In-vitro preliminary screening method of IL-6 / JAK2 / STAT3 signal channel inhibitor

Abstract

The invention belongs to the technical field of biology, and relates to an in-vitro preliminary screening method of an IL-6 / JAK2 / STAT3 signal channel inhibitor. The method comprises the following steps: co-culturing mouse spleen lymphocytes and a to-be-detected compound, and determining the critical concentration of the to-be-detected compound which does not reduce the cell activity; dividing the mouse spleen lymphocytes into a model group, an experimental group and a control group; only 10-20ng / mL of IL-6 is added into the culture solution of the model group, 10-20ng / mL of IL-6 and a to-be-detected compound with critical concentration are added into the culture solution of the experimental group, and no additional substance is added into the culture solution of the control group; if the STAT3 mRNA expression quantity, the STAT3 and p-STAT3 protein expression quantity and the IgG content of the model group are all higher than those of the control group and the experimental group, the compound to be detected is judged to be the IL-6 / JAK2 / STAT3 signal channel inhibitor. The method can reduce resource investment of animal experiments and clinical tests.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an in vitro primary screening method for inhibitors of the IL-6 / JAK2 / STAT3 signaling pathway. Background Art

[0002] The IL-6 / JAK2 / STAT3 signaling pathway is an important signaling pathway that regulates cell growth, survival, and differentiation within cells. This pathway plays a key role in immune responses, inflammatory responses, and tumorigenesis. Abnormal activation of this pathway may lead to the massive release of downstream-regulated cytokines, thereby triggering an inflammatory response; or it may lead to the continuous proliferation, invasion and metastasis of tumor cells, and even immune escape. Therefore, studying inhibitors of the IL-6 / JAK2 / STAT3 signaling pathway not only helps to deeply understand the mechanism of action of this pathway in cell physiological and pathological processes, but also helps to develop new disease treatment strategies and drugs. Summary of the Invention

[0003] In order to promote the research on the IL-6 / JAK2 / STAT3 signaling pathway and the drug development for related diseases, the present invention provides an in vitro primary screening method for inhibitors of the IL-6 / JAK2 / STAT3 signaling pathway. This method uses interleukin 6 (IL-6) to stimulate mouse spleen lymphocytes to construct a cell model, adds the test compound to the cell model, and if the test compound causes a decrease in the expression level of STAT3 mRNA, the expression levels of STAT3 and p-STAT3 proteins, and the IgG content in the cell model, it is determined that the test compound is an inhibitor of the IL-6 / JAK2 / STAT3 signaling pathway.

[0004] The technical solution claimed by the present invention is as follows:

[0005] An in vitro primary screening method for inhibitors of the IL-6 / JAK2 / STAT3 signaling pathway, comprising:

[0006] S1: Co-culturing mouse spleen lymphocytes with test compounds at different concentrations to determine the critical concentration of the test compound that does not reduce cell viability;

[0007] S2: Dividing mouse spleen lymphocytes into a model group, an experimental group, and a control group; adding only 10 - 20 ng / mL of IL-6 to the cell culture medium of the model group, adding 10 - 20 ng / mL of IL-6 and the critical concentration of the test compound to the cell culture medium of the experimental group, and not adding additional substances to the cell culture medium of the control group;

[0008] S3: Cultivate three groups of cells under the same conditions and detect the expression levels of STAT3 mRNA, STAT3 protein, and p-STAT3 protein in the cells, as well as the IgG content in the supernatant of the cell culture medium.

[0009] If the expression levels of STAT3 mRNA, STAT3 protein, p-STAT3 protein, and IgG content in the model group are all higher than those in the control group and the experimental group, it is determined that the test compound is an inhibitor of the IL-6 / JAK2 / STAT3 signaling pathway.

[0010] Preferably, the CCK8 method is used to determine the critical concentration of the test compound that does not reduce cell viability.

[0011] Preferably, the concentration of IL-6 in the cell culture medium of the model group is 10 ng / mL, and the concentration of IL-6 in the cell culture medium of the experimental group is 10 ng / mL.

[0012] Preferably, after culturing the cells for 24 hours, extract the total RNA of the cells and reverse transcribe it into cDNA; using the cDNA as a template, detect the expression level of STAT3 mRNA in the cells by real-time quantitative PCR.

