Application of β-hydroxybutyric acid in the preparation of products that promote hepatocyte proliferation and liver regeneration

Abstract

This invention discloses the application of β-hydroxybutyric acid (BHB) in the preparation of products that promote hepatocyte proliferation and liver regeneration. This invention discovers that β-hydroxybutyric acid can promote liver regeneration and recovery by promoting the proliferation of hepatocytes in the residual liver after partial hepatectomy. Therefore, β-hydroxybutyric acid can be used to prepare products that promote liver regeneration. This invention not only discloses the application of β-hydroxybutyric acid in promoting liver regeneration, providing a drug source for the development of products that promote liver regeneration, but also provides a theoretical basis for subsequent clinical intervention strategies to promote liver regeneration. This helps to improve liver regeneration capacity and avoid poor prognosis and liver failure caused by insufficient liver regeneration capacity after partial hepatectomy or living donor liver transplantation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of β-hydroxybutyric acid in the preparation of products that promote hepatocyte proliferation and liver regeneration. Background Technology

[0002] Partial hepatectomy (PHx) and liver transplantation are currently the most common and effective treatments for various end-stage liver diseases in clinical practice. The recovery process after partial hepatectomy or transplantation is a process of liver regeneration; the liver's strong regenerative capacity is the physiological basis for performing partial hepatectomy and liver transplantation. However, if the regenerative capacity of the remaining liver is delayed or insufficient, it can lead to postoperative liver failure and other complications, ultimately resulting in poor prognosis or death. Effectively improving the regenerative capacity of residual liver tissue is extremely important for improving the treatment effect and prognosis after hepatectomy and transplantation. Therefore, in-depth exploration of the molecular mechanisms, targets, and drug intervention strategies of liver regeneration has significant scientific and clinical value.

[0003] While several drugs have been reported to promote liver regeneration, the mechanisms of liver regeneration are not fully understood, and different drugs have different mechanisms of action. β-hydroxybutyric acid (BHB) is the most abundant ketone body in mammals, readily soluble in water, and has two enantiomers: R / D and S / L. R-BHB is a metabolic product in humans and mice. BHB is primarily produced in the liver through fatty acid oxidation, with smaller amounts potentially produced in other tissues such as the intestines. In vivo, when glucose supply is too low to meet the body's energy needs, fatty acids are mobilized from adipocytes and transported to the liver, where they are metabolized into acetoacetic acid and BHB as an energy source. BHB plays a crucial role in regulating gluconeogenesis and hepatic glucose production.

[0004] BHB is not only an energy substrate for maintaining metabolic homeostasis, but it can also act as a signaling molecule to regulate physiological and pathological processes in the body. Studies have found that BHB levels in the livers of patients with severe alcoholic hepatitis are significantly lower than in the normal group. Mechanistic studies have revealed that BHB can bind to the hydroxycarboxylic acid receptor 2 (Hcar2) on the surface of hepatic macrophages, increasing the transcription of interleukin 10 (IL10) in the liver and promoting the M2 phenotype of intrahepatic macrophages, thereby exerting anti-inflammatory effects and improving alcohol-induced liver injury. Another study found that 12-hour fasting in mice has a protective effect against hepatic ischemia-reperfusion injury. Mechanistic studies showed that 12-hour fasting increases the level of liver bilirubin (BHB) in the liver. BHB upregulates histone-3 acetylation, activates transcription factors forkhead box O1 (FOXO1) and heme oxygenase 1 (HO-1), reduces the expression of high-mobility group box 1 (HMGB1), and inactivates nuclear factor kappa-B (NF-κB) and NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), thereby inhibiting the inflammatory response and improving hepatic ischemia-reperfusion injury. However, there are currently no reports on the use of BHB to promote hepatocyte proliferation and liver regeneration. Summary of the Invention

[0005] The purpose of this invention is to provide the application of β-hydroxybutyric acid in the preparation of products that promote hepatocyte proliferation and liver regeneration.

[0006] The first objective of this invention is to provide the use of β-hydroxybutyric acid in the preparation of products that promote liver regeneration.

