Application of phellinus igniarius extract in preparation of product for inhibiting activity of Wnt signal channel

Abstract

The invention discloses an application of a phellinus igniarius extract in preparation of a product for inhibiting the activity of a Wnt signal channel. Through an in-vitro cell culture model and animal tests, it is verified that the phellinus igniarius extract has the activity of inhibiting a Wnt signal channel and can inhibit tumor cell metastasis; a new thought is provided for lung cancer treatment, a good working foundation is laid for screening of new anti-tumor metastasis drugs, and the lung cancer treatment effect and the survival quality of patients can be improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to the application of a Sanghuang extract in the preparation of a product for inhibiting the activity of the Wnt signaling pathway. Background Art

[0002] Lung cancer is a highly prevalent and highly lethal malignant tumor. Although standard treatment methods can currently partially cure or prolong the lives of patients to varying degrees, most patients ultimately die from tumor metastasis. Therefore, controlling tumor metastasis is the key to improving clinical efficacy, but there is currently a lack of effective therapeutic drugs in clinical practice. Natural products (such as plant and fungal metabolites, etc.) are an important source of new drugs for humans. In recent years, with the promotion of many policies such as the modernization of traditional Chinese medicine in China and the increasing attention to natural products, it has brought an opportunity for the development of safe and effective new anti-tumor drugs. Summary of the Invention

[0003] Aiming at the defects of the prior art, the purpose of the present invention is to provide the application of a Sanghuang extract in the preparation of a product for inhibiting the activity of the Wnt signaling pathway.

[0004] The technical solution of the present invention is as follows: In a first aspect of the present invention, there is provided the application of a Sanghuang extract in the preparation of a product for inhibiting the activity of the Wnt signaling pathway, wherein the Sanghuang extract inhibits the expression of the Wnt signaling pathway effector molecule β-catenin.

[0005] Further, the Sanghuang extract is Sanghuang extraction powder or Sanghuang extraction liquid, and the concentration of the Sanghuang extraction liquid is 1.25 - 5.00 mg / ml.

[0006] In a second aspect of the present invention, there is provided an inhibitor of Wnt signaling pathway activity.

[0007] Further, the active ingredient of the inhibitor includes a Sanghuang extract.

[0008] Further, the Sanghuang extract is Sanghuang extraction powder or Sanghuang extraction liquid.

[0009] Further, the concentration of the Sanghuang extraction liquid is 1.25 - 5.00 mg / ml.

[0010] In a third aspect of the present invention, there is provided the application of an inhibitor of Wnt signaling pathway activity in the preparation of a drug for inhibiting the metastasis of lung cancer cells.

[0011] In a fourth aspect of the present invention, there is provided a drug for inhibiting the metastasis of lung cancer cells.

