Experimental method for treating liver cancer by using oleanolic acid

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an experimental method for treating liver cancer by using oleanolic acid. Comprising an in-vitro experiment method and an in-vivo experiment method. The in-vitro experiment method comprises the following steps: S1, cell culture; s2, cell proliferation and migration; s3, detecting cell apoptosis and a cell cycle; s4, performing immunoblotting; s5, carrying out real-time quantitative PCR; the in-vivo experiment method comprises the following steps: step 1, performing chick embryo chorioallantoic model experiment; and 2, carrying out tumor-bearing experiment, treatment and tissue sampling on the mouse. The application of the oleanolic acid in liver cancer treatment is systematically researched for the first time, it is found that the oleanolic acid has the remarkable effects of inhibiting proliferation, inducing apoptosis, resisting angiogenesis and the like on liver tumors through the synergistic effect of multiple mechanisms, the remarkable curative effect on cancer mice is achieved, and a new medicine choice is provided for liver cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an experimental method for using oleanolic acid in the treatment of liver cancer. Background Technology

[0002] Primary liver cancer (PLC) is a malignant tumor originating in the liver and is the third leading cause of cancer death worldwide. [1-3] In PLCs, hepatocellular carcinoma (HCC) is the most common form, accounting for approximately 85-90% of all PLCs. [4, 5] The causes of HCC are complex, mainly including viral hepatitis. [6] Obesity, alcohol intake [7] and aflatoxin exposure [8] High morbidity and mortality rates of hepatocellular carcinoma (HCC) pose a serious threat to human health. Current clinical interventions for HCC primarily involve radical surgical resection combined with molecularly targeted drugs (such as sorafenib). [9,10] However, due to the rapid increase in HCC incidence, the scarcity of precise therapeutic targets, and the adverse reactions associated with existing drugs, etc.

[11] The treatment of HCC poses a significant clinical challenge, making the exploration of novel therapeutic drugs and their targets an urgent scientific problem to be solved.

[0003] Oleanolic acid (OA, 3β-hydroxy-olea-12-en-28-oic acid), also known as qingtesu, belongs to the pentacyclic triterpenoid class of compounds. As the main active ingredient of the traditional Chinese medicine Ligustrum lucidum, OA is widely distributed in nature, abundant in resources, and exhibits low toxicity, making it a highly anticipated natural product.

[12] In addition to its well-established hepatoprotective effects, its anti-inflammatory, antioxidant, and anti-tumor properties have also been recognized in recent years. [13,14 ] Its effects have been extensively studied. Numerous studies have confirmed that OA can treat liver cancer, colon cancer, and non-small cell lung cancer, among others. [15-17] It exhibits broad-spectrum anti-tumor effects. However, there is still relatively little systematic research and application of oleanolic acid in the treatment of liver cancer, and its exact efficacy, mechanism of action, and appropriate dosing regimen in vivo still need to be explored in depth. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution: An experimental method for using oleanolic acid to treat liver cancer includes an in vitro experimental method and an in vivo experimental method, wherein the in vitro experimental method includes the following steps: S1: Cell culture: Human liver cancer cells SMMC-7721 and human umbilical vein endothelial cells HUVECs were selected and cultured in culture medium and placed in a cell culture incubator at 37°C and 5% CO2. S2: Cell proliferation and migration: Cell proliferation was assessed using the MTT assay and the Cell Count Kit-8 assay. SMMC-7721 and HUVECs cells were seeded in 96-well plates and incubated. The optical density of each well was measured. Cell scratches were created, and cell migration was observed and photographed under a microscope. S3: Detection of apoptosis and cell cycle: To detect apoptosis in SMMC-7721 cells, cells were collected, stained at room temperature in the dark, and then analyzed by flow cytometry. S4: Immunoblotting: Cell and tumor tissue lysates were prepared on ice in a cell lysis buffer containing a mixture of protease inhibitors, followed by centrifugation. Total protein was quantified, and after adding loading buffer, the lysates were denatured, loaded onto an electrophoresis gel, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with skim milk and incubated at 4°C with primary antibodies PI3K, AKT, p-AKT, mTOR, p-mTOR, HIF-1α, VEGF, Bax, Bcl-2, Caspase-3, CCND1, and BIRC5 for 12 hours. Subsequently, the membrane was incubated with secondary antibodies at room temperature for 1 hour. The bands were visualized using a chemiluminescence method with an ultrasensitive luminescent substrate, and automatic exposure and development were performed. Finally, the gray values ​​of the bands were analyzed and the results were statistically analyzed. S5: Real-time quantitative PCR Total RNA was extracted from cells and tumor tissues, and the RNA was reverse transcribed into complementary DNA. Real-time PCR analysis was then performed. The experimental results were normalized with the 18S gene as an internal reference, and the relative mRNA expression level was calculated using the formula 2^(-ΔΔCt). The in vivo experimental method includes the following steps: Step 1: Chicken embryo chorionic allantoic model experiment: Chicken embryos were selected and pretreated. The drug delivery carrier was placed in the center of the CAM membrane. The drug to be tested was added to each group, and an equal amount of sterile saline was added to the control group. After incubation, on the third day of drug administration, the number of surviving chicken embryos was counted, fixative was added, the CAM membrane was cut off, and air-dried for storage. The air-dried CAM membrane was photographed with a camera, and the CAM area and vascular area were calculated. The vascular area ratio and vascular inhibition rate were calculated. Step 2: Tumor-bearing mouse experiment, treatment, and tissue sampling: Kunming rats were selected to establish a liver cancer xenograft model, and the growth volume of the transplanted tumor was calculated. After fixing fresh tumor tissue for 24 hours, it was dehydrated and cleared sequentially, then embedded in paraffin to prepare 4 μm tissue sections. Immunofluorescence staining was performed to observe the staining intensity and localization. The slides were digitally processed to capture images, and finally, the images were further analyzed.

