CN115252599A

CN115252599A - Application of licochalcone A and composition of glabridin and licochalcone A in preparation of medicine for treating colorectal cancer

Info

Publication number: CN115252599A
Application number: CN202210787843.5A
Authority: CN
Inventors: 李婷婷; 付雪艳; 牛阳; 高伟俊
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2022-11-01

Abstract

The invention provides application of licochalcone A and a composition of glabridin and licochalcone A in preparing a medicine for treating colorectal cancer. Belongs to the technical field of anti-tumor biological medicines. The CCK-8 method is used for verifying the inhibitory activity of LCA and CMC on human colon cancer cells, and the survival rate is obviously reduced. The influence of LCA and CMC high and medium dose groups on the apoptosis of human colon cancer cells is detected by adopting a flow cytometry technology, and the apoptosis rate is increased. The influence of LCA and CMC on the migration of human colon cancer cells is researched through a cell scratch experiment, and the cell migration capability can be obviously inhibited. Through Transwell experiments, the influence of LCA and CMC on the invasion capacity of human colon cancer cells is researched, and the number of the invaded cells is obviously reduced. LCA and CMC can inhibit the proliferation of human colon cancer cells in vitro and induce the apoptosis of tumor cells, and the action mechanism of the LCA and the CMC is probably related to the inhibition of the signal pathway of PI 3K/AKT/mTOR.

Description

Application of licochalcone A and composition of glabridin and licochalcone A in preparation of medicine for treating colorectal cancer

Technical Field

The invention belongs to the technical field of anti-tumor biological medicines, and particularly relates to application of licochalcone A and a composition of glabridin and licochalcone A in preparation of a medicine for treating colorectal cancer.

Background

Colorectal cancer is one of common malignant tumors in the digestive tract, the number of the attack is gradually increased, the main means of the combination of surgical operation, radiotherapy, chemotherapy and targeted preparations is adopted, and the problems of short life cycle and poor prognosis exist. The traditional Chinese medicine is used as an auxiliary mode for treating cancer, can improve the prognosis of patients and improve the quality of life of the patients, and is widely accepted clinically. Liquorice is a common traditional Chinese medicine for tonifying qi, and Licorice is found in ancient books of traditional Chinese medicine formulas for treating colon cancer in all ages to be used as a deficiency tonifying medicine in a large amount, and is frequently ranked fourth in all medicines to be used as a core medicine in a formula for treating colon cancer.

Although documents report that licorice crude extracts, flavone or saponin effective parts and monomer compounds have inhibitory effects on tumors such as breast cancer, prostate cancer, liver cancer, stomach cancer, bladder cancer, lung cancer and the like to different degrees, the action mechanism of the traditional Chinese medicine per se, namely multi-component, multi-channel and multi-target point, needs to be further researched, so that the basis and the action mechanism of the drug effect substances of the traditional Chinese medicine are disclosed by modern scientific and technical means, and a basis is further provided for classical clinical application in ancient prescriptions.

Licochalcone a (Licocalcone A, CAS number: 58749-22-7) is a natural phenolic compound, and is shown in formula I. It can be isolated from licorice root and shows in vitro antimalarial, anticancer, antibacterial and antiviral properties (in particular against influenza virus neuraminidase).

In the prior art, for example, a chinese patent with a patent number 201210403351.8 discloses an application of licochalcone a in preparing an anti-cancer drug or a health product, and specifically discloses that licochalcone a can significantly inhibit proliferation and colony formation of 4T1 mouse breast cancer cells, MDA-MB-231 human breast cancer cells, a375 human melanoma cells, AGs human gastric cancer cells, human pancreatic cancer PANC-1, human liver cancer cell strains HepG2, SMMC-7721 and human lung cancer cell strains H2126. However, on the one hand, the mechanism of action of licochalcone a in inhibiting cancer cell proliferation is not clear, and on the other hand, the action of licochalcone a on colon cancer is rarely reported.

Disclosure of Invention

Based on the above, the invention provides an application of licochalcone A in preparing a medicine for treating colorectal cancer.

The invention also provides application of the composition of glabridin and licochalcone A in preparing a medicine for treating colorectal cancer.

The technical scheme for solving the technical problems is as follows:

application of licochalcone A in preparing medicine for treating colorectal cancer is provided.

Preferably, licochalcone A is used as the only active ingredient for preparing the medicine for treating colorectal cancer.

Preferably, the action of licochalcone A leads the expression level of the protein of the cleaned-caspase 3, cleaned-caspase 9 and Bax to be obviously increased, and the expression level of the protein of Bcl-2 and MMP9 to be obviously reduced.

Preferably, in the prepared medicine for treating colorectal cancer, licochalcone A plays a role in regulating a PI3K/Akt/mTOR signaling pathway.

Preferably, licochalcone A is used in a concentration of 5 μ g/mL to 40 μ g/mL in the prepared medicament for treating colorectal cancer.

Application of a composition of glabridin and licochalcone A in preparing medicine for treating colorectal cancer is provided.

Preferably, the composition of glabridin and licochalcone A is used as the only active ingredient for preparing the medicine for treating colorectal cancer.

Preferably, the effect of the composition of glabridin and licochalcone A leads the expression level of the protein of Cleaved-caspase3, cleaved-caspase9 and Bax to be obviously increased, and the expression level of the protein of Bcl-2, MMP2 and MMP9 to be obviously reduced.

Preferably, in the prepared medicament for treating colorectal cancer, the combination of glabridin and licochalcone A plays a role in regulating and controlling PI3K/Akt/mTOR signaling pathways.

Preferably, in the prepared medicine for treating colorectal cancer, the concentration of the composition of glabridin and licochalcone A is 5 μ g/mL-40 μ g/mL.

Compared with the prior art, the invention has at least the following advantages:

the licochalcone A is used for preparing a medicine for treating colorectal cancer, and the inhibitory activity of licochalcone A (LCA) on human colon cancer cells SW480 and SW620 is verified by a CCK-8 method, which shows that the survival rate of each group is obviously reduced after the LCA of 5 mu g/mL, 20 mu g/mL and 40 mu g/mL acts on SW480 and SW620 cells 24h,48h and 72h. The influence of LCA high and medium dose groups (40 mu g/mL and 20 mu g/mL) on the apoptosis of SW480 and SW620 cells of human colon cancer is detected by adopting a flow cytometry technology, and the apoptosis rates of the LCA of 20 mu g/mL and 40 mu g/mL acting on the SW480 and SW620 have significant statistical difference and are increased (P is less than 0.001). The influence of LCA of 40 mu g/mL on the migration of human colon cancer cells SW480 and SW620 is researched through a cell scratch experiment, the migration rate of the LCA acting on the human colon cancer cells SW480 is (44.93 +/-0.49)%, and the LCA has no statistical significance compared with a control group; the SW620 acting on human colon cancer cells has the migration rate of (30.33 +/-0.91)%, has statistical significance (P < 0.001), and can obviously inhibit the cell migration capacity. Through Transwell experiments, the influence of 40 mu g/mL of LCA on the invasion capacity of SW480 and SW620 cells of human colon cancer is researched, the invasion number of the LCA group after being acted on the SW480 is (63.60 +/-8.91), and the LCA group has no statistical significance compared with a control group; the number of invasion after SW620 treatment was (53.6. + -. 6.47), with statistical differences (P < 0.01) and a significant reduction in the number of invading cells. Western blotting experiment is used for detecting the influence of dry prognosis of active ingredients of various groups of liquorice on the expression level of proteins related to metastasis and invasion of human colon cancer SW480 and SW620 cells. LCA medicament is used for dry prognosis, the average of Bax, cleaned-caspase 3 and cleaned-caspase 9 protein expression levels in SW480 cells is remarkably increased (P <0.05, P-woven fabric 0.001 and P-woven fabric 0.001), and the expression levels of Bcl-2, MMP2 and MMP9 proteins are remarkably reduced (P < 0.01); in SW620 cells, the Bax, clear-caspase 3 and clear-caspase 9 protein expression levels were significantly increased (P <0.05, P cover 0.001), and the Bcl-2 and MMP9 protein expression levels were significantly decreased (P < 0.05). LCA can inhibit the proliferation of SW480 and SW620 cells of human colon cancer in vitro and induce the apoptosis of tumor cells, and the action mechanism of the LCA is probably related to the inhibition of the signal pathway of PI 3K/AKT/mTOR.

