Application of Anluo chemical fiber pill in preparation of medicine for inhibiting liver cancer cell proliferation and promoting liver cancer cell apoptosis

Abstract

The invention belongs to the technical field of traditional Chinese medicines, particularly discloses application of an Anluo chemical fiber pill in preparation of medicines for inhibiting liver cancer cell proliferation and promoting liver cancer cell apoptosis, and discloses that the Anluo chemical fiber pill can reduce the cell survival rate of human liver cancer cells Hep G2, Hep 3B and SK-Hep-1 and has time and dose dependence; the Anluo chemical fiber pills can inhibit proliferation, invasion and migration of Hep G2 and Hep 3B cells and induce apoptosis of the Hep G2 and Hep 3B cells at the same time, and the apoptosis mostly occurs at the early stage of cell apoptosis; the Anluo chemical fiber pill can promote apoptosis of Hep G2 and Hep 3B cells by increasing the content of ROS in the Hep G2 and Hep 3B cells, and has dependence on time and dosage, so that the Anluo chemical fiber pill can be used for preparing the medicine for inhibiting liver cancer cell proliferation and promoting apoptosis of liver cancer cells.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, in particular to the application of Anluo Chemical Fiber Pills in preparing drugs for inhibiting the proliferation of liver cancer cells and promoting the apoptosis of liver cancer cells. Background Art

[0002] Anluo Huaxian Pills are mainly used to treat chronic hepatitis and early and middle-stage liver cirrhosis. Clinically, it has been found that it also has a significant effect on liver fibrosis caused by various reasons. Hepatitis, liver fibrosis, and liver cirrhosis can easily develop into liver cancer. As the second most lethal malignant tumor in the world, liver cancer seriously threatens human life and health, and has a long course of disease, a high recurrence rate, easy spread, and difficult treatment. In recent years, traditional Chinese medicine has gradually become a research hotspot for liver cancer treatment, not only because of its long history, but also because of its unique advantages such as multi-pathway, multi-target, and low toxicity.

[0003] Modern pharmacological studies have shown that various Chinese medicinal ingredients in Anluo Chemical Fiber Pills, such as Rehmannia glutinosa, Panax notoginseng, and Atractylodes macrocephala, have anti-tumor activity, but the specific effects and effects of Anluo Chemical Fiber Pills on liver cancer cells are not clear. Summary of the invention

[0004] The purpose of the present invention is to provide the use of Anluo Chemical Fiber Pills in the preparation of drugs for inhibiting the proliferation of liver cancer cells and promoting the apoptosis of liver cancer cells.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] Application of Anluo Chemical Fiber Pills in the preparation of drugs for inhibiting proliferation of liver cancer cells and promoting apoptosis of liver cancer cells.

[0007] Compared with the prior art, the present invention discloses that Anluo Chemical Fiber Pills can reduce the cell survival rate of human liver cancer cells Hep G2, Hep 3B, and SK-Hep-1, and has time and dose dependence; Anluo Chemical Fiber Pills can inhibit the proliferation, invasion and migration of Hep G2 and Hep3B cells, and induce apoptosis of Hep G2 and Hep 3B cells, and most of them occur in the early stage of cell apoptosis; Anluo Chemical Fiber Pills can promote the apoptosis of Hep G2 and Hep 3B cells by increasing the content of ROS in Hep G2 and Hep 3B cells, and has time and dose dependence, indicating that Anluo Chemical Fiber Pills can be used to prepare drugs for inhibiting the proliferation of liver cancer cells and promoting the apoptosis of liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1Effects of Anluo Huaxian Pills (AL) and Compound Blister Capsules (FU) on the proliferation activity of liver cancer cells Hep G2: A. Cell survival rate after 12h of AL and FU treatment; B. Cell survival rate after 24h of AL and FU treatment; C. Cell survival rate after 48h of AL and FU treatment; D. Cell survival rate after AL treatment for different time periods.

[0009] Figure 2 Effects of Anluo Huaxian Pills (AL) and Compound Cantharidin Capsules (FU) on the proliferation activity of liver cancer cells Hep 3B: A. Cell survival rate after 12 hours of AL and FU treatment; B. Cell survival rate after 24 hours of AL and FU treatment; C. Cell survival rate after 48 hours of AL and FU treatment; D. Cell survival rate after AL treatment for different time periods.

[0010] Figure 3 Effects of Anluo Huaxian Pills (AL) and Compound Cantharidin Capsules (FU) on the proliferation activity of liver cancer cell SK-Hep-1: A. Cell survival rate after 12h of AL and FU treatment; B. Cell survival rate after 24h of AL and FU treatment; C. Cell survival rate after 48h of AL and FU treatment; D. Cell survival rate after AL treatment for different time periods.

[0011] Figure 4 Effects of Anluo Huaxian Pills (AL) and Compound Blister Capsules (FU) on the proliferation activity of normal human hepatocytes L-O2: A. Cell survival rate after 12h of AL and FU treatment; B. Cell survival rate after 24h of AL and FU treatment; C. Cell survival rate after 48h of AL and FU treatment; D. Cell survival rate after AL treatment for different time periods.