[0007] A second objective of this invention is to provide the use of β-hydroxybutyric acid in the preparation of products that promote hepatocyte proliferation.

[0008] A third objective of this invention is to provide the application of β-hydroxybutyric acid in the preparation of products that promote liver function recovery.

[0009] The fourth objective of this invention is to provide a drug that promotes liver regeneration.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention has discovered that β-hydroxybutyric acid (β-hydroxybutyric acid) can promote liver regeneration and recovery by stimulating the proliferation of hepatocytes in the residual liver after partial hepatectomy. Furthermore, this invention has also discovered that β-hydroxybutyric acid can promote the recovery of liver biochemical indicators to normal ranges, meaning that β-hydroxybutyric acid can promote liver function recovery. Therefore, this invention seeks protection for the following applications of β-hydroxybutyric acid:

[0012] This invention seeks protection for the use of β-hydroxybutyric acid in the preparation of products that promote liver regeneration.

[0013] Specifically, the β-hydroxybutyric acid promotes liver regeneration by promoting hepatocyte proliferation.

[0014] Specifically, the β-hydroxybutyric acid promotes liver regeneration by promoting hepatocyte proliferation and restoring liver biochemical indicators to normal range.

[0015] More specifically, the β-hydroxybutyric acid promotes hepatocyte proliferation by upregulating the expression of hepatocyte proliferation-related proteins.

[0016] The present invention also claims protection for the use of β-hydroxybutyric acid in the preparation of products that promote hepatocyte proliferation.

[0017] This invention also claims protection for the use of β-hydroxybutyric acid in the preparation of products that promote liver function recovery.

[0018] Specifically, the β-hydroxybutyric acid promotes liver function recovery by helping liver biochemical indicators return to the normal range.

[0019] Specifically, the liver described in this invention is the liver after partial hepatectomy.

[0020] Specifically, the amount of liver removed should not exceed 2 / 3 of the original liver volume.

[0021] Specifically, the products that promote liver regeneration, hepatocyte proliferation, or recovery of liver biochemical indicators include β-hydroxybutyric acid and its pharmaceutically acceptable carrier.

[0022] The present invention also provides a drug for promoting liver regeneration or the recovery of liver biochemical indicators, wherein the drug contains β-hydroxybutyric acid and its pharmaceutically acceptable carrier.

[0023] Specifically, the dosage of β-hydroxybutyric acid is at least 50 mg / kg / day.

[0024] The present invention has the following beneficial effects:

[0025] This invention discovers that β-hydroxybutyric acid (β-hydroxybutyric acid) can promote liver regeneration and recovery by stimulating the proliferation of hepatocytes in the residual liver after partial hepatectomy. β-hydroxybutyric acid can be used to prepare products that promote liver regeneration. This invention not only discloses the application of β-hydroxybutyric acid in promoting liver regeneration, providing a drug source for the development of products that promote liver regeneration, but also provides a theoretical basis for subsequent clinical intervention strategies to promote liver regeneration. It helps improve liver regeneration capacity and avoid poor prognosis and liver failure caused by insufficient liver regeneration capacity after partial hepatectomy or living donor liver transplantation. This has significant clinical implications for improving liver tissue regeneration capacity and promoting postoperative liver regeneration. Attached Figure Description

[0026] Figure 1 The results show the effect of β-hydroxybutyric acid (β-hydroxybutyric acid) on liver regeneration after partial hepatectomy. Figure A shows liver images of mice at different time points after β-hydroxybutyric acid administration; Figure B shows the changes in liver weight of mice at different time points after β-hydroxybutyric acid administration (n=6); Figure C shows the changes in liver / body weight ratio of mice at different time points after β-hydroxybutyric acid administration (n=6); data are expressed as mean ± standard deviation; compared with the Vehicle group, *P<0.05.