[0006] As a preferred embodiment of the experimental method for treating liver cancer with oleanolic acid according to the present invention, the culture medium in S1 is RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0007] As a preferred embodiment of the experimental method for using oleanolic acid to treat liver cancer according to the present invention, the method for measuring the optical density value of each well in S2 is as follows: SMMC-7721 and HUVECs cells are cultured for 24 hours, 48 ​​hours and 72 hours respectively, 10 μl of MTT or CCK-8 solution is added to each well, followed by incubation for 4 hours or 1 hour respectively, and finally, the optical density value of each well is measured at a wavelength of 490 nm or 450 nm using a microplate reader.

[0008] As a preferred embodiment of the experimental method for using oleanolic acid to treat liver cancer according to the present invention, in step S2, the method for creating cell scratches is as follows: A suspension of HUVECs is prepared, and the cell suspension is added to each well to cover the bottom of the well after filtration. Using a ruler as an aid, a black marker pen is used to draw evenly horizontal lines on the back of a 6-well plate, perpendicular to the plate and the horizontal lines on the back, creating scratches along the center line of the wells. The cell culture plate is removed at 0h and 24h after scratching, and the cell migration is recorded. .

[0009] As a preferred embodiment of the experimental method for using oleanolic acid to treat liver cancer according to the present invention, in step S3, the specific method for detection by flow cytometry is as follows: The Cell Cycle Assay Kit is used to detect the cell cycle of SMMC-7721 cells; pre-cooled anhydrous ethanol is slowly added to each group of cells, and after thorough mixing, the cells are placed in a container at 4°C. 0Cells were fixed at 37°C for 24 hours. After fixation, RNase A Reagent was added to fully suspend the cells. 0 Bath in a water bath at 4°C for 30 minutes, then add PI Reagent and mix thoroughly. 0 Incubate at C in the dark for 30 min, and then detect the red fluorescence at an excitation wavelength of 488 nm by flow cytometry.

[0010] As a preferred embodiment of the experimental method for using oleanolic acid to treat liver cancer as described in this invention, the method for chicken embryo pretreatment in step 1 is as follows: after chicken embryos are incubated for 7 days, they are observed under an egg candling lamp to screen out chicken embryos with similar developmental status, and the range of the air cell is drawn. In a clean bench, an ophthalmic forceps is used to carefully knock open a window with a radius of 0.75 cm at the end of the air cell to expose the eggshell membrane of the chicken embryo, which is then torn off.

[0011] In a preferred embodiment of the experimental method for treating liver cancer using oleanolic acid as described in this invention, the calculation method for the vascular area ratio and vascular inhibition rate in step 1 is as follows: ; .