[0012] Figure 5 Effects of Anluo Huaxian Pills (AL) and Compound Blister Capsules (FU) on the proliferation activity of normal human renal epithelial cells 293T: A. Cell survival rate after 12h of AL and FU treatment; B. Cell survival rate after 24h of AL and FU treatment; C. Cell survival rate after 48h of AL and FU treatment; D. Cell survival rate after different time periods of AL treatment.

[0013] Figure 6 The cell counting method was used to detect the proliferation inhibition of Anluo Chemical Fiber Pills (AL) on human liver cancer cells Hep G2 and Hep 3B: A. Cell number result graph of Hep G2 cells; B. Cell number result graph of Hep 3B cells.

[0014] Figure 7 The clone formation experiment was used to detect the effect of Anluo Chemical Fiber Pills (AL) on the proliferation of human liver cancer cells Hep G2 and Hep 3B: A. Cell state result diagram of Hep G2 cells; B. Cell state result diagram of Hep 3B cells.

[0015] Figure 8 The scratch experiment was used to detect the effect of Anluo Chemical Fiber Pills (AL) on the migration of human liver cancer cells Hep G2: A. Result diagram of cell migration status of Hep G2 cells; B. Result diagram of quantitative analysis of Hep G2 cells.

[0016] Fig. 9 The scratch experiment was used to detect the effect of Anluo Chemical Fiber Pills (AL) on the migration of human liver cancer cells Hep 3B: A. Result diagram of cell migration status of Hep 3B cells; B. Result diagram of quantitative analysis of Hep 3B cells.

[0017] Fig.10 Transwell experiment to detect the effect of Anluo Chemical Fiber Pills (AL) on the invasion of human liver cancer cells Hep G2: A. Result diagram of cell invasion status of Hep G2 cells; B. Result diagram of quantitative analysis of Hep G2 cells.

[0018] Fig.11 Transwell experiment to detect the effect of Anluo Huaxian Pills (AL) on the invasion of human liver cancer cells Hep 3B: A. Result diagram of cell invasion status of Hep 3B cells; B. Result diagram of quantitative analysis of Hep 3B cells.

[0019] Fig.12 The ROS experiment was conducted to detect the effect of Anluo Huaxian Pills (AL) on ROS in human liver cancer cells Hep G2.

[0020] Fig.13 The ROS experiment was conducted to detect the effect of Anluo Huaxian Pills (AL) on ROS in human liver cancer cells Hep 3B.

[0021] Fig.14 This is a DAPI experiment to detect the effect of Anluo Huaxian Pills (AL) on apoptosis of human liver cancer cell Hep G2.

[0022] Fig.15 This is a DAPI experiment to detect the effect of Anluo Huaxian Pills (AL) on the apoptosis of human liver cancer cell Hep 3B.

[0023] Fig.16 AnnexinV-PE / 7-AAD flow cytometry experiment was used to detect the effect of Anluo Chemical Fiber Pills (AL) on the apoptosis rate of human liver cancer cells Hep G2: A. Cell flow cytometry results of Hep G2 cells; B. Quantitative analysis results of Hep G2 cells. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0025] The following examples explore the effect of Anluo Huaxian Pills on the proliferation and apoptosis of liver cancer cells through in vitro experiments, and preliminarily explore its possible mechanism. At the same time, the effect of Anluo Huaxian Pills on liver cancer cells is evaluated using Compound Blister Capsules as a reference.

[0026] The experimental materials and reagents used include:

[0027] Experimental cell lines: Human liver cancer cell lines Hep G2, Hep 3B, and SK-Hep-1 were all from Wuhan Pronocell Life Science Co., Ltd., and human normal liver cells L-02 and human normal kidney epithelial cells 293T were from Haixing Biotechnology Co., Ltd.

[0028] (1) Anluo Chemical Fiber Pills are brown pills. Grind them into ultrafine powder with an agate mortar. Weigh 10 mg of Anluo Chemical Fiber Pills powder with an electronic balance and dilute to 40 mL with complete culture medium. The complete culture medium consists of 90% basal culture medium, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (double antibody). Dissolve them fully and prepare a 4 mg / mL stock solution for later use. Before intervening in cells, dilute them with complete culture medium to the corresponding concentration. All of the above are prepared before use.

[0029] (2) Compound Cantharides capsules, the capsules contain brown powder, grind into ultrafine powder with agate mortar, weigh 10 mg of compound Cantharides capsule powder with electronic balance, dilute to 40 mL with complete culture medium, prepare 4 mg / mL stock solution for standby use. Before intervening in cells, dilute it with complete culture medium to the corresponding concentration. All the above are prepared before use.

[0030] DMEM, MEM (containing NEAA) medium ( China), RPMI-1640 medium (Haixing Biotechnology, China), FBS ( China), double antibody ( China), sterile PBS ( China), ready-to-use serum-free non-programmed cryopreservation solution ( China), 0.25% Trypsin-EDTA ( USA).

