WO2022171040A1 - Use of chenodeoxycholic acid or derivative thereof in preparation of egfr and/or stat3 inhibitor - Google Patents

Abstract

Use of chenodeoxycholic acid and a derivative thereof in the preparation of an EGFR and/or STAT3 inhibitor. Chenodeoxycholic acid can be used for the treatment or the synergistic treatment of tumors and immune inflammatory diseases. Use of chenodeoxycholic acid and a combination of a derivative thereof and sorafenib in the preparation of a drug for preventing or treating cancer.

Description

Use of chenodeoxycholic acid or its derivatives in the preparation of inhibitors of EGFR and/or STAT3 technical field

The present invention relates to the technical field of medicine, in particular to the use of chenodeoxycholic acid or its derivatives in the preparation of inhibitors of EGFR and/or STAT3.

Background technique

EGFR (epidermal growth factor receptor, referred to as EGFR, ErbB-1 or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3) and HER4 (erbB4). EGFR is a glycoprotein, which is a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. It belongs to a tyrosine kinase type receptor that penetrates through the cell membrane and is located on the surface of the cell membrane. Upon ligand binding to the epidermal growth factor receptor (EGFR), the receptor undergoes dimerization, which involves both the binding of two cognate receptor molecules (homodimerization) and human EGF Binding of different members of the related receptor (HER) tyrosine kinase family (heterodimerization). EGFR dimerization can activate its intracellular kinase pathway, including activation sites such as Y992, Y1045, Y1068, Y1148 and Y1173. This autophosphorylation can guide downstream phosphorylation, including MAPK, Akt and JNK pathways, to induce cell proliferation. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis. Studies have shown that there is high or abnormal expression of EGFR in many solid tumors. Overexpression or mutational activation of EGFR is involved in the development and progression of many human malignancies.

The Signal Transducer and Activator of Transcription (STAT) protein family is a group of related proteins activated by cytokine receptors, which are involved in important biological processes such as proliferation, differentiation, apoptosis and immune regulation. It is currently known that there are 6 members of the STATs family (STAT1-6). Among them, STAT3 is abnormally expressed and continuously activated in various malignant tumors. It directly or indirectly regulates various oncogenes to affect the occurrence and development of tumors. Maintenance is closely related to self-renewal and chemoradiotherapy resistance. There are three main types of activation pathways of STAT3 signaling pathway: the classical JAK-STAT3 signaling pathway; the receptor-dependent tyrosine kinase (RTKs) pathway; and the non-receptor-dependent tyrosine kinase (No-receptor RTKs) pathway. When activated and phosphorylated by upstream signals, STAT3 aggregates into homologous or heterodimers, enters the nucleus and binds to specific sites of target gene promoters to promote its transcription. Normal STAT3 activation is rapid and short-lived, lasting only a few minutes to a few hours, but there are persistent overactivation of STAT3 in various tumors and induce abnormal expression of genes closely related to cell proliferation, differentiation, and apoptosis. Studies have found that many oncogenic signaling pathways are ultimately concentrated on a set of nuclear transcription factors. Targeting a single transcription factor can block the continuous activation caused by changes in a group of upstream genes. Transcription factors are ideal targets for tumor suppression; Therefore, STAT3 has become one of the most promising targets in cancer therapy.

Currently, many small molecules have been developed to target EGFR or STAT3 inhibitors. Some of these inhibitors have been approved for clinical use. However, no relevant studies have reported the use of chenodeoxycholic acid or its derivatives in the preparation of EGFR and/or STAT3 inhibitors.

SUMMARY OF THE INVENTION

Therefore, the technical problem to be solved by the present invention is the use of chenodeoxycholic acid or its derivatives in the preparation of inhibitors of EGFR and/or STAT3.

For this reason, the invention provides the following technical solutions:

Use of chenodeoxycholic acid or derivatives thereof in the preparation of inhibitors of EGFR and/or STAT3.

In said use, the inhibitor is an antagonist.

Optionally, chenodeoxycholic acid or its derivative modulates the expression level of EGFR protein and/or STAT3 protein to decrease.

Optionally, chenodeoxycholic acid or a derivative thereof binds to EGFR protein and/or STAT3 protein, blocks the activation of EGFR protein and/or STAT3 protein, and downregulates the phosphorylation level of EGFR protein and/or STAT3 protein.

Optionally, chenodeoxycholic acid or a derivative thereof regulates the reduction of the total protein expression levels of EGFR protein and STAT3 protein.

The present invention provides use of chenodeoxycholic acid or a derivative thereof in preparing a medicament for preventing or treating diseases related to EGFR and/or STAT3.

Optionally, the disease includes tumor or immune inflammatory disease;

Optionally, the tumor includes but is not limited to liver cancer, breast cancer, lung cancer, gastric cancer, pancreatic cancer, kidney cancer, brain glioma, bone cancer, ovarian cancer, cervical cancer, head and neck cancer, lymphoma, colorectal cancer Cancer, prostate cancer or leukemia; said immune diseases include but are not limited to psoriasis, neurodermatitis, atopic dermatitis, scleroderma, polyneuritis, lupus erythematosus, amyotrophic lateral sclerosis , multiple sclerosis, Alzheimer's disease, vascular dementia, systemic vasculitis, ulcerative colitis, ankylosing spondylitis, sepsis, rheumatoid arthritis and other immune-inflammatory related diseases.

The present invention provides the use of chenodeoxycholic acid or its derivative in combination with sorafenib in preparing a medicament for preventing or treating cancer.

The present invention provides a pharmaceutical composition comprising chenodeoxycholic acid or a derivative thereof and sorafenib;

Optionally, in the pharmaceutical composition, the concentration of chenodeoxycholic acid is 1 μg/ml, and the concentration of sorafenib is 5 μM.

The present invention provides an EGFR and/or STAT3 inhibitor, using chenodeoxycholic acid or a derivative thereof as an active ingredient;

Optionally, a pharmaceutically acceptable carrier is also included.

The technical scheme of the present invention has the following advantages:

1. Use of chenodeoxycholic acid or its derivatives provided by the present invention in the preparation of inhibitors of EGFR and/or STAT3, experiments have shown that chenodeoxycholic acid is a dual antagonist of EGFR and STAT3 tumor targets , which can be used to develop treatments or synergistic treatments for diseases related to these two clear targets, such as tumors and immuno-inflammatory diseases, tumors such as liver cancer, lung cancer, breast cancer, and immuno-inflammatory diseases such as psoriasis.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the mass spectrometry analysis parameter setting in the embodiment 1 of the present invention;

Figure 2 is the test result in Example 2 of the present invention; in the figure 1 is the HepG 2 protein, 2 is the HepG 2 magnetic bead separation supernatant, 3 is the protein after the HepG 2 magnetic bead separation, and 4 is the HepG 2 control after the magnetic bead separation protein;

Fig. 3 is the SPR principle diagram in the embodiment 3 of the present invention;

Fig. 4 is CM5 chip immobilization EGF R protein result in the embodiment of the present invention 3;

