NF-kappa B inhibitor as well as preparation method and application thereof

Abstract

The invention discloses an NF (nuclear factor)-kappa B inhibitor. The NF-kappa B inhibitor comprises N-trans-p-coumaroyl tyramine; the invention further discloses a preparation method of the NF-kappa B inhibitor. The invention also discloses an application of the NF-kappa B inhibitor in medicines for preventing or treating diseases related to body inflammation. According to the application disclosed by the invention, the influence of the N-trans-p-coumaroyl tyramine on a TLR4 / MyD88 / NF-kappa B inflammation signal channel is detected through an immunofluorescence technology, and a result shows that the N-trans-p-coumaroyl tyramine can be used for reducing the expression of TLR4 and MyD88 proteins and inhibiting the transfer of NF-kappa B proteins to cell nucleuses, so that the N-trans-p-coumaroyl tyramine can be used as an NF-kappa B inhibitor and can be used for preparing an anti-inflammatory drug. The composition is used for preventing or treating body inflammatory response.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an NF-κB inhibitor and a preparation method and application thereof. Background Art

[0002] Inflammatory response is a common pathological process in clinical practice. It is the defense response of the immune system when pathogens invade or tissues are damaged. It is closely related to many chronic diseases such as osteoarthritis, asthma, cardiovascular disease, etc. With the increasing medical, social and economic burden caused by chronic inflammatory diseases from different sources, people's demand for anti-inflammatory drugs is increasing. However, classic anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs, glucocorticoids and opioids, have more or less differences in efficacy and side effects, which makes people urgently need to develop safe and effective new anti-inflammatory analgesic drugs. Although several biological agents targeting different key molecules have made breakthroughs in some autoimmune / inflammatory diseases, they are expensive and can only be obtained by intravenous injection, and long-term side effects often limit their use. Traditional Chinese medicine has incomparable advantages over Western medicine in the treatment of systemic diseases due to its multi-component, multi-target, multi-pathway, overall regulation, and less toxic and side effects. Finding safe and effective anti-inflammatory active ingredients from traditional Chinese medicine has become a new trend in the development of anti-inflammatory drugs.

[0003] N-trans-coumaroyltyramine is a phenylpropanoid phenolic amine compound (molecular formula C 17 H 17 NO 3 ), exists in many traditional Chinese medicines, such as Evodia rutaecarpa of Rutaceae, Thunbergia tenuifolia of Convolvulaceae, Caulis scutellariae of Menispermaceae, Dendrobium candidum of Orchidaceae, etc. Pharmacological studies have shown that N-trans-p-coumaryltyramide has physiological activities such as inhibiting α-glucosidase, inhibiting acetylcholinesterase, anti-oxidation, antibacterial, antiviral, and inhibiting the growth of leukemia cells, but there are few reports on its anti-inflammatory effect. Nuclear factor-κB (NF-κB) is an important nuclear transcription factor in cells. It participates in the body's inflammatory response and immune response, and can regulate cell apoptosis and stress response. Excessive activation of NF-κB is closely related to many human diseases, such as rheumatoid arthritis, inflammatory changes in heart and brain diseases, etc. Summary of the invention

[0004] In order to overcome the above technical problems, the present invention discloses an NF-κB inhibitor and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A NF-κB inhibitor, comprising N-trans-p-coumaryl tyramide.

[0007] The above-mentioned NF-κB inhibitor, wherein the inhibitor inhibits the transfer of NF-κB protein to the cell nucleus, thereby inhibiting the release of inflammatory factors, thereby preventing or treating diseases caused by inflammation in the body.

[0008] A method for preparing an NF-κB inhibitor, the method comprising:

[0009] Step 1, macrophage recovery and culture;

[0010] Step 2, macrophage cryopreservation;

[0011] Step 3, using CCK-8 method to detect cell viability and screen the maximum safe concentration of NF-κB inhibitor;

[0012] Step 4: Detect the expression of TLR4 / MyD88 / NF-κB in cells under the intervention of NF-κB inhibitor by immunofluorescence method.

[0013] In the above-mentioned method for preparing the NF-κB inhibitor, the specific steps of step 1 include:

[0014] The macrophages were taken out of liquid nitrogen and thawed in a 37°C water bath. When the macrophages were nearly thawed, the macrophages were taken out and dried with cotton balls. The macrophages in step 1-1 were disinfected with 75% alcohol and transferred to a clean bench. The cap of the cryopreservation tube was opened, 1 mL of complete medium was added, the mixture was transferred to a centrifuge tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded and resuspended with complete medium, transferred to a 100 mm culture dish, and incubated at 37°C and 5% CO 2 Culture in an incubator, keep observing and change the medium.