[0012] As a preferred embodiment of the experimental method for treating liver cancer with oleanolic acid as described in this invention, the specific method for establishing the liver cancer xenograft model in step 2 is as follows: H22 cells are cultured in batches and diluted to a concentration of 3×107 cells / mL. The cells are injected into the axilla of the left forelimb of Kunming mice. After inoculation, the mice are normally fed under sterile conditions, and their weight is measured every 3 days. After the subcutaneous xenograft tumors of the mice have stabilized, the mice are randomly divided into 4 groups: Con blank control group, OA-LD group, OA-HD group, and SOR positive control group, with 10 mice in each group, half male and half female. Two weeks after administration, the mice are sacrificed 24 hours after the last administration, and the tumor is removed. The subcutaneous tumor is photographed, weighed, and the long and short diameters of the tumor are measured.

[0013] As a preferred embodiment of the experimental method for treating liver cancer using oleanolic acid as described in this invention, in step 2, during immunofluorescence staining, the slides are dewaxed and rehydrated, the antigen is recovered using antigen peeling buffer, and then the slides are blocked in 5% bovine serum albumin and PBS at room temperature for 60 minutes. The primary antibody and secondary antibody are incubated for 16 h and 45 min, respectively, and then the slides are developed with DAB chromogenic solution for 3 min after incubation.

[0014] Compared with the prior art, the beneficial effects of this invention are: This invention is the first to systematically study the application of oleanolic acid in the treatment of liver cancer, and found that oleanolic acid has significant inhibitory effects on liver tumor proliferation, apoptosis induction and anti-angiogenesis through multiple synergistic mechanisms, and has significant therapeutic effects on mice with cancer, providing a new drug option for the treatment of liver cancer. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This figure shows the effect of DMSO, the solvent in an experimental method for treating liver cancer with oleanolic acid, on the proliferation of SMMC-7721 cells. Figure 2 This is a diagram showing the effect of OA on the proliferation of SMMC-7721 cells in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 3 This is a graph showing the effect of OA on HUVEC proliferation in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 4 This is a graph showing the effect of OA on HUVEC migration in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 5 This is a diagram showing the effect of OA on apoptosis in SMMC-7721 cells in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 6 This figure shows the effect of OA on the expression of apoptosis proteins in SMMC-7721 cells in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 7 This is a diagram showing the effect of OA on the cell cycle of SMMC-7721 cells in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 8 This is a graph showing the effect of OA on cyclin expression in SMMC-7721 cells in an experimental method for treating liver cancer according to the present invention. Figure 9 This is a graph showing the effect of OA on the expression of angiogenesis-related proteins in SMMC-7721 in an experimental method for treating liver cancer according to the present invention. Figure 10 This figure shows the effect of OA on apoptosis and cell cycle-related gene transcription levels in SMMC-7721 cells using an experimental method for treating liver cancer according to the present invention. Figure 11 This is a diagram showing the effect of OA on the transcriptional level of HIF-1α-related genes in SMMC-7721 cells in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 12 This is a diagram illustrating the effect of OA on neovascularization of the chorioallantoic membrane in chicken embryos, based on an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 13 This is a graph showing the inhibition rate of OA on angiogenesis in the chorioallantoic membrane of chicken embryos in an experimental method for treating liver cancer according to the present invention. Figure 14 The graph shows the weight change curve of H22 tumor-bearing mice in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 15 This is a graph showing the effect of OA on tumor growth in H22 tumor-bearing mice in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 16 This is an immunohistochemical staining image of tumor tissue from an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 17 This image shows the expression of angiogenesis-related proteins in tumor tissue obtained from an immunohistochemical staining of tumor tissue in an experimental method for treating liver cancer using oleanolic acid according to the present invention. Figure 18 This is a diagram showing the effect of OA on tumor mRNA expression in H22 tumor-bearing mice in an experimental method for treating liver cancer according to the present invention. Figure 19 This figure shows the effect of OA on the expression of tumor angiogenesis-related proteins in H22 tumor-bearing mice in an experimental method for treating liver cancer according to the present invention. Detailed Implementation

[0016] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0017] Secondly, the present invention is described in detail with reference to the schematic diagrams. When describing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include the three-dimensional spatial dimensions of length, width, and depth.