Fig. 5 is chenodeoxycholic acid and EGF R protein affinity determination curve in the embodiment of the present invention 3;

Fig. 6 is the result of fitting curve of affinity of chenodeoxycholic acid and EGF R protein in the embodiment of the present invention 3;

Figure 7 is the result of the effect of chenodeoxycholic acid on the expression of EGFR and STAT3 proteins in Example 4 of the present invention; 1-4 in the figure are normal culture, 5-8 are adding chenodeoxycholic acid;

Fig. 8 is the PASI score trend of imiquimod-induced mice 1-7 days in Example 5 of the present invention;

Fig. 9 is the change situation of the mouse skin lesions of each group of mice in Example 5 of the present invention;

Figure 10 is the HE detection result of each group of mice in Example 5 of the present invention;

Figure 11 is the result of WB detection of mouse tissue in Example 5 of the present invention;

Fig. 12 is the WB detection result in the embodiment of the present invention 6;

Figure 13 is the experimental results in Example 7 of the present invention; in the figure 1 is A549, 2 is A549+CDCA, 3 is HePG2, 4 is HePG2+CDCA, 5 is HUVEC, 6 is HUVEC+CDCA, 7 is MDA231, 8MDA231+ CDCA, 9 is MCF-7, 10 is MCF-7+CDCA.

Detailed ways

The following examples are provided for a better understanding of the present invention, and are not limited to the best embodiments, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by combining with the features of other prior art shall fall within the protection scope of the present invention.

If the specific experimental steps or conditions are not indicated in the examples, it can be carried out according to the operations or conditions of the conventional experimental steps described in the literature in this field. The reagents or instruments used without the manufacturer's indication are all conventional reagent products that can be obtained from the market.

Example 1 Isolation of chenodeoxycholic acid-binding protein

It includes the following steps:

1. Coupling chenodeoxycholic acid to magnetic beads to obtain magnetic beads-chenodeoxycholic acid

1.1 Take an appropriate amount of NHS-magnetic beads and place them on a magnetic separation rack. After the supernatant is completely separated from the precipitate, aspirate and discard the supernatant.

1.2 Add twice the volume of Washing Buffer A to the magnetic beads, resuspend for 15s, and place on a magnetic separation rack. After the supernatant and the precipitate are completely separated, aspirate and discard the supernatant.

1.3 Add an equal volume of protein/drug to be coupled to the magnetic beads (chenodeoxycholic acid in this example), resuspend for 30 s, and place at room temperature for 1 h. If magnetic beads are observed to precipitate, resuspend again (hand shake is sufficient). After placing at room temperature, it was placed in a 4°C refrigerator for 1 hour.

1.4. After placing at 4°C, place it on a magnetic separation rack. After the supernatant is completely separated from the precipitate, collect the supernatant and mark it, and store the supernatant at 4°C.

1.5 Add twice the volume of Blocking Buffer to the magnetic beads, resuspend for 30s, and place on a magnetic separation rack. After the supernatant and the precipitate are completely separated, aspirate and discard the supernatant. Repeat this step 4 times.

1.6. Add twice the volume of Blocking Buffer to the magnetic beads, resuspend for 30s, and place at room temperature for 2h. If magnetic beads are observed to precipitate, resuspend again (hand shake is sufficient).

1.7. After placing at room temperature, place it on a magnetic separation rack. After the supernatant is completely separated from the precipitate, aspirate and discard the supernatant.

1.8. Add twice the volume of water to the magnetic beads, mix well, and place them on a magnetic separation rack. After the supernatant and the precipitate are completely separated, aspirate and discard the supernatant.

1.9. Add twice the volume of 1xPBS to the magnetic beads, mix well, and place on a magnetic separation rack. After the supernatant and the precipitate are completely separated, aspirate and discard the supernatant.

1.10. Add an equal volume of 1xPBS to the magnetic beads, mix well, and store in a 4°C refrigerator. Magnetic beads stored overnight at 4°C can be used in subsequent experiments.

2. Cell Culture Total Protein Extraction

2.1 Hepatocellular carcinoma cell line HEPG2 cell recovery: The cryopreserved cells were rapidly dissolved in a 42°C water bath within 1 min, and placed in a T-25 culture flask for culture. The rat brain microvascular endothelial cells (RBMEC) need to be pre-coated with polylysine. T-25 petri dish for culture;

2.2 Daily maintenance of cells: replace fresh growth medium 24 hours after cell recovery, when the cell density increases to more than 80%, digest with 0.25% trypsin for 2 minutes at room temperature, add growth medium to terminate the reaction, centrifuge at 1000 rpm, and resuspend the cell pellet with cryopreservation solution. Suspend cryopreserved cells or resuspend in growth medium to continue culturing;

2.3 Cell collection: After the digestion was terminated as usual, wash twice with sterile PBS, discard the supernatant and store the cell pellet directly.

2.4 Protein extraction: add PBS at the ratio of 1 ml PBS/10 ⁷ cells, and add protease inhibitors at the same time. The resuspended cells were repeatedly contacted with the buffer; after incubation on ice for 30 minutes, the supernatant was collected by centrifugation at 2000 rpm for 20 minutes and stored at -20°C or -70°C or directly for subsequent experiments.

3. BCA method for protein quantification

3.1 Prepare BCA working solution A: B = 50:1;

3.2 Add 50ul BSA standard substance to each well, the concentrations are 2000, 1000, 500, 250, 125, 62.5, 31.3, 0ug/ml;

3.3 Sample loading: Dilute the sample 5-10 times with PBS, 50ul per well;

3.4 Add 150ul BCA working solution to all detection wells, and incubate at 37°C for 50min after mixing;

3.5 Read the OD value at the wavelength of 570nm by the microplate reader;

3.6 Calculate the total protein concentration in the sample automatically through the standard concentration and OD value software.

4. Protein Isolation

4.1 Mix 100ul total protein extract with compound-magnetic beads in equal volume and incubate at 4°C for 30 minutes, in which the total protein concentration should be above 4mg/ml.

4.2 Separate the magnetic beads by a magnetic stand for 1 minute and collect the supernatant.

4.3 Wash twice with PBS, and separate the magnetic beads for 1 minute each time.

4.4 Discard the PBS washing solution and add 50ul volume of PBS for use.

5 Protein electrophoresis

5.1 Preparation of loading buffer: add 4×LDS and 10×Reducing Agent (RA) buffer according to the sample volume to make the final concentration of LDS and RA to be 1×, and denature in a boiling water bath for 5min.

5.2 Loading amount of protein sample to be detected: 10 μl/well,

The loading sequence is: Marker, total protein, protein after separation by chenodeoxycholic acid magnetic beads, and protein after separation by BSA magnetic beads;

5.3 Electrophoresis conditions: Select the electrophoresis buffer according to the size of the detected protein. When the protein size is >25KD, select the MOPS buffer system, and when the protein size is <25KD, select the MES buffer system.

5.4 Constant voltage of 90V, constant voltage of 120V after about 20min, and the stop time of electrophoresis is determined by pre-stained protein marker.