The composition (CMC) of glabridin and licochalcone A is used for preparing the medicine for treating colorectal cancer, and the inhibitory activity of CMC on human colon cancer cells SW480 and SW620 is verified by a CCK-8 method, which shows that the survival rates of all groups are obviously reduced after CMC of 5 mug/mL, 20 mug/mL and 40 mug/mL acts on SW480 and SW620 cells 24h,48h and 72h. The flow cytometry technology is adopted to detect the influence of CMC high and medium dose groups (40 mug/mL and 20 mug/mL) on the apoptosis of SW480 and SW620 cells of human colon cancer, which shows that the 20 mug/mL and 40 mug/mL CMC has significant statistical difference on the apoptosis rate of SW480 and SW620 and the apoptosis rate is increased (P < 0.001). The influence of CMC (40 mu g/mL) on the migration of human colon cancer cells SW480 and SW620 is researched through a cell scratch experiment, the migration rate of CMC acting on the human colon cancer cells SW480 is (33.62 +/-1.86)%, the migration rate of CMC acting on the human colon cancer cells SW620 is (22.23 +/-0.77)%, and the statistical significance is realized (P < 0.001), and the cell migration capability can be obviously inhibited. Through Transwell experiments, the influence of CMC with the concentration of 40 mu g/mL on the invasion capacity of SW480 and SW620 cells of human colon cancer is researched, the number of the invasion of the CMC group after being acted on the SW480 is (37.80 +/-4.66), the number of the invasion of the CMC group after being acted on the SW620 is (32.80 +/-3.27), the statistical difference is generated (P < 0.01), and the number of the invaded cells is obviously reduced. The influence of the expression level of the proteins related to the metastasis and invasion of human colon cancer SW480 and SW620 cells after CMC intervention is detected through a Western blotting experiment. After CMC medicament is dried, the average of Bax, cleaned-caspase 3 and cleaned-caspase 9 protein expression levels in SW480 cells is obviously increased (P <0.05, P-woven fabric is 0.001), the Bcl-2, MMP2 and MMP9 protein expression levels are all obviously reduced (P < 0.01); bax, cleared-caspase 3 and cleared-caspase 9 protein expression levels were significantly increased in SW620 cells (P <0.05, P < -0.001) and Bcl-2, MMP2 and MMP9 protein expression was significantly decreased (P < 0.05). CMC can inhibit the proliferation of SW480 and SW620 cells of human colon cancer in vitro and induce the apoptosis of tumor cells, and the action mechanism of the CMC is probably related to the inhibition of the signal pathway of PI 3K/AKT/mTOR.

Drawings

FIG. 1 shows the effect of TFGR, LCA, GBN, CMC on the proliferation of SW480 cells measured by the CCK-8 method (A: LCA; B: TFGR; C: GBN; D: CMC; P <0.05,. P < 0.01; n:. On the control).

FIG. 2 shows the CCK-8 assay for the effect of TFGR, LCA, GBN, CMC on the proliferation of SW620 cells (A: LCA; B: TFGR; C: GBN; D: CMC; P < 0.05; P < 0.01; n =3:) compared to the control).

FIG. 3 shows the effect of the active components of licorice on the apoptosis of SW480 and SW620 cells of human colon cancer.

FIG. 4 shows the effect of various experimental groups of active ingredients of Glycyrrhiza on the morphology of human colon cancer SW480, SW620 nuclei (400 ×, shown by arrows, condensed chromatin, or fragmented nuclei).

Figure 5 is the effect of various experimental groups of licorice active ingredients on migration of human colon cancer SW480, SW620 cells (100 ×, p <0.05,. P <0.01. P <0.001, compared to control group).

FIG. 6 shows the effect of TFGR, LCA, CMC, GBN on the ability of human colon cancer SW480, SW620 cells to invade (x. + -. S, n = 3).

Figure 7 is the effect of TFGR, LCA, GBN on human colon cancer SW480 and SW620 cell-associated apoptosis factor protein expression (x ± s, n = 3).

Figure 8 is the effect of CMC on expression of human colon cancer SW480 and SW620 cell-associated apoptosis factor proteins (x ± s, n = 3).

Fig. 9 is a graph of the effect of TFGR, LCA, CMC, GBN on SW480 and SW620 cell-associated apoptosis factor gene expression (x ± s, n = 4).

Figure 10 is the effect of CMC on the expression levels of human colon cancer cells PI3K-Akt-mTOR pathway-associated proteins (. P <0.05,. P <0.01, vs. model group).

Detailed Description

It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict. The technical solutions of the present invention will be further described below with reference to the accompanying drawings of the embodiments of the present invention, and the present invention is not limited to the following specific embodiments.

1.1 Experimental materials

1.1.1 cell lines

Human colon cancer cell lines SW480 and SW620 cells were purchased from Shanghai Luyu Biotech Co. All cells were cultured in L15 medium of 10% Fetal Bovine Serum (FBS), and double antibody (100. Mu.g/mL streptomycin and 100 units/mL penicillin) was added to the medium and placed at 37 ℃ for 5% CO₂Culturing in an incubator. Cells were changed every two days, passaged at a ratio of 1:3, and cells in logarithmic growth phase were used in the experiment.

1.1.2 Experimental animals

SPF-grade BALB/c nude mice, 60 male, 3-4 weeks old, 18.0 + -2.0 g weight. Purchased from beijing huafukang biotech inc, certification No.: NO110322200101294471. Animal experiments were approved by the ethics committee of the animal experiments center of the university of Ningxia medical science.

1.1.3 Experimental drugs

TFGR (UVHPLC ≥ 98%, batch No. 20141015) was purchased from Shanghai-derived leaf Biotech, inc.;

GBN Standard (batch: G006171216) was purchased from Shanghai-derived leaf Biotech, inc.;

LCA standards (batch: P21O8F 46473) were purchased from Shanghai-derived leaf Biotechnology, inc.;

5-Fluorouracil (5-FU) Tianjin Jinyao pharmaceutical Co., ltd, production batch No. 2004201, specification 10 mL: 0.25g;

chloral hydrate, 10%, was purchased from the second hospital, lanzhou university.

1.1.4 Experimental reagents

Each experimental reagent is selected from the commercial experimental reagents meeting the standard requirements.

1.1.5 Instrument and Equipment

1.2 Experimental methods

1.2.1 establishment of human colon cancer cell SW480 tumor-bearing mouse model

The SPF-grade BALB/c nude mice are male, are 3-4 weeks old, have the weight of 18.0 +/-2.0 g, are subjected to adaptive feeding in an experimental animal center of Ningxia medical university after the quarantine is qualified, and are subjected to tumor transplantation and molding by SW480 and SW620 cell strains of the subsequent human colon cancer.

The SW480 and SW620 cell strains of the human colon cancer are cultured and passaged according to a conventional cell culture method. Collecting SW480 cells in good growth state in logarithmic growth phase, and adjusting the concentration of cell suspension to 2 × 10⁶each/mL, subpackaged in syringes, each syringe sucks 0.1mL of cell suspension for standby.

After the skin iodophor at the injection site of the nude mice is disinfected, the prepared cell suspension is injected into the subcutaneous part below the right axilla of the BALB/c nude mice. Conventionally feeding after inoculation, and grouping according to the completely random grouping principle according to the weight of nude mice when the diameter of the tumor nodules to be transplanted is about 4-5 mm: blank control group (0.05% sodium carboxymethylcellulose) 5 mL/kg^-1(ii) a Positive control group (5-FU) 20 mg/kg^-1(ii) a TFGR high, medium and low dose groups were 400mg kg^-1、200mg·kg^-1、100mg·kg^-1(ii) a The content of licochalcone A (LCA) in the high, middle and low dose groups is 40 mg/kg^-1、20mg·kg^-1、5mg·kg^-1(ii) a The high, middle and low dose groups of Glabridin (GBN) are respectively 40mg kg^-1、20mg·kg^-1、5mg·kg^-1(ii) a The composition (CMC) of licochalcone A and glabridin is prepared into high, medium and low dosage groups of 40mg kg^-1、20mg·kg^-1、5mg·kg^-1Wherein, in CMC, the ratio of the content of licochalcone A and glabridin is 1.

The administration mode comprises the following steps: positive control group is injected into abdominal cavity and administered every other day; the blank group and the TFGR, LCA, GBN and CMC high, medium and low dose groups are subjected to intragastric administration once a day at regular time, the dosage is calculated by taking the body weight as a standard each time, and the intragastric administration is continuously carried out for 14 days. After administration, the living behavior of nude mice and the growth of tumor were observed daily. Measuring the diameter of subcutaneous tumor by vernier caliper every 3-4 days, measuring the long axis (A) and short axis (B) of tumor, calculating the volume of transplanted tumor V = A × B²And/2, drawing a tumor volume curve. Groups of miceAfter anesthesia with 10% chloral hydrate on the day after the last administration, the neck is removed for sacrifice, transplanted tumor tissues are stripped and weighed, and the tumor inhibition rate of each group of mice is calculated as follows: tumor inhibition rate (%) = [ model group average tumor mass (g) — administration group average tumor mass (g)]Model group mean tumor mass (g). Times.100%. The materials are quickly obtained, the tissue freshness is ensured, and the tumor body part tissue is taken out and placed in 4% paraformaldehyde for fixation for 24h for use in subsequent pathology experiments. And preserving the remaining part of the tissue in liquid nitrogen for detecting the expression of related proteins and mRNA of the tumor tissue.