[0031] CCK-8 kit ( China), 0.4% trypan blue staining solution ( China), crystal violet (Biyuntian Biotechnology Co., Ltd., China), Matrigel (Xiamen Moji Biotechnology Co., Ltd., China), ROS assay kit (Nanjing Jiancheng, China), DAPI solution ( China), AnnexinV-PE / 7-AADApoptosis Kit( China).

[0032] Cell culture and cell cryopreservation:

[0033] (1) Cell recovery and culture: Take out the frozen cell line from liquid nitrogen, thaw in a 37°C water bath, wipe the cryotube with alcohol cotton, and quickly put the cells into 5 mL of complete culture medium. Centrifuge at 1500 rpm for 5 min, discard the supernatant, add complete culture medium to resuspend, and place in a cell culture bottle. Culture in an incubator at 37°C, saturated humidity, and 5% CO2. When the cell confluence reaches about 80%, digest with trypsin containing EDTA and subculture.

[0034] (2) Cell passaging: Human liver cancer Hep G2, Hep 3B, and SK-Hep-1 cells were cultured in MEM medium containing 10% FBS and 1% double antibody. Human normal liver cells L-02 were cultured in RPMI-1640 medium containing 10% FBS and 1% double antibody. Human normal renal epithelial cells 293T were cultured in DMEM medium containing 10% FBS and 1% double antibody. The cells were placed in an incubator at 37°C, saturated humidity, and 5% CO2 for observation. When the cells covered about 80% of the bottom area of ​​the culture flask, they were passaged and cultured. The passaging ratio of Hep G2, Hep 3B, SK-Hep-1, L-02, and 293T cells was 1:2.

[0035] (3) Passaging steps: Aspirate the original culture medium, rinse twice with 2 mL of sterile PBS, and then add 2 mL of trypsin to digest the cells. The digestion time is about 3 minutes. Dynamically observe the changes in cell morphology under the microscope. When the cell edge shrinks to a circle, add 6 mL of complete culture medium to stop digestion. Centrifuge at 1500 rpm for 5 minutes and add 1 mL of complete culture medium to resuspend. Gently blow 1 mL of resuspended cell liquid to a single cell suspension, pass according to the proportion and continue culturing.

[0036] (4) Cryopreservation: Hep G2, Hep 3B, and SK-Hep-1 cells were cryopreserved in a cryopreservation solution composed of 55% MEM medium, 40% FBS, and 5% DMSO. 293T and L-02 cells were cryopreserved in a ready-to-use serum-free non-programmed cryopreservation solution.

[0037] The specific experiments are as follows:

[0038] 1. CCK-8 experiment

[0039] The CCK-8 assay was used to evaluate the effects of different concentrations of Anluo Huaxian Pills (0, 1, 1.5, 2, 2.5, 3, 4 mg / mL) and Compound Cantharidis Capsules (2 mg / mL) on the safety and growth activity of human liver cancer cell lines Hep G2, Hep 3B, SK-Hep-1, human normal liver cells L-02, and human normal renal epithelial cells 293T. The experiment was divided into a blank group (no cells or drugs were added, only 100 μL of basal culture medium containing 10 μL / well CCK-8 reagent was added), different concentrations of Anluo Huaxian Pills treatment groups, and Compound Cantharidis Capsules treatment groups (2 mg / mL).

[0040] (1) Take cells in the logarithmic growth phase and digest them according to the subculture steps. Count the cells using a cell counter and adjust the cell density to 1×10 4 / hole.

[0041] (2) The cell suspension was inoculated into a 96-well plate. 200 μL of cell suspension was added to each well of the experimental group and 200 μL of complete culture medium was added to each well of the blank group. An appropriate amount of complete culture medium was added around the plate to reduce the effect of water evaporation on the experiment. The plate was placed in an incubator at 37°C, saturated humidity, and 5% CO2 for 24 h.

[0042] (3) After the culture was completed, the original culture medium in the 96-well plate was discarded, and 200 μL of a solution containing different concentrations of Anluo Chemical Fiber Pills (0, 1, 1.5, 2, 2.5, 3, 4 mg / mL) was added. Six replicate wells were set for each drug concentration and placed in a 37°C, 5% CO2 incubator.

[0043] (4) Incubate for 12, 24 and 48 h.

[0044] (5) After incubation, discard the drug-containing culture medium in each well, add 100 μL of basal culture medium containing 10 μL / well CCK-8 reagent, and incubate in an incubator at 37°C, saturated humidity, and 5% CO2 for 1 h.

[0045] (6) The absorbance value was measured at 450 nm using an ELISA instrument, and the cell survival rate was calculated according to the formula: Cell survival rate (%) = [(absorbance value of the group treated with Anluo Chemical Fiber Pills - absorbance value of the blank group) / (absorbance value of the group treated with 0 mg / mL Anluo Chemical Fiber Pills - absorbance value of the blank group)] × 100%.

