Application of heparin dodecaose in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention discloses application of heparin dodecaose in preparation of a medicine for treating hepatocellular carcinoma, and belongs to the technical field of biological medicine. Specifically, the invention relates to application of heparin dodecaose in preparation of a medicine for treating liver cancer. Compared with the prior art, the invention discloses the influence of heparin dodecanose on hypoxia and lipid metabolism change of hepatocellular carcinoma cells when hepatocellular carcinoma occurs, a hypoxia simulation agent cobalt chloride is adopted to simulate a hypoxia environment caused by deep vein thrombosis, and heparin oligosaccharides with different concentrations are adopted to act on hepatocellular carcinoma cells HepG2. The result shows that the heparin dodecaose can inhibit the levels of triglyceride, free fatty acid and ATP in the hypoxia hepatoma cells and significantly down-regulate the expression levels of hypoxia-inducible factors HIF-1alpha, FASN and SREBP-1 protein and mRNA, which indicates that the heparin dodecaose inhibits proliferation of the hepatoma cells by inhibiting lipid metabolism reprogramming of the hepatoma cells under the hypoxia condition. Therefore, the research can provide a new way for preparing the medicine for treating the hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to the use of heparin dodecasaccharide in the preparation of a drug for treating hepatocellular carcinoma. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer mainly derived from the transformation of hepatocytes, ranking third in the world in terms of cancer mortality. The high mortality rate of HCC can be attributed to its late symptoms and resistance to traditional chemotherapeutic drugs. The causes of HCC are complex and diverse, and are usually closely related to chronic liver damage, inflammation and cirrhosis.

[0003] Hypoxia is a hallmark feature of the microenvironment of solid tumors, due to the remodeling of the vascular system and metabolic reprogramming. Hypoxia-inducible factor-1 (HIF-1) is the main regulator of oxygen homeostasis. Previous studies have shown that HIF-1α-mediated genes cause angiogenesis and metastasis, which are crucial for the development and progression of HCC. Fatty acids (FAs) are provided by exogenous FA uptake or de novo synthesis, which can serve as substrates for oxidation and energy production, membrane synthesis, energy storage in the form of triglycerides (TAGs), and signal molecule production, and thus are essential for cell survival and proliferation under normoxic and hypoxic conditions. However, since FA oxidation occurs within the mitochondria, which requires oxygen, under hypoxic conditions, FA metabolism must be modified in order to serve primarily processes other than energy production. Under hypoxic conditions, the transcription factor PPAR γ promotes the uptake of extracellular FAs and the synthesis of TAGs, while the gene of PPAR γ is directly activated by HIF-1α. And in order to maintain de novo synthesis of FAs under hypoxic conditions, cancer cells support the production of FA precursors through HIF-dependent stimulation of reductive glutamine metabolism. Adequate FA supply further supports the activation of Akt and HIF-1-dependent SREBP-1, which in turn upregulates the expression of FASN (fatty acid synthase). Among them, SREBP-1, i.e., sterol regulatory element binding protein-1, is a key transcription factor that regulates lipid synthesis, and plays an important role in liver metabolism, lipogenesis and liver cancer development. Since FA catabolism is impaired under hypoxic conditions, excess free FAs accumulated in cells can cause lipotoxicity. To avoid this, cells can convert FAs into neutral TAGs, which are stored in lipid droplets (LDs) and can serve as the main form of energy reserves. In tumor cells that usually need to grow in a hypoxic microenvironment, these hypoxia-mediated changes in lipid metabolism are particularly important for maintaining the high proliferation rate of cancer cells. Therefore, inhibition of hypoxia-mediated lipid metabolism reprogramming can be a potential therapeutic strategy for hepatocellular carcinoma.