[0012] Further, the drug for inhibiting the metastasis of lung cancer cells includes the inhibitor of Wnt signaling pathway activity.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an application of Sanghuang extract in the preparation of a product for inhibiting the activity of the Wnt signaling pathway. Through in vitro cell culture models and animal experiments, it has been verified that the Sanghuang extract has the activity of inhibiting the Wnt signaling pathway and can inhibit the migration of tumor cells, providing a new idea for the treatment of lung cancer, laying a good working foundation for the screening of new anti-tumor metastasis drugs, and helping to improve the treatment effect of lung cancer and the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent: Figure 1 It is a schematic diagram of the experimental results of the inhibition of tumor cell clones by the Sanghuang extract provided in the experimental examples of the present invention (889DTC refers to primary mouse lung adenocarcinoma cells; H1299 refers to a human non-small cell lung cancer cell line; Figure 1 In FIG. A: It is a schematic diagram of the sensitivity results of 889DTC cells to three different concentrations of the Sanghuang extract; Figure 1 In FIG. B: It is a schematic diagram of the sensitivity results of H1299 cells to three different concentrations of the Sanghuang extract); Figure 2 It is a schematic diagram of the experimental results of cell migration provided in the experimental examples of the present invention (889DTC refers to primary mouse lung adenocarcinoma cells; H1299 refers to a human non-small cell lung cancer cell line; Figure 2 In FIG. A: It is a schematic diagram of the results of the inhibition of in vitro migration of 889DTC cells by the Sanghuang extract; Figure 2 In FIG. B: It is a schematic diagram of the results of the inhibition of in vitro migration of H1299 cells by the Sanghuang extract); Figure 3 It is a schematic diagram of the experimental results of tumor cell stem sphere culture provided in the experimental examples of the present invention (889DTC refers to primary mouse lung adenocarcinoma cells, H1299 refers to a human non-small cell lung cancer cell line; Figure 3 In FIG. A: It is a schematic diagram of the results of the inhibition of the formation of 889DTC tumor cell stem spheres by the Sanghuang extract; Figure 3 In FIG. B: It is a schematic diagram of the results of the inhibition of the formation of H1299 tumor cell stem spheres by the Sanghuang extract); Figure 4 It is a schematic diagram of the experimental results of animal experiments provided in the experimental examples of the present invention (889DTC refers to primary mouse lung adenocarcinoma cells; Figure 4 In FIG. A: It is a schematic diagram of the results of the metastasis of tumor cells to the lungs in mice under a fluorescence microscope; Figure 4 In FIG. B: It is a schematic diagram of the results of the inhibition of the metastasis of tumor cells to the lungs by the Sanghuang extract after intragastric administration of the Sanghuang extract to mice); Figure 5 Schematic diagram of the bioactivity results of the Sanghuang extract provided in the experimental example of the present invention for inhibiting the Wnt signaling pathway (889DTC refers to primary mouse lung adenocarcinoma cells; H1299 refers to a human non-small cell lung cancer cell line; Figure 5 In Figure A: Schematic diagram of the expression level results of Wnt signaling pathway molecules (Wnt3a and Wnt5a) and their target molecules (such as Hoxb9, Lef1, c-myc, and Cyclin D1) after treating mouse 889DTC cells with the Sanghuang extract; Figure 5 In Figure B: Schematic diagram of the results of inhibiting the expression of β-catenin protein after treating 889DTC cells with the Sanghuang extract; Figure 5 In Figure C: Schematic diagram of the results of inhibiting the expression of β-catenin protein after treating H1299 cells with the Sanghuang extract). Detailed implementation manners

[0015] The present invention will be described in detail below in conjunction with specific embodiments.

[0016] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes and improvements can still be made, and these all belong to the protection scope of the present invention.

[0017] First embodiment This embodiment provides a Sanghuang extract, which can inhibit the expression of the Wnt signaling pathway effector molecule β-catenin. This Sanghuang extract is a freeze-dried powder of the aqueous extract of Sanghuang prepared into a Sanghuang extract solution with a concentration of 1.25 mg / ml.

[0018] This embodiment also provides an inhibitor of Wnt signaling pathway activity, and the active ingredient of this inhibitor includes 1.25 mg / ml of the Sanghuang extract solution.

[0019] This embodiment also provides a drug for inhibiting the metastasis of lung cancer cells, and this drug includes an inhibitor of Wnt signaling pathway activity.

[0020] Second embodiment This embodiment provides a Sanghuang extract, which can inhibit the expression of the Wnt signaling pathway effector molecule β-catenin. This Sanghuang extract is a freeze-dried powder of the aqueous extract of Sanghuang prepared into a Sanghuang extract solution with a concentration of 2.50 mg / ml.

[0021] This embodiment also provides an inhibitor of Wnt signaling pathway activity, and the active ingredient of this inhibitor includes 2.50 mg / ml of the Sanghuang extract solution.

[0022] This embodiment also provides a drug for inhibiting the metastasis of lung cancer cells, and the drug comprises a Wnt signaling pathway activity inhibitor.