[0046] The CCK-8 method was used to detect whether Anluo Huaxian Pills had an inhibitory effect on the proliferation of human liver cancer cells. After treating three human liver cancer cells Hep G2, Hep3B and SK-Hep-1 with different concentrations of Anluo Huaxian Pills and Compound Cantharidis Capsule solutions, the cell survival rate was determined. In Hep G2 cells, after 12 hours of treatment with Anluo Huaxian Pills, the cell viability gradually decreased with the increase of the drug concentration. The cell viability was significantly inhibited at the concentrations of 1.5, 2, 2.5, 3, and 4 mg / mL (P<0.001), and the inhibitory effect of the 2 mg / mL Anluo Huaxian Pills treatment group was better than that of the Compound Cantharidis Capsule treatment group ( Figure 1 A); After 24 hours of treatment with Anluo Huaxian Wan, the cell viability gradually decreased with the increase of drug concentration, and the cell viability was significantly inhibited (P<0.001), and the inhibitory effect of the 2mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banyan Capsule treatment group ( Figure 1 B); After 48h of treatment with Anluo Huaxian Wan, the cell viability gradually decreased with the increase of drug concentration, and the cell viability was significantly inhibited (P<0.001), and the inhibitory effect of the 2mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banyan Capsule treatment group ( Figure 1 C in the figure). In Hep 3B cells, after 12h of treatment with Anluo Huaxian Wan, the cell viability gradually decreased with the increase of drug concentration. The cell viability was significantly inhibited at the concentrations of 1.5, 2, 2.5, 3, and 4 mg / mL (P<0.05), and the inhibitory effect of the 2 mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banbian Capsule treatment group ( Figure 2 A); After 24 hours of treatment with Anluo Huaxian Pills, the cell viability gradually decreased with the increase of drug concentration. The cell viability was significantly inhibited at 1.5, 2, 2.5, 3, and 4 mg / mL concentrations (P<0.001), and the inhibitory effect of the 2 mg / mL Anluo Huaxian Pills treatment group was better than that of the Fufang Banyan Capsule treatment group ( Figure 2 B); After 48h of treatment with Anluo Huaxian Wan, the cell viability gradually decreased with the increase of drug concentration, and the cell viability was significantly inhibited (P<0.01), and the inhibitory effect of the 2mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banyan Capsule treatment group ( Figure 2 C in the figure). In SK-Hep-1 cells, after 12 hours of treatment with Anluo Huaxian Wan, the cell viability gradually decreased with the increase of the drug concentration. The cell viability was significantly inhibited at the concentrations of 1.5, 2, 2.5, 3, and 4 mg / mL (P<0.05), and the inhibitory effect of the 2 mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banbian Capsule treatment group ( Figure 3A); After 24 hours of treatment with Anluo Huaxian Pills, the cell viability gradually decreased with the increase of drug concentration. The cell viability was significantly inhibited at 1.5, 2, 2.5, 3, and 4 mg / mL concentrations (P<0.001), and the inhibitory effect of the 2 mg / mL Anluo Huaxian Pills treatment group was better than that of the Fufang Banyan Capsule treatment group ( Figure 3 B); After 48h of treatment with Anluo Huaxian Wan, the cell viability gradually decreased with the increase of drug concentration, and the cell viability was significantly inhibited (P<0.01), and the inhibitory effect of the 2mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banyan Capsule treatment group ( Figure 3 C in Figure 1 D in Figure 2 D in Figure 3 D in the figure, Anluo Huaxian Pills inhibited the proliferation of three human liver cancer cells in a time- and dose-dependent manner. The calculation results show that the half-lethal concentration (IC50) of Hep G2, Hep 3B and SK-Hep-1 cells after 12 hours was 2.527 mg / mL, 3.194 mg / mL and 2.644 mg / mL, respectively, and the IC50 of Hep G2, Hep 3B and SK-Hep-1 after 24 hours was 1.860 mg / mL, 2.208 mg / mL and 2.122 mg / mL, respectively, and the IC50 of Hep G2, Hep 3B and SK-Hep-1 after 48 hours was 1.002 mg / mL, 1.837 mg / mL and 1.846 mg / mL, respectively. The sensitivity of the three human liver cancer cells to Anluo Chemical Fiber Pills are Hep G2, Hep 3B, and SK-Hep-1, respectively. Therefore, the subsequent study will focus on the effects of Anluo Chemical Fiber Pills on Hep G2 and Hep 3B cells.

[0047] In this experiment, the CCK-8 method was used to treat L-02 and 293T cells with different concentrations of Anluo Huaxian Pills and Compound Banhai Capsules for 12, 24 and 48 hours, and then the cell survival rate was determined. In L-02 cells, the cell survival rate was 80.31% after 48 hours of Compound Banhai Capsules treatment; the cell survival rate was 94.77% after 48 hours of 1mg / mL Anluo Huaxian Pills treatment; the cell survival rate was 89.35% after 48 hours of 1.5mg / mL Anluo Huaxian Pills treatment; the cell survival rate was 82.06% after 48 hours of 2mg / mL Anluo Huaxian Pills treatment; the cell survival rate was higher than 78.5% after 48 hours of 2.5mg / mL Anluo Huaxian Pills treatment. Figure 4). In 293T cells, after treatment with Compound Blister Capsule for 48 hours, the cell survival rate was 71.33%; after treatment with 1mg / mL Anluo Fiber Pills for 48 hours, the cell survival rate was 98.46%, and after treatment with 1.5mg / mL Anluo Fiber Pills for 48 hours, the cell survival rate was 84.79%; after treatment with 2mg / mL Anluo Fiber Pills for 48 hours, the cell survival rate was 76.29%, and after treatment with 2.5mg / mL Anluo Fiber Pills for 48 hours, the cell survival rate was higher than 45.91% ( Figure 5 ).