[0013] In some embodiments, a total RNA extraction kit is used to extract the total RNA of the cells, and a reverse transcription kit is used to reverse transcribe the extracted total RNA into cDNA.

[0014] Preferably, the PCR reaction system is: 10 μL of quantitative PCR Mix, 9.2 μL of DNA template, 0.4 μL of 10 μM forward primer, and 0.4 μL of 10 μM reverse primer.

[0015] Preferably, the PCR reaction program is: 95°C for 3 min; 95°C for 5 s, 60°C for 30 s, for 45 cycles.

[0016] Preferably, after culturing the cells for 24 hours, extract the total protein of the cells and detect the expression levels of STAT3 protein and p-STAT3 protein in the cells by Western blot.

[0017] Preferably, after culturing the cells for 3 days, take the supernatant of the cell culture medium and detect the IgG content in the supernatant of the cell culture medium by enzyme-linked immunosorbent assay.

[0018] In some embodiments, a mouse IgG ELISA kit is used to detect the IgG content in the supernatant of the cell culture medium according to the operation manual.

[0019] Preferably, mouse spleen lymphocytes are seeded into cell culture dishes at a density of 5×10 6 cells per well for culture.

[0020] Preferably, the cells are cultured under the conditions of 37 °C and 5% CO2.

[0021] The ability of the method of the present invention to screen for IL-6 / JAK2 / STAT3 signaling pathway inhibitors was verified using quercetin. The results showed that 10 ng / mL interleukin 6 (IL-6) could promote the secretion of IgG, the expression of STAT3 mRNA, the expression of STAT3 protein, and the expression of p-STAT3 protein in mouse spleen lymphocytes, while 80 μM quercetin could inhibit the secretion of IgG, the expression of STAT3 mRNA, the expression of STAT3 protein, and the expression of p-STAT3 protein in mouse spleen lymphocytes. Therefore, the method of the present invention can effectively screen for IL-6 / JAK2 / STAT3 signaling pathway inhibitors.

[0022] The present invention first uses a biological process to screen for complete upstream and downstream inhibitors of IL-6 / JAK2 / STAT3, which can effectively reduce the resource investment in animal experiments and clinical trials and promote the research and development of new drugs. Description of the Drawings

[0023] Figure 1 Shows the effects of different concentrations of quercetin on the viability of mouse spleen lymphocytes; ns indicates no significant difference (P > 0.05), and **** indicates extremely significant difference (P < 0.0001).

[0024] Figure 2 Is the detection result of the IgG content in the supernatant of the culture medium of mouse spleen lymphocytes; * indicates significant difference

[0025] (P < 0.05), ** indicates very significant difference (P < 0.01).

[0026] Figure 3 Is the detection result of the STAT3 mRNA expression level in mouse spleen lymphocytes; ** indicates very significant difference (P < 0.01), and **** indicates extremely significant difference (P < 0.0001).

[0027] Figure 4 Is the detection result of the STAT3 protein expression level and the p-STAT3 protein expression level in mouse spleen lymphocytes; * indicates significant difference (P < 0.05), ** indicates very significant difference (P < 0.01), and *** indicates extremely significant difference (P < 0.001). Detailed Embodiments

[0028] The following examples are provided:

[0029] 1. An in vitro primary screening method for IL-6 / JAK2 / STAT3 signaling pathway inhibitors, which comprises:

[0030] S1: Co-culture mouse spleen lymphocytes with the test compound at different concentrations to determine the critical concentration of the test compound that does not reduce cell viability.

[0031] S2: Divide mouse spleen lymphocytes into a model group, an experimental group, and a control group; add only 10 - 20 ng / mL of IL-6 to the cell culture medium of the model group, add 10 - 20 ng / mL of IL-6 and the critical concentration of the test compound to the cell culture medium of the experimental group, and do not add additional substances to the cell culture medium of the control group.

[0032] S3: Culture the three groups of cells under the same conditions and detect the expression levels of STAT3 mRNA, STAT3 protein, and p-STAT3 protein in the cells, as well as the IgG content in the supernatant of the cell culture medium.

[0033] If the expression levels of STAT3 mRNA, STAT3 protein, p-STAT3 protein, and IgG content in the model group are all higher than those in the control group and the experimental group, then the test compound is determined to be an inhibitor of the IL-6 / JAK2 / STAT3 signaling pathway.