[0023] The third embodiment This embodiment provides a Phellinus linteus extract, which can inhibit the expression of the Wnt signaling pathway effector molecule β-catenin. The Phellinus linteus extract is a freeze-dried powder of the aqueous extract of Phellinus linteus configured into a Phellinus linteus extract solution with a concentration of 5.00 mg / ml.

[0024] This embodiment also provides a Wnt signaling pathway activity inhibitor, and the active ingredient of the inhibitor comprises a Phellinus linteus extract solution with a concentration of 5.00 mg / ml.

[0025] This embodiment also provides a drug for inhibiting the metastasis of lung cancer cells, and the drug comprises a Wnt signaling pathway activity inhibitor.

[0026] Experimental example This experimental example proves that the Wnt signaling pathway activity inhibitor provided by the embodiment has effective anti-tumor activity against lung cancer cells through the following experiments.

[0027] The materials and methods involved in this experimental example are as follows: 1. Lung cancer cells and culture medium Mouse primary lung adenocarcinoma cells 889DTC (kindly provided by the Winslow Laboratory of Stanford University, USA) and human non-small cell lung cancer cell line H1299 (purchased from the Cell Bank Center of the Chinese Academy of Sciences, Shanghai) were cultured and passaged with RPMI-1640 medium, and the medium contained 10% fetal bovine serum and penicillin-streptomycin double antibody.

[0028] Conventional cell culture media and related reagents, and stem cell culture medium reagents such as DMEM / F12 medium, B27 and growth factors (EGF and heat-resistant bFGF) were all purchased from Life Technologies, USA.

[0029] 2. Main experimental reagents The freeze-dried powder of the aqueous extract of Phellinus linteus was provided by Zhejiang Qianjifang Medical Technology Co., Ltd.

[0030] The freeze-dried powder of the aqueous extract of Phellinus linteus was configured into three different concentrations of Phellinus linteus extract solutions, and the concentrations were 5.00 mg / mL, 2.50 mg / mL and 1.25 mg / mL respectively.

[0031] The preparation method of the Phellinus linteus extract solution is as follows: S1. Weigh 2 g of the freeze-dried powder of the aqueous extract of Phellinus linteus, and soak it in 20 mL of distilled water at room temperature for 10 min; S2. Remove residues and other insoluble components by high-speed centrifugation (12,000 g × 5 min) to obtain the mother liquor of the Phellinus igniarius extract (100 mg / mL). S3. Take a certain volume of the mother liquor of the Phellinus igniarius extract, add an appropriate amount of distilled water, and dilute the mother liquor of the Phellinus igniarius extract into Phellinus igniarius extracts with concentrations of 5.00 mg / mL, 2.50 mg / mL, and 1.25 mg / mL respectively. S4. After filtering and sterilizing the three Phellinus igniarius extracts with different concentrations through a 0.22 µm sterile filter, store them at 4°C for later use.

[0032] Hifair ® V Reverse Transcriptase reverse transcriptase and Hieff ® qPCR SYBR Green MasterMix (quantitative PCR premixed solution) was purchased from Shanghai Yeasen Biotechnology Co., Ltd.; β-catenin, GAPDH antibodies, and goat anti-rabbit secondary antibody were all purchased from Proteintech Group, Inc. (USA).