1.2.2 making Paraffin sections

And (2) trimming each fixed group of tumor tissue blocks, putting the tumor tissue blocks into an embedding box, marking the tumor tissue blocks on the embedding box, then putting the tumor tissue blocks into a full-automatic dehydrating machine for dehydrating and waxing, embedding the tumor tissue blocks by using an embedding machine after waxing is finished, slicing the tumor tissue blocks after the wax blocks are completely solidified, wherein the thickness of the slices is 5 mu m, spreading the slices, baking the slices, and storing the slices at 4 ℃.

1.2.3 Observation of HE staining pathology of transplanted tumor tissue of tumor-bearing mice

Performing HE dyeing on the slices, firstly sequentially dewaxing by using dimethylbenzene I and dimethylbenzene II for 10min respectively, dewaxing by using descending alcohol (sequentially absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol, 70% ethyl alcohol and water for 5min respectively), immersing the slices into hematoxylin dye liquor for dyeing for 5min, quickly washing to remove floating color, carrying out hydrochloric acid alcohol differentiation for several seconds, and washing with running water for 15min to turn blue; then immersing the slices into eosin dye liquor for dyeing for 5min, and quickly washing with water to remove floating color; dehydrating with ascending alcohol (sequentially standing 70% ethanol, 80% ethanol, and 90% ethanol for several seconds, respectively standing 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 5 min), soaking the dehydrated slices in xylene I and xylene II for 5min; taking out the slices from xylene, slightly drying the xylene (the slices are not dried), dripping neutral gum, covering a cover glass, and drying the sealed glass in a fume hood. And finally, placing the section on a microscope objective table for image acquisition and observation analysis.

1.2.4 detection of transplanted tumor tissue protein expression Using immunohistochemical method

Dewaxing the slices by using xylene I and xylene II respectively for 10min in sequence, dewaxing by using descending alcohol (absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol respectively for 5min in sequence), and then putting the slices subjected to xylene and gradient alcohol into water for hydration for several minutes; then placing the hydrated slices into a repair box filled with a proper amount of citric acid (pH6.0) repair liquid, placing the slices into a microwave oven (sealed by an adhesive tape to prevent the repair liquid from boiling over), stopping the fire for 8min, keeping the temperature for 7min, naturally cooling after repair, and washing with PBS for 5min (3 times); then putting into 3% hydrogen peroxide for incubation for 25min, washing with PBS for 5min (3 times); the reagent is prevented from flowing away by the aid of the combined pen stroke loop, 10% of normal goat serum is dripped to uniformly cover the tissue, and the tissue is sealed for 30min at room temperature; removing serum, adding primary antibody diluted with PBS in proportion, and placing in a refrigerator at 4 deg.C overnight; the next day, the slices are taken out of the refrigerator and rewarmed for 30min, washed with PBS for 5min (3 times), added with the diluted secondary antibody according to the proportion and incubated for 50min at room temperature, and washed with PBS for 5min (3 times); dropwise adding a DAB color developing solution which is freshly prepared to uniformly cover the tissue, controlling color development under a microscope, and flushing with pure water to stop color development; slicing, re-staining with hematoxylin for 2min, washing with tap water, differentiating with hydrochloric acid and ethanol for several seconds, and bluing with tap water; dehydrating with ethanol in ascending direction (sequentially standing 70% ethanol, 80% ethanol, and 90% ethanol for several seconds, and standing 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 5 min), allowing xylene to be transparent for 5min, sealing with neutral gum, and air drying in a fume hood. And finally, placing the section on a microscope objective table for image acquisition.

1.2.5 Western blotting was used to detect the expression level of transplanted tumor tissue-associated proteins

Cutting the tissue of each group of transplanted tumor into small pieces of about 3mm multiplied by 3mm, respectively placing the small pieces in different 2mL precooled centrifuge tubes, adding 0.5-1mL of prepared precooled lysate into each 100mg of tumor tissue, adding two small steel balls, placing the small pieces in a full-automatic freezing and grinding instrument, grinding for 90s at 60Hz, centrifuging the tissue after cracking at 12000rpm/min at 4 ℃ for 5min, and respectively transferring the supernatant into a new labeled group precooled centrifuge tube to obtain the whole protein extract of the tumor tissue of each treatment group. And (3) detecting the total protein content of the whole protein extracts of each group of tumor tissues by using a BCA protein quantitative kit. Adding the sample buffer solution into the tumor tissue whole protein extracts respectively, adding at a ratio of 4: 1, boiling for 5min to denature the protein, and packaging. And (3) putting the prepared electrophoresis experiment rubber plate into an electrophoresis tank, adding electrophoresis liquid, adding each histone sample, and carrying out constant voltage 120V electrophoresis separation. Transferring the target protein band on the gel to PVDF membrane (activated by methanol) under 200mA, washing 3 times with PBST, 10min each time; the PVDF membrane was then placed in an antibody incubation box, 5% skim milk powder (in PBST) was added and blocked on a shaker at room temperature for 1h, and then the strips were washed once with PBST. Antibody concentrations were prepared using a primary antibody dilution according to the following table. The prepared primary antibody was incubated on the corresponding strip and sealed overnight in a refrigerator at 4 ℃. The next day, primary antibody was recovered. The strips were washed three times with PBST for 10min each. According to the number of bands, a secondary antibody is prepared by using 5% skimmed milk powder according to the table five, and the secondary antibody is incubated for 1h at room temperature. PBST was washed three times for 10min each time after the incubation was completed. And hatching the luminous liquid on the strip, exposing by using a full-automatic chemiluminescence apparatus, and storing strip pictures.

1.2.6 detection of the expression level of mRNA associated with transplanted tumor tissue by q-PCR

(1) Total RNA extraction

Placing the tissue frozen in an ultra-low temperature refrigerator into an RNase-free EP tube with precooling, adding 300 mu L of lysate RL into every 10-20mg of tissue, grinding by using a freeze grinder, and centrifuging for 5min at 12000rpm (about 13400 Xg); taking the supernatant, slowly adding absolute ethyl alcohol with the volume 0.5 times that of the supernatant, and uniformly mixing; transferring the obtained solution and the precipitate into an adsorption column CR3 (the adsorption column is placed in a collecting tube), centrifuging at 12000rpm (about 13400 Xg) for 30s, discarding the waste liquid in the collecting tube, and placing the adsorption column back into the collecting tube; adding 500 μ L deproteinized solution RD, centrifuging at 12000rpm (-13400 Xg) for 30s, removing waste liquid and putting the adsorption column back into the collection tube; adding 500 μ L of cleaning solution RW into the center of the adsorption column CR3, standing for 2min, centrifuging at 4 deg.C and 12000rpm (-13400 Xg) for 30s, discarding the waste liquid, and repeating the steps once; putting the adsorption column back into the collection tube; adding 500 mu L of rinsing liquid RW (containing ethanol) into an adsorption column CR3, standing at room temperature for 2min, centrifuging at 12000rpm (-13400 Xg) for 1min, discarding the waste liquid, putting the adsorption column back into a collection tube, repeatedly adding the rinsing liquid for rinsing once, centrifuging at 12000rpm (-13400 Xg) for 2min, discarding the waste liquid, putting the adsorption column at room temperature for a plurality of minutes, airing the residual rinsing liquid in the material, transferring the adsorption column CR3 into a new RNase-Free EP tube, suspending and dripping 100 mu L of RNase-Free ddH2O into the middle part of an adsorption film, standing at room temperature for 2min, standing at 00120rpm (-13400 Xg) for 2min, obtaining a total RNA extracting solution, and carrying out purity determination.

(2) Reverse transcription to synthesize cDNA

Taking an EP tube filled with a total RNA extracting solution (< 5 mu g), placing the tube on ice, sequentially adding 1 mu L of Oligo (dT) into the tube, adding nucleic-free water into the tube to make the total volume of 12 mu L, performing short centrifugation, placing the tube in a PCR instrument to incubate for 5min at 65 ℃, placing a PCR product on ice to cool after reaction, performing short centrifugation, and adding the following components into an enzyme-free PCR tube according to the specified sequence: 5 × Reaction Buffer (4 μ L), riblock RNase Inhibitor (20U/. Mu.L) (1 μ L), 10mm dNTP Mix (2 μ L), revertAId M-MuLV RT (200U/. Mu.L) (1 μ L). The total volume of the reaction system is 20 mu L, the mixture is gently mixed, the mixture is placed into a PCR instrument for cDNA synthesis after short-time centrifugation, the conditions are that the reaction is incubated for 60min at 42 ℃ and 5min at 70 ℃, and the reaction is stopped. The reaction product is placed in an ice box and is centrifuged for a short time, and the reverse transcribed cDNA can be directly tested or stored in a refrigerator at the temperature of minus 80 ℃.

