Application of beta-hydroxybutyrate in preparation of medicine for preventing or treating hepatocellular carcinoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of beta-hydroxybutyrate in preparation of a medicine for preventing or treating hepatocellular carcinoma. The invention provides an application of beta-hydroxybutyrate in preparation of a medicine for preventing and / or treating hepatocellular carcinoma. The beta-hydroxybutyrate comprises a beta-hydroxybutyrate or a combination of a plurality of beta-hydroxybutyrates. A series of experimental data show that beta-hydroxybutyrate not only can effectively improve fatty degeneration and inflammatory response of hepatocytes, but also can remarkably reduce the risk of occurrence of metabolic dysfunction related hepatocellular carcinoma (HCC), and can inhibit malignant biological characteristics of HCC. The invention provides a powerful scientific basis for the development of novel HCC prevention and treatment medicines, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of β-hydroxybutyrate in the preparation of drugs for the prevention or treatment of hepatocellular carcinoma. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a crucial stage in the progression of metabolic associated fatty liver disease (MAFLD) and can directly develop into hepatocellular carcinoma (HCC). With the continuous rise in the incidence of metabolic diseases, the prevalence of MASH has been increasing year by year, becoming a major pathogenic factor for HCC. However, to date, the mechanisms promoting the progression of MASH to HCC have not been fully elucidated, and corresponding intervention methods are lacking. Therefore, revealing the key regulatory mechanisms of the MASH-HCC transformation and developing novel molecular blocking therapies accordingly has become a critical clinical problem that urgently needs to be solved.

[0003] Lipid metabolism disorders are a key characteristic of MASH and a significant driver of disease progression. Ketone body dysfunction, a crucial component of lipid metabolism disorders, has been shown to be closely associated with MASH progression. Multiple studies have demonstrated that the ketogenic diet can effectively slow MASH progression by improving lipid metabolism, reducing inflammation and oxidative stress, and has been shown to inhibit the development of various tumors. β-hydroxybutyric acid (BHB) is a major component of ketone bodies, serving not only as an energy source but also as an important signaling molecule and epigenetic regulator. Studies have reported reduced BHB synthesis under MASH pathological conditions, with the degree of reduction closely correlated with poor prognosis. However, the specific role and therapeutic potential of BHB in MASH and its progression to HCC remain unclear. Summary of the Invention

[0004] To address the lack of effective drugs for the prevention and treatment of hepatocellular carcinoma in the existing technology, this invention provides the application of β-hydroxybutyrate in the preparation of drugs for the prevention or treatment of hepatocellular carcinoma, specifically including the following technical solutions:

[0005] The use of β-hydroxybutyrate in the preparation of drugs for the prevention and / or treatment of hepatocellular carcinoma.

[0006] Preferably, the β-hydroxybutyrate comprises sodium β-hydroxybutyrate.

[0007] Preferably, the hepatocellular carcinoma includes hepatocellular carcinoma caused by metabolic dysfunction-related steatohepatitis.

[0008] The present invention also provides a medicament for the prevention and / or treatment of hepatocellular carcinoma, wherein the raw materials of the medicament include an active substance and excipients; the active substance includes β-hydroxybutyrate.

[0009] Preferably, the β-hydroxybutyrate comprises one β-hydroxybutyrate or a combination of multiple β-hydroxybutyrates.

[0010] Preferably, the β-hydroxybutyrate comprises sodium β-hydroxybutyrate.

[0011] Preferably, the excipients include pharmaceutically acceptable excipients.

[0012] Preferably, the dosage form of the drug includes one or more of tablets, capsules, pills, oral liquid preparations, granules, powders, and injections.

[0013] Preferably, the method of administration of the drug includes one or more of oral, injection, implantation, external application, spray, and inhalation.