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0019] Example 1 An embodiment of the present invention provides an experimental method for the use of oleanolic acid in the treatment of liver cancer. The in vitro experimental method includes the following steps: S1: Cell culture: Human liver cancer cells SMMC-7721 and human umbilical vein endothelial cells HUVECs were selected and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a cell culture incubator at 37°C and 5% CO2. S2: Cell proliferation and migration: Cell proliferation was assessed using the MTT assay and the Cell Counting Kit-8 (CCK-8) assay. SMMC-7721 and HUVECs cells (approximately 1.2 × 10⁻⁶) were used. 4 Cells per well were seeded into 96-well plates and cultured for 24, 48 and 72 hours, respectively. 10 μl of MTT or CCK-8 solution was added to each well and then incubated for 4 or 1 hour, respectively. Finally, the optical density of each well was measured at a wavelength of 490 nm or 450 nm using a microplate reader. Prepare a suspension of HUVECs, adding an appropriate amount of cell suspension to each well, ensuring it covers the bottom of the well after filtration. Using a ruler as a guide, draw evenly spaced horizontal lines on the back of the 6-well plate with a black marker pen, perpendicular to the horizontal lines on the back of the plate, along the center line of each well. Remove the cell culture plate at 0 h and 24 h after marking, observe under a microscope, and photograph to record cell migration. ; MTT and CCK-8 assays showed that OA significantly inhibited the proliferation of SMMC-7721 and HUVECs cells in a dose-dependent manner. At 40 μM, the inhibitory effect was close to that of the positive control drug SOR.

[0020] Cell migration assays showed that OA inhibited HUVEC migration in a dose-dependent manner. The migration rates of the OA 23 μM and 25 μM groups were similar to those of the SOR group.

[0021] S3: Detection of apoptosis and cell cycle: Apoptosis in SMMC-7721 cells was detected using the Annexin V-FITC / PI apoptosis detection kit. After cell collection, cells were stained with Annexin V labeled with fluorescein isothiocyanate (FITC) and propidium iodide (PI) at room temperature in the dark for 15 minutes, followed by detection by flow cytometry. The Cell Cycle Assay Kit (Red Fluorescence) was used to detect the cell cycle status of SMMC-7721 cells. Cells from each group were collected, and pre-cooled anhydrous ethanol was slowly added dropwise. After thorough mixing, the cells were incubated at 4°C.0 Cells were fixed at 37°C for 24 hours. After fixation, RNase A Reagent was added to fully resuspend the cells, and the cells were incubated at 37°C. 0 Incubate in a water bath at 4°C for 30 min, then add PI Reagent (50 μg / mL) and mix thoroughly. 0 Incubate at C in the dark for 30 min, and then detect red fluorescence at an excitation wavelength of 488 nm by flow cytometry. Annexin V-FITC / PI double staining results showed that OA could induce apoptosis in liver cancer cells. With increasing drug concentration, the proportion of apoptotic cells gradually increased, reaching 38.64 ± 0.25% at 15 μM. Based on this, Western blotting was used to detect the effects of OA on the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3. At 15 μM, the expression of Bax and Caspase-3 proteins significantly increased, while the expression of Bcl-2 protein showed a decreasing trend.

[0022] Flow cytometry results indicated that OA may inhibit the proliferation of hepatocellular carcinoma cells by affecting cell cycle progression. In the 15 μM OA group, the percentages in the S and G2 phases significantly decreased. With increasing OA concentration, the proportion of SMMC-7721 cells in the S and G2 phases significantly decreased. Furthermore, OA treatment of SMMC-7721 cells reduced the expression levels of cell cycle-related proteins CCND1 and BIRC5.

[0023] S4: Immunoblotting: Cell and tumor tissue lysates were prepared on ice in a cell lysis buffer containing a mixture of protease inhibitors, and then centrifuged at 12,000 rpm for 5 min. Total protein was quantified using a BCA kit, and the samples were denatured at 95°C for 10 min after adding loading buffer. Samples (20 μg) were added to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with skim milk and incubated at 4°C for 12 h with primary antibodies PI3K, AKT, p-AKT, mTOR, p-mTOR, HIF-1α, VEGF, Bax, Bcl-2, Caspase-3, CCND1, and BIRC5. Subsequently, the membrane was incubated with secondary antibodies at room temperature for 1 h, and the bands were visualized using chemiluminescence with a hypersensitive luminescent substrate. Automatic exposure and development were performed using a gel imaging system, and the band grayscale values ​​were analyzed and statistically analyzed using ImageJ software.

