Method for researching sensitization effect of gallic acid on HepG2 cell sorafenib chemotherapy

Abstract

The invention discloses a research method of a chemosensitization effect of gallic acid on HepG2 cell sorafenib, and relates to the technical field of biological medicines. The method comprises the following steps: dividing liver cancer HepG2 cells into a control group, a GA group, a Sora group and a GA + Sora group; detecting cell activity, and analyzing a drug combination index; detecting the cell clone forming ability; detecting cell apoptosis; cell scratches and cell migration and invasion ability detection; an analysis process for detecting expression of matrix metalloproteinase 2, matrix metalloproteinase 9 and apoptosis-related protein by using a western blot method shows that the combination of GA and Sora has an obvious inhibiting effect on HepG2 cells, and the combination of GA and Sorafenib is proved to be a promising clinical treatment strategy for liver cancer; the problem that in the prior art, whether combined use of GA and Sora has a synergistic effect on HepG2 cells or not is unclear is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a method for studying the sensitization effect of gallic acid on sorafenib chemotherapy of HepG2 cells. Background Art

[0002] Primary liver cancer is a malignant tumor originating from liver cells or intrahepatic bile duct cells. More than 75% to 85% of primary liver cancers are of the pathological type of hepatocellular carcinoma (HCC) (hereinafter referred to as liver cancer). Radical surgical resection is the main treatment for liver cancer. Due to the insidious onset of liver cancer, 70% of patients are already in the middle or late stages when diagnosed, and lose the opportunity for radical surgical resection. For patients with advanced liver cancer who have large tumors, multiple lesions in the liver, and vascular, lymph node or distant metastases, molecular targeted drug therapy is the standard treatment strategy.

[0003] Sorafenib (Sora) is a first-line molecular targeted drug for the treatment of advanced liver cancer. Studies have shown that Sora can effectively prolong the overall survival time of liver cancer patients and improve the quality of life. The occurrence of drug resistance limits the clinical efficacy of Sora. Finding compounds with anti-tumor activity from medicinal plants and combining them with anti-tumor drugs is a hot topic in anti-tumor treatment research. Gallic acid (GA) is a natural polyphenolic acid found in plants such as Cornus officinalis, Gallic chinensis, Paeonia suffruticosa, and Rheum palmatum. Many studies have shown that GA has anti-cancer activity against a variety of cancer cells, including colorectal cancer, lung cancer, breast cancer, and endometrial cancer. Studies have shown that GA can significantly inhibit the growth, migration and invasion of liver cancer cells, and promote apoptosis and cycle arrest of liver cancer cells. Therefore, although both GA and Sora can affect liver cancer cells, the effect of the combined use of GA and Sora on the proliferation, apoptosis, migration and invasion of human liver cancer HepG2 cells is still a blank area in this field.

[0004] Based on this, the present invention proposes a research method for the sensitization effect of gallic acid on sorafenib chemotherapy of HepG2 cells, so as to observe the effects of the combined use of GA and Sora on the proliferation, apoptosis, migration and invasion of human liver cancer HepG2 cells through in vitro experiments, and to construct a nude mouse liver cancer model in vivo to explore the synergistic effect of the combined use of GA and Sora, so as to solve the problems existing in the above-mentioned prior art, thereby providing a reference for improving the efficacy of Sora in treating liver cancer. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a research method for the sensitizing effect of gallic acid on Sorafenib chemotherapy of HepG2 cells, which proves that GA has a sensitizing effect on Sora chemotherapy, can promote HepG2 cell apoptosis and inhibit cell migration and invasion, and can provide a reference for improving the efficacy of Sora in treating liver cancer; and solves the problem in the prior art that it is unclear whether the combined use of GA and Sora has a synergistic effect on HepG2 cells.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a first technical solution: a method for studying the effect of gallic acid on the chemosensitization of HepG2 cells to sorafenib, comprising:

[0008] Step 1: Cell culture

[0009] Human liver cancer HepG2 cells were placed in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a constant temperature incubator, and subcultured in an incubator at 5% CO2 and 37°C to maintain the cells in a logarithmic growth phase;

[0010] Step 2: Using CCK8 method to detect the cell viability of human liver cancer HepG2 cells cultured in step 1;

[0011] Step 3: grouping the human liver cancer HepG2 cells cultured in step 1, amplifying and detecting the proliferation ability of the human liver cancer HepG2 cells;

[0012] Step 4: Based on the grouping in step 3, the apoptosis rate of human liver cancer HepG2 cells was detected;

[0013] Step 5: Based on the grouping in step 3, the migration ability of human liver cancer HepG2 cells was detected;

[0014] Step 6: Based on the grouping in step 3, the invasion ability of human liver cancer HepG2 cells was detected;

[0015] Step 7: Based on the grouping in step 3, the expression levels of apoptosis-related proteins, MMP-2 and MMP-9 proteins in HepG2 cells were detected.