6. Coomassie Brilliant Blue Staining

6.1 Take out the electrophoresis gel and rinse it with ultrapure water (every step below needs to be performed on a shaking table).

6.2 Add an appropriate amount of Coomassie brilliant blue staining solution to fully cover the gel and stain for 1-4 hours.

6.3 After dyeing, remove the dyeing solution, add an equal volume of decolorizing solution, and decolorize for 1-8 hours. The time can be adjusted according to the strip.

6.4 After decolorization, soak the gel in pure water, take pictures or scan to save the pictures, and confirm whether the compound is separated and the protein bound to it is obtained by comparing the difference between the total protein and the protein band after separation by magnetic beads.

6.5 Perform mass spectrometry identification on the electrophoresis gel to obtain possible binding proteins, and further verify the compound binding proteins by techniques such as immunological methods (immunoblotting or flow cytometry).

7. Mass Spectrometry Identification

7.1 Sample preparation: The status is protein separation strips.

7.2 In-gel enzymatic hydrolysis: freeze-dry the sample after complete decolorization, add 40 μl Trypsin buffer, 37°C for 16-18h.

7.3 Capillary HPLC

Each sample was separated using an Easy nLC 1200 HPLC liquid system with nanoliter flow rates. Buffer: solution A is 0.1% (volume percent) formic acid aqueous solution, and solution B is acetonitrile solution containing 0.1% (volume percent) formic acid. The column was equilibrated with 95% (volume percent) solution A. The sample was loaded into the mass spectrometer pre-column C18 trap column (C18 3μm 0.10×20mm) by the autosampler, and then separated by the analytical column C18 column (C18 1.9μm 0.15×120mm) with a flow rate of 600nl/min. The relevant liquid phase gradients are as follows in Table 1:

Table 1. Liquid Gradient

time (min)	Liquid A (volume percentage)	Liquid B (volume percentage)	Flow rate (nL/min)
0	94%	6%	600
8	90%	10%	600
38	73%	27%	600
54	64%	36%	600
55	5%	95%	600
60	5%	95%	600

7.4 Identification by mass spectrometry

Each sample was subjected to mass spectrometry analysis using a Q Exactive mass spectrometer (Thermo Sceientific) after separation by capillary high performance liquid chromatography. See Figure 1 for parameter settings.

7.5 Data Analysis

Raw mass spectral data

The raw data of mass spectrometry analysis were RAW files, and the software Sequest and Proteome Discoverer (Thermo Scientific) were used for library search and identification.

Search library parameter settings

When searching the database, submit the RAW file to the Sequest server through Proteome Discoverer, select the established database, and then perform database search. The relevant parameters are listed in Table 2 below.

Table 2. Search database parameters

The result filtering parameter is: Peptide FDR≤0.01.

Identification results

The experimental results folder "Mass Spectrometry Identification Results" contains "protein.xlsx" and "peptide.xlsx", which display a visual report. Conclusion: After data analysis, the top 20% proteins of the identification results were selected for literature search and correlation analysis, and the following proteins were finally selected as the ten most relevant proteins, as shown in Table 3 below.

Table 3. Identification results

Annotation of experimental results

Table 4-1 protein (protein identification form) header and annotation

Table 4-2 Peptide (peptide identification form) header and notes

Example 2 Magnetic Bead IP Verification of Chenodeoxycholic Acid Binding Protein

1. Test reagents: (see Table 5 below)

table 5

Reagent name	Manufacturer and article number
Magnetic beads	Thermo Cat.No.88831

Note: Other reagents are prepared from analytical grade reagents

2. Test equipment: (see table 6 below)

Table 6

equipment name	Instrument model	factory
centrifuge	GT16	Xiangyi
vertical mixer	E1677	Topson
Microplate reader	9602	Plan Group

3. Test method

1. Cell protein extraction: The cell culture method is the same as "2. Cell culture total protein extraction" in Example 1, adding PBS at a ratio of 1 ml PBS/10 ⁷ cells, and adding protease inhibitors at the same time. The resuspended cells were repeatedly contacted with the buffer; after 30 minutes of incubation on ice, the supernatant was collected by centrifugation at 2000 rpm for 20 minutes and stored at -20°C or -70°C or directly for subsequent experiments.

2. BCA method for protein quantification

2.1 Prepare BCA working solution A solution: B solution = 50:1

2.2 Add 50ul BSA standard substance to each well, the concentrations are 2000, 1000, 500, 250, 125, 62.5, 31.3, 0ug/ml;

2.3 Sample loading: Dilute the sample 5-10 times with PBS, 50ul per well;

2.4 Add 150ul BCA working solution to all detection wells, and incubate at 37°C for 50min after mixing;

2.5 Read the OD value at the wavelength of 570nm by the microplate reader;

2.6 Automatically calculate the total protein concentration in the sample through the standard concentration and OD value software.

3. Protein separation by drug magnetic beads and WB detection

3.1 Mix 200ug total protein extract with an equal volume of drug-magnetic beads (magnetic beads-chenodeoxycholic acid), and incubate at 4°C for 60 minutes. The protein separation buffer system is PBS buffer.

3.2 The magnetic beads were separated by a magnetic stand for 1 minute, and the supernatant was collected as the IP supernatant.

3.3 Wash 2 times with PBS, and separate the magnetic beads for 1 minute each time.

3.4 Discard the PBS washing solution, separate the protein-chenodeoxycholic acid-magnetic beads by a magnetic stand, and reconstitute the protein-chenodeoxycholic acid-magnetic beads precipitation with SDS electrophoresis loading buffer, and the reconstituted volume is 1/4 total protein volume.

4. WB detection

4.1 The amount of protein sample to be detected: 10ul per well;

4.2 Loading sequence: total protein, magnetic bead supernatant, magnetic bead precipitation, control magnetic bead precipitation

4.3 Summary table of antibody dilution (see Table 7 below)

Table 7

name	Manufacturer's number	target protein size	dilution ratio	species
EGFR	CST, 2232,	175KD	1:1000	rabbit

Stat3

CST, 12640

79,86KD

1:1000

rabbit

4.4 Protein extraction

Pre-cool RIPA protein extraction reagent, add protease inhibitor, add 0.1M PMSF stock solution before protein extraction, the final PMSF concentration is 1mM, and add protease inhibitor at the same time;

Add 1 ml of lysis solution to the cell pellet with 1×10 ⁷ cells, and suspend the cells sufficiently by pipetting with a pipette tip. After completion, incubate on ice for 20 min, centrifuge at 4° C., 13000 rpm, 20 min. After centrifugation, the supernatant was taken and stored in aliquots for testing.

4.5 Protein quantification by BCA method

Prepare BCA working solution A solution: B solution = 50:1, dilute each extracted BSA standard;

Dilute the sample 5-10 times with PBS, add 150ul BCA working solution, and incubate at 37°C for 30min or room temperature for 60min after mixing, and read the OD value with the 570nm wavelength filter of the microplate reader;

4.6 Adjustment of protein concentration: RIPA adjusts the protein concentration. After adding 4× reducing sample buffer, the final concentration of the sample is 2 mg/ml, and it is boiled and denatured for 5 min.