[0024] Western blot results showed that after OA treatment, PI3K, AKT and mTOR proteins were downregulated in SMMC-7721 hepatocellular carcinoma cells, and phosphorylated AKT and phosphorylated mTOR were also significantly reduced; and compared with the Con group, OA reduced the protein expression of HIF-1α and VEGF.

[0025] S5: Real-time quantitative PCR Total RNA was extracted from cells and tumor tissues using TRIzol reagent, and the RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit according to the manufacturer's instructions. Subsequently, real-time PCR analysis was performed using SYBR Green real-time PCR reagent via an ABI Piperism 7300 thermal cycler. Experimental results were normalized using the 18S gene as an internal control. Relative mRNA expression levels were calculated using the formula 2^(-ΔΔCt).

[0026] Experimental Results and Data 1. Cell proliferation and migration assay Please see Figure 1-4 MTT and CCK-8 detection methods ( Figure 1-2 The results showed that OA significantly inhibited the proliferation of SMMC-7721 and HUVECs cells in a dose-dependent manner. At 40 μM, the inhibitory effect was close to that of the positive control drug SOR.

[0027] Cell migration assay ( Figure 3 The results showed that OA inhibited the migration of HUVECs in a dose-dependent manner. The migration rates of the OA 23 μM and 25 μM groups were similar to those of the SOR group.

[0028] RT-qPCR results showed that after SMMC-7721 administration to OA, the transcriptional levels of angiogenesis-related genes HIF-1α, VEGF, and their upstream genes PI3K, AKT, and mTOR were downregulated. This indicates that OA can exert its anti-tumor angiogenesis effect by inhibiting the PI3K / AKT / mTOR / HIF-1α / VEGF signaling pathway, thereby inhibiting tumor growth.

[0029] in, Figure 1 The effect of solvent DMSO on the proliferation of SMMC-7721 cells. (DMSO solvent had no effect on the cells). Figure 2 Effect of OA on the proliferation of SMMC-7721 cells (OA has a significant inhibitory effect on liver cancer cells) (Note: A. Comparison of cell inhibition rate after OA treatment for 24, 48, and 72 hours; B, C, and D are comparisons of OA and SOR treatment for 24, 48, and 72 hours, respectively). Figure 3Effects of OA on HUVEC proliferation (inhibitory effect) (Note: *P<0.05, ***P<0.001); Figure 4 The effect of OA on HUVEC migration (inhibitory effect) (Note: ***P<0.001).

[0030] 2. Detection of apoptosis and cell cycle Please see Figure 5-8 Annexin V-FITC / PI double staining results showed that OA could induce apoptosis in liver cancer cells. With increasing drug concentration, the proportion of apoptotic cells gradually increased, reaching 38.64 ± 0.25% at 15 μM. Based on this, Western blotting was used to detect the effects of OA on the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3. At 15 μM, the expression of Bax and Caspase-3 proteins significantly increased, while the expression of Bcl-2 protein showed a decreasing trend.

[0031] Flow cytometry results indicated that OA may inhibit the proliferation of hepatocellular carcinoma cells by affecting cell cycle progression. In the 15 μM OA group, the percentages in the S and G2 phases significantly decreased. With increasing OA concentration, the proportion of SMMC-7721 cells in the S and G2 phases significantly decreased. Furthermore, OA treatment of SMMC-7721 cells reduced the expression levels of cell cycle-related proteins CCND1 and BIRC5.

[0032] in, Figure 5 Effects of OA on apoptosis of SMMC-7721 cells (OA significantly promotes apoptosis of liver cancer cells) (Note: **P<0.01, ***P<0.001). Figure 6 Effects of OA on the expression of apoptosis proteins in SMMC-7721 cells (increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic proteins) (Note: *P<0.05, **P<0.01, ***P<0.001). Figure 7 Effects of OA on the cell cycle of SMMC-7721 cells (OA's inhibition of cell cycle progression) (Note: *P<0.05, **P<0.01, ***P<0.001); Figure 8 Effects of OA on cyclin expression in SMMC-7721 cells (decreased expression of cell cycle-related proteins) (Note: **P<0.01, ***P<0.001).

[0033] 3. Immunoblotting Please see Figure 9The effect of OA on the expression of angiogenesis-related proteins in SMMC-7721 cells (inhibition of angiogenesis-related protein expression) (Note: *P<0.05, **P<0.01, ***P<0.001). Western blotting results showed that after OA treatment, PI3K, AKT, and mTOR proteins in SMMC-7721 hepatocellular carcinoma cells were downregulated, and phosphorylated AKT and phosphorylated mTOR were also significantly decreased; and compared with the Con group, OA reduced the protein expression of HIF-1α and VEGF.