[0027] Figure 2 The results show the effects of β-hydroxybutyric acid (β-hydroxybutyric acid) administration on liver biochemical parameters after partial hepatectomy. Figure A represents serum AST, ALT, ALP, TBIL, and TBA levels (n=6); Figure B represents H&E staining results of liver tissue (n=3). Data are expressed as mean ± standard deviation. Compared with the Vehicle group, *P<0.05; scale bar = 50 μm.

[0028] Figure 3 The effect of β-hydroxybutyric acid (β-hydroxybutyric acid) on hepatocyte proliferation after partial hepatectomy was shown in Figure A, which represents Ki67 staining results in liver tissue; Figure B represents the quantitative results of Ki67-positive cells (n=3); data are expressed as mean ± standard deviation; compared with the Vehicle group, *P<0.05, **P<0.01; scale bar = 50 μm.

[0029] Figure 4 The results show the effect of β-hydroxybutyric acid (β-hydroxybutyric acid) on the expression of hepatocyte proliferation-related proteins after partial hepatectomy. Figure A shows the expression results of CCNA1, CCND1, CCNE1, and CDK4 proteins in liver tissue, and Figure B shows the quantitative results of proliferation-related protein expression (n=3). Data are expressed as mean ± standard deviation. Compared with the Vehicle group, *P<0.05. Detailed Implementation

[0030] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0032] β-Hydroxybutyric acid used in the embodiments of the present invention was purchased from MedChemExpress (MCE), and the product catalog number is HY-113378.

[0033] The experimental animals used in the embodiments of the present invention were C57BL / 6 mice, male, 7 weeks old, weighing 20-23 g, purchased from Guangdong Medicilon Biotechnology Co., Ltd., and the animal certificate number is SCXK (Guangdong) 2020-0054; after inspection and quarantine, they were raised in the Experimental Animal Center of Southern Medical University; the standard experimental conditions for raising: temperature 22-25 °C, humidity 55-70%, light and dark cycles of 12 h each, light from 8:00 to 20:00; dark from 20:00 to 8:00 the next day, free access to water and food, and adaptively fed with ordinary feed provided by the Experimental Animal Center of Southern Medical University for 1 week; the animal research protocol complied with the requirements of the Experimental Animal Ethics Committee of Southern Medical University.

[0034] The experimental data of the present invention were all statistically analyzed as mean ± standard error (Mean ± S.D.), and SPSS 20.0 software was used for statistical analysis of the data; the gray scale detection of protein bands was processed using Image J 1.53 software; one-way ANOVA was used for comparison of means and variance analysis of multiple samples, and Student's t test was used for comparison analysis between two groups; GraphPad Prism 8.0 software was used for chart drawing; P < 0.05 represents statistical significance between the two, *P < 0.05, **P < 0.01, ***P < 0.001.

[0035] Example 1 β-Hydroxybutyric acid accelerates the recovery of liver weight in mice after partial hepatectomy

[0036] 1. Experimental method

[0037] (1) Preparation of β-hydroxybutyric acid (BHB) solution

[0038] 200 mg of BHB was added to 20 mL of PBS, fully dissolved and mixed to prepare a 10 mg / mL BHB intraperitoneal injection solution; aliquoted into 1.5 mL centrifuge tubes and stored at -20 °C, avoiding repeated freezing and thawing.

[0039] (2) Partial hepatectomy (PHx) surgery

[0040] This invention utilizes partial hepatectomy to construct a 2 / 3 PHx mouse model. The surgical steps are as follows:

[0041] 1) All surgical instruments and mouse sutures were autoclaved.

[0042] 2) Mouse anesthesia: Mice were anesthetized by intraperitoneal injection of 3.5% sodium pentobarbital at a dose of 350 mg / kg;

[0043] 3) Opening the abdomen: Fix the mouse’s limbs to the surgical board with medical tape, remove the hair from the chest and abdomen, disinfect the abdomen with iodine, and use surgical scissors to make an incision about 1 cm below the xiphoid process along the midline of the abdomen.