4.7 WB detection experiment of target protein

Loading amount of protein sample to be detected: 10ug/well; electrophoresis conditions: select the electrophoresis buffer according to the size of the detected protein, when the protein size is >25KD, select the MOPS buffer system, when the protein size <25KD, select the MES buffer system; Constant voltage of 90V, constant voltage of 120V after about 20min, the stop time of electrophoresis is determined by pre-stained protein marker; fast membrane transfer instrument: automatic program, 0.45um pore size PVDF membrane, membrane transfer time 12 minutes; blocking: the membrane is completely immersed in 5% Shake gently for 30 minutes at room temperature in nonfat dry milk-TBS; primary antibody incubation: dilute the primary antibody with 5% nonfat dry milk-TBS, incubate at room temperature for 10 minutes, and leave at 4°C overnight; take out the membrane from 4 degrees the next day and incubate at room temperature for 30 minutes; wash Membrane: wash the membrane 5 times with TBST, 3 min each time;

Secondary antibody incubation: Dilute the secondary antibody with 5% nonfat dry milk-TBS, goat anti-rabbit IgG (H+L) HRP, 1:20000, shake gently at room temperature for 40min, wash the membrane: wash the membrane 6 times with TBST, 3min each time; use ECL The PVDF membrane was soaked and reacted for 3-5min, and the exposure device was exposed for 1s and 60s, respectively. 5. Test results

The test results are shown in Figure 2. If the target band in the total protein and the magnetic bead precipitation sample is positive, the drug and the target protein are combined with each other. If the target band in the magnetic bead precipitation sample is negative, the drug does not interact with the target protein. Binding, the target protein in the supernatant of the magnetic beads may be positive or negative due to protein excess. The target band in the control magnetic bead precipitation should be negative, weakly positive or weaker than the magnetic bead precipitation sample. Conclusion: The drug chenodecholic acid is an antagonist of EGFR and STAT3 proteins, and can directly bind to EGFR and STAT3 proteins.

Embodiment 3 SPR experiment of chenodeoxycholic acid and EGFR protein

1. The principle of surface plasmon resonance

The principle of Surface Plasmon Resonance (SPR) is that when the incident light is incident on the interface of two media with different refractive indices (such as gold or silver coating on the glass surface) at a critical angle, it can cause the resonance of metal free electrons. As a result, the electrons absorb the light energy, so that the reflected light is greatly weakened within a certain angle. Among them, the incident angle at which the reflected light disappears completely within a certain angle is called the SPR angle. The SPR varies with the surface refractive index, which in turn is proportional to the mass of biomolecules bound to the metal surface. Therefore, the specific signal of the interaction between biomolecules can be obtained by obtaining the dynamic change of the SPR angle during the biological reaction (as shown in Figure 3).

2. Purpose

The purpose of this study was to determine the affinity of chenodeoxycholic acid with human EGFR protein using SPR technique.

3. Materials

3.1 Test product information:

Table 8 Information on test substances and protein

Table 8 Continued

article number	batch number	sample date
NA	151010	20200727
EGR-H5222	A1541-191015F1-Bulk	20200727

3.2 Equipment consumables information:

BiacoreT200 (GE Healthcare Life Sciences, GE)

Chip Series S Sensor Chip CM5 (Item No.: BR-1008-30, GE);

3.3 Reagent information:

Table 9 Reagent Information Sheet

4. Method:

4.1 Reagent preparation

Running reagents: containing 2 mM potassium dihydrogen phosphate (KH ₂ PO ₄ ), 137 mM sodium chloride (NaCl), 10 mM disodium hydrogen phosphate dodecahydrate (Na ₂ HPO ₄ .12H ₂ O), 2.7 mM potassium chloride (KCl) ), 0.05% (volume) Tween-20 (Tween-20), 5% DMSO;

Amino Coupling Kit (Cat. No. BR100050, GE), including: 115mg N-hydroxysuccinimide (NHS), 750mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt acid (EDC) and 10.5 mL of 1 M ethanolamine (pH 8.5). Add 10 mL of deionized water to each tube of EDC and NHS, respectively, and store in aliquots at -18°C to a lower temperature. The shelf life is two months. (Refer to GE Amino Coupling Instruction Manual "22-0510-62AG").

4.2 Chip Preparation

EGFR protein was diluted to 30 μg/mL with immobilization reagent (10 mM sodium acetate, pH 4.5). First, the surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL/min for 420 s. Next, 30 μg/mL of EGFR protein was injected into the experimental channel (FC4) at a flow rate of 10 μL/min in a fixed amount of about 10000 RU (resonance signal). Finally, the chip was blocked with 1 M ethanolamine at 10 μL/min for 420 s. The reference channel (FC3) performs the same operation as the test channel (FC4).

4.3 Solvent correction

Solvent correction was performed with 4.5% and 5.8% DMSO (Table 10) and calibration curves (Table 11) prepared.

Table 10, 4.5% and 5.8% DMSO configuration methods

	4.5% DMSO	5.8% DMSO
1 x PBS	9.5mL	9.5mL
100% DMSO	0.45mL	0.58mL
Final volume	~10mL	~10mL

Table 11 Calibration curve configuration method

Buffer/Vial

1

2

3

4

5

6

7

8

4.5% DMSO (μL)	0	200	400	600	800	1000	1200	1400
5.8% DMSO (μL)	1400	1200	1000	800	600	400	200	0

4.4 Analyte Multicycle Analysis

Chenodeoxycholic acid was first diluted 20 times with dilution buffer (1×PBS, 0.05%Tween20) to make the DMSO content 5%, and then diluted with running reagent (1×PBS, 0.05%Tween20, 5%DMSO) , the concentrations were 500, 250, 125, 62.5, 31.25, 15.625, 7.813, and 0 μM, respectively. The diluted chenodeoxycholic acid was sequentially injected into the experimental channel and the reference channel at a flow rate of 30 μL/min, the binding time was 60s, and the dissociation time was 90s.

4.5 Others:

All operation steps are carried out in the running reagent, and the analytical reagents of SPR need to be filtered and degassed.

5. Results

Data analysis: KD values for each antibody were calculated using Biacore T200 analysis software, see Table 12 below. The reference channel (FC3) is used for background subtraction. The results of immobilizing EGF R protein on the CM5 chip are shown in Figure 4, and the results of the affinity determination curve and fitting curve between chenodeoxycholic acid and EGFR protein are shown in Figure 5 and Figure 6.

Table 12 Affinity test results of chenodeoxycholic acid and EGF R protein

6 Conclusion

EGFR, as the most popular tumor target, was re-validated by SPR experiment. The selected EGFR is a commercial protein, which is a recombinant protein, and the expressed region is the extracellular ligand binding domain. The experimental results show that: Chenodeoxycholic acid can directly bind to the extracellular ligand-binding domain of human EGFR protein detected by Biacore T200, and the mechanism of its drug activity is further clarified that it binds to the extracellular ligand-binding domain of EGFR protein to block EGFR activation. exert drug activity.

