase and aspartate aminotransferase activities

[0061] After the animal model is established, anesthetize the mice by intraperitoneal injection of 10 mg / mL pentobarbital sodium at a dose of 10 mL / kg. Collect the whole blood of the mice by cardiac puncture into a clean 1.5-mL centrifuge tube, let it stand at room temperature for 2 h, and centrifuge at 4000 rpm for 15 min. The obtained supernatant is the mouse serum. Aliquot the mouse serum, 50 μL per tube, and store it at -80 °C for later use.

[0062] Operate according to the kit instructions as follows: During the measurement, take out a tube of serum and thaw it at 4°C, and pre-warm the substrate solution at 37°C. Add 20 μL of the substrate solution and 5 μL of serum to the measurement wells, while only add 20 μL of the substrate solution to the control wells. React at 37°C for 30 min. After the reaction, add 20 μL of 2,4-dinitrophenylhydrazine to the measurement wells, and add 20 μL of 2,4-dinitrophenylhydrazine and 5 μL of serum sample to the control wells. React at 37°C for 20 min. Add 200 μL of 0.4 mol / L sodium hydroxide solution to both the control wells and the measurement wells, gently shake the 96-well plate horizontally to mix evenly, place at room temperature for 15 min, measure the OD value of each well at a wavelength of 510 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and use the absolute OD value (OD value of the measurement wells minus the OD value of the control wells) to check the standard curve to obtain the corresponding ALT / AST activity units.

[0063] 7. Statistical analysis

[0064] The data are expressed as mean ± SEM. The statistical differences between two samples are analyzed by independent samples t-test, and the statistical differences between multiple samples are analyzed by one-way analysis of variance (one-way ANOVA).

[0065] The differences are statistically significant at *p < 0.05 and **p < 0.01.

[0066] III. Experimental results

[0067] Figure 1 In it, 1A is the expression level of fibrosis genes characterized by qRT-PCR experiments during the activation of LX2. The experimental results show that 0.5 mM BHB can significantly reduce the transcription of fibrosis-related genes ACTA2, COL1A1, COL3A1, CTGF, and FN caused by the activation of LX2, and shows a certain concentration dependence; 1B is a protein immunoblotting experiment to characterize the protein expression levels of α-SMA and COL1A1 during the activation of LX2. The results show that TGF-β can increase the protein expression levels of α-SMA and COL1A1 in LX-2. After treatment with BHB, the protein levels of α-SMA and COL1A1 are significantly reduced ( Figure 3 B). The experimental results suggest that BHB can counteract the activation of LX-2 cells induced by TGF-β.

[0068] Figure 2In it, 2A shows the expression levels of fibrosis genes during the spontaneous activation of primary mouse hepatic stellate cells characterized by qRT-PCR experiments. The experimental results showed that compared with the blank group, the relative mRNA expression levels of Acta2, Col1a1, Col3a1, Ctgf, and Fn genes in the BHB (0.5 mM, 5 mM) groups were significantly decreased. 2B is the qualitative analysis of the α-SMA expression level in primary mouse hepatic stellate cells by immunofluorescence staining technique, and the α-SMA positive reaction product is green fluorescence. The experimental results still showed that compared with the control group, BHB could significantly inhibit the expression of α-SMA.

[0069] Figure 3 In it, 3A and 3B show the AST and ALT levels in the serum of the mouse BDL model. The experimental results showed that the serum ALT level in the model group was more than 10 times that of the control group, and the AST level was also significantly increased, indicating that the modeling was successful; compared with the model group, the serum AST and ALT levels in the administration group were significantly decreased; 3C is the hematoxylin & eosin staining (Hematoxylin&eosin staining, H&E) and Sirius Red staining of mouse liver tissue sections, and the results showed that BHB significantly improved the liver inflammatory infiltration and abnormal excessive collagen deposition caused by BDL; 3D is the detection of the mRNA expression levels of Acta2, Col1a1, Col3a1, Ctgf, and Tgfb1 fibrosis genes by qRT-PCR technology. The experimental results showed that the mRNA expression levels of Acta2, Col1a1, Col3a1, Ctgf, and Tgfb1 genes in the liver tissue of the BDL group mice were significantly increased, and BHB could significantly inhibit the mRNA expression levels of fibrosis genes.

[0070] Figure 4 In it, 4A and 4B show the AST and ALT levels in the serum of the CCl4-induced chronic liver fibrosis model in mice. The experimental results showed that the serum ALT level in the model group was more than 10 times that of the control group, and the AST level was also significantly increased, indicating that the modeling was successful. Compared with the model group, the serum AST and ALT levels in the administration group were significantly decreased; 4C is the H&E and Sirius Red staining of mouse liver tissue sections, and the results showed that BHB significantly improved the liver inflammatory infiltration and abnormal excessive collagen deposition caused by CCl4; 4D is the detection of the mRNA expression levels of Acta2, Col1a1, Col3a1, Ctgf, and Tgfb1 fibrosis genes by qRT-PCR technology. The experimental results showed that the mRNA expression levels of Acta2, Col1a1, Col3a1, Ctgf, and Tgfb1 genes in the liver tissue of the BDL group mice were significantly increased, and BHB could significantly inhibit the mRNA expression levels of fibrosis genes.

[0071] Example 2: Sodium β-hydroxybutyrate for treating liver fibrosis

[0072] For the experimental materials and methods, refer to Example 1, and β-hydroxybutyric acid was replaced with the same dose of sodium β-hydroxybutyrate.

[0073] Figure 5 In it, 5A and 5B are the AST and ALT levels in the serum of the mouse BDL model. The experimental results show that the serum ALT level in the model group is more than 10 times that of the control group, and the AST level also increases significantly, indicating that the modeling is successful; compared with the model group, the serum AST and ALT levels in the administration group are significantly reduced; 5C is the H&E and Sirius red staining of the mouse liver tissue sections, and the results show that BHB sodium salt significantly improves the liver inflammatory infiltration and abnormal excessive collagen deposition caused by BDL.

[0074] Figure 6 In it, 6A and 6B are the AST and ALT levels in the serum of the CCl4-induced chronic liver fibrosis model in mice. The experimental results show that the serum AST and ALT levels in the model group are more than 10 times that of the control group, indicating that the modeling is successful. Compared with the model group, the serum AST and ALT levels in the administration group are significantly reduced; 6C is the H&E and Sirius red staining of the mouse liver tissue sections, and the results show that BHB sodium salt significantly improves the liver inflammatory infiltration and abnormal excessive collagen deposition caused by CCl4

[0075] The above experiments prove that β-hydroxybutyric acid and its pharmaceutically acceptable salts can significantly inhibit liver fibrosis with excellent effects.

[0076] The function of the above examples is to specifically introduce the substantial content of the present invention. However, those skilled in the art should know that the protection scope of the present invention should not be limited to this specific example.

Claims

1. Use of β-hydroxybutyric acid or a pharmaceutically acceptable salt thereof for preparing a drug for treating liver fibrosis.

2. The use according to claim 1, wherein: The drug uses β-hydroxybutyric acid or a pharmaceutically acceptable salt thereof as an active ingredient and is made into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: The carrier is in solid, liquid or semi-solid form.

4. The use according to claim 2, characterized in that: The dosage form includes injections, tablets, capsules and drops.

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