[0034] 4. Real-time quantitative PCR Please see Figure 10-11 RT-qPCR results showed that after SMMC-7721 administration to OA, the transcriptional levels of apoptosis-related genes Bax and Caspase-3 were upregulated, while the transcriptional level of Bcl-2 was downregulated. Furthermore, cycle marker genes CCND1 and BIRC5 showed a downregulation trend.

[0035] The transcriptional levels of genes related to angiogenesis, such as HIF-1α and VEGF, as well as their upstream genes PI3K, AKT, and mTOR, were downregulated. This indicates that OA can exert its anti-tumor angiogenesis effect by inhibiting the PI3K / AKT / mTOR / HIF-1α / VEGF signaling pathway, thereby inhibiting tumor growth.

[0036] in, Figure 10 Effects of OA on apoptosis and cell cycle-related gene transcription levels in SMMC-7721 cells (increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic proteins; decreased expression of cell cycle-related genes) (Note: *P<0.05, **P<0.01, ***P<0.001). Figure 11 Effects of OA on the transcriptional levels of HIF-1α-related genes in SMMC-7721 cells (inhibition of gene expression in the PI3K / AKT / mTOR / HIF-1α / VEGF signaling pathways) (Note: **P<0.01, ***P<0.001).

[0037] Example 2 An embodiment of the present invention provides an experimental method for the use of oleanolic acid in the treatment of liver cancer. The in vivo experimental method includes the following steps: Step 1: Chicken Embryo Villi Allantoic Model (CAM) Experiment After 7 days of incubation, chicken embryos were observed under an egg candling lamp to select those with similar developmental stages, and the air cell area was marked. In a clean bench, a 0.75 cm radius window was carefully opened at the air cell end using ophthalmic forceps to expose the eggshell membrane, which was then removed. The drug delivery carrier was placed in the center of the CAM membrane, and the test drug was added to each group (the control group received an equal volume of sterile saline). The embryos were then placed back into the incubator for further incubation.

[0038] On the third day after administration, the sterile breathable tape was torn off, and the number of surviving chicken embryos was counted. The embryos were fixed in a methanol:acetone (1:1) fixative for 20 min, the CAM membrane was cut off, and the membrane was flattened and air-dried in a six-well plate containing distilled water for storage.

[0039] The CAM membrane was photographed after air drying, and the CAM area and blood vessel area were calculated using ImageJ software. The blood vessel area ratio and blood vessel inhibition rate were also calculated.

[0040] ;

[0041] Step 2: Tumor-bearing mouse experiment, treatment, and tissue sampling A liver cancer xenograft model was established using Kunming rats. H22 cells were cultured in batches and diluted to a concentration of 3 × 10⁷ cells / mL. The cells were then injected subcutaneously into the left forelimb axilla of Kunming rats. After inoculation, the mice were fed normally under sterile conditions, and their weight was measured every 3 days. Once the subcutaneous xenograft tumors in the mice had stabilized, they were randomly divided into four groups: Con group (blank control), OA (LD) group, OA (HD) group, and SOR group (positive control), with 10 mice in each group (half male and half female). Two weeks after drug administration, the mice were sacrificed 24 hours after the last administration. The tumor masses were removed, photographed, weighed, and their long and short diameters were measured to calculate the tumor growth volume.

[0042] Fresh tumor tissue was fixed in 4% paraformaldehyde for 24 h, then subjected to graded ethanol dehydration and xylene clearing, followed by paraffin embedding to prepare 4 μm tissue sections. For immunofluorescence staining, slides were dewaxed and rehydrated, and antigen was recovered using antigen peeling buffer. Slides were then blocked in 5% bovine serum albumin (BSA) and PBS at room temperature for 60 min. Primary and secondary antibodies were incubated for 16 h and 45 min, respectively. After incubation, DAB (Dako) staining solution was applied for 3 min, and staining intensity and localization were observed under a microscope. Slides were digitized using a Pannoracic MIDI scanner, and images were captured using CaseViewer slide management software. Finally, ImageJ 1.49v software was used for further image analysis.