[0044] 4) Resection of liver lobes: Gently squeeze the sides of the mouse's incision to expel the left anterior lobe, right anterior lobe, and left posterior lobe from the abdominal incision. Place a 4-0 suture at the base of the left anterior lobe and surgically tie it. Carefully cut away the ligated liver lobe and excess suture with scissors, and aspirate blood from the abdominal cavity with a cotton swab. The right anterior lobe and left posterior lobe are removed in the same way as the left anterior lobe; approximately 2 / 3 of the total liver volume is removed.

[0045] 5) Suturing: Use forceps to lift the peritoneum and skin on both sides of the opening, and use 9mm staples to fasten the peritoneum and skin on both sides of the opening together; inject 1mL of physiological saline subcutaneously into the mouse and place it in a warm environment to promote its awakening.

[0046] Surgical precautions:

[0047] 1) When tying the roots of the three leaves, be sure to tie them tightly, otherwise the liver will bleed and the mouse will die. However, be careful not to tie them too tightly and cause the liver leaves to rot.

[0048] 2) When ligating the roots of the three leaves, be careful not to ligate the gallbladder, otherwise it may cause the gallbladder to rupture and affect the liver system of mice;

[0049] 3) Aseptic technique must be maintained during the surgical procedure to prevent postoperative infection that could lead to the death of the mice;

[0050] 4) After ligating the liver lobe, remove as much excess liver tissue as possible to reduce the amount of residual liver tissue. However, avoid cutting the ligation suture, otherwise it will cause liver bleeding in mice, leading to death.

[0051] (3) Animal experimental protocol

[0052] Male C57BL / 6 mice were randomly divided into two groups: the PHx-treated PBS group (Vehicle group) and the PHx-treated β-hydroxybutyric acid group (BHB group). The daily food intake and body weight of the mice were recorded during the experiment.

[0053] On days 1-3 of the experiment, mice in the Vehicle group were injected intraperitoneally with PBS, and mice in the β-hydroxybutyric acid group were injected intraperitoneally with BHB (50 mg / kg / d); the injection volume was 0.1 mL / 10 g.

[0054] On day 4 of the experiment, mice in both groups underwent PHx surgery and continued to receive intraperitoneal injections of PBS or BHB (50 mg / kg / d) until the end of the experiment; the injection volume was 0.1 mL / 10 g.

[0055] During the experiment, the daily food intake and body weight of the mice were recorded. Six mice were randomly selected from each group on postoperative days 0, 1, 2, and 5. The weight of the mice at these time points was measured and recorded. Feces from each mouse were collected, flash-frozen in liquid nitrogen, and then stored at -80°C. The mice were euthanized by enucleating the eyeballs, dislocating the cervical spine, and then dissecting the abdomen. The liver was removed, necrotic tissue was removed, and the surface blood was rinsed with physiological saline. The surface moisture was absorbed with filter paper, the liver weight was measured and recorded, and a three-dimensional photograph of the liver was taken. Each liver sample was taken from the same lobe and roughly divided into two identical pieces. One piece was fixed in 4% paraformaldehyde solution for subsequent H&E and Ki67 staining experiments. The remaining liver tissue was placed in EP tubes and stored at -80°C. The small intestine, cecum, cecal contents, and colon / rectum were removed, placed in EP tubes, and stored at -80°C.

[0056] 2. Experimental Results

[0057] This invention investigated the effect of β-hydroxybutyrate (BHB) administration on liver regeneration after PHx surgery using a 2 / 3 partial hepatectomy (PHx) mouse model. The results are as follows: Figure 1 As shown; Figure 1 In the image, A represents the liver images of mice at different time points after BHB administration; Figure 1 B in the figure represents the changes in liver weight in mice at different time points after BHB administration (n=6); Figure 1 In the figure, C represents the change in liver / body weight ratio in mice at different time points after BHB administration (n=6). Figure 1It was found that at different time points after PHx surgery, the liver weights of mice in the Vehicle group and BHB group were: 0d (0.38±0.02g, 0.37±0.05g), 1d (0.44±0.04g, 0.48±0.03g), 2d (0.47±0.07g, 0.56±0.07g), and 5d (0.63±0.05g, 0.70±0.05g); the liver / body weight ratios were: 0d (1.8%), 1.7%, 1d (2%), 1d (3%), 1d (4%), 1d (5%), 1d (6%), 1d (7%), 1d (8%), 1d (9%), 1d (1 ... The liver volume, liver weight, and liver / body weight ratio of the two groups of mice increased with the duration of PHx surgery (1 day, 2.3%), 2 days (2.3%, 2.9%), and 5 days (3.1%, 3.6%). It was found that the liver volume, liver weight, and liver / body weight ratio of both groups increased with the duration of PHx surgery. At 1 day post-PHx surgery, there were no significant differences in liver volume, liver weight, and liver / body weight ratio between the two groups. However, at 2 and 5 days post-surgery, the liver volume, liver weight, and liver / body weight of the BHB group were significantly increased compared to the Vehicle group, indicating that BHB can accelerate liver weight recovery after PHx surgery in mice.