[0014] The present invention also provides a method for preparing the drug as described above, characterized by comprising the following steps: mixing the active substance and excipients to obtain the drug; wherein the active substance comprises β-hydroxybutyrate.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention belongs to the field of biomedical technology, specifically relating to the application of β-hydroxybutyrate in the preparation of drugs for the prevention or treatment of hepatocellular carcinoma. This invention provides the application of β-hydroxybutyrate in the preparation of drugs for the prevention and / or treatment of hepatocellular carcinoma. A series of experimental data demonstrate that β-hydroxybutyrate can not only effectively improve steatosis and inflammatory responses in hepatocytes, but also significantly reduce the risk of metabolic dysfunction-related hepatocellular carcinoma (HCC) and inhibit the malignant biological characteristics of HCC. This invention provides strong scientific evidence for the development of novel drugs for the prevention and treatment of HCC and has broad prospects for clinical application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The molecular structural formula of BHB;

[0019] Figure 2 The molecular structural formula of NaBHB is shown below.

[0020] Figure 3 The inhibitory effect of NaBHB on the occurrence of MASH-related HCC in mice is shown in Figure A, which is a schematic diagram of the process of constructing a mouse model of MASH-related HCC; Figure B shows the differences in liver tumors among different groups of mice; and Figure C shows the statistical results of the number of liver tumors among different groups of mice.

[0021] Figure 4 The inhibitory effect of NaBHB on tumor growth in tumor-bearing nude mice is shown in Figure 1. A represents the inhibitory effect of NaBHB on tumor size in tumor-bearing nude mice, and B represents the tumor growth curve of a subcutaneous tumor model in nude mice treated with NaBHB.

[0022] Figure 5 The results show the inhibitory effect of NaBHB on the malignant phenotype of hepatocellular carcinoma cells; where A and B show the inhibitory effects of NaBHB on the proliferation of human hepatocellular carcinoma cell line Huh7 and mouse hepatocellular carcinoma cell line Hepa1-6, respectively; C shows the inhibitory effect of NaBHB on the migration and invasion ability of hepatocellular carcinoma cell line Huh7; and D shows the inhibitory effect of NaBHB on the migration and invasion ability of hepatocellular carcinoma cell line Hepa1-6.

[0023] Figure 6 This demonstrates the inhibitory effect of NaBHB on the growth of human liver cancer organoids. Detailed Implementation

[0024] This invention provides the use of β-hydroxybutyrate in the preparation of medicaments for the prevention and / or treatment of hepatocellular carcinoma.

[0025] In one embodiment, the β-hydroxybutyrate comprises one β-hydroxybutyrate or a combination of multiple β-hydroxybutyrates. In another embodiment, the β-hydroxybutyrate of this invention comprises sodium β-hydroxybutyrate. In yet another embodiment, the hepatocellular carcinoma includes hepatocellular carcinoma caused by metabolic dysfunction-associated steatohepatitis. In the prior art, although some techniques mention the relationship between β-hydroxybutyrate and MASH, the pathological mechanisms of MASH and HCC are significantly different. The occurrence of MASH mainly involves factors such as lipid metabolism, inflammatory response, and oxidative stress, while the occurrence of HCC is related to more complex gene mutations, the carcinogenesis process of hepatocytes, and changes in the tumor microenvironment. Therefore, although BHB may have an inhibitory effect on MASH by improving lipid metabolism and suppressing inflammation, its therapeutic effect on HCC cannot be directly inferred.

[0026] This invention also provides a medicament for the prevention and / or treatment of hepatocellular carcinoma, the medicament comprising an active substance and excipients; the active substance is β-hydroxybutyrate. As one embodiment, the β-hydroxybutyrate comprises sodium β-hydroxybutyrate. As one embodiment, the dosage of the β-hydroxybutyrate is 80–120 mg / kg based on mouse body weight when the target organism is a mouse. As another embodiment, when the target organism is a mouse, the dosage of the β-hydroxybutyrate based on mouse body weight can be any one or an intermediate value of any two of the following: 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, and 120 mg / kg. As one embodiment, the excipients comprise pharmaceutically acceptable excipients. In another embodiment, the excipients include any one or more of solvents, propellants, solubilizers, suspending agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, flow aids, pressure aids, flavoring agents, preservatives, coating agents, and fragrances. In another embodiment, the dosage form of the drug includes one or more of tablets, capsules, pills, oral liquid preparations, granules, powders, and injections. In another embodiment, the route of administration of the drug includes one or more of oral, injection, implantation, external application, spray, and inhalation. In a specific embodiment, the dosage form of the drug is an injection, and the route of administration includes injection.