[0016] In a preferred embodiment, the process of detecting the cell viability of human liver cancer HepG2 cells using the CCK8 method comprises:

[0017] (1) 5×10 3 HepG2 cells / well were seeded on a 96-well plate, and 100 μL of culture medium containing different drugs was replaced after 24 h of culture;

[0018] Among them: GA group was 0, 25, 50, 100, 150, 200, 300 μmol·l -1 The Sora group was treated with 0, 1, 2, 5, 10, 20, 40 μmol·l -1 The combined application group was treated with 70 μmol·l -1 GA (IC30) + Sora were used in combination; in the combined application group, the concentration gradient of Sora was 1, 2, 5, 10, 20, 40 μmol·l -1 ;

[0019] (2) An equal amount of culture medium without drugs and cells was set up as a blank control group, with 5 replicate wells in each group. After 48 h of drug intervention, the absorbance value was measured at 450 nm using an ELISA reader to calculate the cell proliferation inhibition rate.

[0020] In a preferred embodiment, the process of amplifying and detecting the proliferation ability of human liver cancer HepG2 cells includes:

[0021] (1) 1×10 3 HepG2 cells were seeded in 6-well plates and divided into four groups: control group (Control); GA group (70 μmol·l -1 GA); Sora group (2 μmol / L Sora); GA+Sora group (70 μmol·l -1 GA+2μmol·l -1 Sora);

[0022] (2) The cells were cultured in a CO2 incubator for 14 days, fixed with 4% paraformaldehyde solution, washed three times with PBS buffer, stained with crystal violet, and the number of colonies with more than 10 cells was counted under a microscope. The experiment was repeated three times.

[0023] In a preferred embodiment, the process of detecting the apoptosis rate of human liver cancer HepG2 cells includes:

[0024] Based on the grouping in step 3, 2×10 5 HepG2 cells / well were seeded in 6-well plates, and cells were collected after 48 hours of intervention with different drugs. Cells were stained strictly according to the staining instructions of the kit, and the cell apoptosis rate was detected and calculated by flow cytometry;

[0025] Wherein: cell apoptosis rate = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%; the experiment was repeated 3 times.

[0026] In a preferred embodiment, the process of detecting the migration ability of human liver cancer HepG2 cells comprises:

[0027] Based on the grouping in step 3, 1×106 HepG2 cells / well were inoculated in a 6-well plate. After culturing for 24 hours, the bottom of the 6-well plate was scratched vertically with a 200 μL sterile pipette tip and treated with drugs. After 0 hours and 24 hours of drug addition, the plates were observed and photographed under an inverted microscope. The scratch distance was analyzed using Image J software to calculate the cell migration rate.

[0028] Wherein: cell migration rate = (0h scratch spacing - 24h scratch spacing) / 0h scratch spacing × 100%; the experiment was repeated 3 times.

[0029] In a preferred embodiment: the process of detecting the invasion ability of human liver cancer HepG2 cells includes:

[0030] On the basis of the grouping in step 3, HepG2 cells treated with different drugs for 48 h were inoculated in the upper chamber, and 600 μL of complete culture medium containing 20% ​​FBS was added to the lower chamber. After 24 h, the chamber was taken out, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet staining solution for 5 min. The cells in the upper chamber were wiped off with a cotton swab, and the chamber was observed under a microscope after drying to count the number of cells that penetrated the membrane.

[0031] In a preferred embodiment, the process of detecting the expression levels of HepG2 cell apoptosis-related proteins, MMP-2 and MMP-9 proteins comprises:

[0032] (1) Based on the grouping in step 3, cells were collected 48 h after drug intervention, and a certain volume of RIPA lysis buffer (containing 1% PMSF) was added according to the number of cell pellets. The cells were lysed on ice for 30 min, centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected. The protein concentration was detected by BCA protein quantitative detection kit;

[0033] (2) Take 30 μg of protein for SDS-PAGE electrophoresis and transfer to PVDF membrane, block with 5% BSA for 4 h, prepare primary antibody according to the antibody instruction manual and incubate at 4°C overnight;

[0034] (3) Wash the membrane three times with TBST buffer, add 1:1000 diluted secondary antibody, incubate at room temperature for 2 h, wash the membrane three times, develop with chemiluminescent reagent and take pictures;

[0035] (4) Image J software was used to analyze the grayscale values ​​of the bands. The ratio of the grayscale value of the target protein band to the grayscale value of β-actin band was used to represent the relative expression level of the target protein. The experiment was repeated 3 times.