[0015] In the above-mentioned method for preparing the NF-κB inhibitor, the complete culture medium is prepared by a penicillin-streptomycin mixed solution, fetal bovine serum and α-MEM liquid culture medium in a volume ratio of 1:10:89.

[0016] In the above-mentioned method for preparing the NF-κB inhibitor, the specific steps of step 2 include:

[0017] Take the macrophages in step 1, and when the macrophages are cultured to 80%, digest the cells with trypsin, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fetal bovine serum and dimethyl sulfoxide in a volume ratio of 9:1 to resuspend, transfer to a cryopreservation tube, mark it, transfer to -80°C after overnight at -20°C, and then transfer to a liquid nitrogen tank for long-term cryopreservation.

[0018] In the above-mentioned method for preparing the NF-κB inhibitor, the specific steps of step 3 include:

[0019] The macrophages from step 2 were plated at 1×10 4 100 μL was inoculated into each well at a density of 10 cells / mL. When the macrophages grew to 50-60%, 100 μL of NF-κB inhibitor was added into each well at a series of concentrations of 200, 100, 50, 25, and 12.5 μM to treat the macrophages for 24 hours, in parallel for 3 times.

[0020] Then 10 μL of CCK-8 solution was added to each well, and cell viability was measured after 2 h;

[0021] The absorbance was measured at 450 nm using a microplate reader;

[0022] The in vitro safety of NF-κB inhibitors was evaluated according to cell survival rate = (OD experimental group / OD control group) × 100%, and the maximum safe concentration of NF-κB inhibitors was screened as the drug concentration for subsequent experiments.

[0023] In the above-mentioned method for preparing the NF-κB inhibitor, in step 3, the method for preparing the NF-κB inhibitor comprises:

[0024] Accurately weigh an appropriate amount of N-trans-p-coumaryl tyramide and place it in a brown sterile 1.5 mL EP tube. Add a certain amount of DMSO to prepare a 200 mM NF-κB inhibitor and store it at -20°C for later use.

[0025] In the above-mentioned method for preparing the NF-κB inhibitor, the specific steps of step 4 include:

[0026] Step 4-1, seeding board:

[0027] After sterilizing the cell slides on flame, place them in a 24-well plate and add 75% alcohol for sterilization for 30 min. After sterilization, wash them three times with PBS buffer.

[0028] HepG2 cells were plated in 24-well plates at a density of 2 × 10 4 500 μL was inoculated into each well at a density of 10 cells / mL, and cell growth was observed;

[0029] Step 4-2, drug administration:

[0030] When the cells grow to about 50%, add NF-κB inhibitor and continue culturing;

[0031] Step 4-3; Fixation:

[0032] After 24 h of NF-κB inhibitor intervention, the 24-well plate was removed, the cell culture medium was discarded, 200 μL of PBS buffer was added to each well, and the cells were washed after standing for 5 min. After repeating 3 times, 200 μL of 4% paraformaldehyde was added to each well and fixed for 30 min at room temperature.

[0033] Step 4-4, closure:

[0034] After fixation, 200 μL of PBS buffer was added again for washing three times, each time for 5 min;

[0035] After washing, the cells were treated with 200 μL 0.5% Triton X-100 for 5 min;

[0036] After the treatment, the cells were washed three times with PBS buffer, each time for 5 min;

[0037] 1% bovine serum albumin was added to the 24-well plate and blocked at room temperature for 30 min;

[0038] After blocking, wash with PBS buffer three times, 5 min each time;

[0039] Step 4-5, incubate with primary antibody:

[0040] TLR4\MyD88\NF-κB were diluted with 5% bovine serum albumin, and the diluted primary antibody was added to each well, 200 μL, and incubated at 4°C overnight. The primary antibody was recovered and washed three times with PBS buffer, each time for 5 min;

[0041] Steps 4-6, incubation with secondary antibody:

[0042] The secondary antibody was diluted with 0.5% bovine serum albumin, and 200 μL was added to each well. The cells were incubated at room temperature for 1 h, and the secondary antibody was recovered and washed three times with PBS buffer for 5 min each time.