[0027] As one embodiment, the present invention also provides a method for preparing the drug for the prevention and / or treatment of hepatocellular carcinoma as described above, comprising the steps of: dissolving the active substance in a solvent, mixing, and obtaining the drug. As one embodiment, the active substance and its dosage are as described above, and will not be repeated here.

[0028] To further illustrate the present invention, the application of the β-hydroxybutyrate provided by the present invention in the preparation of drugs for the prevention or treatment of hepatocellular carcinoma is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] The chemical structural formula of β-hydroxybutyric acid described in the background section of this invention is as follows: Figure 1 As shown; the sodium β-hydroxybutyrate (NaBHB) used in the embodiments of the present invention was purchased from Sigma-Aldrich, product code #54965, and the chemical structural formula of NaBHB is shown below. Figure 2As shown. NaBHB was prepared into a 159 mM (20 mg / mL) solution and stored. The specific preparation method is as follows: Weigh 600 mg of NaBHB powder, add 30 mL of dd water to a 50 mL centrifuge tube, shake for 1 min, and then heat in a 37°C water bath for 20–30 min. Dissolve completely by ultrasonic cleaning for 10 min × 3 times. After standing for 20 min, use… Filter the solution through a 0.22μm syringe filter, dispense into 1.5mL centrifuge tubes, seal and label, and store at -80℃.

[0030] The mice used in this embodiment were C57BL / 6J mice and BALB / cNude nude mice, both purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and raised under specific pathogen-free (SPF) conditions. The cell lines used in this embodiment were: human hepatocellular carcinoma cell line Huh7 and mouse hepatocellular carcinoma cell line Hepa1-6. Both cell lines were cultured in DMEM medium (iCell) containing 1.5 g / L sodium bicarbonate supplemented with 10% fetal bovine serum (ExCell) and 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO2.

[0031] Example 1: NaBHB inhibits the evolution of MASH to HCC in mice.

[0032] Model Construction: Female C57BL / 6J pregnant mice were raised and bred under specific pathogen-free (SPF) conditions. Newborn male pups were randomly divided into three groups: normal group, model group, and experimental group, with 8 mice in each group. On day 2 after birth, mice in the model and experimental groups received a single subcutaneous injection of 100 μg streptozotocin (STZ) in their backs, while mice in the normal group received an equal amount of dimethyl sulfoxide (DMSO) as a control. After 4 weeks, mice in the model and experimental groups were fed a high-fat diet (HFD), while the normal group was fed a normal diet. After 8 weeks, mice in the experimental group received an intraperitoneal injection of sodium β-hydroxybutyrate (NaBHB, concentration 20 mg / mL, dosage: 100 mg / kg based on mouse body weight, every other day), while the normal and model groups received an equal dose of saline. Mice were monitored for body weight and blood glucose weekly, and euthanized at the end of week 20. The procedure for constructing a mouse HCC model is as follows: Figure 3 As shown in Figure A.

[0033] Experimental results are as follows Figure 3 As shown in Table 1, the results demonstrate that this embodiment successfully constructed a MASH-related HCC mouse model capable of simulating the evolution of MASH into HCC. Figure 3(See Figure A). Histological sections stained with hematoxylin and eosin (HE) from mice were prepared using standard histological section preparation methods. Comparison of the gross morphology of the livers and HE-stained histological sections from different groups of mice showed that at week 20, significant carcinogenesis occurred in the liver tissue of the model group mice. After NaBHB intervention, the incidence and number of MASH-related HCCs were significantly reduced (p<0.05) (see Figure A). Figure 3 (See Tables B, C, and 1).

[0034] Table 1. Number of tumors in mice from different groups.