[0034] 2. The method according to Example 1, wherein the CCK8 method is used to determine the critical concentration of the test compound that does not reduce cell viability.

[0035] 3. The method according to Example 1 or 2, wherein the concentration of IL-6 in the cell culture medium of the model group is 10 ng / mL, and the concentration of IL-6 in the cell culture medium of the experimental group is 10 ng / mL.

[0036] 4. The method according to any one of Examples 1 - 3, wherein after culturing the cells for 24 hours, extract the total RNA of the cells and reverse transcribe it into cDNA; use the cDNA as a template to detect the expression level of STAT3 mRNA in the cells by real-time quantitative PCR.

[0037] 5. The method according to Example 4, wherein the PCR reaction system is: 10 μL of quantitative PCR Mix, 9.2 μL of DNA template, 0.4 μL of 10 μM forward primer, and 0.4 μL of 10 μM reverse primer.

[0038] 6. The method according to Example 4, wherein the PCR reaction program is: 95°C for 3 min; 95°C for 5 s, 60°C for 30 s, for 45 cycles.

[0039] 7. The method according to any one of Examples 1 - 6, wherein after culturing the cells for 24 hours, extract the total protein of the cells and detect the expression levels of STAT3 protein and p-STAT3 protein in the cells by Western blot assay.

[0040] 8. The method according to any one of Examples 1-7, wherein after culturing the cells for 3 days, the supernatant of the cell culture medium is taken, and the IgG content in the supernatant of the cell culture medium is detected by enzyme-linked immunosorbent assay.

[0041] 9. The method according to any one of Examples 1-8, wherein mouse spleen lymphocytes are seeded into a cell culture dish at 5×10 6 cells per well for culture.

[0042] 10. The method according to any one of Examples 1-9, wherein the cells are cultured under the conditions of 37 °C and 5% CO2.

[0043] The present invention will be further illustrated with reference to the following examples. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0044] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be commercially obtained or prepared according to the conventional methods in the art, and the specification is laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and relevant experimental manuals or manufacturer's instructions can be referred to.

[0045] Example 1

[0046] Verification of the in vitro primary screening method for IL-6 / JAK2 / STAT3 signaling pathway inhibitors

[0047] The following experiment uses quercetin to verify the screening effect of the in vitro primary screening method of the present invention on IL-6 / JAK2 / STAT3 signaling pathway inhibitors.

[0048] Reagents: Lymphocyte separation solution was purchased from Shenzhen Dakewei Biotechnology Co., Ltd. (DAKEWE). RPMI 1640 medium was purchased from Cibco. Quercetin was purchased from TargetMol, CAS No.: 117-39-5, molecular formula: C 15 H 10 O7. Interleukin 6 (IL-6) was purchased from ABclonal, product number: RP01321.

[0049] The GenBank accession number of mouse IgG is AAA51107. The GenBank accession number of mouse STAT3 gene is 20848. The GenBank accession number of mouse STAT3 protein is AAA19452. The GenBank accession number of mouse p-STAT3 protein is P42227.

[0050] The experimental results were analyzed by one-way analysis of variance. ns indicates no significant difference, P<0.05 indicates a significant difference, P<0.01 indicates a very significant difference, P<0.001 indicates an extremely significant difference, and P<0.0001 indicates an extremely significant difference.

[0051] 1. Isolation and culture of mouse spleen lymphocytes

[0052] Female BALB / C mice aged 8-10 weeks were obtained from the Experimental Animal Center of Chongqing Medical University. The mice were sacrificed by cervical dislocation. After spraying the whole body of the mice with alcohol, the spleens of the mice were taken out in a laminar flow hood. The mouse spleens were placed in a 35 mm culture dish, and 4-5 mL of lymphocyte separation medium (DAKEWE) was added. The mouse spleens were ground with the piston of a 5 mL syringe to obtain a separation medium containing spleen cells. A 70 μm cell strainer was placed in a 15 mL centrifuge tube, and the separation medium containing spleen cells was transferred to the centrifuge tube through the cell strainer. 500 μL to 1 mL of RPMI1640 medium was overlaid on the liquid surface of the centrifuge tube to keep the liquid surface boundary distinct. The centrifuge tube was centrifuged at 3000 g for 30 min at room temperature. After centrifugation, the culture solution was stratified. The lymphocyte layer was aspirated, 10 mL of RPMI1640 medium was added, and the mixture was inverted and washed. Centrifugation was carried out at 1000 g for 10 min to collect mouse spleen lymphocytes, and a Rewod cell counting plate and a cell counter were used for cell counting. The isolated mouse spleen lymphocytes were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum (BDBIO) and 1% penicillin-streptomycin solution (Beyotime Biotechnology) in a humidified atmosphere of 37 °C and 5% CO2.