[0036] In a preferred embodiment: the research method further comprises establishing a nude mouse xenograft tumor model; the process of establishing a nude mouse xenograft tumor model comprises:

[0037] (1) HepG2 cells were collected and prepared into a cell suspension with phosphate buffered saline. 0.2 mL of the cell suspension was subcutaneously injected into the lower right back of nude mice.

[0038] Wherein: the 0.2mL cell suspension contains 5×10 6 HepG2 cells;

[0039] (2) Six days after tumor implantation, the mice were divided into four groups according to the order of tumor size: control group, GA group, Sora group, and GA+Sora group, with 7 mice in each group. The GA group received 80 mg / kg GA, the Sora group received 20 mg / kg Sora, and the GA+Sora group received 80 mg / kg GA + 20 mg / kg Sora. The drugs were administered once every other day according to the above drug concentrations, and the control group received normal saline.

[0040] Among them: When administering, the intragastric volume is calculated according to the standard of 0.1mL / 10g;

[0041] (3) Monitor the activity and general condition of nude mice, and measure tumor size and body weight once a week. According to the formula, tumor volume = long diameter × short diameter 2 / 2, estimate the tumor volume; after 21 days of drug intervention, weigh the tumor.

[0042] In a preferred embodiment: the research method further includes a statistical analysis process, and the statistical analysis process specifically includes:

[0043] (1) Calculate the 30% cell growth inhibition concentration IC of the drug using Graphpad Prism 7.0 software 30 and half inhibitory concentration IC 50 ;

[0044] (2) SPSS 20.0 software was used for data analysis. The experimental data were expressed as (x±s). One-way analysis of variance was used for comparison among the groups, and LSD-t test was used for further pairwise comparison. P < 0.05 was considered statistically significant.

[0045] The present invention provides a second technical solution: application of gallic acid in combination with Sorafenib to enhance the chemotherapy sensitization of HepG2 cells.

[0046] Compared with the prior art, the present invention provides a method for studying the effect of gallic acid on the chemosensitization of HepG2 cells to sorafenib, which has the following beneficial effects:

[0047] 1. In the research method of the present invention on the sensitization effect of gallic acid on sorafenib chemotherapy of HepG2 cells, the synergistic inhibitory effect of GA combined with sorafenib on the proliferation of HepG2 cells was first observed. The results showed that the IC 50 The values ​​were (9.87±0.98) μmol·l -1 , and 70 μmol·l -1 GA(IC 30 ) After combined use, IC 50 Reduced to (2.06±0.35)μmol·l -1 , and different concentrations of Sora and 70μmol·l -1 The CI values ​​of GA combined use were all less than 1, which proved that GA and Sora both had inhibitory effects on the proliferation of liver cancer HepG2 cells, and the combined inhibitory effect of the two drugs on HepG2 cells was more obvious.

[0048] 2. This method measured the tumor growth of the GA monotherapy group (80 mg / kg), Sora monotherapy group (20 mg / kg) and the GA (80 mg / kg) and Sora (20 mg / kg) combination group in a nude mouse liver cancer transplant tumor model. The results showed that compared with the control group, GA or Sora monotherapy group, the tumor volume of the combination group was significantly reduced, and the combination of GA and Sora had the best anti-tumor effect. The results of in vitro and in vivo experiments both suggested that GA combined with Sora had a synergistic anti-liver cancer effect, which could reduce the dose of sorafenib and reduce adverse reactions, which has important guiding significance for clinical medication.

[0049] 3. The present invention obtains that GA and Sora exert synergistic effects on anti-liver cancer effects both in vivo and in vitro through the above research methods. These results suggest that the combined use of GA and sorafenib will be a promising clinical treatment strategy for liver cancer; and solves the problem in the prior art that it is unclear whether the combined use of GA and Sora has a synergistic effect on HepG2 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a flow chart of the research method of the gallic acid on the sorafenib chemotherapy sensitization effect of HepG2 cells of the present invention;

[0051] Figure 2 This is a graph showing the clone formation ability of each group of cells observed by crystal violet staining;

[0052] Figure 3 This is a graph showing the apoptosis rate of each group of cells detected by flow cytometry in the present invention;

[0053] Figure 4 This is a picture of the cell migration ability of each group detected by the cell scratch experiment of the present invention (×40);

[0054] Figure 5 This is a picture of the cell invasion ability of each group detected by the Transwell experiment of the present invention (×100);

[0055] Figure 6 This is the protein expression diagram of MMP-2, MMP-9, Bcl-2, Bax and Cleaved caspase-3 in HepG2 cells of the present invention;

[0056] Figure 7 This is a graph showing the effect of the combination of GA and Sora on tumor growth in nude mice.