[0004] Previous studies have shown that in many cancer types, silencing or interfering with the expression of target genes required for lipid accumulation by HIFs leads to reduced proliferative potential and chemotherapy resistance under hypoxic conditions. Intervention in HIF-dependent lipid metabolism reprogramming can indeed effectively inhibit cancer cell proliferation. Therefore, research on hypoxia pathways will contribute to the treatment of HCC. Cobalt chloride is a commonly used chemical hypoxia mimic; its mechanism involves cobalt inactivating ferrous chelate enzymes through intracellular ion exchange, thereby inhibiting cellular oxygen utilization and ultimately inducing cellular hypoxia under normoxic conditions. Summary of the Invention

[0005] In view of the shortcomings of the prior art, this invention proposes the use of heparin dodecanose in the preparation of drugs for treating hepatocellular carcinoma.

[0006] This invention reveals the effects of heparin dodecanose on hypoxia and lipid metabolism changes in hepatocellular carcinoma (HCC) cells during the development of HCC. Using cobalt chloride, a hypoxia mimic, to simulate the hypoxic environment induced by deep vein thrombosis, different concentrations of heparin oligosaccharides were applied to HepG2 HCC cells. The results showed that heparin dodecanose inhibited the levels of triglycerides, free fatty acids, and ATP in hypoxic HCC cells and significantly downregulated the expression levels of hypoxia-inducible factors HIF-1α, FASN, and SREBP-1 proteins and mRNA. This indicates that heparin dodecanose inhibits HCC cell proliferation by suppressing lipid metabolism reprogramming under hypoxic conditions, thereby inhibiting HCC progression. This research could provide a new approach for the preparation of drugs to treat HCC.

[0007] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to the use of heparin dodecanose in the preparation of a medicament for treating liver cancer.

[0008] Optionally, the treatment of liver cancer includes inhibiting hypoxia-mediated lipid metabolism reprogramming.

[0009] Optionally, the treatment of liver cancer includes: inhibiting the levels of free fatty acids and / or triglycerides in liver cancer cells.

[0010] Optionally, the treatment of liver cancer includes: inhibiting the level of adenosine triphosphate (ATP) in liver cancer cells.

[0011] Optionally, the treatment of liver cancer includes inhibiting the transcription and / or expression of lipid synthesis genes FASN, SREBP-1, and HIF-1α proteins.

[0012] Optionally, the structural formula of the heparin dodecaose is: .

[0013] Optionally, the concentration of heparin dodecanose in the drug is configured such that the concentration of heparin dodecanose in the liver cancer cell environment reaches 0.01 μM to 1 μM.

[0014] A second aspect of the invention relates to a medicament for treating liver cancer, comprising heparin dodecaose.

[0015] The beneficial effects of this invention are: The heparin dodecanose described in this invention can be used as an active ingredient in the treatment of hepatocellular carcinoma, providing a new option for the preparation of drugs for treating hepatocellular carcinoma. The heparin dodecanose of this invention inhibits the abnormal proliferation and ATP levels of hypoxic hepatocellular carcinoma cells, inhibits the levels of triglycerides and free fatty acids in hypoxic hepatocellular carcinoma cells, and significantly downregulates the expression levels of hypoxia-inducible factors HIF-1α, FASN, and SREBP-1 proteins and mRNA, thus inhibiting lipid metabolism reprogramming in hypoxic hepatocellular carcinoma cells. Therefore, heparin dodecanose can be used to prepare drugs for the treatment of hepatocellular carcinoma. Attached Figure Description

[0016] The invention will now be further described with reference to the accompanying drawings.