[0035] normal group Model group NaBHB treatment group 0 5 2 0 4 3 0 6 4 0 8 4 0 5 3 0 3 5 0 4 1 0 6 0

[0036] Example 2: NaBHB inhibits the growth of HCC in tumor-bearing nude mice.

[0037] Methods for establishing a mouse tumor-bearing model:

[0038] (1) Preparation of Hepa1-6 cell suspension: Hepa1-6 cells were digested with trypsin, centrifuged at 800 rpm, and the culture medium was removed as described above. The cells were resuspended in PBS and centrifuged again to remove residual culture medium. After cell counting, the suspension was adjusted to 1×10⁶ cells / mL. 7 A suspension of cells / mL.

[0039] (2) Establishment of tumor-bearing mouse model: Twelve BALB / cNude nude mice were randomly divided into experimental group and control group, with six mice in each group. When the nude mice were 5 weeks old, Hepa1-6 cell suspension (100 μL / mouse) was injected subcutaneously into the axilla of the nude mice.

[0040] (3) Tumor growth observation: After inoculation of cells, the growth of mouse tumors was observed regularly, and the time of tumor appearance and growth characteristics were recorded. Drug administration was started when the tumor was palpable.

[0041] (4) Administration regimen: Mice in the experimental group were given intraperitoneal injections of NaBHB (concentration of 20 mg / mL, dosage: 100 mg / kg based on mouse body weight, every other day), while mice in the control group were given an equal volume of physiological saline intraperitoneally. Each mouse was administered the medication once daily for 12 consecutive days. Tumor size was measured every 3 days. The method for measuring tumor size was as follows: on live mice, the width and diameter of the subcutaneous tumor were measured using calipers to a palpable extent, and the tumor volume was calculated using the following formula:

[0042] Tumor volume = length × width 2 / 2.

[0043] Experimental results are as follows Figure 4As shown in Table 2, tumor-bearing models were successfully established in all nude mice, and the tumors became palpable approximately 6 days after cell inoculation. Compared with the control group, the experimental group mice showed significantly slower tumor growth and significantly smaller tumor volume after 12 consecutive days of intraperitoneal injection of NaBHB (p<0.05), indicating that NaBHB effectively inhibited tumor growth.

[0044] Table 2. Tumor volume in mice at different time points (mm) 3 )

[0045]

[0046]

[0047] Example 3: The ability of NaBHB to inhibit the proliferation, migration, and invasion of liver cancer cells in vitro.

[0048] Experimental methods:

[0049] 1. Cell Counting Kit-8 (CCK-8) Experiment

[0050] Detection principle: Mitochondrial dehydrogenases in living cells can reduce WST-8 in the CCK-8 reagent to water-soluble yellow formazan. Within a certain cell number range, the amount of formazan produced is directly proportional to the number of living cells, while dead cells do not have this function. Therefore, the number of living cells can be indirectly reflected by measuring the absorbance (OD value) at 450 nm. This method has been widely used in cell proliferation, antitumor drug screening, and cytotoxicity detection.

[0051] Experimental steps: (1) Human liver cancer cell line Huh7 and mouse liver cancer cell line Hepa1-6 were digested with trypsin and centrifuged to collect the cells. The collected cells were resuspended in their respective culture media, counted, and diluted to 1×10⁻⁶. 4 (2) Inoculate the cell suspension in a 96-well plate with 100 μL per well. Design five concentration groups: 0 mM, 20 mM, 40 mM, 60 mM and 80 mM. Design three replicates for each concentration group and set up a blank group. (3) Place the culture plate in an incubator (37℃, 5% CO2) overnight. After the cells adhere well, aspirate the culture medium and add 0 mM, 20 mM, 40 mM, 60 mM and 80 mM NaBHB to each well of the culture plate, and incubate for 72 h. (4) Add 10 μL of LCK-8 solution to each well, gently shake the culture plate to mix, and continue incubating for 1 h. (5) Measure the absorbance (OD value) at 450 nm using a microplate reader.