[0048] In summary, we finally selected Hep G2 and Hep 3B cells as the research objects, 24 and 48 h as the treatment duration, and 1 mg / mL, 1.5 mg / mL and 2 mg / mL as the treatment concentrations of Anluo Chemical Fiber Pills used in subsequent experiments.

[0049] 2. Cell growth counting

[0050] (1) Collect cells and count the cells at 1×10 per well. 4 Hep G2 and Hep 3B cells were inoculated into 6-well plates at a cell number of 10 cells / mL and incubated for 24 h to allow them to adhere.

[0051] (2) Hep G2 and Hep 3B cells were treated with solutions containing different concentrations of Anluo Huaxian Pills (0, 1, 1.5, and 2 mg / mL) and Fufang Banbian Capsules (2 mg / mL) and incubated for culture.

[0052] (3) Incubate for 24 and 48 h.

[0053] (4) After the incubation is completed, the cells are digested with trypsin and evenly perfused with sterile PBS. After trypan blue staining, the cells are counted using a cell counter. Three wells with different concentrations are set at each time point.

[0054] After treatment with different concentrations of Anluo Huaxian Pills and Fufang Banhai Capsules for 24 and 48 hours, the proliferation of Hep G2 cells was significantly inhibited by 1, 1.5 and 2 mg / mL Anluo Huaxian Pills compared with the 0 mg / mL Anluo Huaxian Pills treatment group at 48 hours (P<0.01), and the inhibitory effect of 1.5 mg / mL and 2 mg / mL Anluo Huaxian Pills treatment groups was better than that of Fufang Banhai Capsules treatment group ( Figure 6 A in the figure); In Hep 3B cells, the 1, 1.5, and 2 mg / mL Anluo Huaxian Wan treatment groups all significantly inhibited the proliferation of Hep 3B cells (P<0.05), and the effect of the 2 mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banbian Capsule group ( Figure 6 The above results show that Anluo Huaxian Pills can inhibit the proliferation of human liver cancer cells Hep G2 and Hep 3B.

[0055] 3. Plate colony formation experiment

[0056] (1) Collect cells and count the cells at 5×10 per well. 3 The cells were seeded in six-well plates and incubated for 24 h to allow them to adhere to the plate.

[0057] (2) Hep G2 and Hep 3B cells were treated with different concentrations of Anluo Huaxian Pills (0, 1, 1.5, 2 mg / mL) and Compound Cantharidin Capsules (2 mg / mL) and incubated.

[0058] (3) Culture the cells for 14 days, during which time the culture medium was replaced regularly and cell growth was observed under a microscope at any time.

[0059] (3) When most monoclones grew to >50 cells, discard the original culture medium, gently wash each well twice with sterile PBS, dry at room temperature, and fix with 4% paraformaldehyde for 20 min.

[0060] (4) After fixation, discard the 4% paraformaldehyde, wash once with sterile PBS, and stain with 0.1% crystal violet at room temperature for 30 min.

[0061] (5) After staining, wash twice with sterile PBS, dry, and take photos and count. Set up 3 wells for different concentration treatments.

[0062] After culturing Hep G2 and Hep 3B cells with different concentrations of Anluo Huaxian Pills and Fufang Banbian Capsules for 14 days, Figure 7 As shown in A, in Hep G2 cells, the number of cell clones in the 0 mg / mL Anluo Huaxian Wan treatment group was larger and the colonies formed were larger. Compared with the 0 mg / mL Anluo Huaxian Wan treatment group, the number of Hep G2 cell clones in the other concentrations of Anluo Huaxian Wan treatment groups was significantly reduced and the colonies formed were smaller, showing a certain dose-dependent change. Moreover, the inhibitory effect of the 2 mg / mL Anluo Huaxian Wan treatment group was better than that of the Compound Cantharidin Capsule treatment group. Figure 7 As shown in Figure B, in Hep 3B cells, the number of cell clones in the 0mg / mL Anluo Huaxian Wan treatment group was larger and the colonies formed were larger. Compared with the 0mg / mL Anluo Huaxian Wan treatment group, the number of Hep 3B cell clones in the other concentrations of Anluo Huaxian Wan treatment groups was significantly reduced, and the colonies formed were smaller, showing a certain dose-dependent change. The inhibitory effect of the 2mg / mL Anluo Huaxian Wan treatment group was better than that of the Fufang Banyan Capsule treatment group. The above results show that Anluo Huaxian Wan treatment can significantly inhibit the colony formation of Hep G2 and Hep 3B cells and inhibit their proliferation ability.

[0063] 4. Scratch test

[0064] (1) Collect cells and count the cells at 1×10 per well. 7 Hep G2 and Hep 3B cells were inoculated into 6-well plates at a concentration of 1 cell / mL and incubated for 24 h to allow them to adhere to the plate and grow.