[0017] Figure 1 Statistical analysis results of data from the MTT assay to determine the effect of heparin dodecanose on the proliferation of HepG2 cells; Figure 2 Figure showing the results of Oil Red O staining assay to determine the effect of oleic acid on lipid droplet accumulation in HepG2 cells; Figure 3 Statistical analysis results of data on the effect of different concentrations of OA on the viability of HepG2 cells in the CCK-8 assay; Figure 4 Statistical analysis of data from triglyceride detection experiments to determine the effect of heparin dodecanose on triglyceride levels in HepG2 cells; Figure 5 Statistical analysis of data to determine the effect of heparin dodecanose on the free fatty acid content of HepG2 cells in the free fatty acid detection experiment; Figure 6 Statistical analysis of data from ATP content detection experiments to determine the effect of heparin dodecanose on ATP levels in HepG2 cells; Figure 7 Statistical analysis of data from qPCR experiments to determine the effect of heparin dodecanose on FASN and SREBP-1 mRNA levels in HepG2 cells; Figure 8 Protein bands and grayscale analysis results for determining the effect of heparin dodecanose on FASN protein expression levels in HepG2 cells using Western blotting experiments; Figure 9Protein bands and grayscale analysis results for determining the effect of heparin dodecanose on SREBP-1 protein expression level in HepG2 cells using Western blotting experiments; Figure 10 The image shows the protein bands and grayscale analysis results used in Western blotting experiments to determine the effect of heparin dodecanose on the expression level of HIF-1α protein in HepG2 cells. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In the following examples, OA refers to oleic acid; TG refers to triglycerides; FFA refers to free fatty acids; and ATP refers to adenosine triphosphate. In the following results figures, C refers to Control; M refers to Model; and HDO refers to heparin dodecaose.

[0020] The heparin dodecanose of this invention is a heparin oligosaccharide fragment with a molecular weight of 3.2 KD obtained by cleaving heparin with nitrite and then separating and purifying it using a Bio-gel P6 molecular sieve. Its structural formula is as follows: The commonly used concentration of cobalt chloride to induce cellular hypoxia is 10-300 μM. In this invention, based on the relevant literature "The use of cobalt chloride as a chemical hypoxia model" and cell differentiation, 50 μM cobalt chloride was selected for subsequent experiments. Under this concentration treatment, the expression of the hypoxia signaling factor HIF-1α in liver cancer cells was significantly increased.

[0021] Example 1: MTT assay to detect the effect of heparin dodecaose on the proliferation of HepG2 cells Step 1: Culture liver cancer cells (HepG2 cells, ATCC) to 90% confluence, digest with 0.25% (w / v) trypsin (Biosharp) containing 0.02% (w / v) EDTA to prepare a cell suspension, mix thoroughly, and then use 1×10 4Cells were seeded at a density of cells / well in 96-well plates and cultured at 37°C until cells adhered and reached an appropriate density. Cells were divided into a control group, a model group (treated with 50 μM CoCl2 to simulate a hypoxic tumor environment), and different concentrations of heparin dodecanose (0.01 μM, 0.1 μM, and 1 μM, each containing the same concentration of CoCl2 as the model group). Each group had six replicates. The culture medium for each group was replaced with FBS-free DMEM medium (Gibco, high-glucose DMEM medium) and cultured at 37°C for 24 h. 50 μM CoCl2 and different concentrations of heparin dodecanose were all prepared using FBS-free DMEM medium.

[0022] Step 2: Add 20 μL of 5 mg / mL MTT solution (GLPBIO) to each well and incubate at 37°C for 4 h. Carefully aspirate the culture medium from the wells, add 150 μL of DMSO to each well, and shake on a shaker for 10 min to dissolve the crystals. After mixing, measure the absorbance of each well at 490 nm (Multiskan FC microplate reader, Thermo). Statistical analysis of the obtained data was performed using Graph Pad Prism 5.0 software. Quantitative analysis results are shown below. Figure 1 As shown.

[0023] Depend on Figure 1 It can be seen that the proliferation of HepG2 cells is promoted under hypoxic environment simulation, while the proliferation of HepG2 cells is inhibited after treatment with different concentrations of heparin dodecaose compared with the model group, with 0.1 μM heparin dodecaose showing the best inhibitory effect.

[0024] Example 2: Oil Red O staining assay to detect the effect of oleic acid on lipid droplet accumulation in HepG2 cells Step 1: After routine digestion of HepG2 cells that are in good growth condition and have reached 90% confluence, adjust the density to 5 × 10⁶ cells / year. 5 Cells were seeded at a density of 100 cells / mL in 6-well plates. When the cell density was suitable and the cells were in good condition, the cells were divided into a blank group and groups with different concentrations of OA (0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM). All groups were cultured in DMEM medium (Gibco, high-glucose DMEM medium) containing FBS and cultured at 37°C for 24 h.