(3) RT-qPCR (reverse transcription-quantitative polymerase chain reaction) for detecting mRNA (messenger ribonucleic acid) expression quantity related to transplanted tumor tissue

The reference gene is selected as beta-actin, and the mRNA primers of the target gene are shown in the following table:

each set of RT-qPCR primer sequences

Separately adding 90-100 μ L of RNase-Free ddH to each treatment group of reverse-transcribed cDNA samples₂And O, diluting the sample to be moderate in concentration, placing each group of samples in a special eight-connection pipe, and configuring a reaction system according to the following table: aceQ Universal SYBR qPCR Master Mix (10. Mu.L), ROX Passive Reference Dye (50X) (0.5. Mu.L), each cDNA sample (2. Mu.L), PCR Forward Primer 0.4. Mu.L (0.5. Mu.L), PCR Reverse Primer 0.4. Mu.L (0.5. Mu.L), RNase-FreedH₂O(0.5μL)。

The reaction system is put into an ABI stepone plus real-time fluorescence quantitative PCR instrument, and the reaction conditions are set as follows:

after the reaction is finished, the relative expression level of the target mRNA is obtained, and the expression level is determined by 2^-△△CtThe method calculates the expression condition of the target gene relative to the internal reference to obtain the relative quantitative detection of the gene to be measured, and the expression quantity is expressed by multiples.

1.2.7 cell culture

(1) Cell resuscitation

The cell strain frozen in the liquid nitrogen is taken out and quickly thawed, then transferred to a sterile 10mL centrifuge tube prepared in advance by a pipette gun, and put into a centrifuge for centrifugation (1000rpm, 5 min), and the supernatant is discarded after the centrifugation is finished. Then adding 1mL of cell culture medium containing 10% fetal calf serum, repeatedly blowing until the cell culture medium becomes single cell suspension, transferring the single cell suspension into a cell culture bottle, adding 5-6mL of complete culture medium into the cell culture bottle, placing the cell culture bottle in a 5% carbon dioxide incubator, and adjusting the temperature of the incubator to 37 ℃ for cell culture.

(2) Cell passage

And passage can be realized when the cell density is observed to be multiplied by about 85% under a mirror. The spent medium in the cell culture flask was discarded, rinsed with 3mL PBS and repeated 3 times. Then adding 1mL of 0.25% trypsin digestion solution into the bottle, slightly shaking to enable the digestion solution to be uniformly distributed in the bottle, placing the culture bottle into an incubator for several minutes, taking out the culture bottle, observing under a rear mirror until cells become round, immediately adding 3mL of complete culture medium to stop digestion, slightly blowing the bottle wall by using a liquid transfer gun until most adherent cells basically fall off, transferring the cell suspension to 15mL of a centrifuge tube, centrifuging at 1000rpm for 5min, and discarding the supernatant. Taking 1mL of complete culture medium to resuspend cells, blowing and beating the cells into single cell suspension, counting, dividing the cell suspension into 2-3 culture bottles according to the specific conditions of an experiment, respectively adding 5-7mL of complete culture medium into each bottle, and culturing in a 5% carbon dioxide incubator at 37 ℃.

(3) Cell cryopreservation

Taking cells with the density of 60-70% in logarithmic phase, digesting and blowing the cells into single cell suspension by using 0.25% trypsin digestive juice according to the conditions in cell passage, transferring the single cell suspension into a 10mL centrifuge tube, centrifuging at 1000rpm for 5min, removing a culture medium, adding 1mL of serum-free cryopreservation solution, uniformly blowing, transferring into a cell cryopreservation tube again, and marking. The freezing tube is placed in a cell freezing box and placed in a refrigerator at the temperature of minus 80 ℃, and the freezing tube is transferred to a liquid nitrogen tank after one night and can be stored for a long time.

1.2.8 preparation of drug stock solution

TFGR was weighed to 100mg/mL and diluted by fold to 50, 25, 12.5mg/mL. LCA and GBN are precisely weighed and respectively dissolved in DMSO solution to prepare mother solution, and the mother solution is filtered by a 0.22 mu m microporous filter membrane and is placed at 4 ℃ for later use. Each group of drugs was diluted with complete medium to final concentrations of 100, 50, 25, 12.5. Mu.g/ml before each cell administration.

Drug configuration for subsequent experiments: LCA, GBN and CMC are prepared according to the above method, and the final administration concentrations are 40, 20, 10 and 5 mu g/mL respectively, and the final administration concentrations of TFGR extract are 100, 75, 50 and 25 mu g/mL.

1.2.9 flow cytometry detection of Effect of TFGR, LCA, GBN, CMC on apoptosis of human Colon cancer cells

Cells were seeded in 6-well plates and after overnight culture cells were dosed. Cells were treated with different concentrations of LCA (20. Mu.g/mL, 40. Mu.g/mL), GBN (20. Mu.g/mL, 40. Mu.g/mL), CMC (20. Mu.g/mL, 40. Mu.g/mL), total flavonoids (75. Mu.g/mL, 100. Mu.g/mL), cultured in a cell incubator at 37 ℃ and saturated humidity for 24 hours, collected by digestion with EDTA-free trypsin, centrifuged at 2000rpm for 5 minutes at room temperature, washed twice with cold PBS, and suspended in 400. Mu.L of 1 × Binding Buffer at a concentration of about 1 × 10⁶Per mL; adding 5 μ L Annexin V-FITC into the cell suspension, mixing, incubating at 2-8 deg.C in the dark for 15min, adding 10 μ L PI, mixing, and incubating at 2-8 deg.C in the dark for 5min; detecting on a flow cytometer in 1h by a machine, and analyzing the total apoptosis rate.

1.2.10hoechst 33258 fluorescent staining method for observing influence of TFGR, LCA, GBN and CMC on cell nucleus morphology of human colon cancer

Soaking common clean cover glass in 70% ethanol for 5min or longer, washing with cell culture medium PBS for 3 times, and washing with cell culture solution for 1 time. Coverslips were placed in 6-well plates and seeded into cells for overnight culture at a cell density of about 50% -80%. Each group of drug (40. Mu.g/mL LCA, 40. Mu.g/mL GBN, 40. Mu.g/mL CMC, 100. Mu.g/mL TFGR) was administered to treat the cells, the culture was aspirated, 0.5mL of fixative was added, and fixation was performed for 10min or overnight at 4 ℃. Removing the fixing solution, washing with PBS for 2 times, 3min each time, discarding the liquid, adding 0.5mL Hoechst33258 staining solution, staining for 5min, and fully contacting by manual shaking. Wash 2 times with PBS for 3min each time, then block with anti-quench blocking solution. The staining was observed with a fluorescence microscope and images were collected.

1.2.11 scratch test to examine the influence of TFGR, LCA, GBN and CMC on the migration ability of human colon cancer cells

Selecting cells in logarithmic growth phase, culturing each group of cells to 80% density according to 1 × 10⁵The cells are plated in a/well culture box, incubated, observed after adherence and marked on the back of the plate, scratched with a 10 μ L sterile tip perpendicular to the six-well plate at the marked position, the original medium is discarded, and the cells are treated by adding each group of drug-containing medium (40 μ g/mL LCA, 40 μ g/mL GBN, 40 μ g/mL CMC, 100 μ g/mL TFGR). And (3) placing each group of cells in an incubator at 37 ℃ for culturing for 0h and 24h respectively, then taking a picture for recording, determining the healing distance of the scratch, and repeating each group of experiments for 3 times.

1.2.12Transwell experiment for detecting influence of TFGR, LCA, GBN and CMC on human colon cancer cell invasion capacity

Firstly, the Matrigel gel was thawed overnight at 4 ℃, the Matrigel gel was placed on ice on a clean bench, the gel 1.

Inoculating 60mm of logarithmic growth phase cells³In the petri dish, the original culture medium was aspirated when the density reached 80%, and rinsed with PBSTwice, trypsinized, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cell suspension was prepared by adding serum-free medium to the tube and counted, 200. Mu.L of the cell suspension was aspirated and added to the upper chamber of the transwell chamber, and the number of cells was 3X 10⁴The cells were placed in 24-well plates, the medium was aspirated after adherence, the FBS-free drug-containing medium was added to the upper chamber (40. Mu.g/mL LCA, 40. Mu.g/mL GBN, 40. Mu.g/mL CMC, 100. Mu.g/mL TFGR) for treatment of the cells for 24h, and 20. Mu.g/mL FBS medium was added to the lower chamber 500. Mu.L per well, and the cells were cultured in a cell culture incubator. The medium was aspirated off, the Transwell chamber was washed 2 times with PBS, 4% paraformaldehyde was fixed at room temperature for 30min (200. Mu.L in the upper chamber and 500. Mu.L in the lower chamber), the fixative was discarded, PBS was washed 2 times, 0.1% crystal violet stain was added for staining for 15min, the staining solution was discarded, and PBS was washed 2 times. Counting the cells invading to the lower layer of the microporous membrane under a microscope, selecting 5 fields for each sample to count the number of the cells, and processing the data.