[0057] Among them: Figure 7 Figure A is a diagram of the tumor tissues of each group of nude mice; Figure B is a curve diagram of the changes in tumor volume of each group of nude mice; Figure C is a curve diagram of the changes in tumor weight of each group of nude mice DETAILED DESCRIPTION

[0058] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] See also Figure 1-Figure 7 , the present invention provides a technical solution:

[0060] A method for studying the effect of gallic acid on the sensitization of HepG2 cells to sorafenib chemotherapy, which is used to observe the effects of the combined use of GA and Sora on the proliferation, apoptosis, migration and invasion of human liver cancer HepG2 cells through in vitro experiments, and to construct a nude mouse liver cancer model in vivo to explore whether the combined use of GA and Sora has a synergistic effect; including:

[0061] Step 1: Material preparation

[0062] Human hepatocellular carcinoma HepG2 cell line was purchased from Jiangsu Keyi Biotechnology Co., Ltd.; Sora and GA were purchased from MCE, USA; high-glucose DMEM medium and fetal bovine serum were purchased from Gibco, USA; rabbit anti-human B-cell lymphoma-2 proto-oncogene (B-cell lymphoma-2, Bcl-2) antibody, rabbit anti-human Bcl-2-associated X protein (Bcl-2-associated X, Bax) antibody, rabbit anti-human cleaved caspase-3 antibody, mouse anti-human matrix metalloproteinase-2 (MMP-2) antibody, and rabbit anti-human matrix metalloproteinase-9 (MMP-9) antibody were purchased from Abcam, USA; horseradish peroxidase-labeled goat anti-rabbit secondary antibody and horseradish peroxidase-labeled goat anti-mouse secondary antibody were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; CCK8 kit and cell apoptosis detection kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; Transwell cell culture plates and matrix gel were purchased from Corning, USA; CO2 cell culture incubator was purchased from Thermo, USA, and inverted microscope was purchased from Olympus, Japan; flow cytometer was purchased from Beckman Coulter, USA; and microplate reader and SDS-PAGE protein electrophoresis instrument were purchased from Bio-Rad, USA.

[0063] Step 2: Method

[0064] Step 1: Cell culture

[0065] Human hepatocarcinoma HepG2 cells were placed in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a constant temperature incubator (5% CO2, 37°C) for routine subculture to maintain the cells in the logarithmic growth phase.

[0066] Step 2: CCK8 assay to detect cell viability

[0067] 5×10 3 HepG2 cells were seeded on 96-well plates, and 100 μL of medium containing different drugs was replaced after 24 h of culture. The GA group was treated with 0, 25, 50, 100, 150, 200, and 300 μmol·l -1 The Sora group was treated with 0, 1, 2, 5, 10, 20, 40 μmol·l -1 The combined application group was treated with 70 μmol·l -1 GA(IC30)+Sora(1, 2, 5, 10, 20, 40 μmol·l -1) were used in combination; an equal amount of culture medium without drugs and cells was set as a blank control group, with 5 replicates in each group. After 48 hours of drug intervention, the absorbance value (OD) was detected at 450nm by a microplate reader, and the cell proliferation inhibition rate was calculated; the combination index (CI) was calculated by CompuSyn software.

[0068] Step 3: Amplify and detect the proliferation ability of human liver cancer HepG2 cells

[0069] 1×10 3 HepG2 cells were seeded in 6-well plates and divided into four groups: control group (Control); GA group (70 μmol·l -1 GA); Sora group (2 μmol / L Sora); GA+Sora group (70 μmol·l -1 GA+2μmol·l -1 Sora); placed in a CO2 incubator for 14 days, fixed with 4% paraformaldehyde solution, washed three times with PBS buffer, stained with crystal violet, and counted the number of colonies with more than 10 cells under a microscope; the experiment was repeated three times;

[0070] The experimental results of the inhibitory effect of GA and Sora on the proliferation of HepG-2 cells are shown in Table 1: Different concentrations of GA, Sora, and the combination of the two drugs all have an inhibitory effect on the proliferation of HepG2 cells in a concentration-dependent manner; the IC values ​​of GA and Sora on HepG2 cells for 48 h are 50 The values ​​were: (123.47±5.16)μmol·l -1 and (9.87±0.98)μmol·l -1 , Sora and 70 μmol·l -1 GA(IC 30 ) after combined use, IC 50 Reduced to (2.06±0.35)μmol·l -1 ; CompuSyn software was used to calculate the CI value. Different concentrations of Sora and 70 μmol·l -1 The CI values ​​of GA combined use were all less than 1, indicating that the combined use of GA and Sora had a synergistic effect. According to the drug combination selection principle (CI>1 antagonism, CI=1 superposition, 0.6<CI<1 synergy, CI<0.6 strong synergy), 70 μmol·l -1 GA and 2 μmol·l -1 Sora is the drug concentration of the combined drug group in the subsequent experiments, with an action time of 48 h;

[0071] Table 1: Inhibitory effect of the combination of gallic acid and sorafenib on the proliferation of HepG2 cells

[0072]

[0073] Note: *Compared with the same concentration of Sora group, t=-50.051~-3.131, P<0.05;

[0074] The experimental results of the effect of GA and Sora on the proliferation ability of HepG2 cells were as follows: after 14 days of cell culture in each group, the number of cell clones in the GA+Sora group (73.0±7.6) was significantly lower than that in the control group (234.0±20.4), the GA group (147.0±12.1) and the Sora group (129.3±13.3) (P<0.05). Figure 2 shown.