[0025] Step 2: Slowly aspirate the old culture medium and wash once with PBS. Fix with 4% paraformaldehyde fixative for 10 min, then rinse twice with PBS. Cover the cells with an appropriate amount of staining and washing buffer for 20 s, discard the staining and washing buffer, add an appropriate amount of Oil Red O staining working solution, and stain for 20-40 min. After staining, remove the Oil Red O staining working solution, add an appropriate amount of staining and washing buffer, let stand for 30 s, then remove the staining and washing buffer, and wash twice with PBS. Add an appropriate amount of PBS to evenly cover the cells, then observe and photograph under a microscope. Experimental results are as follows: Figure 2 As shown.

[0026] Depend on Figure 2 The results showed that, compared with the control group, lipid droplet accumulation in HepG2 cells was significantly increased after treatment with different concentrations of oleic acid, with 0.5 mM oleic acid showing the best induction effect.

[0027] Example 3: CCK-8 assay to detect the effect of different concentrations of OA on the viability of HepG2 cells Step 1: After routine digestion of HepG2 cells that are in good growth condition and have reached 90% confluence, the HepG2 cells are then... 5 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37 °C in a 5% CO2 incubator until approximately 80% confluence. The old culture medium was then discarded. Cells were divided into a control group and groups with different concentrations of oleic acid (0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM), with 6 replicates per group. All groups were cultured in DMEM medium (Gibco, high-glucose DMEM) containing FBS, and cultured at 37 °C for another 24 h.

[0028] Step 2: Add 10 μL of CCK-8 solution to each well, taking care not to introduce air bubbles into the wells to avoid interfering with the readings. Incubate the plate in an incubator for 1–4 h. Before reading the plate, gently mix on a shaker for 1 min to ensure uniform color distribution. Measure the absorbance at 450 nm using a microplate reader. Statistically analyze the data using Graph Pad Prism 5.0 software. The analysis results are as follows: Figure 3 As shown.

[0029] Depend on Figure 3 The results showed that, compared with the control group, 0.1-0.2 mM oleic acid had no significant effect on HepG2 cell viability, while 0.3-0.5 mM oleic acid significantly inhibited HepG2 cell viability. Therefore, 0.2 mM oleic acid was used as the induction concentration in subsequent experiments. The results of this example confirm that the interference of oleic acid at appropriate concentrations on cell viability can be ignored, ensuring the rigor of the experimental conclusions in subsequent examples.

[0030] Example 4: Effect of triglyceride test kit on triglyceride content in HepG2 cells Step 1: After routine digestion of HepG2 cells that are in good growth condition and have reached 90% confluence, adjust the density to 5 × 10⁶ cells / year. 5 Cells were seeded at a density of 10 cells / mL into 6-well plates. Once the cells adhered and covered approximately 70%-80% of the bottom of each well, the old culture medium was discarded, and 0.2 mM OA complete medium was added. The cells were then cultured at 37°C for 24 h. Cells were divided into three groups: a control group, a model group (treated with 50 μM CoCl2), and groups with different concentrations of heparin-dodecanoic acid (0.01 μM, 0.1 μM, 1 μM, each containing the same concentration of CoCl2 as the model group). The culture medium for each group was replaced with FBS-free DMEM medium (Gibco, high-glucose DMEM medium), and the cells were cultured at 37°C for 24 h.

[0031] Step 2: Discard the old culture medium, digest the cells as usual, transfer the cell suspension to an EP tube, centrifuge at 1000 rpm for 10 min, discard the supernatant, and retain the cell pellet. Wash 1-2 times with isotonic buffer, centrifuge at 1000 rpm for 10 min, discard the supernatant, and retain the cell pellet. Add 0.2-0.3 mL of homogenizing medium to homogenize, place in an ice bath and sonicate at 300 W for 5 s at a time, with 30 s intervals between each sonication. Repeat 3-5 times to obtain a cell homogenate. Measure the cell homogenate according to the instructions of the triglyceride (TG) test kit (Nanjing Jiancheng Bioengineering Institute, model number). Refer to Table 1 for the measurement conditions, and set up 3 replicates for each group.