1.2.1 Western blotting detection of influence of TFGR, LCA, GBN and CMC on expression of human colon cancer cell apoptosis-related proteins

After the adherent tumor cells absorb the culture medium, cold PBS of a 10mL/150mm culture plate is respectively added for washing twice, and the culture solution is removed as much as possible by shaking for a plurality of times each time; scraping adherent cells by using a cell scraper, transferring the cells and the culture solution into a centrifuge tube, centrifuging for 10min at 800 Xg, discarding supernatant, and centrifuging for 5min at 800 Xg twice by using cold PBS of 10mL/150mm culture plate; adding 10 mu L of phosphatase inhibitor, 1 mu L of protease inhibitor and 5 mu L of 100mM PMSF into each 1mL of cold Lysis Buffer, mixing uniformly, and storing on ice for a plurality of minutes for later use; after the cells are washed, transferring the cells to a new precooled centrifuge tube, and adding 1mL of the prepared cold lysine Buffer; placing on a table of a shaking table at 4 deg.C, shaking vigorously for 30s, placing on ice for 4min, and repeating for 5 times; then, the mixture was centrifuged at 12000rpm at 4 ℃ for 5min to obtain the supernatant as a whole protein extract. And (3) detecting the total protein content of the whole protein extracts of each group of tumor tissues by using a BCA protein quantitative kit. Each treatment histone was tested according to 1.2.5 method.

1.2.14qRT-PCR detection of influence of TFGR, LCA, GBN and CMC on human colon cancer cell apoptosis-related mRNA level

Adherent cells aspirate medium, wash cells with PBS, aspirate PBS, digest cells normally, transfer cell solution to RNase-Free centrifuge tube, centrifuge at 300 × g for 5min, collect cell pellet, and remove supernatant. mu.L of lysate RL was added to the tube and all solutions were transferred to the filtration column CS and centrifuged at 12000rpm (-13400 Xg) for 2min to collect the filtrate. Adding 350 mu L of 70% ethanol into the filtrate, uniformly mixing, transferring the obtained solution and the precipitate into CR3, centrifuging for 1min at 12000rpm (about 13400 Xg), pouring off the waste liquid in the collecting tube, putting the adsorption column CR3 back into the collecting tube, adding 350 mu L of deproteinized liquid RW1 into the adsorption column, centrifuging for 1min at 12000rpm (about 13400 Xg), discarding the waste liquid, and putting the adsorption column back into the collecting tube; adding 80 μ L DNase I working solution (adding 70 μ L RDD solution into 10 μ L DNase I storage solution, mixing well) into the center of an adsorption column CR3, standing at room temperature for 15min, adding 350 μ L deproteinizing solution RW1 into the adsorption column, centrifuging at 12000rpm (-13400 Xg) for 1min, discarding waste liquid, and placing the adsorption column back into a collection tube; adding 500 μ L of rinsing solution RW (containing ethanol) into an adsorption column CR3, standing at room temperature for 2min, centrifuging at 12000rpm (-13400 Xg) for 1min, putting the adsorption column back into a collection tube, repeatedly adding the rinsing solution for rinsing once, centrifuging at 12000rpm (-13400 Xg) for 2min, discarding waste liquid, putting the adsorption column at room temperature for several minutes, airing, transferring the adsorption column CR3 into a new RNase-Free EP tube, dropwise adding 100 μ L of RNase-Free ddH2O in the middle of an adsorption membrane in a hanging manner, standing at room temperature for 2min, centrifuging at 12000rpm (-13400 Xg) for 2min to obtain a cell total RNA extracting solution, and performing purity determination. Reverse transcription of cDNA and PCR experiments are referred to 1.2.6 methods.

1.2.15 reverse virtual screening technique was used to investigate potential targets and signaling pathways of GBN and LCA against colon cancer

Completely analyzing a target crystal structure corresponding to a disease gene by using a whole target library of a reverse virtual screening platform, wherein the whole target library comprises 2119 active sites of 140 targets; the part target library, the corresponding target structure part of the disease gene was resolved, and the total includes 5487 active sites of 506 targets. And searching and obtaining structural formula files of LCA and GBN by using a DrugBank database, submitting to a reverse virtual platform for screening, carrying out molecular virtual docking, scoring the binding energy of the compound and the protein, and returning to a result of 200 before scoring. A larger absolute value of the score indicates more stable binding of the drug to the target. And (5) comparing the results with ProteinBank and KEGG databases to perform target prediction and pathway analysis.

1.2.16LCA, GBN and CMC in vitro colon cancer resistant proteomics research

The experiment was divided into 8 groups, 4 of which were human colon cancer SW480 cells and 4 of which were human colon cancer SW620 cells. SW480 cells were processed in the manner of blank control, 40. Mu.g/mL LCA, 40. Mu.g/mL GBN, 40. Mu.g/mL CMC, and 100. Mu.g/mL TFGR, and each drug group was processed for 24h. SW620 cells were treated in the same manner as SW480 cells. Cell culture methods are described in 1.2.7. Protein extraction and quantification were as described above.

Enzymolysis and marking:

reductive alkylation and enzymatic hydrolysis: a100. Mu.g protein sample was taken and the volume was made up to 90. Mu.L with lysis buffer. Adding TCEP reducing agent with final concentration of 10 mmol/L, and reacting at 37 deg.C for 60min. Adding iodoacetamide with final concentration of 40mmol/L, and reacting at room temperature in dark for 40min. Precooled acetone (acetone: sample volume ratio = 6) was added to each tube, and the pellet was centrifuged at 20 ℃ for 4h and 10000 Xg for 20 min. The sample was dissolved well with 50mmol/L TEAB and Trypsin was added at a mass ratio of 1. TMT labeling and mixing samples TMT reagent (Thermofisiher) was removed at-20 ℃ and returned to room temperature, acetonitrile was added, vortex centrifuged and a tube of TMT reagent was added per 100. Mu.g of polypeptide and the sample labels are given in the table below. Incubating for 2h at room temperature; hydroxylamine was added and the reaction was carried out at room temperature for 15min, and the same amount of labeled product was mixed in one tube and dried by vacuum concentration.

Sequence listing of samples from each group

RPLC one-dimensional separation: the polypeptide samples were reconstituted with UPLC loading buffer and subjected to high pH liquid phase separation using a reverse phase C18 column acquisition UPLC BEH C18 column1.7 μm,2.1mm × 150mm (Waters, USA). Phase A of 2% acetonitrile (adjusted to pH10 with ammonia water), phase B of 80% acetonitrile (adjusted to pH10 with ammonia water), 0-1.9 min,100% A; 2-17min, 0-5 percent of C; 17-18 min,5% -10% B;18 to 35.5min,10 to 30 percent of B;35.5 to 38min,30 to 36 percent of B; 38-39min, 36% -42%; 39-40min, 42-100%; 40 to 44min,100 percent of B; 44-45min, 100% -0%, see the following table. The ultraviolet detection wavelength is 214nm, the volume flow is 200 mu L/min, and the elution time is 48min. According to the peak shape and time, 20 fractions are collected and combined into 10 fractions, and the 10 fractions are concentrated by vacuum centrifugation.

UPLC loading elution gradient

And (3) identifying the protein by liquid chromatography tandem mass spectrometry: the second dimension was analyzed using nanoliter liquid chromatography tandem mass spectrometry (Easy-nLC 1200 in combination with Q _ active HF-X mass spectrometer). The peptide fragments were dissolved in mass spectrometric loading buffer and separated by C18 chromatography (75 μm × 25cm, thermo, USA) for 120min at a volume flow of 300 μ L/min. EASY-nLC liquid phase gradient elution, phase A2% acetonitrile (plus 0.1% formic acid), phase B80% acetonitrile (plus 0.1% formic acid) gradient as shown in the following table. The MS and MS/MS acquisitions were switched automatically, with mass spectral resolutions of 70K and 35K, respectively. MS full sweep (m/z 350-1500) acquisition mode: DDA, cycle time:2s; selecting a parent ion top20 for secondary fragmentation, wherein the resolution of a primary mass spectrum is as follows: 120000. TurboTMT (smart acquisition improves resolution of reporter ion isotopes); maximum injection time: 20ms, fragmentation mode: HCD; secondary resolution: 45000, maximum implant time: 86ms, fixed first mass:110m/z; minimum AGC target: 8e3, intense threshold:8.3e4 dynamically excludes time 40s.