[0053] 2. Cell viability analysis

[0054] The CCK8 method was used to detect the effect of quercetin on the viability of mouse spleen lymphocytes. The CCK8 kit (Beyotime Biotechnology) was used for detection according to the operation manual. Mouse spleen lymphocytes were seeded into 96-well plates at a density of 10 5 cells / well. The experimental groups included a quercetin treatment group, a negative control group, and a blank control group. In the cell culture wells of the quercetin treatment group, quercetin solutions were added to make the final concentrations of quercetin in the cell culture medium 80 μM, 160 μM, 320 μM, and 640 μM, respectively. In the cell culture wells of the negative control group, RPMI1640 culture medium was added. The cell culture wells of the blank control group were not treated. After culturing for 24 h, CCK8 solution was added to all cell culture wells at a volume of 10 μL / well. After incubation for 2 h, the OD 450 .

[0055] The results are as Figure 1As shown: 80 μM quercetin had no significant effect on the activity of mouse spleen lymphocytes.

[0056] 3.ELISA analysis

[0057] Mouse spleen lymphocytes were collected at a rate of 5×10 6 Cells / well were plated into six-well cell culture dishes (Sevier), and IL6+quercetin group, IL6 group and blank control group were set up. IL6+quercetin group: interleukin 6 (IL-6) was added to the cell culture wells to a final concentration of 10 ng / mL, and quercetin was added to a final concentration of 80 μM. IL6 group: IL-6 was added to the cell culture wells to a final concentration of 10 ng / mL. Blank control group: The corresponding volume of RPMI1640 culture medium was added to the cell culture wells. After 3 days of culture, the IgG content in the supernatant of the cell culture fluid was detected using the mouse IgG ELSIA kit (Bioswamp, product number MU30005) according to the operating manual. The specific steps are as follows:

[0058] (1) Dilution of standard: prepare 6 small test tubes and number them in sequence. First, add 150μL of standard diluent to each small test tube, then take 150μL of the original concentration standard and add it to a numbered test tube, mix thoroughly; then take 150μL from the test tube and add it to the second test tube, mix thoroughly; then take 150μL from the test tube and add it to the third test tube, mix thoroughly; then take 150μL from the test tube and add it to the fourth test tube, mix thoroughly; then take 150μL from the test tube and add it to the fifth test tube, mix thoroughly; the sixth test tube is used as standard No. 0. After dilution, the concentrations of the standards in each tube are: 96μg / mL, 48μg / mL, 24μg / mL, 12μg / mL, 6μg / mL, 0μg / mL. Set standard wells on the enzyme-labeled plate and add 50μL of standards of different concentrations in sequence.

[0059] (2) Sample addition: Set up blank wells (blank control wells do not contain samples, enzyme-labeled reagents, and biotin-labeled anti-IgG antibodies, and the rest of the steps are the same) and test sample wells. First add 40 μL of sample to the test sample well on the enzyme-labeled plate, and then add 10 μL of biotin-labeled anti-IgG antibodies. When adding samples, add the samples to the bottom of the wells on the enzyme-labeled plate, try not to touch the well walls, and gently shake to mix.

[0060] (3) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0061] (4) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0062] (5) Liquid preparation: Dilute the 30-fold concentrated washing solution with 30-fold distilled water and set aside.

[0063] (6) Washing: Carefully remove the sealing plate film, discard the liquid, centrifuge to dry, fill each well with washing solution, let stand for 30 seconds and then discard. Repeat this 5 times and pat dry.

[0064] (7) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake and mix well, and develop color at 37 °C in the dark for 10 minutes.

[0065] (8) Termination: Add 50 μL of termination solution to each well to terminate the reaction (at this time, the color immediately changes from blue to yellow).