[0032] Table 1 Measurement conditions Step 3: Take the remaining cell suspension and determine the protein concentration of the cell suspension according to the instructions of the BCA Protein Quantitative Kit (Hangzhou Fude Biotechnology Co., Ltd., model FD2001).

[0033] Step 4: Calculate the results according to the triglyceride (TG) test kit instructions. Statistically analyze the data using Graph Pad Prism 5.0 software. The analysis results are as follows: Figure 4 As shown.

[0034] Depend on Figure 4 The results showed that HepG2 cells exhibited increased triglyceride levels under hypoxic conditions, but decreased triglyceride levels after treatment with different concentrations of heparin dodecaose compared to the model group, with 1 μM heparin dodecaose showing the best inhibitory effect.

[0035] Example 5: Detection of the effect of heparin dodecanose on the free fatty acid content of HepG2 cells using a free fatty acid assay kit Step 1: After routine digestion of HepG2 cells that are in good growth condition and have reached 90% confluence, adjust the density to 5 × 10⁶ cells / year. 5 Cells were seeded at a density of 10 cells / mL into 6-well plates. Once the cells adhered and covered approximately 70%-80% of the bottom of each well, the old culture medium was discarded, and 0.2 mM OA complete medium was added. The cells were then cultured at 37°C for 24 h. Cells were divided into three groups: a control group, a model group (treated with 50 μM CoCl2), and groups with different concentrations of heparin-dodecanoic acid (0.01 μM, 0.1 μM, 1 μM, each containing the same concentration of CoCl2 as the model group). The culture medium for each group was replaced with FBS-free DMEM medium (Gibco, high-glucose DMEM medium), and the cells were cultured at 37°C for 24 h.

[0036] Step 2: Discard the old culture medium, routinely digest the cells, and transfer the cell suspension to an EP tube. Centrifuge at 1000 rpm for 10 min, discard the supernatant, and retain the cell pellet. Wash 1-2 times with isotonic buffer, centrifuge at 1000 rpm for 10 min, discard the supernatant, and retain the cell pellet. Add 0.2-0.3 mL of homogenizing medium to homogenize, place in an ice bath, and sonicate at 300 W for 5 seconds at 30-second intervals, repeating 3-5 times to obtain a cell homogenate. Measure the cell homogenate according to the instructions of the free fatty acid (FFA) assay kit (Nanjing Jiancheng Bioengineering Institute, model number). Refer to Table 2 for the measurement conditions, and set up 3 replicates for each group.

[0037] Table 2 Measurement conditions Step 3: Take the remaining cell suspension and determine the protein concentration of the cell suspension according to the instructions of the BCA Protein Quantitative Kit (Hangzhou Fude Biotechnology Co., Ltd., model FD2001).

[0038] Step 4: Calculate the results according to the instructions for the Free Fatty Acid (FFA) test kit. Statistically analyze the data using Graph Pad Prism 5.0 software. The analysis results are as follows: Figure 5 As shown.

[0039] Depend on Figure 5 The results showed that HepG2 cells exhibited increased free fatty acid content under hypoxic conditions, but decreased free fatty acid content compared to the model group after treatment with different concentrations of heparin dodecaose, with 1 μM heparin dodecaose showing the best inhibitory effect.

[0040] Example 6: Detection of the effect of heparin dodecanose on ATP levels in HepG2 cells using an ATP assay kit Step 1: After routine digestion of HepG2 cells that are in good growth condition and have reached 90% confluence, adjust the density to 5 × 10⁶ cells / year. 5 Cells were seeded at a density of 10 cells / mL into 6-well plates. Once the cells adhered and covered approximately 70%-80% of the bottom of each well, the old culture medium was discarded, and 0.2 mM OA complete medium was added. The cells were then cultured at 37°C for 24 h. Cells were divided into three groups: a control group, a model group (treated with 50 μM CoCl2), and groups with different concentrations of heparin-dodecanoic acid (0.01 μM, 0.1 μM, 1 μM, each containing the same concentration of CoCl2 as the model group). The culture medium for each group was replaced with FBS-free DMEM medium (Gibco, high-glucose DMEM medium), and the cells were cultured at 37°C for 24 h.