[0043] Steps 4-7, nuclear staining:

[0044] Nuclear staining was performed with 200 μL DAPI staining solution, and the cells were incubated at room temperature in the dark for 10 min. After the incubation, the DAPI staining solution was discarded and the cells were washed three times with PBS buffer, each time for 5 min.

[0045] Steps 4-8, DAPI mounting:

[0046] After taking out the cell slide and drying it, drop a drop of anti-fluorescence quenching mounting medium on the slide, stick the cell slide with the cell side on the mounting medium, and fix the cell slide with neutral resin;

[0047] Steps 4-9, microscopic examination:

[0048] After sealing, the slides were protected from light and stored at 4°C, and then examined under a microscope after the neutral resin solidified.

[0049] A use of an NF-κB inhibitor in a drug for preventing or treating inflammation-related diseases in the body.

[0050] The beneficial effects of the present invention are as follows: the present invention detects the influence of N-trans-p-coumaryltyramide on the TLR4 / MyD88 / NF-κB inflammatory signaling pathway through immunofluorescence technology (IF), and the results show that N-trans-p-coumaryltyramide can reduce the expression of TLR4 and MyD88 proteins and inhibit the transfer of NF-κB protein to the cell nucleus, indicating that N-trans-p-coumaryltyramide can be used as an NF-κB inhibitor for preventing or treating inflammatory responses of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0052] Figure 1 The effect of N-trans-p-coumaryltyramide on the activity of RAW 264.7 at different concentrations in the present invention; wherein, n = 3, *P < 0.05 compared with the control group;

[0053] Figure 2 The expression analysis of TLR4 / MyD88 in RAW264.7 cells in the present invention; wherein A is the immunofluorescence image of TLR4 / MyD88, B is the expression analysis of TLR4 in cells, and C is the expression analysis of MyD88 in cells; n=3, scale bar is 10uM, ##P<0.01 compared with the control group; **P<0.01 compared with the LPS-treated group;

[0054] Figure 3 This is the nuclear localization analysis of NF-κB in RAW264.7 cells in the present invention; wherein A is the immunofluorescence image of NF-κB; B is the ratio analysis of NF-κB in the nucleus and cytoplasm; n=3, scale bar is 10uM, ##P<0.01 compared with the control group; **P<0.01 compared with the LPS-treated group. DETAILED DESCRIPTION

[0055] The present invention is further described below by means of specific embodiments to make the technical solution of the present invention easier to understand and grasp, rather than to limit the present invention.

[0056] In this embodiment, unless otherwise specified, all reagents can be purchased on the market.

[0057] In this embodiment, unless otherwise specified, the methods used are all conventional methods.

[0058] 1. Materials and Instruments

[0059] 1.1 Experimental Materials

[0060] Penicillin-streptomycin mixed solution (double antibody), RAW 264.7 cell line, α-MEM liquid culture medium (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.); Lipopolysaccharide (LPS) (Batch No.: 220518; Manufacturer: Sigma, USA); Fetal bovine serum (FBS) (Beijing Quanshijin Biotechnology Co., Ltd.); Dimethyl sulfoxide (DMSO) (Batch No.: F514BA0010, Shenggong Biotechnology (Shanghai) Co., Ltd.); CCK-8 kit (Shanghai Biyuntian Biotechnology Co., Ltd.); BSA; Cell crawling (Biosharp); Anti-fluorescence attenuation mounting medium, PBS buffer, PBST, DAPI solution (Solerbo); Trypsin cell digestion solution (Gibco); Bovine serum albumin VBSA (Albumin Bovine, BSA), Dylight 594 (Goat Anti-Rabbit IgG), Dylight 488 (Goat Anti-Rabbit IgG)(H+L)(Earthox, USA); p-coumaric acid, trans-p-coumaryl tyramide (batch number: AF20041951, batch number: AF22032709, purity>98%, Chengdu Aifa Biotechnology Co., Ltd.), salicylic acid (batch number: 0854-9501, purity>98%, China Food and Drug Administration), N-trans-feruloyl tyramide (batch number: RFS-F03302108016, purity>97%, Chengdu Ruifensi Biotechnology Co., Ltd.); TLR4 (Hangzhou Huaan Biotechnology Co., Ltd., China); MyD88 (Wuxing Sewell Biotechnology Co., Ltd., China); NF-κB (Affinity, Australia).