[0033] Experiment 3.1 Cell colony survival assay: First, inoculate a certain number of tumor cells into a 6 cm culture dish, and then treat them with Phellinus igniarius extracts with different concentrations (5.00 mg / mL, 2.50 mg / mL, and 1.25 mg / mL) for 48 hours; after changing to fresh medium, continue to culture the cells for 7 - 10 days, then discard the medium, fix and stain with a staining solution (containing 10% acetic acid, 20% methanol, and 0.25% Coomassie Brilliant Blue G-250) for 20 min, then rinse with running water and air dry. Calculate the number of cell colonies according to the standard that cell colonies with more than 50 cells are positive colonies to determine the survival ability of tumor cells. 3.2 Cell migration experiment: First, pre-equilibrate the transwell chamber. Add serum-free medium to the transwell chamber and the lower chamber respectively (100 μL in the upper chamber and 600 μL in the lower chamber), and equilibrate in a 37°C incubator for more than 2 h to promote the adhesion of tumor cells; digest the tumor cells with trypsin, collect the digested cells by low-speed centrifugation, wash them 2 times with PBS to fully remove the serum; resuspend the cells with serum-free medium to make the cell concentration 4×10 5cells / mL. Add 100 μl of serum-free cell suspension to the upper chamber, and 600 μl of medium containing 15% FBS to the lower chamber. Use forceps to place the transwell chamber into the Chamber. Incubate the cells with Sanghuang extracts at different concentrations (5.00 mg / mL, 2.50 mg / mL, and 1.25 mg / mL) for 6 - 24 h (the incubation time is determined according to the migration characteristics of tumor cells, usually between 6 - 24 hours, to observe the effect of Sanghuang extracts on cell migration ability); Remove the transwell chamber, wipe the cells on the upper surface of the chamber membrane thoroughly with a clean and moist cotton swab, and wash once with PBS; Fix with pre-cooled methanol for 10 min, stain with 0.1% crystal violet for 30 min, wash the crystal violet with tap water, air dry, and then observe, count, and photograph under an inverted microscope.

[0034] 3.3 Tumor stem cell sphere culture assay: Digest the tumor cells into single cells with an appropriate amount of 0.05% trypsin, neutralize with RPMI-1640 medium containing 10% FBS, then centrifuge at low speed (400 g × 2 min) to collect the cells, and discard the supernatant; Wash twice with PBS (pH 7.4) to fully remove the serum, and then resuspend and count with PBS (pH 7.4); Further dilute with tumor stem cell sphere medium (DMEM / F12, containing 0.4% BSA, B27 (1x), 20 ng / mL EGF, 10 ng / mL bFGF, 0.5 mM HEPES) to a cell suspension with a concentration of 2000 / ml. In an ultra-low attachment 24-well culture plate, add 1 mL of cell suspension and Sanghuang extracts at different concentrations (5.00 mg / mL, 2.50 mg / mL, and 1.25 mg / mL) to each well. Incubate in an incubator for 3 - 4 days, then supplement 500 μl of tumor stem cell sphere medium to each well, and then continue to culture until the end point; Finally, photograph and record under a light microscope and count the tumor stem cell spheres (a cell cluster containing more than 30 cells can be counted as 1 tumor stem cell sphere).

[0035] 3.4 Real-time fluorescence quantitative PCR detection: Collect 889DTC cells according to the conventional method for total RNA extraction by the TRIZOL method; Reverse transcribe using Hifair ® V ReverseTranscriptase reverse transcriptase according to the instructions; Perform PCR quantification using the primers of each gene (see Table 1) and Hieff ® qPCRSYBR Green Master Mix, and finally analyze the expression difference of the test sample relative to the calibration sample by the 2 -ΔΔct method.

[0036] 3.5 Western blot detection: 3.5.1 Operation procedure Protein sample preparation → Electrophoresis → Transfer membrane → Antibody incubation → Color development and photography 3.5.2 Method 1) Protein sample preparation (1) Harvest cells (primary lung adenocarcinoma cells of 889DTC mice; H1299 human non-small cell lung cancer cell line).

[0037] ① Carefully aspirate the cell culture medium, rinse the adherent cells once with pre-cooled PBS, and then discard the washing solution.

[0038] ② Add an appropriate amount of PBS, gently scrape the cells to one end of the petri dish with a cell scraper, transfer the cell suspension to a new labeled EP tube, centrifuge at 1,500 rpm for 5 min to collect the cell pellet, repeat the PBS wash 2 times, place the EP tube on ice after washing with PBS.