Example 4 The effect of chenodeoxycholic acid on the expression of EGFR and STAT3 proteins

The cell culture method is the same as "2. Extraction of total protein from cell culture" in Example 1

1.1 Cell grouping

1.1.1 Cell line: HePG2 (human hepatocellular carcinoma cell line) after routine digestion was terminated, adjust the cell density to 1×10 ⁶ cells/ml, spread it in a 100mm diameter cell culture dish, 10ml/dish, and continue to routinely culture for 24h.

1.1.2 When the cell confluence reaches 60-70%, discard the original medium, replace the medium containing 5 μg/ml chenodeoxycholic acid, and set up a normal culture group at the same time, and continue to culture for 1 h;

1.1.3 Cell collection: After the digestion is terminated as usual, wash twice with sterile PBS, discard the supernatant and store the cell pellet directly.

1.2 Protein extraction

1.2.1 Pre-cool RIPA protein extraction reagent, add 0.1M PMSF stock solution before protein extraction, the final PMSF concentration is 1mM, and add protease and phosphorylated protease inhibitors at the same time;

1.2.2 The cell pellet was resuspended in an appropriate amount of lysis buffer, and ice bathed for 30 minutes;

1.2.3 Centrifuge at 10000rpm at 4°C for 10min, collect the supernatant, which is the total protein, and store in aliquots for testing.

1.3 Protein quantification by BCA method

1.3.1 Prepare BCA working solution A solution: B solution = 50:1

1.3.2 Add 25ul BSA standard to each well, the concentrations are 2000, 1000, 500, 250, 125, 62.5, 31.3, 0ug/ml;

1.3.3 Sample loading: Dilute the sample 5-10 times with PBS, 25ul per well;

1.3.4 Add 150ul BCA working solution to all detection wells, and incubate at 37°C for 30min after mixing;

1.3.5 Read the OD value at the wavelength of 570nm by the microplate reader;

1.3.6 Automatically calculate the total protein concentration in the sample through the standard concentration and OD value software.

1.4 WB detection

1.4.1 Adjustment of protein concentration: Calculate and adjust the protein concentration, add 4 × LDS and 10 × RA buffer to make the concentration of each sample the same, and denature in a boiling water bath for 5 minutes.

1.4.2 Loading amount of protein sample to be detected: 10ul per well, containing 13ug protein;

1.4.3 Electrophoresis conditions: Select the electrophoresis buffer according to the size of the detected protein. When the protein size is more than 25KD, select the MOPS buffer system. When the protein size is less than 25KD, select the MES buffer system; constant voltage 90V, constant voltage after about 20min 120V, the stop time of electrophoresis is determined by pre-stained protein marker;

1.4.4 Wet transfer method, transfer conditions: 0.45um pore size PVDF membrane, fully soaked in methanol and equilibrium solution before use; when the protein molecular weight> 90KD, the membrane transfer instrument is set to Long mode, 30KD < protein molecular weight < 90KD, The membrane transfer instrument is set to Stand mode, and when the protein molecular weight is less than 30KD, the membrane transfer instrument is set to Short mode;

1.4.5 Blocking: immerse the membrane completely in 5% nonfat dry milk-TBS and shake gently for 30min at room temperature;

1.4.6 Primary antibody incubation: Immerse the membrane in 5% skimmed milk powder-TBS diluted primary antibody, make corresponding records, incubate at room temperature for 30 minutes, and place at 4°C overnight;

Table 13. Summary of antibody dilutions

name	Manufacturer's number	target protein size	dilution ratio	species
EGFR	CST, 2232,	175KD	1:1000	rabbit
P-EGFR	CST, 3777	175KD	1:1000	rabbit
STAT3	CST, 12640	79,86KD	1:1000	rabbit
P-STAT3	CST, 9145	79,86KD	1:1000	rabbit

1.4.7 The next day, take out the membrane from 4°C, incubate at room temperature for 30 minutes, and wash the membrane: TBST wash the membrane 3 times, 5 minutes each time;

1.4.8 Secondary antibody incubation: Immerse the membrane in 5% skimmed milk powder-TBS diluted secondary antibody, at a dilution ratio of 1:5000, shake gently at room temperature for 1-4 hours, wash the membrane: wash the membrane with TBST 3 times, 5 min each time;

1.4.9 After adding ECL to the PVDF film, the reaction was carried out in the dark for 3-5 minutes, and the exposure time was 1s and 60s with the eBlot exposure meter;

1.4.10 Select images with appropriate exposure time, and perform gray value analysis through Image J software.

1.5 Formal experiment of internal reference protein WB

1.5.1 Wash the membrane with Stripping Buffer, and wash the membrane at 37°C for 30 minutes (if the molecular weight difference between the target protein and the internal reference protein is more than 10K, this step of stripping buffer can be omitted);

1.5.2 Wash the membrane: wash the membrane 3 times with deionized water;

1.5.3 Wash the membrane: wash the membrane 3 times with TBST, 3 min each time;

1.5.4 Fully immerse the membrane in 5% skimmed milk powder-TBS and shake gently for 30min at room temperature;

1.5.5 Incubate the internal reference: select the appropriate internal reference antibody according to the type of sample, dilute the antibody with 5% nonfat dry milk-TBS, 1:5000-10000, incubate at room temperature for 30 minutes, then incubate at 4°C overnight or at 37°C for 2h;

1.5.6 Wash the membrane: wash the membrane 3 times with TBST, 5 min each time;

1.5.7 Secondary antibody incubation: Dilute the secondary antibody with 5% nonfat dry milk-TBS, goat anti-mouse IgG(H+L) HRP, 1:5000, shake gently at room temperature for 1h;

1.5.8 Wash the membrane: wash the membrane 3 times with TBST, 5 min each time;

1.5.9 After adding ECL to the PVDF film, the reaction was performed in the dark for 3-5 minutes, and the exposure time was 1s and 60s with the eBlot exposure instrument;

1.5.10 Select images with appropriate exposure time, and perform gray value analysis through Image J software.

2. Experimental results

The experimental results are shown in Figure 7. The protein expressions of EGFR and STAT3 were down-regulated after adding chenodeoxycholic acid. Conclusion: WB results verify that the drug chenodeoxycholic acid acts as a dual antagonist of STAT3 and EGFR proteins, and the mechanism of its drug activity is as follows:

(1) Chenodeoxycholic acid blocks the activation of EGFR and STAT3 by binding to EGFR and STAT3 proteins, and the phosphorylation levels of EGFR and STAT3 proteins are down-regulated;

(2) The expression levels of the total proteins of EGFR and STAT3 proteins were also down-regulated due to the influence of the drug activity of chenodeoxycholic acid.