[0041] Step 2: Perform routine cell digestion, centrifuge to collect the cell pellet into an EP tube, add 300-500 µL of cold double-distilled water, and sonicate in an ice bath at 300 W for 5 seconds at 30-second intervals, repeating 3-5 times to obtain a cell homogenate. Then, heat the cell suspension in a boiling water bath for 10 minutes, vortex to mix for 1 minute, and then proceed according to the ATP content test kit (Nanjing Jiancheng Biotechnology Institute, model A095-1-1). The measurements were performed according to the instructions. The measurement conditions are shown in Table 3, and three replicates were set up for each group.

[0042] Table 3 Measurement conditions Step 3: Take the remaining cell suspension and determine the protein concentration of the cell suspension according to the instructions of the BCA Protein Quantitative Kit (Hangzhou Fude Biotechnology Co., Ltd., model FD2001).

[0043] Step 4: Calculate the results according to the ATP content test kit instructions. Statistically analyze the data using Graph Pad Prism 5.0 software. The analysis results are as follows: Figure 6 As shown.

[0044] Depend on Figure 6 The results showed that HepG2 cells had increased ATP levels under hypoxic simulated conditions, but decreased ATP levels after treatment with different concentrations of heparin dodecaose compared to the model group, with 1 μM heparin dodecaose showing the most significant effect.

[0045] Example 7: qPCR detection of the effect of heparin dodecanose on the transcription of FASN and SREBP-1 genes in HepG2 cells Step 1: After routine digestion of HepG2 cells that are in good growth condition and have reached 90% confluence, adjust the density to 5 × 10⁶ cells / year. 5Cells were seeded at a density of 10 cells / mL into 6-well plates. Once the cells adhered and covered approximately 70%-80% of the bottom of each well, the old culture medium was discarded, and 0.2 mM OA complete medium was added. The cells were then cultured at 37°C for 24 h. Cells were divided into three groups: a control group, a model group (treated with 50 μM CoCl2), and groups with different concentrations of heparin-dodecanoic acid (0.01 μM, 0.1 μM, 1 μM, each containing the same concentration of CoCl2 as the model group). The culture medium for each group was replaced with FBS-free DMEM medium (Gibco, high-glucose DMEM medium), and the cells were cultured at 37°C for 24 h.

[0046] Step 2: Discard the culture medium, perform routine digestion, transfer to EP tubes, centrifuge, and discard the supernatant. Extract total RNA using TRIzol (Solarbio). After drying the RNA precipitate, add 40 μL of ddH2O and vortex at room temperature to completely dissolve it. Measure the RNA concentration using a Nanodrop 2000 (Thermo Scientific) and store at -80℃ for later use.

[0047] Step 3: Reverse transcription was performed using a reverse transcription kit (Abcam). The obtained cDNA was stored at -20℃ for later use. Relevant human mRNA sequences were searched using NCBI, and primers were designed and synthesized online by Sangon Biotech. qPCR analysis was performed using the ChamQ UniversalSYBR qPCR Master Mix kit (Novizan).

[0048] Following the qPCR reaction system setup shown in Table 4, add each component to the tube on ice, mix thoroughly, and then centrifuge. Place the reaction tubes on the qPCR instrument in sequence and set the reaction program as follows: Pre-denaturation: 95℃ for 30 seconds; Cyclic reaction: 95℃ for 10s, 60℃ for 60s, 95℃ for 15s.

[0049] After the reaction, Ct values ​​were obtained, and mRNA levels were calculated using the 2-ΔΔt method. Data were processed using GraphPad Prism 5.0 software, and the results are as follows: Figure 7 , Figure 8 As shown.