[0058] Example 2: β-hydroxybutyric acid accelerates the recovery of liver biochemical indicators to normal in mice after partial hepatectomy.

[0059] 1. Experimental Methods

[0060] (1) Serum biochemical index detection

[0061] Blood samples collected from the Vehicle and BHB groups were left to stand at room temperature for 40 minutes, then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant serum was collected for biochemical index detection. The levels of aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL), and total bile acid (TBA) in the serum were detected using an automated serum biochemistry analyzer.

[0062] (2) H&E staining of liver tissue

[0063] Liver tissue was fixed in 4% paraformaldehyde for 24 hours and then sent to Wuhan Sewell Technology Co., Ltd. for paraffin embedding, dehydration, sectioning and H&E staining.

[0064] 2. Experimental Results

[0065] This invention investigated the effect of β-hydroxybutyrate (BHB) administration on liver function after PHx surgery using a 2 / 3 partial hepatectomy (PHx) mouse model. The results are as follows: Figure 2 As shown; Figure 2In this context, A represents the serum levels of AST, ALT, ALP, TBIL, and TBA (n=6). Figure 2 B in the table represents the H&E staining results of liver tissue (n=3); data are expressed as mean ± standard deviation; *P<0.05 compared with the Vehicle group; scale bar = 50μm. Serum biochemical index detection showed that the levels of AST, ALT, ALP, TBIL, and TBA in serum samples of both groups reached their peak 1 day after PHx, and then gradually returned to their initial levels with liver regeneration, but there was no significant difference between the two groups; however, compared with the Vehicle group, BHB administration significantly reduced the serum ALT level on postoperative day 2 (480.3±82.8U / L vs. 334.3±66.1U / L) and the ALP level on postoperative day 1 (200.3±15.6U / L vs. 165.3±17.5U / L). Figure 2 (A) Liver tissue H&E staining results showed no significant damage to hepatocytes and hepatic sinusoids. Figure 2 (B in the text); The above results indicate that administration of BHB accelerates the recovery of liver biochemical indicators to normal in mice after PHx surgery.

[0066] Example 3: β-hydroxybutyric acid promotes hepatocyte proliferation in mice after partial hepatectomy.

[0067] 1. Experimental Methods

[0068] (1) Ki67 staining of liver tissue

[0069] Ki67 protein is expressed in the cell nucleus, and its level can accurately reflect the level of hepatocyte proliferation. The Ki67 staining process in liver tissue is shown below:

[0070] 1) Dewaxing and rehydration: Place the paraffin sections in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min → 50% anhydrous ethanol / 50% xylene for 1 min → anhydrous ethanol I for 1 min → anhydrous ethanol II for 1 min → 95% ethanol for 2 min → 80% ethanol for 2 min → 70% ethanol for 2 min. Remove the sections and wash them with distilled water 3 times, 5 min each time.

[0071] 2) Antigen retrieval: Add 0.01 mol / L citric acid solution to the staining vat to cover the slides, heat in a microwave oven on high until boiling, then cover and heat on medium-low for 11 minutes. After the process is complete, remove the slides and allow them to cool to room temperature.