[0039] ③ Add an appropriate amount of lysis buffer to the EP tube, pipette up and down to resuspend the cell pellet, place it on ice for lysis for 30 min, then centrifuge at 13,000 rpm for 15 min at 4°C, and transfer the supernatant to a new labeled EP tube.

[0040] (2) Determine the protein concentration by BCA method Experimental principle: The peptide bond structure in protein molecules can form a complex with Cu 2+ and reduce Cu 2+ to Cu + in an alkaline environment. The BCA reagent can specifically and sensitively bind to Cu + to form a stable colored complex, which has the maximum absorbance value at 562 nm. The color depth of this complex is proportional to the protein concentration, and the protein content can be determined according to the absorbance value.

[0041] The standard solution is bovine serum albumin (BCA), and the measurement range is 0.02 μg - 2.00 μg / μL.

[0042] ① According to the required number of sample wells, add BCA working solution to the 96-well plate, 200 μL / well. Take 10 μL of the standard curve sample and 2 μL of the test sample and add them to the 96-well plate respectively, and mix well.

[0043] ② Cover the plate and incubate at 37°C for 30 min.

[0044] ③ Read the absorbance value at 562 nm with a spectrophotometer, obtain the standard curve through EXCEL, and calculate the protein concentration of the sample.

[0045] (3)Protein sample preparation ① Add 5xloading buffer and sample volume in a ratio of 1:4, and mix well (to facilitate loading, the sample can be adjusted to the same concentration with 1xloading).

[0046] ② Boil in a metal bath at 100 °C for 10 min (Note: Tissue samples generally need to be boiled for 30 min). To prevent the tube lid from bursting due to high temperature, an ice pack can be added above the shaker heater to cool down. After boiling, let it stand for a while until the EP tube cools down, then centrifuge at 4 °C and 13,000 rpm for 10 min. The sample preparation is completed and can be stored at -40 °C or proceed to the next experiment.

[0047] 2) Electrophoresis (1)Preparation of SDS-PAGE gel Clean and dry the glass plates and combs before preparation. Align the glass plates and clamp them tightly in the clip, then vertically clamp them on the rack to prepare for pouring the gel. (During operation, make sure the two glasses are aligned to avoid gel leakage).

[0048] The volume of a separating gel is approximately 4.6 mL. After preparing the separating gel, add 1 mL of anhydrous ethanol for liquid sealing. After about 30 min, when an obvious demarcation line appears, pour out the ethanol and blot it dry, then add the stacking gel. The volume of a stacking gel is approximately 1 mL, then insert the comb, slowly insert it from left to right to prevent gel splashing.

[0049] (Note: For BioRad 1.0 mm plates, the maximum loading volume per well of a 15-well comb is approximately 33 uL, and that of a 10-well comb is approximately 55 uL, which needs to be added with a micro-injection needle. If using a pipette tip, for a 15-well comb, it is about 15 uL, and for a 10-well comb, it is about 25 - 30 uL.) (2)Loading Take out the protein sample from -40°C. After thawing, mix it well by shaking. Heat it at 100°C for 5 - 6 minutes, centrifuge at 12,000 rpm for 10 minutes, and then load the sample. Before loading, prepare the gel. Rinse the stacking gel with water and place it in the electrophoresis tank. (The small glass plate faces inward and the large glass plate faces outward. If only running one gel, a plastic plate should be padded on the other side of the tank with the side with words facing outward.) Fill the inner tank with running buffer to completely cover the loading wells without leakage, then load the sample. The general loading amount is 30 μg - 50 μg. Reserve 1 - 2 wells for loading the marker. Adjust the loading volume of the marker as appropriate, and different loading amounts of the marker can be used to distinguish the gel. The volume of each well of the sample is supplemented with 1×loading buffer to prevent the large-volume loading wells from squeezing the small-volume wells.