[0043] Experimental Results and Data 1. Chicken embryo chorioallantoic model (CAM) experiment Please see Figure 12-13 After administration of OA, the number of CAM vessels decreased, the vessels became thinner and exhibited abnormal morphology, and the vessels became tortuous and ruptured. As the concentration of OA increased, the angiogenesis inhibition rate also increased synchronously, showing a concentration-dependent effect, indicating that OA has an anti-angiogenic effect.

[0044] in, Figure 12 Effects of OA on angiogenesis in the chorioallantoic membrane of chicken embryos (OA has an anti-angiogenic effect) (Note: ***P<0.001). Figure 13 The inhibitory effect of OA on neovascularization of the chorioallantoic membrane of chicken embryos (Note: ***P<0.001).

[0045] 2. Mouse tumor-bearing experiment and immunohistochemistry Please see Figure 14-19 Compared with the Con group, the tumor volume of the OA group was significantly reduced; and compared with the positive drug SOR group, the tumor inhibition rate of the OA group was increased.

[0046] Immunohistochemical staining results showed that, compared with the Con group, the expression of PI3K, AKT, mTOR, HIF-1α, and VEGF proteins in the xenograft tissue of the OA-treated group was reduced. This indicates that OA can inhibit angiogenesis in xenograft tissue by regulating the PI3K / AKT / mTOR / HIF-1α / VEGF pathway, further confirming that OA can inhibit angiogenesis in xenograft tissue in vivo.

[0047] in, Figure 14 The curve of body weight change in H22 tumor-bearing mice; Figure 15 Effect of OA on tumor growth in H22 tumor-bearing mice (tumor volume was significantly reduced after OA administration) (Note: *P<0.05, **P<0.01, ***P<0.001); Figure 16 Immunohistochemical staining of tumor tissue (Note: *P<0.05, **P<0.01, ***P<0.001); Figure 17 Immunohistochemical staining images of tumor tissue show the expression of angiogenesis-related proteins (inhibition); Figure 18 Effects of OA on tumor mRNA expression in H22 tumor-bearing mice (inhibition of gene expression in the PI3K / AKT / mTOR / HIF-1α / VEGF signaling pathways) (Note: *P<0.05, ***P<0.001); Figure 19 Effects of OA on the expression of tumor angiogenesis-related proteins in H22 tumor-bearing mice (inhibition of angiogenesis-related protein expression) (Note: *P<0.05, **P<0.01, ***P<0.001).

[0048] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. An experimental method for using oleanolic acid to treat liver cancer, comprising in vitro and in vivo experimental methods, characterized in that, The in vitro experimental method includes the following steps: S1: Cell culture: Human liver cancer cells SMMC-7721 and human umbilical vein endothelial cells HUVECs were selected and cultured in culture medium and placed in a cell culture incubator at 37°C and 5% CO2. S2: Cell proliferation and migration: Cell proliferation was assessed using the MTT assay and the Cell Count Kit-8 assay. SMMC-7721 and HUVECs cells were seeded in 96-well plates and incubated. The optical density of each well was measured. Cell scratches were created, and cell migration was observed and photographed under a microscope. S3: Detection of apoptosis and cell cycle: To detect apoptosis in SMMC-7721 cells, cells were collected, stained at room temperature in the dark, and then analyzed by flow cytometry. S4: Immunoblotting: Cell and tumor tissue lysates were prepared on ice in a cell lysis buffer containing a mixture of protease inhibitors, followed by centrifugation. Total protein was quantified, and after adding loading buffer, the lysates were denatured, loaded onto an electrophoresis gel, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with skim milk and incubated at 4°C with primary antibodies PI3K, AKT, p-AKT, mTOR, p-mTOR, HIF-1α, VEGF, Bax, Bcl-2, Caspase-3, CCND1, and BIRC5 for 12 hours. Subsequently, the membrane was incubated with secondary antibodies at room temperature for 1 hour. The bands were visualized using a chemiluminescence method with an ultrasensitive luminescent substrate, and automatic exposure and development were performed. Finally, the gray values ​​of the bands were analyzed and the results were statistically analyzed. S5: Real-time quantitative PCR Total RNA was extracted from cells and tumor tissues, and the RNA was reverse transcribed into complementary DNA. Real-time PCR analysis was then performed. The experimental results were normalized with the 18S gene as an internal reference, and the relative mRNA expression level was calculated using the formula 2^(-ΔΔCt). The in vivo experimental method includes the following steps: Step 1: Chicken embryo chorionic allantoic model experiment: Chicken embryos were selected and pretreated. The drug delivery carrier was placed in the center of the CAM membrane. The drug to be tested was added to each group, and an equal amount of sterile saline was added to the control group. After incubation, on the third day of drug administration, the number of surviving chicken embryos was counted, fixative was added, the CAM membrane was cut off, and air-dried for storage. The air-dried CAM membrane was photographed with a camera, and the CAM area and vascular area were calculated. The vascular area ratio and vascular inhibition rate were calculated. Step 2: Tumor-bearing mouse experiment, treatment, and tissue sampling: Kunming rats were selected to establish a liver cancer xenograft model, and the growth volume of the transplanted tumor was calculated. After fixing fresh tumor tissue for 24 hours, it was dehydrated and cleared sequentially, then embedded in paraffin to prepare 4 μm tissue sections. Immunofluorescence staining was performed to observe the staining intensity and localization. The slides were digitally processed to capture images, and finally, the images were further analyzed.