[0072] 3) PBS rinsing: After the sections have cooled to room temperature, discard the antigen retrieval solution, add PBS buffer to cover the sections, and rinse 3 times on a horizontal shaker at 80 r / min for 5 min each time;

[0073] 4) Endogenous peroxidase blockade: After rinsing with PBS buffer, blot off excess PBS with filter paper, mark the tissue with an immunohistochemistry pen, cover the tissue with 3% hydrogen peroxide solution, place in a humidified chamber, and incubate at room temperature in the dark for 15 min; rinse 3 times with PBS buffer on a horizontal shaker at 80 r / min for 5 min each time.

[0074] 5) Blocking: Add 10% goat serum to the enclosure and block in a humidified chamber at room temperature for 1 hour;

[0075] 6) Primary antibody incubation: Wipe away excess goat serum with filter paper, being careful not to touch the tissue. Dilute the antibody according to the primary antibody instructions, drop the diluted antibody solution onto the tissue, and incubate overnight at 4°C in a humidified chamber.

[0076] 7) Secondary antibody incubation: Gently shake off the primary antibody from the slide, place it in the staining jar, add PBS buffer and wash 3 times on a shaker, 5 min each time; blot off the excess PBS on the slide with filter paper, add the corresponding secondary antibody and incubate at room temperature in a humidified chamber for 1 h, then wash 3 times on a shaker with PBS buffer, 5 min each time.

[0077] 8) Preparation of DAB colorimetric solution: During slide rinsing, prepare DAB working solution by adding one drop of solution A to every 1 mL of solution B, and prepare it fresh for each use.

[0078] 9) DAB staining: Add DAB working solution to the tissue and observe under a microscope until the positive area develops color. Record the time required for staining. After the staining is complete, place the section in a staining jar containing distilled water to stop the staining process.

[0079] 10) Hematoxylin staining of cell nuclei: Immerse the sections in hematoxylin staining solution for 1-5 seconds, rinse with running water until the color does not change, then differentiate with hydrochloric acid alcohol for 1-2 seconds, wash with tap water, then soak the sections in 1% ammonia water for 3 minutes to regain blue color, and wash with tap water again.

[0080] 11) Dehydration and mounting: Place the sections in the following order of dehydration: 70% ethanol for 2 min → 80% ethanol for 2 min → 95% ethanol for 2 min → anhydrous ethanol I for 1 min → anhydrous ethanol II for 1 min → 50% anhydrous ethanol / 50% xylene for 1 min → xylene I for 5 min → xylene II for 5 min → xylene III for 5 min. Remove the sections from the xylene and immediately add neutral resin to the tissue. Cover with a coverslip, avoiding air bubbles during mounting. Allow the sections to dry and photograph them under a microscope.

[0081] 2. Experimental Results

[0082] The effects of β-hydroxybutyric acid (BHB) administration on hepatocyte proliferation after partial hepatectomy (PHx) are as follows: Figure 3 As shown; Figure 3In the diagram, A represents the Ki67 staining result of liver tissue. Figure 3 B in the table represents the quantitative results of Ki67 positive cells (n=3); data are expressed as mean ± standard deviation; compared with the Vehicle group, *P<0.05, **P<0.01; scale bar = 50μm. Figure 3 It was found that Ki67 expression was not observed in the liver tissue of mice in the Vehicle and BHB groups 1 day after PHx surgery; 2 days after surgery, the number of Ki67 positive cells in the BHB group reached its peak (24.3% positive cells), which was significantly higher than that in the Vehicle group (11.5% positive cells); 5 days after PHx surgery, the number of Ki67 positive cells decreased significantly, but the number in the BHB group (10.3% positive cells) was still higher than that in the Vehicle group (4.2% positive cells); the results indicate that administration of BHB significantly increased the proliferation of hepatocytes in mice after PHx.

[0083] Example 4: β-hydroxybutyrate upregulates the expression of proliferation-related proteins in the liver of mice after partial hepatectomy.