[0050] (3) Gel running Add the remaining running buffer to the outer electrophoresis tank, cover the lid, carefully check the positive and negative poles for matching (the red plug is the positive pole corresponding to the red jack of the power supply; similarly, the black plug corresponds to the black jack of the power supply), then turn on the power. The voltage for the upper layer of the gel can be 70V; for the lower layer of the gel, it can be 120V. When the red marker appears, immediately adjust the voltage to 120V until the bromophenol blue in the protein sample runs to the bottom of the separation gel, and then proceed with membrane transfer.

[0051] 3) Membrane transfer (1) PVDF membrane pretreatment: Before membrane transfer, soak the PVDF membrane (5.8 cm × 8.5 cm) in pure methanol for 5 s, and then soak it in the transfer buffer.

[0052] (2) Gel extraction: Perform membrane transfer before the marker corresponding to the smallest molecular weight of the required protein runs out. Under the tap water, use a plastic blade to pry open the two plates and remove all the stacking gel.

[0053] (3) Set up the membrane transfer device: Side view (Note: This is only applicable to the sandwich wet transfer method) (Note: When the PVDF membrane covers the gel, there must be no bubbles. When inserting the transfer clip into the transfer tank, pay attention that the black side of the clip faces the black side of the tank, white to red, and carefully check whether the electrodes of the tank correspond to the outer tank.) Put ice cubes into the outer tank, pour all the transfer buffer into the line up to Blotting, cover the lid, and pay attention to the electrode correspondence. Place the tank in a foam box and fill it with ice around and tamp it down.

[0054] Turn on the power, keep a constant current of 180 mA for 2 h (for domestic power supplies, for BioRad power supplies, it can be 400 mA for 100 min), and recycle the remaining methanol.

[0055] 4) Antibody Incubation (1)Ponceau S prestained membrane: After the transfer is completed, place the membrane in Ponceau S solution for 5 min, wash it once with TBST, and observe the transfer effect. (Note: This process is reversible. The red dye on the membrane can be removed by washing it several times with deionized water.) (2)Blocking: Place the membrane in 5% skim milk (50 mL / membrane), with the front side up, i.e., the side that was in contact with the gel. Shake it on a shaker for 2 h for blocking. If the background is relatively high after the preliminary experiment, the blocking time can be appropriately extended, and it can be blocked overnight at 4°C as needed.

[0056] (3)Prepare primary antibody: Prepare it with 5% BSA in TBST according to the ratio in the instruction manual. Approximately 3 - 4 mL is needed for one small trough.

[0057] (4)Cut the membrane: Wash the blocked membrane with TBST and cut it according to the molecular weight of the target protein. (Note: Leave enough space at the upper and lower edges of the membrane corresponding to the molecular weight of the target protein to avoid cutting off the target protein.) (5)Primary antibody incubation: Place the cut membrane into the corresponding primary antibody dilution solution and incubate it overnight at 4°C, or for 3 - 4 h at room temperature as needed.

[0058] (6)Wash the membrane: Recover the primary antibody and store it at -40°C. Wash the membrane 4 times with TBST, 8 min each time.

[0059] (7)Prepare secondary antibody: Prepare it with 5% BSA in TBST according to the ratio in the instruction manual. Approximately 3 - 4 mL is needed for one small trough. (Note: When preparing the secondary antibody, be sure to check the species origin of the primary antibody.) (8)Secondary antibody incubation: Incubate at room temperature for 1 - 2 h.

[0060] (9)Wash the membrane: Recover the secondary antibody and store it at -40°C. Wash the membrane 4 times with TBST, 8 min each time.

[0061] 5) Development and Photographing (1)Prepare developing solution: For both domestic and imported ones, prepare it by mixing black and white in a 1:1 ratio, 200 μL per membrane.

[0062] (2)Scan the membrane: First, take the membrane out of TBST, gently press it with a tissue to absorb the liquid, place it in the center of the iron tray of LAS4000 (a chemiluminescence detector produced by GE), add the pre-prepared developing solution, tilt it left and right so that the membrane is just completely wetted without the liquid overflowing, and put it into the machine for development. Generally, the development parameters are set as: Auto, Super. The exposure time for one membrane is about 1 s - 15 s.