[0075] Step 4: FITC-Annexin V / PI double staining flow cytometry to detect cell apoptosis

[0076] Based on the grouping in step 3, 2×10 5 HepG2 cells / well were seeded in 6-well plates. After 48 hours of intervention with different drugs, the cells were collected and stained strictly according to the staining instructions of the kit. The cell apoptosis rate was detected and calculated by flow cytometry; cell apoptosis rate = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%; the experiment was repeated 3 times;

[0077] The experimental results of the effect of GA and Sora combined on HepG2 cell apoptosis were as follows: Annexin V-FITC / PI double staining experimental results ( Figure 3 ), the apoptosis rates of cells in the control group, GA group, Sora group and GA+Sora group were (1.98±20.4)%, (15.17±1.56)%, (18.65±1.48)% and (34.60±5.36)%, respectively; compared with the control group, the apoptosis rates of cells in the GA group, Sora group and GA+Sora group were significantly increased (P<0.05); compared with the GA group and Sora group, the apoptosis rate of cells in the GA+Sora group was significantly increased (P<0.05).

[0078] Step 5: Cell scratch assay to detect cell migration ability

[0079] Based on the grouping in step 3, 1×10 6 HepG2 cells / well were inoculated in 6-well plates. After culturing for 24 hours, the bottom of the 6-well plates was scratched vertically with a 200 μL sterile pipette tip and treated with drugs. After 0 hours and 24 hours of drug addition, the plates were observed and photographed under an inverted microscope. The scratch distance was analyzed by Image J software to calculate the cell migration rate; cell migration rate = (0 hour scratch distance - 24 hour scratch distance) / 0 hour scratch distance × 100%; the experiment was repeated 3 times;

[0080] The experimental results of the effect of GA and SORA on the migration ability of HepG2 cells were as follows: 24 hours after drug treatment, the migration rates of cells in the control group, GA group, Sora group and GA+Sora group were (55.59±5.08)%, (29.34±4.36)%, (21.80±5.16)% and (6.47±2.75)%, respectively. Compared with the control group, the migration rates of cells in the GA group, Sora group and GA+Sora group were decreased (P<0.05); there was no significant difference in the migration rates between the GA group and the Sora group (P>0.05); compared with the GA group and the Sora group, the migration rate of cells in the GA+Sora group was decreased (P<0.05); see Figure 4 .

[0081] Step 6: Transwell assay to detect cell invasion ability

[0082] On the basis of the grouping in step 3, HepG2 cells treated with different drugs for 48 hours were inoculated in the upper chamber, and 600 μL of complete medium containing 20% ​​FBS was added to the lower chamber. After 24 hours, the chamber was taken out, fixed with 4% paraformaldehyde for 15 minutes, and stained with crystal violet staining solution for 5 minutes. The cells in the upper chamber were wiped off with a cotton swab, and the chamber was observed under a microscope after drying, and the number of cells penetrating the membrane was counted;

[0083] The results showed that the combined use of GA and Sora had an effect on the invasion ability of HepG2 cells: 24 hours after the Transwell experiment, the numbers of HepG2 cells penetrating the membrane in the control group, GA group, Sora group and GA+Sora group were (223.7±13.0), (168.3±10.9), (155.3±29.1) and (62.7±19.7), respectively. Compared with the control group, the numbers of cells penetrating the membrane in the GA group, Sora group and GA+Sora group were decreased (P<0.05); compared with the GA group and Sora group, the number of cells penetrating the membrane in the GA+Sora group was significantly decreased (P<0.05); see Figure 5 .

[0084] Step 7: Western blotting to detect the expression levels of apoptosis-related proteins, MMP-2 and MMP-9 proteins in HepG2 cells

[0085] On the basis of the grouping in step 3, cells were collected 48 hours after drug intervention, and a certain volume of RIPA lysis buffer (containing 1% PMSF) was added according to the number of cell pellets, lysed on ice for 30 minutes, centrifuged at 4°C and 12000r / min for 15 minutes, and the supernatant was collected. The protein concentration was detected by BCA protein quantitative detection kit; 30 μg of protein was taken for SDS-PAGE electrophoresis and transferred to PVDF membrane, blocked with 5% BSA for 4 hours, and the primary antibody was prepared according to the antibody instructions and incubated at 4°C overnight; the membrane was washed 3 times with TBST buffer and a 1:1000 diluted secondary antibody was added, incubated at room temperature for 2 hours, and the membrane was washed 3 times with chemiluminescent reagent for development and photographed; the gray value of the band was analyzed by Image J image analysis software, and the relative expression level of the target protein was expressed by the ratio of the gray value of the target protein band to the gray value of the β-actin band; the experiment was repeated 3 times;