[0065] (2) Use a 200 μL pipette tip to draw three vertical lines on the bottom of a 6-well plate, add sterile PBS and rinse slowly and carefully three times to wash away the cell debris that was drawn, so as to facilitate photography and observation. The cells were divided into groups according to the treatment conditions, and different concentrations of Anluo Huaxian Pills (0, 1, 1.5, 2 mg / mL) serum-free culture medium and Fufang Banbian Capsule (2 mg / mL) serum-free culture medium were added.

[0066] (3) Incubate for 24 and 48 h.

[0067] (4) After incubation is completed, take photos and observe.

[0068] (5) The scratch healing area was measured using Image J. Cell migration rate (wound healing rate) = (initial scratch area - scratch area at time t) / initial scratch area. Three wells were set for treatment with different concentrations at each time point.

[0069] After treating Hep G2 and Hep 3B cells with different concentrations of Anluo Huaxian Pills and Fufang Banbian Capsules for 24 and 48 h, the Figure 8 A in Fig. 9 As shown in A, in the two groups of cells, the scratches of the cells in the 48h 0mg / mL Anluo Chemical Fiber Pills treatment group were the narrowest, and the scratches could heal quickly, indicating that the cells migrated quickly; while the scratches of the cells in the other concentrations of Anluo Chemical Fiber Pills treatment group were wider, and the scratches could not be effectively healed. Moreover, the width of the scratches became wider as the concentration increased, indicating that the migration speed of Hep G2 and Hep 3B cells slowed down with the increase of Anluo Chemical Fiber Pills concentration. The cell migration rate was quantitatively analyzed by calculating the change in scratch area, and the results are shown in Figure 8 A in Figure 1 shows that compared with the 0 mg / mL Anluo Huaxian Wan treatment group, the 1, 1.5, and 2 mg / mL Anluo Huaxian Wan treatment groups significantly reduced the migration of Hep G2 cells (P<0.001), and the migration of the 2 mg / mL Anluo Huaxian Wan treatment group was significantly better than that of the Fufang Banbian Capsule treatment group. Fig. 9In B, in Hep 3B cells, compared with the 0mg / mL Anluo Huaxian Wan treatment group, the 1, 1.5, and 2mg / mL Anluo Huaxian Wan treatment groups also significantly reduced the migration of Hep 3B cells (P<0.001), and the migration of the 2mg / mL Anluo Huaxian Wan treatment group was significantly better than that of the Fufang Banyan Capsule treatment group. The above results show that Anluo Huaxian Wan can inhibit the growth of Hep G2 and Hep 3B by inhibiting the migration ability of Hep G2 and Hep 3B, and there is a certain dose dependence.

[0070] 5. Transwell experiment

[0071] (1) Take out the Matrigel gel stored at -80°C in advance, embed it on ice, and place it in a 4°C refrigerator overnight to allow it to fully melt.

[0072] (2) Pre-cool the Transwell chamber, pipette tips, centrifuge tubes, and EME-free basal culture medium at -20°C.

[0073] (3) Dilute Matrigel with serum-free basal medium and mix it evenly using a pre-cooled pipette tip. Be careful not to form bubbles during mixing. Take 80 μL of the diluted Matrigel and add it to the pre-cooled Transwell chamber to ensure that the bottom of the chamber is fully covered.

[0074] (4) Incubate in an incubator at 37°C, saturated humidity, and 5% CO2 for 3 h to allow the Matrigel to polymerize into a thin film.

[0075] (5) After incubation, the excess liquid in the upper chamber was discarded and 100 μL of EME basal medium was added to each well and incubated for 30 min to hydrate the basement membrane.

[0076] (6) Starve the cells for 24 h in advance, digest them with trypsin, resuspend them in EME basal medium, and count the cells.

[0077] (7) Add 200 μL of 1×10 5 500 μL of complete medium containing 10% FBS was added to the lower chamber and incubated for 4 h.

[0078] (8) After the incubation, the original culture medium was discarded, and Hep G2 and Hep 3B cells were treated with solutions containing different concentrations of Anluo Huaxian Pills (0, 1, 1.5, 2 mg / mL) and Fufang Banbian Capsules (2 mg / mL) and incubated.

[0079] (9) Incubate for 24 and 48 h.

[0080] (10) After the incubation is completed, discard the original culture medium, gently wipe off the cells that have not penetrated the upper layer, wash them with sterile PBS solution, pay attention to the intensity of washing, repeat twice, and air dry.

[0081] (11) After air drying, add 600 μL of 4% paraformaldehyde for fixation for 30 min.

[0082] (12) After fixation, the cells were washed three times with sterile PBS.

[0083] (13) After washing, 0.1% crystal violet stain was added and stained for 10 min.

[0084] (14) After staining, the cells were washed three times with sterile PBS for 2 min each time and air-dried.

[0085] (15) After air drying, observe the cells using a cell imaging device, randomly select 5 fields of view, take photos and record, and calculate the number of cells that have not invaded the lower chamber. Set 3 wells with different concentrations at each time point.