Example 5 Chenodeoxycholic acid in the treatment of psoriasis demonstrates the therapeutic effect of CDCA as a STAT3 antagonist on immune inflammatory diseases

As a classic immune inflammatory disease, the pathogenesis of psoriasis is still unclear and related to both genetic immunity and inflammatory pathways. Many literatures have proved that STAT3 is a potential target of psoriasis, and STAT3 is the most downstream of the classic inflammatory pathway-IL6 pathway. indicator, therefore, using psoriasis as an example to demonstrate the value of chenodeoxycholic acid in the treatment of immune-inflammatory diseases.

1. Experimental method

1. Animal experiments:

1.1. 24 male BALB/c mice, weighing 18-20 g, 6-8 weeks old, methotrexate tablets (Shanghai Xinyi Pharmaceutical Co., Ltd.), 5% imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd.) Industry LLC), animal modeling, grouping, and drug administration.

1.2. Before the experiment, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg), the back was dehaired, with an area of about 2cm×3cm, and the mice were reared in a single cage after dehairing.

1.3. According to the random number table method, the mice were randomly divided into blank control group (C group), model group (M group), chenodeoxycholic acid drug group (chenodeoxycholic acid group referred to as E group), methylamine Pterin group (MTX group), 6 mice in each group: mice in group C were smeared with Vaseline on their backs daily, and mice in the other three groups were smeared with 5% imiquimod cream 62.5 mg daily on their backs; Once, 0.2 mL each time, for 6 consecutive days, group C and group M were given 0.2 mL of 0.9% (mass fraction) sodium chloride injection, and group MTX was given 1 mg·kg ^-1 ·d ^-1 methotrexate tablet solution 0.2 mL; Group E was given 0.2 mL of 1 mg·kg ^-1 ·d ^-1 drug solution, respectively.

2. Photograph and score the lesion area

Photographs were taken every day and the PASI score was performed at the same time, the change trend of the PASI score of the mice in each group was drawn, and the changes of the skin lesions of the mice were dynamically observed.

3.HE detection

3.1 Measure the thickness of the skin lesions of the mice. On the 7th day of the modeling, the back skin of the mice was fixed in formalin, dehydrated and embedded;

3.2 Slice with a microtome, and the thickness of the paraffin section is 4 microns;

3.3 Bake the sheet at 60℃ for 1 hour;

3.4 Xylene I was dewaxed for 10 minutes;

3.5 Xylene II dewaxing for 10 minutes;

3.6 Gradient alcohol to water: 100% alcohol for 5 minutes, 100% alcohol for 5 minutes, 95% alcohol for 5 minutes, 80% alcohol for 5 minutes, and tap water for 5 minutes;

3.7 Hematoxylin staining of nuclei for 5min (newly configured hematoxylin depends on the situation);

3.8 Rinse the excess hematoxylin with running water and differentiate in the differentiation solution for 1-2 seconds;

3.9 Rinse with running water for 5 minutes;

3.10 Eosin staining for 10 minutes;

3.11 Gradient alcohol rehydration: 80% alcohol for 1-2 seconds, 95% alcohol for 10-20 seconds, 100% alcohol for 3 minutes, 100% alcohol for 5 minutes, xylene II transparent for 5 minutes, xylene I transparent for 5 minutes (if not completely transparent) , the time can be appropriately extended);

3.12 Seal the film with neutral gum;

3.13 Observe and take pictures under the microscope.

4. WB detection of mouse tissue: the method is the same as the WB detection method in Example 4.

2. Experimental results

Analysis of results: The results are shown in Figure 11 ("+" in the figure represents the drug group with chenodeoxycholic acid added, and "-" represents the M group without chenodeoxycholic acid), which proves that the activation of STAT3 protein, a popular target of psoriasis, is affected by Blocking, phosphorylated stat3 expression was downregulated. As shown in Figures 8-10, pharmacodynamic indicators (pathology, PASI score, phenotype after medication) proved that the drug chenodeoxycholic acid has a therapeutic effect on psoriasis model mice, and is better than the positive drug.

Example 6 Selection of liver cancer cell line HEPG2 for synergistic treatment of chenodeoxycholic acid and sorafenib to prove that it is used as an EGFR antagonist in the treatment of tumors

The test reagents are shown in Table 14 below:

Table 14

The test equipment is shown in Table 15:

Table 15

equipment name	Instrument model	factory
CO2 incubator	MCO-15AC type	SANYO
Inverted microscope	XDS-2B type	Chongqing Optoelectronics
clean bench	SW-CJ-1D type	Jiangsu Tongjing
Vortex Shaker	QL-902	Haimen Qilin Bell Instrument Manufacturing Co., Ltd.
Microplate reader	MultiSkan3	Therno scientific
Electrophoresis	Mini Ge Tank	Therno scientific
Film transfer instrument	Mini Blot Module	Therno scientific
centrifuge	TG-16	Xiangyi

experiment method

1. Cell culture and routine maintenance

1.1 Cell recovery

Quickly dissolve the frozen cells in a 42°C water bath within 1 min, and place them in a T-25 culture flask for culture;

1.2 Daily maintenance of cells

24h after recovery, replace with fresh growth medium. When the cell density increases to more than 80%, digest with 0.25% trypsin for 2 min at room temperature, add growth medium to terminate the reaction, centrifuge at 1000 rpm, and resuspend the frozen cells in the cell pellet with cryopreservation solution or use Resuspend in growth medium and continue to culture; Note: HepG2 cell growth medium: 90% high glucose 1640 medium + 10% fetal bovine serum (FBS)

Cell freezing medium: 50% growth medium + 40% FBS + 10% DMSO

2. Cell Model

2.1 After the cells are cultured to a sufficient amount, digest with 0.25% trypsin for 2 minutes at room temperature, stop the cells in the growth medium, and centrifuge at 1000 rpm for 5 minutes;

2.2 Adjust the density of 1640 cells to 1×10 ⁵ cells/ml with growth medium, and spread the cells in a 24-well cell culture plate, 500 μl per well; for cell scratching experiments, cells are spread in a 6-well cell culture plate , 1.5ml per well; for WB detection, cells were spread in a 100mm diameter cell culture dish, 8ml/dish.

2.3 is divided into the following 4 groups:

A normal culture group

B treated cells with 1μg/ml chenodeoxycholic acid for 24h

C cells were treated with 5 μM sorafenib for 24 h

D. Cells were treated with 1μg/ml chenodeoxycholic acid + 5μM sorafenib for 24h

3. The WB detection method is the same as the WB detection method in Example 4.

4. MTT value-added detection

4.1 After the cell culture results, discard the supernatant and add 10μl MTT and 100ul serum-free medium respectively;

37℃ for 4h;

4.2 Discard the reaction solution, add 10% SDS to dissolve the cell pellet, and read the absorbance value at 570 nm;

4.3 Calculation of cell proliferation inhibition rate by OD value

Cell proliferation inhibition rate=100%×(OD value of control group-OD value of experimental group)/OD value of control group

5. Transient cell transfection and sequence screening

5.1. After the cells were terminated by routine digestion, adjust the cell density to 1×10 ⁶ cells/ml, spread them in a 6-well cell culture plate at 1500 μl/dish, and continue to routinely culture for 24 hours.