[0086] The results showed that the combined use of GA and Sora could affect the expression of MMP-2, MMP-9 and apoptosis-related proteins in HepG2 cells as follows: compared with the control group, the expression levels of MMP-2 and MMP-9 in the GA group, Sora group and GA+Sora group were decreased (P<0.05), the expression levels of apoptosis-related proteins Bax and Cleaved caspase-3 were increased (P<0.05), and the expression of Bcl-2 was decreased (P<0.05); compared with the GA group and Sora group, the expression levels of MMP-2 and MMP-9 in the GA+Sora group were significantly decreased (P<0.05), the expression levels of apoptosis-related proteins Bax and Cleaved caspase-3 were increased (P<0.05), and the expression of Bcl-2 was decreased (P<0.05); see Figure 6 and Table 2;

[0087] Table 2: Expression levels of MMP-2, MMP-9, Bcl-2, Bax and Cleaved caspase-3 proteins in HepG2 cells

[0088]

[0089]

[0090] Note: * P < 0.05, compared with the control group; △ P < 0.05, compared with the GA group; # P<0.05, compared with the Sora group.

[0091] Step 8: Establishment of nude mouse xenograft tumor model

[0092] Thirty SPF male nude mice (6 weeks old, body weight 18-22 g) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd., Animal Production License No.: SCXK (Ji) 2020-0002, Laboratory Animal Use License No.: SYXK (Ji) 2021-0020; HepG2 cells were collected, phosphate buffered saline was prepared into a cell suspension, and 0.2 mL (containing 5×10 6 HepG2 cell suspension was placed on the lower right back of nude mice; 6 days after tumor implantation, the mice were divided into 4 groups according to the order of tumor size: control group, GA group, Sora group and GA+Sora group, with 7 mice in each group; the in vivo administration dose and method of GA and Sora were referred to the literature "SHI CJ, ZHENG YB, PAN FF, et al. Gallic acid suppressed by anLncRNA MALAT1-Wnt / β-Catenin axis in hepatocellular carcinoma[J]. FrontPharmacol, 2021, 12: 708967. DOI: 10.3389 / fphar.2021.708967" and "LI ZJ, DAI HQ, HUANGXW, et al. Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma[J]. Acta Pharmacol Sin, 2021, 42(2): 301-310.DOI: 10.1038 / s41401-020-0478-3”, the mice were given drugs according to the concentrations of GA group (80 mg / kg GA), Sora group (20 mg / kg Sora), and GA+Sora group (80 mg / kg GA+20 mg / kg Sora); the drugs were given once every other day according to the above drug concentrations (the oral volume was calculated according to the standard of 0.1 mL / 10 g), and the control group was given normal saline; the activity and general condition of the nude mice were monitored, and the tumor size and body weight were measured once a week. According to the formula, tumor volume = long diameter × short diameter 2 / 2, estimated tumor volume; after 21 days of drug intervention, the nude mice were killed and the tumors were weighed;

[0093] The effects of GA combined with Sora on tumor growth in nude mice were as follows: During the feeding process, the spirit, diet and defecation of the four groups of nude mice were relatively normal, and their weight also increased during the feeding process. The GA group and the GA+Sora group gained more weight, followed by the Sora group, but compared with the control group, there was no statistically significant difference (P>0.05); compared with the control group, the GA group, the Sora group and the GA+Sora group all significantly inhibited the growth and tumor size of nude mice tumors (P<0.05); compared with the Sora group alone, the volume and weight of the tumors in the GA+Sora group were significantly reduced (P<0.05, see Figure 7 ).

[0094] Among them, Figure 7 Figure A is a diagram of the tumor tissues of nude mice in each group; Figure B is a curve diagram of the changes in tumor volume of nude mice in each group; and Figure C is a curve diagram of the changes in tumor weight of nude mice in each group.