[0086] In the Transwell experiment, cells were treated with different concentrations of Anluo Huaxian Pills and Fufang Banbian Capsules for 24 and 48 hours. Fig.10 A in Fig.11 As shown in A, in the two groups of cells, the number of cells that passed through Matrigel in the 0 mg / mL Anluo Huaxian Wan treatment group at 48 h was large, while the number of cells that passed through Matrigel in the other concentrations of Anluo Huaxian Wan groups was small, and there was a certain dose dependence. Fig.10 In B, in Hep G2 cells, compared with the 0 mg / mL Anluo Huaxian Wan treatment group, the 1, 1.5, and 2 mg / mL Anluo Huaxian Wan treatment groups could significantly reduce the cell invasion of Hep G2 cells (P<0.001), and the effect of the 2 mg / mL Anluo Huaxian Wan treatment group was more obvious than that of the Youfufang Banbian Capsule treatment group. Fig.11 In B, in Hep 3B cells, compared with the 0mg / mL Anluo Huaxian Wan treatment group, the 1, 1.5, and 2mg / mL Anluo Huaxian Wan treatment groups can significantly reduce the cell invasion in Hep 3B cells (P<0.001), and the effect of the 2mg / mL Anluo Huaxian Wan treatment group is more obvious than that of the Fufang Banbian Capsule treatment group. The above results show that Anluo Huaxian Wan can inhibit the invasion of Hep G2 and Hep 3B.

[0087] 6. ROS Experiment

[0088] (1) Collect cells and count the cells at 1×10 per well. 6Hep G2 and Hep 3B cells were inoculated into 6-well plates at a concentration of 1 cell / mL and incubated for 24 hours to allow them to adhere to the plate and grow.

[0089] (2) Hep G2 and Hep 3B cells were treated with solutions containing different concentrations of Anluo Huaxian Pills (0, 1, 1.5, and 2 mg / mL) and Compound Cantharidin Capsules (2 mg / mL) and incubated for culture.

[0090] (3) Incubate for 24 and 48 h.

[0091] (4) Add DCFH-DA probe. Add 1 mL of diluted DCFH-DA probe to each well (DCFH-DA probe: serum-free culture medium = 1:1500), and incubate in an incubator at 37°C, saturated humidity, and 5% CO2 for 30 min.

[0092] (5) After incubation, the cells were washed twice with sterile PBS to fully remove the DCFH-DA probe that did not enter the cells.

[0093] (6) Place under a fluorescence microscope, with an excitation wavelength of 525 nm, observe the results and take photos. Set 3 wells with different concentrations at each time point.

[0094] After treating cells with different concentrations of Anluo Huaxian Pills and Fufang Banbian Capsules for 24 and 48 hours, the Fig.12 As shown in the figure, in Hep G2 cells, the green fluorescence intensity of the 0 mg / mL Anluo Huaxian Wan treatment group was weak. Compared with the 0 mg / mL Anluo Huaxian Wan treatment group, the green fluorescence of the other concentrations of Anluo Huaxian Wan treatment groups became stronger and stronger, showing a certain dose-dependency, indicating that Anluo Huaxian Wan can increase the content of ROS in Hep G2 cells after treatment, and the intracellular green fluorescence intensity of the 2 mg / mL Anluo Huaxian Wan treatment group was stronger than that of the Compound Cantharidin Capsule treatment group. In Hep 3B cells, as shown in the figure, Fig.13 As shown in the figure, the green fluorescence intensity of the 0mg / mL Anluo Huaxian Wan treatment group was weak. Compared with the 0mg / mL Anluo Huaxian Wan treatment group, the green fluorescence in Hep 3B cells increased with the increase of Anluo Huaxian Wan concentration, indicating that the intracellular ROS content increased, indicating that Anluo Huaxian Wan can increase the intracellular ROS content of Hep3B cells after treatment, and the intracellular green fluorescence intensity of the 2mg / mL Anluo Huaxian Wan treatment group was stronger than that of the Fufang Banyan Capsule treatment group. The results show that Anluo Huaxian Wan can promote the apoptosis of Hep G2 and Hep 3B cells by increasing the content of ROS in Hep G2 and Hep 3B cells.

[0095] 7. DAPI experiment

[0096] (1) Collect cells and count the cells at 1×10 per well. 6Hep G2 and Hep 3B cells were inoculated at 100 cells / mL in 6-well plates and incubated for 24 hours to allow them to adhere to the plate and grow.

[0097] (2) Hep G2 and Hep 3B cells were treated with solutions containing different concentrations of Anluo Huaxian Pills (0, 1, 1.5, and 2 mg / mL) and Compound Cantharidin Capsules (2 mg / mL) and incubated for culture.

[0098] (3) Incubate for 24 and 48 h.

[0099] (4) Add DAPI dye solution. Add a small amount of DAPI dye solution to each well plate to cover the sample.

[0100] (5) Stain at room temperature for 10 minutes.

[0101] (6) After staining, discard the DAPI stain in the 6-well plate and gently wash three times with sterile PBS, each time for 5 minutes.

[0102] (7) Place the sample under a fluorescence microscope with an excitation wavelength of 360 nm, observe the results and take photos. Set three treatment wells with different concentrations at each time point.