[0084] 1. Experimental Methods

[0085] This invention further examined the expression levels of cyclin A1 (CCNA1), D1 (CCND1), E1 (CCNE1), and cyclin-dependent kinase 4 (CDK4) in liver tissues from the Vehicle and P. distasonis groups after partial hepatectomy. CCNA1 and CCND1 expression are key markers of hepatocytes entering the S phase of the cell cycle; CCNE1 and CDK4 regulate the transition from G1 to S phase of the cell cycle. The main steps of protein immunoblotting are as follows:

[0086] (1) Extraction of total protein from liver tissue

[0087] 1) Remove the liver tissue from the -80℃ freezer and thaw it on ice;

[0088] 2) Mark the homogenizing tube and place 2 ceramic beads in the homogenizing tube;

[0089] 3) Weigh about 30 mg of liver tissue, put it into a homogenization tube, add 500 μL of protein RIPA lysis buffer (add PMSF and phosphatase inhibitor at a ratio of 1:100 before use), and tighten the cap of the homogenization tube.

[0090] 4) Place the homogenizing tube into the homogenizer and homogenize twice;

[0091] 5) After homogenization, let stand on ice for 20 minutes, then transfer the supernatant to a new 1.5 mL centrifuge tube and centrifuge at 4°C and 16000 g for 30 minutes.

[0092] 6) After centrifugation, transfer 200 μL of supernatant to a new 1.5 mL centrifuge tube and determine the protein concentration using a BCA kit.

[0093] (2) Protein concentration determination by BCA method

[0094] 1) Dilute the 2000 μg / mL bovine serum albumin standard with PBS buffer to 1000, 500, 250, 125 and 62.5 μg / mL, respectively;

[0095] 2) Take 10 μL of the original sample solution to be tested, add 290 μL of PBS buffer, and dilute the sample 30 times;

[0096] 3) Prepare BCA working solution according to the sample volume of +2. Mix BCA reagent A and reagent B in a ratio of 50:1, and then add the BCA working solution to the 96-well plate (avoid generating air bubbles), 200 μL / well.

[0097] 4) Add 25 μL of diluted protein standard and sample to the corresponding wells of the 96-well plate and gently tap to mix.

[0098] 5) Place the 96-well plate in a 37℃ water-jacketed electric thermostatic incubator and react for 30 minutes;

[0099] 6) Use a multi-functional microplate reader to measure the OD values ​​of the standard and the sample to be tested at a wavelength of 562 nm;

[0100] 7) Plot a standard curve based on the concentration of diluted protein standards and the measured OD values, and then calculate the protein concentration of the sample to be tested based on the curve;

[0101] 8) Based on the calculated protein concentration, dilute the protein concentration to 2 μg / μL with double-distilled water and 5× loading buffer. Place the prepared protein sample in a dry thermostat at 100℃ and heat for 10 min to denature the protein. After cooling to room temperature, store in a -80℃ refrigerator.

[0102] (3) Protein immunoblotting experiment

[0103] 1) Prepare the separating gel, flatten the liquid surface with anhydrous ethanol, and wait 30 minutes for the separating gel to solidify;

[0104] 2) Discard the anhydrous ethanol and blot off the excess anhydrous ethanol with filter paper; prepare the stacking gel, carefully and quickly add it to the gel plate, trying to avoid generating air bubbles, then insert a 15-well comb, wait 30 minutes until the stacking gel solidifies, immerse the prepared gel in the electrophoresis solution, and store it in a refrigerator at 4°C for later use.

[0105] 3) Sample loading: Take the protein sample out of the -80℃ freezer and place it in a 100℃ dry thermostat for 2 minutes to thaw the sample. Vortex to mix and then briefly separate the gel plate. Place the gel plate into the electrophoresis rack, add the electrophoresis buffer, and gently pull out the 15-well comb. Use the sample pipette tip to add 15μL of protein sample and 3μL of protein marker to the corresponding lane, and place it in the electrophoresis tank to prepare for electrophoresis.