[0066] (9) Measurement: Zero with the blank well, measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be carried out within 15 minutes after adding the termination solution.

[0067] (10) Detection range: 1.2 μg / mL - 96 μg / mL, sensitivity: ≤0.24 μg / mL.

[0068] The results are as Figure 2 shown: 10 ng / mL interleukin 6 (IL-6) can induce the secretion of IgG by mouse spleen lymphocytes, and 80 μM quercetin can inhibit the secretion of IgG.

[0069] 4. RT-qPCR analysis

[0070] Seed mouse spleen lymphocytes at 5×10 6 cells / well into a six-well cell culture dish (Saiweier), and set up the IL6 + quercetin group, IL6 group and blank control group. IL6 + quercetin group: Add interleukin 6 (IL-6) to the cell culture well to a final concentration of 10 ng / mL, and at the same time add quercetin to a final concentration of 80 μM. IL6 group: Add IL-6 to the cell culture well to a final concentration of 10 ng / mL. Blank control group: Add the corresponding volume of RPMI1640 culture medium to the cell culture well. After culturing for 24 h, extract the total RNA of the cells in each group using an RNA extraction kit (Beyotime Biotechnology).

[0071] The steps for total RNA extraction are as follows:

[0072] (1) Sample preparation: Pipette the cell culture medium into a 1.5 mL centrifuge tube, centrifuge at 2000 g for 10 min, discard the supernatant, add 300 μL of lysis buffer, and gently pipette 8 - 10 times until the solid suspension dissolves and the solution becomes clear.

[0073] (2) Add an equal volume of binding solution to the lysis buffer and gently invert and mix 3 - 5 times.

[0074] (3) Transfer the mixture (including the precipitate) into the purification column, centrifuge at 12,000 g for 30 seconds, discard the liquid in the collection tube. When the volume of the lysis solution is greater than 300 μL, after adding an equal volume of binding solution, the total volume will exceed the capacity of the purification column. In this case, it should be passed through the column in two batches.

[0075] (4) Add 600 μL of Wash Buffer I, centrifuge at 12,000×g for 30 seconds, discard the liquid in the collection tube.

[0076] (5) Add 600 μL of Wash Buffer II, centrifuge at 12,000×g for 30 seconds, discard the liquid in the collection tube.

[0077] (6) Repeat step (5) once.

[0078] (7) Centrifuge at the highest speed (about 14000 - 16000×g) for 2 minutes to remove the residual liquid.

[0079] (8) Place the RNA purification column into the RNA elution tube provided in this kit, add 30 - 50 μL of elution buffer, let it stand at room temperature for 2 - 3 minutes, and centrifuge at the highest speed for 30 seconds. The resulting solution is the purified RNA.

[0080] (9) Detect the RNA concentration using a Nano instrument.

[0081] Reverse transcribe the extracted total RNA into cDNA using a reverse transcription kit (ABclonal Technology). The reverse transcription reaction system and reaction conditions are shown in Table 1 and Table 2.

[0082] Table 1: Reverse transcription reaction system

[0083] Reagent Volume 4×ABScript Neo RT Master Mix 5 μL 20×gDNA Remover Mix 1 μL Total RNA 10 pg - 1 μg <![CDATA[Nuclease-free H2O]]> Make up to 20 μL

[0084] Table 2: Reverse transcription reaction conditions

[0085] Temperature Time 37℃ 2 min 55℃ 15 min 85℃ 5 min 4℃ Maintain

[0086] Using the synthesized cDNA as a template, perform real-time PCR using SYBE (ABclonal Technology). The primers for amplifying the STAT3 gene were purchased from Beyotime Biotechnology, and the product number is QM05286S. The primers for amplifying the β-actin gene (internal reference) were purchased from Aikerui Biotech, and the product number is AG11724. Use the 2 -(ΔΔCT) method to quantify the PCR results. The reaction system and reaction conditions for real-time PCR are shown in Table 3 and Table 4.

[0087] Table 3: Real-time PCR reaction system

[0088] Reagent Volume 2×Universal SYBR Green Fast qPCR Mix 10 μL DNA Template 9.2 μL Forward Primer (10 μM) 0.4 μL Reverse Primer (10 μM) 0.4 μL

[0089] Add the above PCR reaction system into a non-enzymatic eight-strip tube, and repeat each sample in triplicate wells.