[0052] Experimental results are as follows Figure 5 As shown in Table A and Tables 3-4. Figure 5 Figure A shows the dose-response curves of NaBHB on the cell viability of human hepatocellular carcinoma line Huh7 and mouse hepatocellular carcinoma line Hepa1-6. The NaBHB concentrations used in the experiments were 0 mM, 20 mM, 40 mM, 60 mM, and 80 mM. The results showed that at a concentration of 80 mM, the survival rate of Huh7 cells was 19%, and the survival rate of Hepa1-6 cells was 14%, indicating that NaBHB significantly inhibited the activity and growth of both Huh7 and Hepa1-6 cells.

[0053] Table 3. Cell growth in the NaBHB and Huh-7 groups.

[0054] NaBHB(mM) 0 20 40 60 80 Blank OD value (1) 0.304 0.290 0.248 0.197 0.188 0.151 OD value (2) 0.312 0.277 0.263 0.223 0.172 0.154 OD value (3) 0.334 0.251 0.258 0.222 0.202 0.164

[0055] Table 4. Cell growth in the NaBHB group and the Hepa1-6 group.

[0056] NaBHB(mM) 0 20 40 60 80 Blank OD value (1) 0.224 0.230 0.214 0.192 0.184 0.172 OD value (2) 0.220 0.237 0.207 0.197 0.178 0.176 OD value (3) 0.257 0.228 0.230 0.198 0.188 0.176

[0057] 2. Plate cloning experiment

[0058] Detection Principle: A colony refers to a cell population formed after a single cell has proliferated for more than six generations in vitro, forming a visible colony or clone. At this stage, each clone is 0.3–1.0 mm in size and contains more than 50 cells. By counting the colony formation rate, the proliferation potential of a single cell can be quantitatively analyzed. Colony formation on a plate, applied to adherent cells, is an effective method for determining the proliferative capacity of a single cell.

[0059] Experimental steps: (1) Take the human liver cancer cell line Huh7 and the mouse liver cancer cell line Hepa1-6 in the logarithmic growth phase, digest them with trypsin and pipette them into single cells, and suspend them in 10% fetal bovine serum medium for later use. (2) Dilute the cell suspension and seed them into six-well plates at an appropriate cell density (500 cells per well). Disperse them evenly and set up a control group and an experimental group. The experimental group was given 60mM NaBHB intervention, and the control group was given the same volume of PBS. (3) Incubate in a 37℃ 5% CO2 incubator for 2 weeks, changing the medium every 3 days. When visible clones appear in the culture dish, stop the culture. (4) Carefully wash twice with PBS, add 5mL of pure methanol, fix for 15min, then remove the fixative, add an appropriate amount of 0.1% crystal violet staining for 20-30min, slowly wash away the staining solution with running water, and air dry. (5) Use ImageJ software to count clones and calculate the clone formation rate; clone formation rate = (number of clones / number of seeded cells) × 100%.

[0060] Experimental results are as follows Figure 5The results showed that NaBHB treatment significantly reduced the number and size of Huh7 and Hepa1-6 cell colonies, indicating that NaBHB has a significant inhibitory effect on the proliferation and colony formation of liver cancer cells.

[0061] 3. Transwell migration / invasion experiments

[0062] Detection Principle: The Transwell chamber consists of a porous polycarbonate membrane and two chambers, upper and lower. Cells are seeded in the upper chamber, while the lower chamber contains components that induce cell migration. Cells migrate through the pores in the membrane to the lower chamber, mimicking the behavior of cells crossing the basement membrane and extracellular matrix in vivo. There are two types of assays, depending on whether matrix gel is added to the chamber: migration assay (no matrix gel added) and invasion assay (matrix gel added, in which case cells not only migrate through the porous membrane but also undergo degradation or invasion through the extracellular matrix layer coated on top of the membrane).