[0050] Table 4 qPCR reaction system Primers were purchased from Shanghai Sangon Biotech, and their sequences are shown in Table 5. Table 5 Primer Sequences Depend on Figure 7The results showed that under hypoxic simulated conditions, the mRNA expression of FASN and SREBP-1 in HepG2 cells increased, indicating an increase in gene transcription levels. However, after treatment with different concentrations of heparin dodecaose (0.01 μM, 0.1 μM, and 1 μM), the mRNA expression of FASN and SREBP-1 decreased compared with the model group, indicating that heparin dodecaose can inhibit the gene transcription of FASN and SREBP-1 to a certain extent.

[0051] Example 8: Western blotting detection of the effect of heparin dodecanose on the expression of FASN, SREBP-1 and HIF-1α proteins in HepG2 cells Step 1: After routine digestion of HepG2 cells, adjust the density to 5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / mL into 6-well plates. Once the cells adhered and covered approximately 70%-80% of the bottom of each well, the old culture medium was discarded, and 0.2 mM OA complete medium was added. The cells were then cultured at 37°C for 24 h. Cells were divided into three groups: a control group, a model group (treated with 50 μM CoCl2), and groups with different concentrations of heparin-dodecanoic acid (0.01 μM, 0.1 μM, 1 μM, each containing the same concentration of CoCl2 as the model group). The culture medium for each group was replaced with FBS-free DMEM medium (Gibco, high-glucose DMEM medium), and the cells were cultured at 37°C for 24 h.

[0052] Step 2: After routine cell digestion, transfer the cells to EP tubes and centrifuge, discarding the supernatant. Resuspend the cell pellet in pre-chilled PBS and transfer to 1.5 mL EP tubes. Centrifuge at 3000 rpm and 4°C for 10 min (Biofuge Stratos high-speed refrigerated centrifuge, Thermo Fisher Scientific, USA). Repeat once with pre-chilled PBS. After discarding the supernatant, carefully aspirate any remaining liquid, add pre-prepared RIPA lysis working solution (Beyotime), and resuspend by pipetting. Lyse thoroughly on ice for at least 30 min, vortexing every 5 min (XW-80A miniature vortex mixer, Shanghai Huxi Analytical Products Co., Ltd.). After lysis, centrifuge at 12000 rpm and 4°C for 10 min, and slowly aspirate the white precipitate at the bottom of the EP tube to obtain total cell protein.

[0053] Step 3: Determine the protein content in the sample using the BCA method. Calculate the loading volume based on a protein loading amount of 30 μg according to the standard curve. Add an appropriate amount of 5× protein loading buffer (Novizan) to each sample protein, boil in a water bath for 5 min to activate the protein, cool to room temperature, and store at -20℃ for later use. Prepare the gel according to the rapid gel preparation kit (Novizan), and allow it to solidify at room temperature. Remove the sample, vortex and pipette to mix, and load the samples sequentially according to the loading volume. Perform electrophoresis at a constant voltage of 160V at room temperature until the bromophenol blue reaches the bottom of the gel, then stop electrophoresis. After electrophoresis, refer to the protein standard (Novizan) to take the gel containing the target protein and place it in 1× transfer buffer (prepared by dissolving 2.9 g glycine, 0.38 g SDS, 5.8 g Tris in distilled water and adjusting the volume to 800 mL, then adding 200 mL methanol). Measure the size of the gel cut on the glass plate and cut out 0.45μm PVDF films (purchased from Beyotime) of the same size, and activate them in methanol for 60s.