[0090] Table 4: real-time PCR reaction conditions

[0091]

[0092] The results are as Figure 3 shown: 10 ng / mL interleukin-6 (IL-6) can cause an increase in the expression of STAT3 mRNA in mouse spleen lymphocytes, and 80 μM quercetin can inhibit the expression of STAT3 mRNA.

[0093] 5. Western blot analysis

[0094] Seed mouse spleen lymphocytes at 5×10 6 cells / well into a six-well cell culture dish (Saiweier), and set up the IL6 + quercetin group, IL6 group, and blank control group. IL6 + quercetin group: Add interleukin-6 (IL-6) to the cell culture wells to a final concentration of 10 ng / mL, and at the same time add quercetin to a final concentration of 80 μM. IL6 group: Add IL-6 to the cell culture wells to a final concentration of 10 ng / mL. Blank control group: Add the corresponding volume of RPMI1640 culture medium to the cell culture wells. After culturing for 24 h, extract the total protein for Western blot analysis.

[0095] The steps for total protein extraction are as follows:

[0096] (1) Take out the cell plate from the ice, transfer the cell culture medium to a 1.5 mL centrifuge tube, centrifuge at 2000 g for 10 min, and discard the supernatant; repeat the above operation to collect more cells.

[0097] (2) Wash the cell pellet with PBS 3 times, 2000 g, 3 min each time.

[0098] (3) Prepare the cell lysate by mixing RIPA (Solarbio): protease and phosphatase inhibitor mixture (Beyotime Biotechnology) at a ratio of 50:1. Add 50 μL of the lysate to each tube of cells and incubate on ice for 10 minutes.

[0099] (4) Ultrasonic treatment: Work at 60 W for 5 s and rest for 2 s, for 5 cycles.

[0100] (5) Centrifuge at 12000 r / min and 4 °C for 5 minutes, collect the supernatant, and record the volume.

[0101] The protein concentration was determined using the BCA method (Beyotime Biotechnology). The specific operation steps are as follows:

[0102] (1) Preparation of protein standards: a) Add 0.8 mL of protein standard preparation solution to a tube of protein standard (20 mg BSA), and after complete dissolution, prepare a 25 mg / mL protein standard solution. b) Take an appropriate amount of the 25 mg / mL protein standard and dilute it to a final concentration of 0.5 mg / mL.

[0103] (2) Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution by mixing 50 volumes of BCA reagent A with 1 volume of BCA reagent B (50:1), and mix well.

[0104] (3) Protein concentration determination: a) Add the standards at 0, 1, 2, 4, 8, 12, 16, 20 μL to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL respectively. b) Add an appropriate volume of the sample to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, add standard diluent to make up to 20 μL. c) Add 200 μL of BCA working solution to each well, incubate at 37 °C for 20 - 30 minutes, and measure OD 562 .

[0105] The operation steps of Western Blot analysis are as follows:

[0106] (1) Add different volumes of loading buffer (White Shark) to the protein samples according to the protein concentration to make the final protein concentration consistent, and then boil.

[0107] (2) After installing the 8% FuturePAGE TM (ACE Biotechnology) precast gel in the electrophoresis tank, add the supporting electrophoresis buffer, add each protein sample to the wells, and add protein marker (Beyotime Biotechnology) on the left and right.

[0108] (3) Electrophoresis: 80 V for 20 min, then 140 V for 30 min.

[0109] (4) Transfer: After electrophoresis, take out the gel and place it in the electrotransfer buffer, cut off the excess part, and place the gel on the electrotransfer clamp lined with sponge; cut the PVDF membrane (Millipore) to the same size as the gel, activate it with methanol, cover it on the gel, use a roller to remove air bubbles, then place a sponge and another electrotransfer clamp on it and clamp tightly, then put it into the electrophoresis apparatus, pour in the electrotransfer buffer (Seville), and transfer at 400 mA for 30 min.

[0110] (5) Blocking: After the transfer was completed, the PVDF membrane was taken out and soaked in ddH2O. The bands were cut according to the positions indicated by the marker. The bands were blocked with 5% non-fat milk powder (White Shark) for 1 h.

[0111] (6) Incubation with primary antibodies: Monoclonal antibody against β-actin (Hua'an Biotech, product number EM21002), monoclonal antibody against STAT3 (ABclonal, product number A19566), monoclonal antibody against p-STAT3 (Abmart, product number T56566S), overnight at 4 °C.