[0095] Step 9: Statistical analysis

[0096] Graphpad Prism 7.0 software was used to calculate the 30% cell growth inhibition concentration (IC 30 ) and 50% inhibition concentration (IC 50 ); SPSS 20.0 software was used for data analysis. The comparison among the groups was performed by one-way analysis of variance, and the further pairwise comparison was performed by LSD-t test, with P < 0.05 indicating statistically significant differences;

[0097] The above experimental results prove that:

[0098] 1.GA and Sora both have an inhibitory effect on the proliferation of liver cancer HepG2 cells, and the combined inhibitory effect of the two drugs on HepG2 cells is more obvious. The IC value of Sora on HepG2 cells for 48 hours is 50 The values ​​were (9.87±0.98) μmol·l -1 , and 70 μmol·l -1 GA(IC 30 ) After combined use, IC 50 Reduced to (2.06±0.35)μmol·l -1 , and different concentrations of Sora and 70μmol·l -1The CI values ​​of GA combined use were all less than 1. To further confirm the synergistic anti-liver cancer effect of GA and Sora in vivo, this study measured the tumor growth of GA monotherapy (80 mg / kg), Sora monotherapy (20 mg / kg) and GA (80 mg / kg) and Sora (20 mg / kg) combined use groups in nude mouse liver cancer transplant tumor models. The results showed that compared with the control group, GA or Sora monotherapy group, the tumor volume of the combined use group was significantly reduced, and the combination of GA and Sora had the best anti-tumor effect. The results of in vitro and in vivo experiments both suggest that GA combined with Sora has a synergistic anti-liver cancer effect, which can reduce the dose of sorafenib and reduce adverse reactions, which has important guiding significance for clinical medication.

[0099] 2.GA and Sora alone have weak inhibition on the migration and invasion of HepG2 cells, but combined use can significantly reduce the apoptotic migration and invasion of HepG2 cells. Extracellular matrix degradation and penetration of the basement membrane are key steps in the invasion and metastasis of liver cancer. Matrix metalloproteinases (MMPs) can degrade components such as type IV collagen, laminin and elastin in the basement membrane and extracellular matrix, thereby promoting the migration and invasion of cancer cells to adjacent and distal tissues. Studies have shown that MMP-2 and MMP-9 are closely related to the invasion, metastasis and prognosis of liver cancer. Western blot analysis results showed that the expression levels of MMP-2 and MMP-9 in the combination group were significantly reduced. The combination of GA and Sora can inhibit the migration and invasion of liver cancer cells by reducing the expression of MMP-2 and MMP-9 proteins.

[0100] In summary, the present invention has obtained that GA and Sora have synergistic effects in both in vivo and in vitro anti-liver cancer effects, and this synergistic effect may be related to promoting apoptosis of liver cancer cells and inhibiting migration and invasion. These results suggest that the combined use of GA and sorafenib will be a promising clinical treatment strategy for liver cancer. The research team will further explore the mechanism of GA's sensitization to sorafenib chemotherapy from the perspectives of cell migration, invasion signaling pathways and animal experiments, in order to provide experimental basis for clinical improvement of liver cancer treatment effects.

[0101] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0102] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for studying the effect of gallic acid on the chemosensitization of HepG2 cells with sorafenib, characterized in that: include: Step 1: Cell culture Human liver cancer HepG2 cells were placed in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a constant temperature incubator, and subcultured in an incubator at 5% CO2 and 37°C to maintain the cells in a logarithmic growth phase; Step 2: Detecting the cell viability of the human liver cancer HepG2 cells cultured in step 1; Step 3: grouping the human liver cancer HepG2 cells cultured in step 1, amplifying and detecting the proliferation ability of the human liver cancer HepG2 cells; Step 4: Based on the grouping in step 3, the apoptosis rate of human liver cancer HepG2 cells was detected; Step 5: Based on the grouping in step 3, the migration ability of human liver cancer HepG2 cells was detected; Step 6: Based on the grouping in step 3, the invasion ability of human liver cancer HepG2 cells was detected; Step 7: Based on the grouping in step 3, the expression levels of apoptosis-related proteins, MMP-2 and MMP-9 proteins in HepG2 cells were detected.

2. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The process of detecting the cell viability of human liver cancer HepG2 cells using the CCK8 method includes: (1) 5×10 3 HepG2 cells / well were seeded on a 96-well plate, and 100 μL of culture medium containing different drugs was replaced after 24 h of culture; Among them: GA group was 0, 25, 50, 100, 150, 200, 300 μmol·l -1 The Sora group was treated with 0, 1, 2, 5, 10, 20, 40 μmol·l -1 The combined application group was treated with 70 μmol·l -1 GA (IC30) + Sora were used in combination; in the combined application group, the concentration gradient of Sora was 1, 2, 5, 10, 20, 40 μmol·l -1 ; (2) An equal amount of culture medium without drugs and cells was set up as a blank control group, with 5 replicate wells in each group. After 48 h of drug intervention, the absorbance value was measured at 450 nm using an ELISA reader to calculate the cell proliferation inhibition rate.

3. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The process of amplifying and detecting the proliferation ability of human liver cancer HepG2 cells includes: (1) 1×10 3 HepG2 cells were seeded in 6-well plates and divided into four groups: control group (Control); GA group (70 μmol·l - 1 GA); Sora group (2 μmol / L Sora); GA+Sora group (70 μmol·l -1 GA+2μmol·l -1 Sora); (2) The cells were cultured in a CO2 incubator for 14 days, fixed with 4% paraformaldehyde solution, washed three times with PBS buffer, stained with crystal violet, and the number of colonies with more than 10 cells was counted under a microscope. The experiment was repeated three times.

4. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The process of detecting the apoptosis rate of human liver cancer HepG2 cells includes: Based on the grouping in step 3, 2×10 5 HepG2 cells / well were seeded in 6-well plates, and cells were collected after 48 hours of drug intervention. Cells were stained strictly according to the staining instructions of the kit, and the cell apoptosis rate was detected and calculated by flow cytometry; Wherein: cell apoptosis rate = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%; the experiment was repeated 3 times.

5. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The process of detecting the migration ability of human liver cancer HepG2 cells includes: Based on the grouping in step 3, 1×10 6 HepG2 cells / well were inoculated in a 6-well plate. After culturing for 24 hours, the bottom of the 6-well plate was scratched vertically with a 200 μL sterile pipette tip and treated with drugs. After 0 hours and 24 hours of drug addition, the plates were observed and photographed under an inverted microscope. The scratch distance was analyzed using Image J software to calculate the cell migration rate. Wherein: cell migration rate = (0h scratch spacing - 24h scratch spacing) / 0h scratch spacing × 100%; the experiment was repeated 3 times.

6. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The process of detecting the invasion ability of human liver cancer HepG2 cells includes: On the basis of the grouping in step 3, HepG2 cells treated with different drugs for 48 h were inoculated in the upper chamber, and 600 μL of complete culture medium containing 20% ​​FBS was added to the lower chamber. After 24 h, the chamber was taken out, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet staining solution for 5 min. The cells in the upper chamber were wiped off with a cotton swab, and the chamber was observed under a microscope after drying to count the number of cells that penetrated the membrane.

7. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The process of detecting the expression levels of apoptosis-related proteins, MMP-2 and MMP-9 proteins in HepG2 cells includes: (1) Based on the grouping in step 3, cells were collected 48 h after drug intervention, and a certain volume of RIPA lysis buffer (containing 1% PMSF) was added according to the number of cell pellets. The cells were lysed on ice for 30 min, centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected. The protein concentration was detected by BCA protein quantitative detection kit; (2) Take 30 μg of protein for SDS-PAGE electrophoresis and transfer to PVDF membrane, block with 5% BSA for 4 h, prepare primary antibody according to the antibody instruction manual and incubate at 4°C overnight; (3) Wash the membrane three times with TBST buffer, add 1:1000 diluted secondary antibody, incubate at room temperature for 2 h, wash the membrane three times, develop with chemiluminescent reagent and take pictures; (4) Image J image analysis software was used to analyze the gray value of the bands, and the ratio of the gray value of the target protein band to the gray value of the β-actin band was used to represent the relative expression level of the target protein. The experiment was repeated 3 times.

8. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The research method also includes establishing a nude mouse xenograft tumor model; The process of establishing a nude mouse xenograft tumor model includes: (1) HepG2 cells were collected and prepared into a cell suspension with phosphate buffered saline. 0.2 mL of the cell suspension was subcutaneously injected into the lower right back of nude mice. Wherein: the 0.2mL cell suspension contains 5×10 6 HepG2 cells; (2) Six days after tumor implantation, the mice were divided into four groups according to the order of tumor size: control group, GA group, Sora group, and GA+Sora group, with 7 mice in each group. The GA group received 80 mg / kg GA, the Sora group received 20 mg / kg Sora, and the GA+Sora group received 80 mg / kg GA+20 mg / kg Sora. The drugs were administered once every other day according to the above drug concentrations, and the control group was given normal saline. Among them: When administering, the intragastric volume is calculated according to the standard of 0.1mL / 10g; (3) Monitor the activity and general condition of nude mice, and measure tumor size and body weight once a week. According to the formula, tumor volume = long diameter × short diameter 2 / 2, estimate the tumor volume; after 21 days of drug intervention, weigh the tumor.

9. The method for studying the effect of gallic acid on the chemosensitization of HepG2 cells by sorafenib according to claim 1, characterized in that: The research method also includes a statistical analysis process, which specifically includes: (1) Calculate the 30% cell growth inhibition concentration IC of the drug using Graphpad Prism 7.0 software 30 and half inhibitory concentration IC 50 ; (2) SPSS 20.0 software was used for data analysis. The experimental data were expressed as (x±s). One-way analysis of variance was used for comparison among the groups, and LSD-t test was used for further pairwise comparison. P < 0.05 was considered statistically significant.

10. Application of gallic acid combined with Sorafenib to enhance chemotherapy sensitization of HepG2 cells.

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