EASY-nLC liquid phase gradient

Searching a library by using protein:

the original file of the mass spectrometer is analyzed by a ProteDiscoverTM Software 2.4, and a database adopted for searching a database is NCBInr. The False Discovery Rate (FDR) of peptide Fragment identification in the library searching process is set to be that the FDR is less than or equal to 0.01, the Mass tolerance (Precursor Mass tolerance) of the peptide Fragment is set to be 20ppm, the Mass tolerance (Fragment Mass tolerance) of the ion Fragment is set to be 0.02Da, and at least one specific peptide Fragment is contained. The protein co-detected in this item was calculated for the significance P value of the difference between samples using the t.test function in the R language, and for the Fold difference between groups (FC). Significance test P is less than 0.05, and the protein with the difference multiple more than 1.2 times is the protein with difference expression.

Bioinformatics analysis: GO (gene ontology, http:// gene ontology. Org /) is selected to carry out GO annotation analysis functional annotation analysis on all differential proteins from three aspects of biological processes, cell components and molecular functions; the KEGG (Kyoto encyclopedia of genes and genes, http:// www. Genes. Jp/KEGG//) pathway database was used to analyze the metabolic pathways and signaling pathways involved in differential proteins.

1.2.17 statistical methods

Data processing was performed using SPSS 26.0 statistical software. The data obtained from the experiment and the data obtained from the measurement are all expressed as (mean. + -. Standard deviation). The mean comparison between the two sets of samples was performed using the t test (if σ)₁ ²≠σ₂ ²Then the t' test is used), and the mean-average comparison between multiple groups of samples is performed by One-way ANOVA test (if σ. Is₁ ²≠σ₂ ²Dunnett-t test) with P <0.05 as significant.

1.3 results of the experiment

1.3.1 verification of inhibition of TFGR, LCA, CMC and GBN on proliferation of SW480 and SW620 cells of human colon carcinoma by CCK-8 cytotoxicity assay

The experimental results are shown in fig. 1 and fig. 2. The inhibitory activity of TFGR, LCA, GBN and CMC on human colon cancer cells SW480 and SW620 was verified by the CCK-8 method. The effect of the drug groups with different concentrations on the proliferation of the tumor cells of the two cells in different time periods is measured, and the inhibition results are shown in fig. 1 and fig. 2. Compared with a control group, each dosage group of the licorice total flavonoids has statistical significance (P is less than 0.01), can inhibit the proliferation of SW480 cells and SW620 cells to different degrees, has certain dosage dependence and time dependence as the time is prolonged and the inhibition effect is gradually enhanced along with the increase of the dosage, and selects each high dosage group of a 24h treatment group for subsequent experiments because the 24h treatment group has obvious inhibition effect on the proliferation. The proliferation inhibition rates of the four drug groups at different doses for 24h are different, and the inhibition rate of 20 mu g/mL LCA is (91.16 +/-0.69)%, when the four drug groups act on SW480 cells as shown in figure 1; the inhibition rate of 20 mu g/mL GBN (34.85 +/-10.04)%; the inhibition rate of 20 mu g/mL CMC is (79.05 +/-3.50)%; 50. the inhibition rate of the mu g/mL TFGR (81.76 +/-1.46)%, wherein the inhibition rate of the monomeric compound LCA at the same level concentration is higher than that of the GBN, and the difference compared with a control group has statistical significance (p <0.05, p < -0.01). The inhibition rate of 20. Mu.g/mL LCA when acting on SW620 cells was (84.62. + -. 0.87)%; inhibition of 20. Mu.g/mL GBN (92.34. + -. 0.24)%; the inhibition rate of 20 mu g/mL CMC is (93.87 +/-0.54)%; the inhibition rate of 50 μ g/mL TFGR (81.74. + -. 1.76)% (see FIG. 2), and both LCA and GBN were inhibited at the same concentration. Compared with SW480 cells, SW620 cells have stronger sensitivity to drugs LCA, GBN and CMC and higher cell proliferation inhibition rate, and the SW620 cells are obviously inhibited in proliferation and the SW480 cells are lower in inhibition rate than SW620 under the action of the drugs with the same concentration. The subsequent action mechanism and proteomics experiment adopt 20 mu g/mL and 40 mu g/mL of monomer compounds and compatible groups and 75 mu g/mL and 100 mu g/mL of licorice total flavonoids groups for carrying out apoptosis experiment, and the action time is 24h.

1.3.2TFGR, LCA, CMC and GBN Effect on apoptosis of human Colon cancer cells

The flow cytometry technology is adopted to detect LCA, GB) and CMC high and medium dose groups (40 mu g-mL)^-1And 20. Mu.g.mL^-1) High and Medium dose groups of TFGR (100. Mu.g.mL)^-1And 75. Mu.g.mL^-1) The effect on apoptosis of human colon cancer cells SW480, SW620 is shown in FIG. 3. The apoptosis rate of SW480 cells in the control group is only (1.13 +/-0.11)%, and the apoptosis rate of SW620 cells in the control group is only (1.79 +/-0.15)%.

20μg·mL^-1Apoptosis rate of LCA on SW480 onlyIs (2.39+0.17)%, and has no significant difference compared with a SW480 cell of a control group; the apoptosis rate of the SW620 cells is (4.56 +/-0.26)%, and compared with the SW620 cells in a control group, the apoptosis rate is increased (p)<0.01)。40μg·mL^-1The apoptosis rate of LCA acting on SW480 is only (5.59 +/-0.66)%, the apoptosis rate acting on SW620 cell is (11.56 +/-1.44)%, and the LCA has obvious statistical difference (p) compared with the apoptosis rate of each control group cell<0.001 And the apoptosis rate of the cells is increased in different degrees after the medicine is taken. 20. Mu.g/mL^-1The apoptosis rate of GBN on SW480 is only (7.77 +/-0.28)%, and compared with SW480 cells in a control group, the GBN has significant statistical significance, and the apoptosis rate is increased (p)<0.01 ); the apoptosis rate of the SW620 cells is (6.35 +/-0.53)%, and compared with the SW620 cells in a control group, the apoptosis rate has no statistical significance. 40. Mu.g/mL^-1The apoptosis rate of GBN on SW480 is only (16.35 +/-0.88)%, the apoptosis rate on SW620 is (16.38 +/-1.17)%, and the difference is significant and statistically different (p) compared with the apoptosis rate of each control group<0.001 And the apoptosis rate of the cells is increased in different degrees after the medicine is taken. 20. Mu.g/mL^-1The apoptosis rate of CMC acting on SW480 is only (10.50 +/-0.77)%, the apoptosis rate acting on SW620 cell is (12.55 +/-0.13)%, and the apoptosis rate is increased (p) compared with each control group of cells with significant statistical significance<0.05，p<0.01)。40μg·mL^-1The apoptosis rate of CMC acting on SW480 is only (18.54 +/-1.16)%, while the apoptosis rate of CMC acting on SW620 is (27.90 +/-2.06)%, which has significant statistical significance compared with each control group of cells, and the apoptosis rate is significantly increased (p)<0.001)。75μg·mL^-1The apoptosis rate of TFGR on SW480 is only (17.29 +/-0.94)%, and compared with SW480 cells in a control group, the apoptosis rate is not obviously different; the apoptosis rate of SW620 cells was (15.84. + -. 0.71)%, 100. Mu.g/mL^-1The apoptosis rate of TFGR on SW480 is only (33.78 + -2.50)%, and the apoptosis rate on SW620 cells is (32.97 + -0.51)%. The cells of each dose group have significant statistical significance compared with the cells of each control group, and the apoptosis rate is significantly increased (p)<0.001). The experimental result shows that the medium dose groups of GBN, CMC and TFGR can induce the SW480 of the human colon cancer cell to be apoptotic; the intermediate dose groups of LCA, CMC and TFGR can all induce SW620 of human colon cancer cells to witherDeath; LCA, GBN, CMC high dose group (40. Mu.g.mL)^-1) And high dose group of TFGR (100. Mu.g.mL)^-1) Can obviously induce human colon cancer SW480 cells and SW620 cells to generate apoptosis. All subsequent experiments were performed by selecting high dose group for each group.

1.3.3 influence of TFGR, LCA, CMC and GBN on morphology of human Colon cancer nuclei

The effect of the drug on the nuclear morphology was examined after staining with Hoechst33258 fluorochrome and the results are shown in figure 4. The observation under a microscope shows that the cell nucleuses of the SW480 and SW-620 cells of the human colon cancer of the control group show weak blue fluorescence and have complete nuclear structures. LCA, GBN, CMC high dose group (40. Mu.g.mL)^-1) And high dose group of TFGR (100. Mu.g.mL)^-1) Dense and thick staining of cell nuclei appears in each group, the cell nuclei show bright blue fluorescence, and the cell nuclei show apoptosis characteristic changes in different degrees: the nucleosome is reduced, the nucleus is fragmented, the cell is not broken completely, and the apoptotic bodies are generated in individual groups (the specific nucleosome fragmentation, the arrow in the nucleosome-compaction diagram). From the cytomorphological point of view, LCA, GBN, CMC and TFGR can induce the apoptosis of SW480 and SW620 cells of human colon cancer to different degrees.