[0063] Experimental steps: (1) The Transwell experiment was conducted in a polycarbonate filter chamber (pore size: 8.0 μm, Corning, USA). The invasion experiment was performed by coating the filter chamber with Matrigel (Corning, USA). (2) The experiment set up a control group and an experimental group. The experimental group was treated with 60 mM NaBHB for 72 h, and the control group was given the same volume of PBS. The cells were digested with enzymes according to the method described above and then resuspended in serum-free medium (the cells were starved for 12-24 h before preparing the cell suspension to remove the influence of serum). (3) 500 μL of medium containing 10% FBS was added to the lower chamber of the 24-well plate. The Transwell chamber was placed in the 24-well plate with tweezers (note to avoid and remove air bubbles generated between the lower culture medium and the chamber). Then, 200 μL of serum-free cell suspension (1.5 × 10⁻⁶) was added to each well. 5 Huh7 cells / well, 1.5 × 10⁶ 5 (1-6 Hepa cells / well) were transferred to the upper chamber of the Transwell plate; after incubation for 24h (migration) and 48h (invasion), fixation and staining were performed. (4) Wash twice with PBS, add 4% paraformaldehyde to the clean wells of the 24-well plate, immerse the bottom of the chamber in the solution, and fix for 10-15min. (5) Wash the chamber and the inside once with PBS, stain with 0.1% crystal violet for 5-10min, wash the chamber and the inside three times with PBS to remove the crystal violet that has not bound to the cells, gently wipe the upper side of the chamber with a cotton swab to remove non-migrating or non-invading cells; after proper air drying, take images through a 400× microscope and use ImageJ software to perform quantitative analysis of the cells in the images.

[0064] Experimental results are as follows Figure 5As shown in Figures C and D, NaBHB inhibits the migration and invasion of Huh7 and Hepa1-6 cells: Treatment with 60 mM NaBHB significantly inhibited the migration ability of Huh7 and Hepa1-6 cells after 24 h; and significantly inhibited their invasion ability after 48 h. These results confirm the inhibitory effect of NaBHB on the migration and invasion of Huh7 and Hepa1-6 cells.

[0065] Example 4: BHB inhibits the growth of human liver cancer organoids in vitro.

[0066] Construction of human hepatocellular carcinoma organoids (Hepatocellular carcinoma organoid kit purchased from BioGenous Biotechnology Co., Ltd.) TM Hepatocellular Carcinoma Organoid Kit (Catalog: K2105-HCC) is prepared as follows: (1) Collect primary human hepatocellular carcinoma tissue blocks (about the size of a soybean) in a 15 mL centrifuge tube containing primary tissue transport and preservation solution, and maintain a low temperature environment of 4°C until arrival at the laboratory. (2) Evaluate the composition of the obtained tissue fragments. If fat or muscle tissue is present, remove it as much as possible using surgical scissors and forceps. (3) Hold the tissue block with sterile forceps and wash it 2-3 times in a culture dish containing tumor organoid basal culture medium. (4) Prepare 10 mL of tumor tissue digestion solution in a 15 mL centrifuge tube (the digestion solution is a component of the kit, including 9.5 mL of hepatocellular carcinoma organoid basal culture medium A + 0.5 mL of hepatocellular carcinoma organoid supplement B). (5) Place the tissue block in a 5 mL centrifuge tube, add 3-5 mL of tumor tissue digestion solution, and cut the tumor tissue into 1-3 mm pieces using sterile tissue scissors. 3(6) Transfer the suspension to a 15 mL centrifuge tube containing tumor tissue digestion solution, place it in a cell culture incubator, digest at 37°C for 30 min, and remove the centrifuge tube every 5 min to shake thoroughly. (7) Add fetal bovine serum (FBS) to the digested tissue suspension to a final concentration of approximately 2% (205 μL) to terminate the digestion. (8) Filter the tissue suspension using a cell filter (100 μm), centrifuge the filtrate at 4°C and 300 g for 3 min, and retain the precipitate. (9) Add 2-3 mL of erythrocyte lysis buffer to resuspend, gently pipette to mix, maintain at room temperature for 1 min, then centrifuge at 4°C and 300 g for 3 min, and retain the precipitate. (10) Add 3-5 mL of tumor organoid basal culture medium to resuspend, pipette to mix, centrifuge at 4°C and 300 g for 3 min, and retain the precipitate. (11) Resuspend the cells in 3-5 mL of tumor organoid basal culture medium, mix by pipetting, and count and observe cell viability using a small amount of trypan blue staining solution. (12) Centrifuge the remaining cell suspension at 4°C and 300 g for 3 min, and retain the precipitate. (13) Calculate the amount of matrix gel to be added according to the ratio of 25-30 μL of matrix gel per well for every 10,000 cells (the matrix gel should be melted at 4°C beforehand and the temperature should be maintained), and mix on ice throughout the process (the pipetting action should be gentle, and air bubbles should be avoided; mixing at room temperature should be completed within 15 s), and place on ice after mixing. (14) Use a pipette to aspirate the mixture into the cell culture plate, add 25-30 μL of cell suspension to each well (place the pipette in the center of the well, without touching the sidewall of the culture well), and then place the culture plate in a 37°C, 5% CO2 cell culture incubator to solidify for 15 min. (15) Prepare 10 mL of Hepatocellular Carcinoma Organoid Kit (the raw materials for the Hepatocellular Carcinoma Organoid Kit are the components in the kit, including 9.76 mL of Hepatocellular Carcinoma Organoid Basic Culture Medium A + 200 μL of Hepatocellular Carcinoma Organoid Supplement B + 40 μL of Hepatocellular Carcinoma Organoid Supplement C). (16) After solidification, add 500 μL of Hepatocellular Carcinoma Organoid Kit to each well along the sidewall of the culture well, and then place the culture plate in a 37℃, 5% CO2 cell culture incubator for culture. The experiment set up a NaBHB dosing group (60 mM) and a control group (same volume of PBS intervention). The organoid growth was observed every 2-3 days and the culture medium was changed. The culture was carried out for a total of 14 days.