5.2. Add 2ug of shEGFR plasmid (origene, TR320326) to 100μl of serum-free medium and mix well, as a solution;

5.3. Add 100 μl serum-free medium to 4 μl Lipo2000 and mix well, as liquid b;

5.4. After mixing liquid a and liquid b, place at room temperature for 15 minutes;

5.5. Add 800 μl of serum-free medium, discard the original cell culture medium, and slowly add the mixture of liquid a and liquid b to the cells;

5.6. Change the growth medium after 4-8h;

After 48 hours, the cells were collected by WB method (the same method as before) to detect the expression of stat3 to confirm the knockdown sequence.

6-Cell Stable Cell Line Construction

6.1. Repeat steps 2.1-2.6;

6.2. 24h after transfection, the cells were digested and resuspended, and then plated in a 96-well cell culture plate, 3 cells/well, 100 μl/well;

6.3. 48h after transfection, add growth medium containing 2μg/ml Puromycin, continue to culture, replace the medium every 3-5 days, and continue to culture for more than 8 weeks.

7. Cell Grouping

A HepG2-shNC-con

B HepG2-shNC+1μg/ml chenodeoxycholic acid

C HepG2-shNC 5 μM sorafenib

D HepG2-shNC+1μg/ml chenodeoxycholic acid+5μM sorafenib

E HepG2-shEGFR-con

F HepG2-shEGFR+1μg/ml chenodeoxycholic acid

G HepG2-shEGFR+5μM sorafenib

H HepG2-shEGFR+1 μg/ml chenodeoxycholic acid and 5 μM sorafenib

8. The method of MTT detection of cell proliferation in stable transfection cell line is the same as above

9. Experimental results

9.1. The WB test results are shown in Figure 12

9.2. Cell proliferation test results

Table 16. Results of the value-added test of chenodeoxycholic acid combined with sorafenib

Table 17. Comparative detection results after transfection of shEGFR

Result analysis:

(1) WB results showed that the addition of chenodeoxycholic acid blocked EGFR activation, and the phosphorylation level of EGFR decreased;

(2) The cell proliferation results of chenodeoxycholic acid combined with sorafenib showed that chenodeoxycholic acid could improve the inhibitory effect of sorafenib on tumor cells;

(3) Transfection and knockdown of EGFR, and the results of cell proliferation in stable transfection cell lines showed that knockdown of EGFR could also improve the inhibitory effect of sorafenib on tumor cells;

(4) Transfection and knockdown of EGFR, the results of cell proliferation of stable transfection cell line showed that the combination of chenodeoxycholic acid and sorafenib did not improve the inhibitory effect of sorafenib on cells after knockdown of EGFR, proving that chenodeoxycholic acid Acid exerts pharmacological activity by antagonizing EGFR.

CONCLUSION: Chenodeoxycholic acid, as an EGFR antagonist, has a synergistic therapeutic effect on tumors, which can improve the efficacy of first-line treatment drugs such as sorafenib, and improve the sensitivity of sorafenib. The mechanism of action of the drug is to antagonize the EGFR protein. achieved by inhibiting EGFR activation. Example 7 Universality of chenodeoxycholic acid CDCA as an EGFR antagonist to block EGFR activation

1. The experimental reagents are shown in Table 18:

Table 18

2. The experimental instruments are shown in Table 19 below:

Table 19

equipment name	Instrument model	factory
Vortex Shaker	MIX-28	Ningbo Yinzhou Qun'an Experimental Instrument Co., Ltd.
Cryogenic benchtop centrifuge	TGL-16	Hunan Xiangyi Laboratory Instrument Development Co., Ltd.
ELISA analyzer	DNM-9602 type	Beijing Pulang New Technology Co., Ltd.
Electrophoresis	Mini Ge Tank	Thermo scientific
Film transfer instrument	eBlot ™ L1	GenScript
shaker	NYC-80	Taizhou Nuomi Medical Technology Co., Ltd.
Exposure meter	eBlot	Shanghai Ebot

3. Experimental method

3.1. Recovery of A549, HePG2, HUVEC, MDA231, MCF-7 cells: quickly dissolve the frozen cells in a 42°C water bath within 1 min, and place them in a T-25 culture flask for culture

3.2. Daily maintenance of cells: replace the fresh growth medium 24h after cell recovery, and wait until the cell density increases.

When added to more than 80%, 0.25% trypsin was digested at room temperature for 2 minutes, and then the growth medium was added to terminate the reaction, centrifuged at 1000 rpm, and the cell pellet was resuspended in cryo-preserved cells or resuspended in growth medium to continue culturing;

Note: A549, HePG2, MDA231, MCF-7 cell culture medium: 90% (volume percentage) 1640 medium + 10% (volume percentage) fetal bovine serum (FBS);

HUVEC cell culture medium: 90% (volume percent) DMEM/F12 medium + 10% (volume percent) fetal bovine serum (FBS);

Cell freezing solution: 50% (volume percentage) growth medium+40% (volume percentage) FBS+10% (volume percentage) DMSO;

3.3 Cell grouping

3.3.1 Cell lines: A549 (human lung cancer cell line), HePG2 (human liver cancer cell line), MDA231 (human breast cancer cell line), HUVEC (human umbilical vein endothelial cell), MCF-7 (human breast cancer cell line) After the conventional digestion was terminated, the cell density was adjusted to 1×10 ⁶ cells/ml, and the cells were spread in a cell culture dish with a diameter of 100 mm at 10 ml/dish, and the conventional culture was continued for 24 h.

3.3.2 When the cell confluence reaches 60-70%, discard the original medium, replace the medium containing 5 μg/ml chenodeoxycholic acid, and set up a normal culture group at the same time, and continue to culture for 1 h;

3.3.3 Cell collection: After the digestion is terminated as usual, wash twice with sterile PBS, discard the supernatant and store the cell pellet directly.

3.4 Protein extraction

3.4.1 Pre-cool RIPA protein extraction reagent, add 0.1M PMSF stock solution before protein extraction, the final PMSF concentration is 1mM, and add protease and phosphorylated protease inhibitors at the same time;

3.4.2 The cell pellet was resuspended with an appropriate amount of lysis buffer, and ice bathed for 30 minutes;

3.4.3 Centrifuge at 10,000 rpm at 4°C for 10 min, collect the supernatant, which is the total protein, and store in aliquots for testing.

3.5 Protein quantification by BCA method

3.5.1 Prepare BCA working solution A solution: B solution = 50:1

3.5.2 Add 25ul BSA standard substance to each well, the concentrations are 2000, 1000, 500, 250, 125, 62.5, 31.3, 0ug/ml;

3.5.3 Sample loading: Dilute the sample 5-10 times with PBS, 25ul per well;

3.5.4 Add 150ul BCA working solution to all detection wells, and incubate at 37°C for 30min after mixing;

3.5.5 Read the OD value at the wavelength of 570nm by the microplate reader;

3.5.6 Automatically calculate the total protein concentration in the sample through the standard concentration and OD value software.