[0063] (3) Photograph: After the immune blotting bands of the film become obvious in the machine, click save, save the results in your own folder, and make marks.

[0064] 3.6 Animal experiments Inoculate nude mice aged 6 - 8 weeks with mouse tumor cells 889DTC by tail vein injection method (1×10 5 cells / mouse); Inject BALB / c nude mice by tail vein (1×10 5 cells / mouse), then orally administer two groups of mice (6 mice in each group) with Sanghuang extract at two different concentrations (100 mg / kg and 200 mg / kg) once a day for 5 consecutive days, a total of 5 doses; 2 weeks after inoculation with tumor cells, euthanize the mice by CO2 euthanasia method, then excise the lungs, observe the occurrence of lung metastasis of tumor cells under a stereomicroscope, and take pictures and save them. The above experimental content and operation methods have been approved by the Animal Experiment Ethics Committee of Nanjing Hospital Affiliated to Nanjing Medical University (The Third Clinical Medical College).

[0065] 3.7 Statistical analysis Use SPSS 21.0 statistical software to process the data. Measurement data that conforms to the normal distribution is expressed as x ± s. The independent sample t - test is used for comparison between two groups, and one - way analysis of variance is used for comparison among multiple groups. P < 0.05 is considered statistically significant.

[0066] Results and analysis 4.1 Analysis of the results of cell clone survival assay In this experimental example, the sensitivity of 889DTC cells and H1299 cells to 3 different concentrations of Sanghuang extract was detected by cell clone survival assay. The results showed that the treatment with Sanghuang extract could significantly inhibit the clonal growth of tumor cells, and this inhibitory effect was significantly concentration - dependent (see Figure 1 ). These results at least confirm at the cell culture level that Sanghuang has a significant killing effect on lung cancer cells.

[0067] 4.2 Analysis of the results of cell migration experiment The migratory and invasive properties of tumor cells are important conditions for their ability to metastasize distantly. In this experimental example, the effect of the Sanghuang extract on the migratory ability of tumor cells was evaluated by a cell transwell migration assay. According to the sensitivity of tumor cells to Sanghuang extracts at different concentrations, two different concentrations (2.50 mg / mL and 1.25 mg / mL) of Sanghuang extracts were used to treat tumor cells, and then the effect of the Sanghuang extract on the migratory ability of lung cancer cells was identified by a transwell migration assay. The results showed that Sanghuang extracts at both concentrations effectively inhibited the in vitro migration of 889DTC cells and H1299 cells, and this inhibitory effect was significantly concentration-dependent (see Figure 2 ).

[0068] 4.3 Analysis of the results of the tumor stem cell sphere culture assay Tumor stem cells are a very small number of cancer cells present in malignant tumor tissues that have stem cell characteristics (the ability of self-renewal, unlimited proliferation, and multi-directional differentiation), and they are closely related to tumorigenesis, drug resistance, and metastasis. To evaluate the potential inhibitory or killing effect of the Sanghuang extract on tumor stem cells, in this experimental example, tumor cells were treated with two different concentrations of the Sanghuang extract, and then the effect of the Sanghuang extract on the formation of tumor stem cell spheres was evaluated by a tumor stem cell sphere culture experiment. The results showed that the Sanghuang solution at concentrations of 2.50 mg / mL and 1.25 mg / mL could significantly inhibit the size and number of tumor stem cell spheres, showing an obvious concentration-dependence. These results indicate that Sanghuang has certain inhibitory activity on tumor cell stemness under cell culture conditions (see Figure 3 ).