[0067] Experimental results are as follows Figure 6 As shown, NaBHB treatment significantly inhibited the growth of human hepatocellular carcinoma organoids. After NaBHB intervention, the volume of human hepatocellular carcinoma organoids was significantly reduced, providing strong experimental evidence for the potential of NaBHB in the treatment of hepatocellular carcinoma. Further in-depth research into its mechanism of action and clinical application value is warranted.

[0068] In summary, β-hydroxybutyrate has broad application prospects in the fields of tumor prevention and anti-tumor drugs. Furthermore, the β-hydroxybutyrate of this invention exhibits unique advantages: it is easily accepted by patients, inexpensive, and readily available. β-hydroxybutyrate is expected to change the market landscape of existing tumor chemotherapy drugs, gradually becoming a clinically viable drug that can be taken long-term, with the potential to effectively prevent HCC and inhibit tumor growth, metastasis, and invasion.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of β-hydroxybutyrate in the preparation of drugs for the prevention and / or treatment of hepatocellular carcinoma.

2. The application as described in claim 1, characterized in that, The β-hydroxybutyrate includes sodium β-hydroxybutyrate.

3. The application as described in claim 1, characterized in that, The hepatocellular carcinoma includes hepatocellular carcinoma caused by metabolic dysfunction-related steatohepatitis.

4. A drug for the prevention and / or treatment of hepatocellular carcinoma, characterized in that, The raw materials of the drug include active substances and excipients; the active substances include β-hydroxybutyrate.

5. The drug as described in claim 4, characterized in that, The β-hydroxybutyrate includes one β-hydroxybutyrate or a combination of multiple β-hydroxybutyrates.

6. The drug as described in claim 4, characterized in that, The β-hydroxybutyrate includes sodium β-hydroxybutyrate.

7. The drug as described in claim 4, characterized in that, The excipients include pharmaceutically acceptable excipients.

8. The drug as described in claim 4, characterized in that, The dosage form of the drug includes one or more of the following: tablets, capsules, pills, oral liquid preparations, granules, powders, and injections.

9. The drug as described in claim 4, characterized in that, The drug can be administered via one or more of the following methods: oral, injection, implantation, topical, spray, and inhalation.

10. A method for preparing the drug according to any one of claims 4 to 9, characterized in that, The process includes the following steps: mixing the active substance and excipients to obtain the drug; The active substance includes β-hydroxybutyrate.

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