2. The experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, The culture medium in S1 is RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

3. The experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, The method for measuring the optical density value of each well in S2 is as follows: SMMC-7721 and HUVECs cells are cultured for 24 hours, 48 ​​hours and 72 hours respectively. 10 μl of MTT or CCK-8 solution is added to each well, followed by incubation for 4 hours or 1 hour respectively. Finally, the optical density value of each well is measured at a wavelength of 490 nm or 450 nm using a microplate reader.

4. The experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, In step S2, the method for creating cell scratches is as follows: Prepare a suspension of HUVECs, add the cell suspension to each well, and after filtration, spread it to cover the bottom of the well. Using a ruler as an aid, draw evenly horizontal lines on the back of the 6-well plate with a black marker pen, perpendicular to the horizontal lines on the back of the plate, along the center line of the wells. Remove the cell culture plate at 0h and 24h after scratching, and then record the cell migration. .

5. The experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, The specific method for detecting by flow cytometry in the S3 is that the SMMC-7721 cell cycle condition is detected by using a Cell Cycle Assay Kit kit, pre-cooled anhydrous ethanol is slowly added to collect the cells in each group, and after being fully mixed, the cells are placed in 4 0 C refrigerator for 24 h, after the fixation is completed, RNase A Reagent is added to fully suspend the cells, and the cells are placed in 37 0 C water bath for 30 min, then PI Reagent is added and fully mixed, and the cells are placed in 4 0 C avoid light incubation for 30 min, and the red fluorescence at an excitation wavelength of 488 nm is detected by flow cytometry.

6. The experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, The method for pre-treating chicken embryos in step 1 is as follows: after incubating chicken embryos for 7 days, observe them under an egg candling lamp, select chicken embryos with similar developmental status, and draw the range of the air cell. In a clean bench, use ophthalmic forceps to carefully knock open a window with a radius of 0.75 cm at the end of the air cell to expose the eggshell membrane of the chicken embryo, and then tear it off.

7. An experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, The calculation methods for the vessel area ratio and vessel inhibition rate in step 1 are as follows: ； 。 8. An experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, In step 2, the specific method for establishing the liver cancer xenograft model is as follows: H22 cells are cultured in batches and diluted to a concentration of 3×107 cells / mL. The cells are injected into the left forelimb axilla of Kunming mice. After inoculation, the mice are fed normally under sterile conditions, and their weight is measured every 3 days. After the subcutaneous xenograft tumors in the mice have stabilized, the mice are randomly divided into 4 groups: Con blank control group, OA-LD group, OA-HD group, and SOR positive control group, with 10 mice in each group, half male and half female. Two weeks after administration, the mice are sacrificed 24 hours after the last administration, and the tumor is removed. The subcutaneous tumor is photographed, weighed, and the long and short diameters of the tumor are measured.

9. An experimental method for using oleanolic acid to treat liver cancer according to claim 1, characterized in that, In step 2, during immunofluorescence staining, the slides are dewaxed and rehydrated, and the antigen is recovered using antigen peeling buffer. Then, the slides are blocked in 5% bovine serum albumin and PBS at room temperature for 60 minutes. The primary antibody and secondary antibody are incubated for 16 hours and 45 minutes, respectively. After incubation, DAB chromogenic solution is used for 3 minutes for color development.

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