[0106] 4) Electrophoresis: After loading the sample, cover the transfer tank and turn on the power (note the positive and negative terminals: red to red, black to black). Electrophoresis is performed at a constant voltage of 70V for 30 minutes. Then, the voltage is changed to 120V and electrophoresis is continued. Electrophoresis can be stopped when the blue indicator band of the loading buffer reaches the bottom of the gel plate.

[0107] 5) Electroporation: Immerse the cut PVDF membrane in methanol to activate it; remove the gel plate, cut off the target protein gel, and place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the electroporation clamp from negative to positive electrode in that order, taking care to avoid air bubbles between the membrane and the gel; turn on the power supply and electroporate under a constant current of 230A; set the electroporation time according to the molecular weight of the protein: 75 min for proteins below 70KD, 90 min for proteins above 70KD, 120 min for proteins above 90KD, and 150 min for proteins above 120KD.

[0108] 6) Sealing: After the transfer is completed, remove the transfer clamp and place the PVDF membrane in 5% skim milk. Seale it on a horizontal shaker at room temperature for 1 hour.

[0109] 7) Primary antibody incubation: After blocking, remove the PVDF membrane, rinse with TBST to remove residual milk, place it in the corresponding primary antibody dilution solution, and incubate overnight at 4°C;

[0110] 8) Secondary antibody incubation: Remove the membrane, wash it 3 times with TBST for 10 min each time; incubate with the corresponding secondary antibody at room temperature for 1 h; then remove the membrane again and wash it 3 times with TBST for 10 min each time.

[0111] 9) Development: Turn on the chemiluminescence analyzer and allow the instrument to cool to the working temperature; mix equal volumes of solutions A and B to make a developing solution, use filter paper to absorb excess TBST on the PVDF membrane, immerse it completely in the developing solution for 5-10 seconds, and then place it in the chemiluminescence analyzer for development.

[0112] 2. Experimental Results

[0113] The effects of β-hydroxybutyric acid (BHB) administration on the expression of hepatocyte proliferation-related proteins in liver tissue after partial hepatectomy (PHx) are as follows: Figure 4 As shown; Figure 4 In this context, A represents the protein expression results of CCNA1, CCND1, CCNE1, and CDK4 in liver tissue. Figure 4 B in the table represents the quantitative results of proliferation-related protein expression (n=3); data are expressed as mean ± standard deviation; compared with the Vehicle group, *P<0.05, **P<0.01. Figure 4 The results showed that, compared to the Vehicle group, CCNA1 protein expression in the BHB group significantly increased by 1.7-fold on day 2 post-PHx surgery, and decreased to initial levels on day 5 post-surgery. CCND1 protein expression was significantly upregulated by 2.0-fold on day 1 post-PHx surgery; CCNE1 protein expression significantly increased on both days 2 and 5 post-PHx surgery, upregulated by 2.1-fold and 1.9-fold, respectively. However, CDK4 protein expression showed no significant difference at any post-operative time point. These results indicate that BHB administration can upregulate the expression of proliferation-related proteins in the liver after PHx surgery.

[0114] This invention analyzed liver tissue using Ki67 staining and protein immunoblotting to determine whether β-hydroxybutyrate (β-hydroxybutyrate) promotes liver regeneration after PHx surgery by promoting hepatocyte proliferation. The results indicate that β-hydroxybutyrate can promote hepatocyte proliferation and thus liver regeneration by upregulating the expression of proliferation-related proteins in the liver after PHx surgery.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of β-hydroxybutyric acid as the sole active ingredient in the preparation of products promoting liver regeneration, characterized in that, The liver in question is the liver after partial hepatectomy, with the removed portion not exceeding 2 / 3 of the original liver volume.

2. The application according to claim 1, characterized in that, The β-hydroxybutyric acid promotes liver regeneration by promoting hepatocyte proliferation and restoring liver biochemical indicators to normal.

3. The application according to claim 2, characterized in that, The β-hydroxybutyric acid promotes hepatocyte proliferation by upregulating the expression of hepatocyte proliferation-related proteins.

4. The application according to any one of claims 1 to 3, characterized in that, The product includes β-hydroxybutyric acid and its pharmaceutically acceptable carrier.

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