1.3.4TFGR, LCA, CMC and GBN Effect on migration of human Colon cancer cells

Study of LCA, GBN, CMC high dose groups (40. Mu.g. ML) by cell scratch experiments^-1) And high dose group of TFGR (100. Mu.g.mL)^-1) The effect on the migration of SW480 and SW-620 human colon cancer cells, the results are shown in FIG. 5. The mobility of the SW480 cells of the control group after being scratched for 24 hours is respectively (45.14 +/-1.92)%, and the scratches are quickly healed; the migration rates of LCA, GBN, CMC and TFGR groups on SW480 of human colon cancer cells are respectively (44.93 +/-0.49)%, (34.10 +/-2.34)%, (33.62 +/-1.86)%, and (24.09 +/-3.68)%; the migration rates of SW620 cells in the control group after being scratched for 24 hours are respectively (39.01 +/-3.95%), and the scratches are quickly healed; the migration rates of LCA, GBN, CMC and TFGR groups on SW620 of human colon cancer cells were (30.33. + -. 0.91%), 25.11. + -. 0.73%, (22.23. + -. 0.77%) and (19.01. + -. 1.35%) respectively. LCA was statistically insignificant compared to the SW480 control and statistically significant compared to the SW620 control for mobility (p)<0.05 Can be used forReducing the migratory ability of SW620 for scratch healing. The mobility of the GBN group compared to the mobility of the control group of two cell lines, respectively, was statistically significant (p)<0.01，p<0.001 Can remarkably inhibit the migration ability of cells. The mobility of the CMC group was statistically significant (p) compared to the mobility of the control group of two cell lines<0.01，p<0.001 Can remarkably inhibit the migration ability of cells. The mobility of the TFGR group compared with the mobility of the control group of two cell lines, respectively, was statistically significant (p)<0.001 SW480 and SW620 cells migration ability can be significantly inhibited. The experimental result shows that GBN, CMC and TFGR can obviously inhibit the migration of SW480 and SW620 cells of human colon cancer and slow the healing of scratches; LCA can only inhibit human colon cancer cell SW620 cell migration.

1.3.5 Effect of TFGR, LCA, CMC and GBN on the ability to invade human Colon cancer cells

The LCA, GBN, CMC high dose group (40. Mu.g.mL) was studied by Transwell experiments^-1) And high dose group of TFGR (100. Mu.g.mL)^-1) The effect on the ability of SW480 and SW-620 cells to invade human colon cancer cells, the results are shown in FIG. 6. The invasion number of the SW480 cell control group of the human colon cancer is (71.00 +/-8.72), the invasion number of the LCA group after acting on the SW480 treatment is (63.60 +/-8.91), the invasion number of the GBN treatment group is (51.00 +/-6.48), the invasion number of the CMC treatment group is (37.80 +/-4.66) and the invasion number of the TFGR treatment group is (33.8 +/-4.82); the number of invasion of SW620 cell control group of human colon cancer was (65.80. + -. 8.44), the number of invasion of LCA group after SW620 treatment was (53.6. + -. 6.47), the number of invasion of GBN treatment group was (51.00. + -. 6.56), the number of invasion of CMC treatment group was (32.80. + -. 3.27), and the number of invasion of TFGR treatment group was (30.0. + -. 4.18). The LCA treated group was statistically not different from the SW480 control group and was statistically different from the SW620 control group (p)<0.001 Reduction in the number of attacks after action; the GBN treated group was statistically different from the SW480 and SW620 control groups (p)<0.01， p<0.001 ) the number of invading cells is significantly reduced; the CMC-treated group and the TFGR-treated group were statistically significantly different from the SW480 and SW620 control groups, respectively (p)<0.001 ) the number of invading cells is significantly reduced. The experimental result shows that GBN, CMC and TFGR can obviously inhibit the invasive ability of SW480 and SW620 cells of human colon cancer, and the number of the invasive cells is obviousDecrease; LCA can only significantly inhibit SW620 cell invasion capacity of human colon cancer cells.

1.3.6TFGR, LCA, CMC and GBN Effect on expression levels of proteins involved in apoptotic metastasis invasion of human Colon cancer cells

Western blotting experiment is used for detecting the influence of dry prognosis of active ingredients of various groups of liquorice on the expression level of proteins related to metastasis and invasion of human colon cancer SW480 and SW620 cells. The results are shown in FIG. 7, compared to the control group; LCA drug dry prognosis, in SW480 cells, the expression levels of Bax, cleaned-caspase 3 and cleaned-caspase 9 proteins are obviously increased (P <0.01, P <0.001 and P < -0.001), the expression levels of Bcl-2, MMP2 and MMP9 proteins are obviously reduced (P <0.001, P < -0.01 and P < -0.001); the expression levels of Bax, clear-caspase 3 and clear-caspase 9 proteins in SW620 cells are obviously increased (P <0.01, P <0.001 and P < -0.001), and the expression levels of Bcl-2 and MMP9 proteins are obviously reduced (P <0.001 and P < -0.01). (see A in FIG. 7).

Compared with a control group, after GBN medicament is dried, the average level of Bax, cleaned-caspase 3 and cleaned-caspase 9 protein expression in SW480 cells is obviously increased (P <0.05, P < -0.001 and P < -0.001), and the expression level of Bcl-2 and MMP9 protein is obviously reduced (P < 0.01); the levels of Bax, cleared-caspase 3, cleared-caspase 9 protein expression were significantly increased (P < 0.001), and Bcl-2, MMP9 protein expression were significantly decreased (P < 0.05) in SW620 cells (see FIG. 7, panel B).

Compared with a control group, the TFGR drug-dried prognosis shows that the levels of Bax, cleaned-caspase 3 and cleaned-caspase 9 protein expression are remarkably increased in SW480 cells (P < 0.001), the expression levels of Bcl-2, MMP2 and MMP9 proteins are remarkably reduced (P <0.001, P < -0.01 and P < -0.001); bax, cleaned-caspase 3, cleaned-caspase 9, bcl-2 protein expression levels were significantly increased in SW620 cells (P < 0.001), and MMP2, MMP9 protein expression levels were significantly decreased (P <0.001, P < -0.01) (see FIG. 7, C).

Compared with a control group, CMC drug dry prognosis has obviously increased Bax, cleaned-caspase 3 and cleaned-caspase 9 protein expression levels in SW480 cells (P < 0.001), and obviously decreased Bcl-2, MMP2 and MMP9 protein expression levels (P <0.001, P < -0.01 and P < -0.001); in SW620 cells, the average of Bax, cleared-caspase 3, cleared-caspase 9 and Bcl-2 protein expression was significantly increased (P < 0.001), and the expression levels of MMP2 and MMP9 proteins were significantly decreased (P <0.001, P < -0.01 and P < -0.01) (see FIG. 8).

The results show that GBN, LCA, CMC and TFGR can promote the apoptosis of human colon cancer SW480 and SW620 cells through Caspase apoptosis pathways, and GBN and TFGR can also inhibit the migration and invasion of the human colon cancer SW480 and SW620 cells.

1.3.7 effects of tfgr, LCA, CMC and GBN on mRNA expression levels associated with apoptotic metastasis invasion of human colon cancer cells qRT-PCR results showed that LCA drug stem prognosis, caspase3, caspase9, bax gene levels were significantly increased in SW480 cells (P <0.001, P < -0.01, P < -0.05), MMP2 gene levels were significantly decreased (P < 0.05) compared to controls; the gene levels of Caspase3 and Caspase9 were significantly increased (P < 0.001), and the gene levels of Bcl-2 and MMP2 were significantly decreased (P < 0.001; P-woven 0.001) in SW620 cells (see FIG. 9, panel A).

Compared with the control group, after GBN drug dry prognosis, the levels of Caspase3, caspase9 and Bax genes are obviously increased in SW480 cells (P <0.001, P-woven 0.001 and P-woven 0.05) and the level of MMP2 genes is obviously reduced (P < 0.01); the gene levels of Caspase3, caspase9 and Bax were significantly increased (P < 0.001) and the gene levels of Bcl-2 and MMP2 were significantly decreased (P < 0.001) in SW620 cells (see B in FIG. 9).