[0054] Place the sponge, filter paper, adhesive strip, and PVDF membrane in the following order: Place in the electroporation tank, fill with 1× transfer buffer, and transfer at a constant current of 200mA for 90 min in an ice-water bath. Prepare 5% skim milk powder as blocking buffer in advance. After transfer, place in the blocking buffer and gently shake at room temperature for 3 h. After blocking, place in the primary antibody dilution buffer corresponding to the target protein (Anti-HIF-1αAntibody / Anti-NOX4-Antibody, ZhengNeng Biotechnology) and incubate overnight at 4°C. The next day, remove the PVDF membrane and wash it three times with 1% TBST solution, shaking for 10 min each time. Place in the secondary antibody dilution buffer corresponding to the primary antibody (Goat anti-Rabbit IgGHRP label, ZhengNeng Biotechnology) and incubate at room temperature for 60 min. Remove the PVDF membrane and wash it three times with 0.1% TBST solution, shaking for 10 min each time. Prepare the working solution by mixing solutions A and B from the ECL chemiluminescence reagent kit (Tianneng) at a 1:1 volume ratio. After absorbing moisture from the PVDF membranes, they were placed in the exposure area. 200 μL of luminescent working solution was evenly dropped onto the surface of each PVDF membrane. The Tanon 5200 fully automated chemiluminescence analysis system was used for exposure and imaging. The imaging results and grayscale analysis results are as follows: Figure 9 , Figure 10 As shown.

[0055] Depend on Figure 9 , Figure 10It was found that under hypoxic simulated conditions, the expression of FASN, SREBP-1, and HIF-1α proteins in HepG2 cells increased. However, after treatment with different concentrations of heparin dodecaose, the expression of FASN, SREBP-1, and HIF-1α proteins was inhibited to varying degrees compared with the model group. Among them, 0.01 μM heparin dodecaose showed the best effect in inhibiting FASN protein expression, 0.01 μM heparin dodecaose showed the best effect in inhibiting SREBP-1 protein expression, and 0.1 μM heparin dodecaose showed the best effect in inhibiting HIF-1α protein expression.

[0056] Based on the experiments and conclusions of the above embodiments, this invention reveals that heparin dodecanose can possess potential value in treating liver cancer by inhibiting hypoxia-mediated lipid metabolism reprogramming. When used as a drug, the drug comprises heparin dodecanose and pharmaceutically acceptable excipients. Commonly used pharmaceutically acceptable excipients include diluents, surfactants, lubricants, antioxidants, binders, colorants, emulsifiers, etc. The dosage form of the drug is an injection or a lyophilized injection. The heparin dodecanose of this invention is a heparin oligosaccharide obtained by cleaving heparin with nitrite and then separating and purifying it using Bio-gel P6 and Bio-gel P4 molecular sieves. Pharmaceutically acceptable excipients can be added to prepare it into commonly used pharmaceutical dosage forms for clinical use, such as heparin oligosaccharide injections or heparin oligosaccharide lyophilized injections.

[0057] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific medicament of the present invention used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, general health condition, and medical history of the patient being treated, and similar factors known in the medical field.

[0058] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0059] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. Application of heparin dodecanose in the preparation of drugs for the treatment of liver cancer.

2. The use of heparin dodecanose according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The treatment for liver cancer includes inhibiting hypoxia-mediated lipid metabolism reprogramming.

3. The use of heparin dodecanose according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The treatment for liver cancer includes: inhibiting the levels of free fatty acids and / or triglycerides in liver cancer cells.

4. The use of heparin dodecanose according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The treatment for liver cancer includes inhibiting the level of adenosine triphosphate (ATP) within liver cancer cells.

5. The use of heparin dodecanose according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The treatment for liver cancer includes inhibiting the transcription and / or expression of lipid synthesis genes FASN, SREBP-1, and HIF-1α proteins.

6. The use of heparin dodecanose according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The structural formula of the heparin dodecanose is: 。 7. The use of heparin dodecanose according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The concentration of heparin dodecanose in the drug is configured such that the concentration of heparin dodecanose in the liver cancer cell environment reaches 0.01 μM to 1 μM.

8. A drug for treating liver cancer, comprising heparin dodecaose.

9. The medicament for treating liver cancer according to claim 8, characterized in that, The structural formula of heparin dodecanose is: 。 10. The medicament for treating liver cancer according to claim 8, characterized in that, The concentration of heparin dodecanose in the drug is configured such that the concentration of heparin dodecanose in the liver cancer cell environment reaches 0.01 μM to 1 μM.

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