3.6 WB detection

3.6.1 Adjustment of protein concentration: Calculate and adjust the protein concentration, add 4×LDS and 10×RA (Reducing Agent) buffer to make the concentration of each sample the same, and denature in a boiling water bath for 5 minutes.

3.6.2 Loading amount of protein sample to be detected: 10ul per well, containing 13ug protein;

3.6.3 Electrophoresis conditions: select the electrophoresis buffer according to the size of the detected protein. When the protein size is more than 25KD, select the MOPS buffer system. When the protein size is less than 25KD, select the MES buffer system; constant voltage 90V, constant voltage after about 20min 120V, the stop time of electrophoresis is determined by pre-stained protein marker;

3.6.4 Wet transfer method, transfer conditions: PVDF membrane with a pore size of 0.45um, fully soaked in methanol and equilibrium solution before use; when the protein molecular weight is greater than 90KD, the transfer instrument is set to Long mode, and when 30KD < protein molecular weight <90KD, The membrane transfer instrument is set to Stand mode, and when the protein molecular weight is less than 30KD, the membrane transfer instrument is set to Short mode;

3.6.5 Blocking: completely immerse the membrane in 5% nonfat dry milk-TBS and shake gently for 30min at room temperature;

3.6.6 Primary antibody incubation: Immerse the membrane in 5% skimmed milk powder-TBS diluted primary antibody, make corresponding records, incubate at room temperature for 30 minutes, and leave at 4°C overnight; see Table 20 below;

Table 20. Summary of antibody dilutions

name	Manufacturer's number	target protein size	dilution ratio	species
P-EGFR	CST, 3777,	175KD	1:1000	rabbit

3.6.7 The next day, take out the membrane from 4 degrees, incubate at room temperature for 30 minutes, and wash the membrane: TBST wash the membrane 3 times, 5 minutes each time;

3.6.8 Secondary antibody incubation: Immerse the membrane in 5% skimmed milk powder-TBS diluted secondary antibody at a dilution ratio of 1:5000, shake gently at room temperature for 1-4 hours, wash the membrane: wash the membrane with TBST 3 times, 5 min each time;

3.6.9 After adding ECL to the PVDF film, the reaction was performed in the dark for 3-5min, and the exposure time was 1s and 60s with the eBlot exposure meter;

3.6.10 Select images with appropriate exposure time, and perform gray value analysis through Image J software.

3.7 Formal experiment of internal reference protein WB

3.7.1 Wash the membrane with Stripping Buffer, and wash the membrane at 37°C for 30 minutes (if the molecular weight difference between the target protein and the internal reference protein is more than 10K, this step of stripping buffer can be omitted);

3.7.2 Wash the membrane: wash the membrane 3 times with deionized water;

3.7.3 Wash the membrane: wash the membrane 3 times with TBST, 3 min each time;

3.7.4 Fully immerse the membrane in 5% skimmed milk powder-TBS and shake gently for 30min at room temperature;

3.7.5 Incubate the internal reference: select the appropriate internal reference antibody according to the type of sample, dilute the antibody with 5% nonfat milk powder-TBS, 1:5000-10000, incubate at room temperature for 30 minutes, then place it at 4°C overnight or incubate at 37°C for 2h;

3.7.6 Wash the membrane: wash the membrane 3 times with TBST, 5 min each time;

3.7.7 Secondary antibody incubation: Dilute secondary antibody with 5% nonfat dry milk-TBS, goat anti-mouse IgG(H+L) HRP, 1:5000, shake gently for 1h at room temperature;

3.7.8 Wash the membrane: wash the membrane 3 times with TBST, 5 min each time;

3.7.9 After adding ECL to the PVDF film, the reaction was performed in the dark for 3-5min, and the exposure time was 1s and 60s with the eBlot exposure meter;

3.7.10 Select images with appropriate exposure time, and perform gray value analysis through Image J software.

4. Experimental results

The experimental results are shown in Figure 13. Except for human breast cancer cell MCF7, the activation of EGFR was blocked and the phosphorylation level of EGFR protein was reduced in different cell lines after adding chenodeoxycholic acid.

MCF7 cell line is a HER2-positive cell line. Both EGFR (also known as HER1) and HER2 are members of the epidermal growth factor receptor family. HER2 itself can dimerize with EGFR (HER1) and activate EGFR (HER1). Effects of HER2-positive cell lines on EGFR, chenodeoxycholic acid did not inhibit EGFR activation in MCF7 cell lines.

Conclusions Summary: The drug chenodeoxycholic acid is universal as an antagonistic inhibitor of EGFR and can be used for the treatment or synergistic treatment of EGFR-related diseases.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (10)

1. Use of chenodeoxycholic acid or derivatives thereof in the preparation of inhibitors of EGFR and/or STAT3.
2. The use according to claim 1, wherein the inhibitor is an antagonist.
3. The use according to claim 1 or 2, characterized in that chenodeoxycholic acid or a derivative thereof regulates and reduces the expression level of EGFR protein and/or STAT3 protein.
4. The use according to claim 3, characterized in that, chenodeoxycholic acid or its derivatives bind to EGFR protein and/or STAT3 protein, block the activation of EGFR protein and/or STAT3 protein, and downregulate EGFR protein and/or STAT3 protein protein phosphorylation levels.
5. The use according to claim 3, wherein the total protein expression levels of EGFR protein and STAT3 protein regulated by chenodeoxycholic acid or its derivatives are reduced.
6. Use of chenodeoxycholic acid or a derivative thereof in the preparation of a medicament for preventing or treating diseases related to EGFR and/or STAT3.
7. The use according to claim 6, wherein the disease comprises a tumor or an immune inflammatory disease;

   Optionally, the tumor includes but is not limited to liver cancer, breast cancer, lung cancer, gastric cancer, pancreatic cancer, kidney cancer, brain glioma, bone cancer, ovarian cancer, cervical cancer, head and neck cancer, lymphoma, colorectal cancer Cancer, prostate cancer or leukemia; said immune diseases include but are not limited to psoriasis, neurodermatitis, atopic dermatitis, scleroderma, polyneuritis, lupus erythematosus, amyotrophic lateral sclerosis, multiple Sexual sclerosis, Alzheimer's disease, vascular dementia, systemic vasculitis, ulcerative colitis, ankylosing spondylitis, sepsis or rheumatoid arthritis.
8. Use of chenodeoxycholic acid or its derivative in combination with sorafenib in the preparation of a medicament for preventing or treating cancer.
9. A pharmaceutical composition, comprising chenodeoxycholic acid or a derivative thereof and sorafenib;

   Optionally, in the pharmaceutical composition, the concentration of chenodeoxycholic acid is 1 μg/ml, and the concentration of sorafenib is 5 μM.
10. An EGFR and/or STAT3 inhibitor, characterized in that the active ingredient is chenodeoxycholic acid or a derivative thereof;

    Optionally, a pharmaceutically acceptable carrier is also included.

Images