[0103] After treating cells with different concentrations of Anluo Huaxian Pills and Fufang Banbian Capsules for 24 and 48 hours, the Fig.14 As shown, in Hep G2 cells, compared with the 0 mg / mL Anluo Huaxian Wan treatment group, the cell nuclei in the 1, 1.5, and 2 mg / mL treatment groups were shrunken and fragmented, and the cytoplasm was concentrated and darkly stained, showing strong blue fluorescence. Compared with the Fufang Banbian Capsule treatment group, the medium blue color of the 2 mg / mL Anluo Huaxian Wan treatment group was stronger. Fig.15 As shown, in Hep 3B cells, compared with the 0mg / mL Anluo Huaxian Wan treatment group, the 1, 1.5, and 2mg / mL Anluo Huaxian Wan treatment groups can also shrink and fragment the Hep 3B nuclei, condense and darken the cytoplasm, and enhance the blue fluorescence staining. Compared with the Compound Blister Capsule treatment group, the 2mg / mL Anluo Huaxian Wan treatment group has a deeper staining. The results show that Anluo Huaxian Wan can promote the apoptosis of Hep G2 and Hep 3B cells.

[0104] 8. AnnexinV-PE / 7-AAD double staining experiment

[0105] (1) Collect cells and count the cells at 1×10 per well. 6 Hep G2 cells were inoculated into 6-well plates at a concentration of 100 cells / mL and incubated for 24 h to allow them to adhere to the plate and grow.

[0106] (2) Hep G2 cells were treated with solutions containing different concentrations of Anluo Huaxian Pills (0, 1, 1.5, 2 mg / mL) and Compound Cantharidin Capsules (2 mg / mL) and incubated for 24 h.

[0107] (3) After incubation, trypsin was used for digestion, and the cells were collected by centrifugation at 1500 rpm for 5 min and then washed once with pre-cooled sterile PBS.

[0108] (4) Add 100 μL of diluted 1× Annexin V Binding Buffer to resuspend the cells.

[0109] (5) Add 2.5 μL of Annexin V-PE and 2.5 μL of 7-ADD Reagent (final concentration 100 μg / μL)

[0110] (6) After gentle vortex mixing, incubate at room temperature in the dark for 20 min.

[0111] (7) After incubation in the dark, add 400 μL of diluted 1× Annexin V Binding Buffer and mix the sample.

[0112] (8) Flow cytometry detection and analysis. Set 3 wells with different concentrations at each time point.

[0113] AnnexinV-PE / 7-AAD flow cytometry was used to further explore the effect of Anluo Huaxian Pills on Hep G2 cells. Fig.16 In A, in the 0mg / mL Anluo Chemical Fiber Pills treatment group, the apoptosis rate was 5.8%, late apoptosis was 3.1%, and early apoptosis was 2.7%; at 1mg / ml, the apoptosis rate was 18.1%, late apoptosis was 5.5%, and early apoptosis was 12.6%; at 1.5mg / ml, the apoptosis rate was 55.6%, late apoptosis was 9.1%, and early apoptosis was 46.5%; at 2mg / ml, the apoptosis rate increased to 81.8%, late apoptosis was 12.6%, and early apoptosis was 69.2%. The apoptosis rate showed a certain dose-dependent change, and the early apoptosis rate of cells was higher than the late apoptosis rate. The results showed that after the Hep G2 cells were treated with Anluo Chemical Fiber Pills, apoptosis mainly occurred in the early stage, and a few occurred in the late stage. Fig.16 In B, in Hep G2 cells, compared with the 0 mg / mL Anluo Huaxian Wan treatment group, the 1, 1.5, and 2 mg / mL Anluo Huaxian Wan treatment groups all increased the intracellular apoptosis rate (P<0.001), and the cell apoptosis rate of the 1.5 and 2 mg / mL Anluo Huaxian Wan treatment groups was higher than that of the Fufang Banbian Capsule treatment group. Therefore, it shows that Anluo Huaxian Wan can induce apoptosis of liver cancer cells and most of them occur in the early stage of apoptosis.

[0114] In summary, Anluo Chemical Fiber Pills can reduce the cell survival rate of human liver cancer cells Hep G2, Hep 3B, and SK-Hep-1, and it is time- and dose-dependent; Anluo Chemical Fiber Pills can inhibit the proliferation, invasion and migration of Hep G2 and Hep 3B cells, and induce apoptosis of Hep G2 and Hep 3B cells, and most of them occur in the early stage of cell apoptosis; Anluo Chemical Fiber Pills can promote the apoptosis of Hep G2 and Hep 3B cells by increasing the content of ROS in Hep G2 and Hep 3B cells, and it is time- and dose-dependent, indicating that Anluo Chemical Fiber Pills can be used to prepare drugs that inhibit the proliferation of liver cancer cells and promote the apoptosis of liver cancer cells.

[0115] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Application of Anluo Chemical Fiber Pills in the preparation of drugs for inhibiting the proliferation of liver cancer cells and promoting the apoptosis of liver cancer cells.

Images