[0069] 4.4 Analysis of the results of the animal experiment In view of the above experimental results showing that Sanghuang has significant activities in inhibiting the survival, migration, and stemness of lung cancer cells, the inhibitory effect of Sanghuang on the distant metastasis of lung cancer cells was further evaluated by an animal tumor metastasis model. To better simulate the effect of tumor cell distant metastasis in vivo, a highly metastatic primary lung adenocarcinoma cell line 889DTC (genotype k-ras G12D ; p53 KO) was used as the tumor cell model, and BALB / c nude mice were injected via the tail vein. Then, the mice were orally administered with two different concentrations of the Sanghuang extract; two weeks after inoculating the tumor cells, the lungs of the animals were taken to detect the metastasis of tumor cells to the lungs. Since the tumor cells carried a red fluorescent label, it was directly observed under a fluorescence microscope that the oral administration of the Sanghuang extract to the mice could effectively inhibit the metastasis of tumor cells to the lungs, and its anti-tumor metastasis effect showed a certain dose-dependence (see Figure 4 ).

[0070] 4.5 Analysis of the results of real-time fluorescence quantitative PCR detection and Western blot detection To reveal the molecular mechanism of the anti-tumor metastasis of Phellinus igniarius, in the previous study, mice were orally administered with the extract of Phellinus igniarius, and then the metabolites in the blood of mice were analyzed by combining metabolomics detection technology and metabolite database. As a result, a phenolic small molecule with rich content was found; through pharmacological molecular docking experiments, it was found that this phenolic small molecule could bind to the key molecules of the Wnt signaling pathway. Based on this, it was speculated that the anti-tumor metastasis activity of Phellinus igniarius was closely related to this phenolic molecule. In view of the potential association between the above-mentioned phenolic small molecule and the Wnt signaling pathway, after treating mouse lung cancer cells 889DTC with the extract of Phellinus igniarius, the expression levels of the molecules of the Wnt signaling pathway and its downstream target genes in tumor cells were detected by quantitative PCR to identify the effect of Phellinus igniarius on the activity of the Wnt signaling pathway. The results showed that: after the tumor cells were treated with the extract of Phellinus igniarius, the expression levels of the Wnt signaling pathway molecules (Wnt3a and Wnt5a) and their target molecules (such as Hoxb9, Lef1, c-myc and Cyclin D1) were all down-regulated to varying degrees (see Figure 5 A); further, the expression level of the Wnt signaling pathway effector molecule β-catenin was identified by Western blot technology. The results showed that Phellinus igniarius could significantly inhibit the expression of β-catenin protein after treating tumor cells (see Figure 5 B, Figure 5 C). These results indicate that Phellinus igniarius can inhibit the activity of the intracellular Wnt signaling pathway after treating tumor cells.

[0071] The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of a Phellinus linteus extract in the preparation of a product for inhibiting the activity of the Wnt signaling pathway, characterized in that, The Sanghuang extract inhibits the expression of the Wnt signaling pathway effector molecule β-catenin.

2. The application according to claim 1, wherein The Sanghuang extract is Sanghuang extraction powder or Sanghuang extraction liquid, and the concentration of the Sanghuang extraction liquid is 1.25 - 5.00 mg / ml.

3. The application according to claim 1, characterized in that, The product is an inhibitor of Wnt signaling pathway activity or a drug for inhibiting the metastasis of lung cancer cells.

4. A Wnt signaling pathway activity inhibitor, characterized in that, The active ingredient of the inhibitor includes the Sanghuang extract.

5. The Wnt signaling pathway activity inhibitor according to claim 4, wherein The Sanghuang extract is Sanghuang extraction powder or Sanghuang extraction liquid.

6. The Wnt signaling pathway activity inhibitor according to claim 4, wherein The concentration of the Sanghuang extraction liquid is 1.25 - 5.00 mg / ml.

7. Use of the Wnt signaling pathway activity inhibitor according to any one of claims 4 - 6 in the preparation of a drug for inhibiting the metastasis of lung cancer cells.

8. A drug for inhibiting the metastasis of lung cancer cells, characterized in that, Includes the Wnt signaling pathway activity inhibitor according to claim 7.

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