[0112] (7) Incubation with secondary antibodies: The PVDF membrane was washed three times with TBST, 5 min each time; HRP-labeled goat anti-mouse IgG antibody (Hua'an Biotech, product number HA1006) was added to the β-actin band, and HRP-labeled goat anti-rabbit IgG antibody (Hua'an Biotech, product number HA1001) was added to the STAT3 and P-STAT3 bands, incubated at room temperature for one hour.

[0113] (8) Development: After detecting the bands using a chemiluminescent ECL assay kit (Beyotime Biotechnology), the chemiluminescent bands were imaged using an imaging system (Bio-Rad, Hercules, CA, USA).

[0114] The results were as Figure 4 shown: 10 ng / mL interleukin-6 (IL-6) could cause an increase in the protein expression levels of STAT3 and p-STAT3 (Tyr705) in mouse spleen lymphocytes, and 80 μM quercetin could inhibit the increase in the protein expression levels of STAT3 and p-STAT3 (Tyr705).

[0115] In summary, the IgG content in the supernatant of the culture medium of mouse spleen lymphocytes, the STAT3 mRNA expression level in mouse spleen lymphocytes, and the protein expression levels of STAT3 and p-STAT3 (Tyr705) in the IL6 group were all higher than those in the blank control group and the IL6 + quercetin group, indicating that quercetin can be used as an inhibitor of the IL-6 / JAK2 / STAT3 signaling pathway.

Claims

1. An in vitro primary screening method for an IL-6 / JAK2 / STAT3 signaling pathway inhibitor, which includes: S1: Co-culture mouse spleen lymphocytes with test compounds at different concentrations to determine the critical concentration of the test compound that does not reduce cell viability; S2: Divide mouse spleen lymphocytes into a model group, an experimental group, and a control group; add only 10 - 20 ng / mL of IL-6 to the cell culture medium of the model group, add 10 - 20 ng / mL of IL-6 and the critical concentration of the test compound to the cell culture medium of the experimental group, and do not add additional substances to the cell culture medium of the control group; S3: Culture the three groups of cells under the same conditions and detect the STAT3 mRNA expression level, STAT3 protein expression level, and p-STAT3 protein expression level in the cells, as well as the IgG content in the supernatant of the cell culture medium; If the STAT3 mRNA expression level, STAT3 protein expression level, p-STAT3 protein expression level, and IgG content in the model group are all higher than those in the control group and the experimental group, then the test compound is determined to be an IL-6 / JAK2 / STAT3 signaling pathway inhibitor.

2. The method according to claim 1, characterized in that, Use the CCK8 method to determine the critical concentration of the test compound that does not reduce cell viability.

3. The method according to claim 1, wherein The concentration of IL-6 in the cell culture medium of the model group is 10 ng / mL, and the concentration of IL-6 in the cell culture medium of the experimental group is 10 ng / mL.

4. The method according to claim 1, characterized in that After culturing the cells for 24 hours, extract the total RNA of the cells and reverse transcribe it into cDNA; use the cDNA as a template to detect the STAT3 mRNA expression level in the cells by real-time quantitative PCR.

5. The method according to claim 4, characterized in that, The PCR reaction system is: 10 μL of quantitative PCR Mix, 9.2 μL of DNA template, 0.4 μL of 10 μM forward primer, and 0.4 μL of 10 μM reverse primer.

6. The method according to claim 4, wherein The PCR reaction program is: 95°C for 3 min; 95°C for 5 s, 60°C for 30 s, for 45 cycles.

7. The method according to claim 1, wherein After culturing the cells for 24 hours, extract the total protein of the cells and detect the STAT3 protein expression level and p-STAT3 protein expression level in the cells by Western blot.

8. The method according to claim 1, characterized in that After culturing the cells for 3 days, take the supernatant of the cell culture medium and detect the IgG content in the supernatant of the cell culture medium by enzyme-linked immunosorbent assay.

9. The method according to any one of claims 1-8, characterized in that, Mouse spleen lymphocytes were seeded into cell culture dishes at a density of 5×10 6 cells per well and cultured.

10. The method according to claim 9, wherein Culture the cells under the conditions of 37°C and 5% CO2.

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