Compared with the control group, TFGR drug-dried prognosis shows that the levels of Caspase3, caspase9 and Bax genes are remarkably increased in SW480 cells (P <0.01, P < -0.001), the levels of Bcl-2 and MMP2 genes are remarkably reduced (P <0.01, P < -0.001); caspase3, caspase9, bax gene levels were significantly elevated in SW620 cells (P <0.05, P-knot 0.001), bcl-2, MMP2 gene levels were significantly decreased (P <0.01, P-knot 0.05); (see FIG. 9, panel C).

Compared with a control group, after CMC drug dry prognosis, the levels of Caspase3, caspase9 and Bax genes are obviously increased in SW480 cells (P <0.01, P < -0.001 and P < -0.001), the levels of Bcl-2 and MMP2 genes are obviously reduced (P <0.01 and P < -0.001); caspase3, caspase9, bax gene levels were significantly increased in SW620 cells (P <0.05, P < -0.001), bcl-2, MMP2 gene levels were significantly decreased (P <0.01, P < -0.05); (see D in FIG. 9).

The results show that GBN, LCA, CMC and TFGR have influence on the expression level of mRNA related to apoptosis and metastasis invasion of human colon cancer cells.

Further, a Western Blot method is used for observing the expression result of the PI3K-Akt-m TOR pathway related protein regulated and controlled by the CMC drug in SW480 and SW620 cells

Compared with the control group, after the CMC drug is subjected to dry treatment, the expression levels of PI3K, AKT, mTOR, P-PI3K, P-AKT and P-mTOR proteins in SW480 and SW620 cells are obviously reduced (P < 0.05); see fig. 9. The Western Blot method results suggest: the CMC drug can promote the apoptosis of SW480 and SW620 cells through PI 3K-AKT-mTOR.

1.3.8 reverse virtual screening technique is used to discuss potential action targets and signal pathways of Glabridin (GBN) and licochalcone A (LCA) against colon cancer, and the results show that:

(1) the protein is not only a protein with up-regulated expression in colorectal cancer cells, but also a candidate protein acting on a signal path, 13 candidate targets screened by glabridin and 16 candidate protein targets of LCA. The final prediction of LCA and GBN possible protein targets for 8, BRAF, TGFBR1, CSNK2A1, PPARD, CDK2, HSP90AB1, RXRA, and MAPK14. These targets are mostly concentrated on the PI3K/AKT, mTOR, MAPK, wnt signaling pathways.

(2) Comparing gene information in a Cancer Cell Line database CCLE (Cancer Cell Line Encyclopedia), and comparing GBN and LCA screened targets with SW480 Cell gene information to obtain 7 target genes; can be aligned with gene information in SW620 cells, and has 5 target genes. The target genes are compared with candidate target genes which are screened by GBN and LCA, are not only proteins with up-regulated expression in colorectal cancer cells, but also act on a signal pathway, three target genes can be compared, namely PPARD, TGFBR1 and HSP90AB1, and the target genes participate in Wnt, TGF-beta, MAPK, PI3K/AKT signal pathways.

1.3.9TFGR, GBN, LCA, CMC results of proteomics research for resisting colon cancer in vitro

The expression difference of the licoflavone active ingredient after acting on human colon cancer cells is detected by using a proteomics technology, and bioinformatics analysis is carried out on the differentially expressed protein to conjecture an acting signal path, so that the potential acting mechanism of the licoflavone active ingredient against colon cancer is discussed. The results show that:

(1) a total of 57965 peptides, 6750 proteins, were identified. The SW620 cell control group of human colon cancer is compared with the administration group (LCA, GBN and TFGR) and 183 proteins are different among the LCA groups, wherein 31 proteins are up-regulated and 152 proteins are down-regulated in the administration group; 150 proteins are different between GBN groups, wherein 43 proteins are up-regulated by the administration group, 107 proteins are down-regulated by the administration group, and 180 proteins are different between TFGR groups, wherein 62 proteins are up-regulated by the administration group, and 118 proteins are down-regulated by the administration group. Compared with a control group of SW480 cells of human colon cancer, 165 proteins are differentiated among the LCA groups in the administration groups (LCA, GBN and TFGR), wherein 29 proteins are up-regulated and 136 proteins are down-regulated in the administration groups; 55 proteins are different among GBN groups, wherein 23 proteins are up-regulated and 32 proteins are down-regulated in the administration group; there are 200 TFGR group differential proteins, 66 up-regulated proteins and 134 down-regulated proteins.

(2) The GO function annotation analysis result shows that compared with a SW620 cell control group, the LCA group differential protein carries out GO annotation analysis on 21 genes participating in biological processes, 3 cell components and 11 molecular functions; GBN group differential protein GO is annotated and analyzed, 19 pieces participate in biological processes, 2 pieces of cell components and 8 pieces of molecular functions; the TFGR group differential protein is subjected to GO annotation analysis, 15 genes participating in biological processes, 2 cell components and 11 molecular functions are obtained. Comparing with SW480 control group, carrying out GO annotation analysis on LCA group differential protein to participate in 18 biological processes, 3 cell components and 14 molecular functions; differential protein GO between GBN groups is annotated and analyzed to participate in 16 biological processes, 2 cell components and 7 molecular functions; the TFGR group differential protein was analyzed by GO annotation and involved in 2 cellular components and 12 molecular functions in the biological process.

(3) The KEGG functional analysis result shows that LCA, GBN and TFGR respectively act on the differential protein KEGG annotation analysis among groups after SW620 cells, and the differential protein is shown to participate in 193 KEGG paths in total.

(4) The total 382 differential proteins of each treatment group control human colon cancer SW620 cell group participate in 260 KEGG metabolic pathways, 29 differential proteins in a main pathway Signal transduction pathway are further subjected to KEGG pathway analysis, the number of the 29 differential proteins participating in cancer, mTOR and PI3K-Akt Signal pathways is the largest, and the expression of other Signal transduction related proteins except GTP, heat shock stress protein and intercellular adhesion molecules in the drug treatment group is reduced in different amplitudes. The total number of the differential proteins of each treatment group is 348 compared with a human colon cancer SW480 cell group and participate in 225 KEGG metabolic pathways, and the KEGG pathway analysis is further carried out on the 27 differential proteins in the Signaltrasduction pathway, so that the number of the differential proteins in the two signal pathways of cancer and PI3K-Akt is the largest, the expressions of the rest signal conduction related proteins except for heme oxidase-1 and ARF-6 proteins are reduced in different amplitudes, and the drug treatment has better callback effect on the signal pathways. The regulation of some important proteins in these proteins is related to the apoptosis, metastasis and invasion of colon cancer.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of licochalcone A in preparing medicine for treating colorectal cancer is provided.

2. Use of licochalcone a according to claim 1 for the preparation of a medicament for the treatment of colorectal cancer, wherein licochalcone a is used as the sole active ingredient for the preparation of a medicament for the treatment of colorectal cancer.

3. Use of licochalcone a for the preparation of a medicament for the treatment of colorectal cancer according to claim 1 or 2, wherein licochalcone a is effective in significantly increasing the level of expression of clear-caspase 3, clear-caspase 9, bax protein and significantly decreasing the expression of Bcl-2, MMP9 protein.

4. Use of licochalcone a according to claim 3 for the preparation of a medicament for the treatment of colorectal cancer, wherein licochalcone a acts by modulating the PI3K/Akt/mTOR signaling pathway in the prepared medicament for the treatment of colorectal cancer.

5. The application of licochalcone A for preparing a drug for treating colorectal cancer according to claim 3, wherein the licochalcone A is used at a concentration of 5 μ g/mL-40 μ g/mL in the prepared drug for treating colorectal cancer.

6. Application of a composition of glabridin and licochalcone A in preparing medicine for treating colorectal cancer is provided.

7. Use of a combination of glabridin and licochalcone a according to claim 6 for the preparation of a medicament for the treatment of colorectal cancer, wherein the combination of glabridin and licochalcone a is used as the sole active ingredient for the preparation of a medicament for the treatment of colorectal cancer.

8. The use of a combination of glabridin and licochalcone a for the manufacture of a medicament for the treatment of colorectal cancer according to claim 6 or 7, wherein the effect of the combination of glabridin and licochalcone a is to significantly increase the expression level of clear-caspase 3, clear-caspase 9, bax protein and significantly decrease the expression of Bcl-2, MMP9 protein.

9. Use of a combination of glabridin and licochalcone a for the manufacture of a medicament for the treatment of colorectal cancer according to claim 8, wherein the combination of glabridin and licochalcone a acts by modulating the PI3K/Akt/mTOR signaling pathway in the manufactured medicament for the treatment of colorectal cancer.

10. The use of the composition of glabridin and licochalcone A according to claim 8, in the preparation of a medicament for treating colorectal cancer, wherein the use concentration of the composition of glabridin and licochalcone A in the prepared medicament for treating colorectal cancer is 5 μ g/mL-40 μ g/mL.