[0061] 1.2 Main instruments

[0062] Cellometer Mini fully automatic cell counter (Nexcelom Bioscience, USA); ClassⅡType B12 biological safety cabinet (Esco, Singapore); LSM900 laser confocal microscope (Zeiss, Germany).

[0063] 1.3 Preparation of reagents and drugs

[0064] Accurately weigh an appropriate amount of N-trans-p-coumaryltyramide and place it in a brown sterile 1.5 mL EP tube. Add a certain amount of DMSO to prepare a 200 mM drug stock solution and store it at -20 °C for later use.

[0065] The complete culture medium used in this embodiment is prepared by a penicillin-streptomycin mixed solution, fetal bovine serum and α-MEM liquid culture medium in a volume ratio of 1:10:89.

[0066] 2. Methods

[0067] 2.1 Cell recovery and culture

[0068] RAW264.7 macrophages were cultured according to the instructions.

[0069] The macrophages were taken out of liquid nitrogen and thawed in a 37°C water bath. When the macrophages were nearly thawed, the macrophages were taken out and dried with cotton balls. The macrophages were disinfected with 75% alcohol and transferred to a clean bench. The cap of the cryopreservation tube was opened, 1 mL of complete medium was added, mixed and transferred to a centrifuge tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded and resuspended with complete medium, transferred to a 100 mm culture dish, and incubated at 37°C and 5% CO 2 Culture in an incubator, keep observing and change the medium.

[0070] 2.2 Cell cryopreservation

[0071] When the macrophages are cultured to 80%, digest the cells with trypsin, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) in a volume ratio of 9:1 to resuspend, transfer to a cryopreservation tube, mark it, transfer to -80°C after overnight at -20°C, and then transfer to a liquid nitrogen tank for long-term cryopreservation.

[0072] 2.3CCK-8 detection of drug safety concentration

[0073] The macrophages were plated at 1×10 4 100 μL was inoculated into each well at a density of 10 cells / mL. When the macrophages grew to 50-60%, 100 μL of NF-κB inhibitor was added into each well at a series of concentrations of 200, 100, 50, 25, and 12.5 μM to treat the macrophages for 24 hours, in parallel for 3 times.

[0074] Then 10 μL of CCK-8 solution was added to each well, and cell viability was measured after 2 h;

[0075] The absorbance was measured at 450 nm using a microplate reader;

[0076] The in vitro safety of NF-κB inhibitors was evaluated according to cell survival rate = (OD experimental group / OD control group) × 100%, and the maximum safe concentration of NF-κB inhibitors was screened as the drug concentration for subsequent experiments.

[0077] 2.4 Immunofluorescence detection of cell TLR4 / MyD88 / NF-κB expression under drug intervention

[0078] 2.4.1 Type of board:

[0079] After sterilizing the cell slides on flame, place them in a 24-well plate and add 75% alcohol for sterilization for 30 min. After sterilization, wash them three times with PBS buffer.

[0080] HepG2 cells were plated in 24-well plates at a density of 2 × 10 4 500 μL was inoculated into each well at a density of 10 cells / mL, and cell growth was observed.

[0081] 2.4.2 Administration:

[0082] When the cells grew to about 50%, they were treated with drugs and cultured according to the following groups: (1) blank group; (2) LPS group; (3) N-trans-p-coumaryltyramide (25 μM) + LPS.

[0083] 2.4.3 Fixed:

[0084] After 24 hours of drug intervention in 2.4.2, the 24-well plate was removed, the cell culture medium was discarded, 200 μL PBS buffer was added to each well, and the cells were washed after standing for 5 minutes. After repeating 3 times, 200 μL 4% paraformaldehyde was added to each well and fixed at room temperature for 30 minutes.

[0085] 2.4.4 Closure:

[0086] After fixation, 200 μL of PBS buffer was added again for washing three times, each time for 5 min;

[0087] After washing, the cells were treated with 200 μL 0.5% Triton X-100 for 5 min;

[0088] After the treatment, the cells were washed three times with PBS buffer, each time for 5 min;

[0089] 1% bovine serum albumin (BSA) was added to the 24-well plate and blocked at room temperature for 30 min;

[0090] After blocking, wash with PBS buffer for 3 times, 5 min each time.

[0091] 2.4.5 Incubate with primary antibody:

[0092] According to the instructions of the primary antibody, TLR4\MyD88\NF-κB was diluted with 5% bovine serum albumin BSA, and the diluted primary antibody was added to each well, 200 μL, and incubated at 4°C overnight. The primary antibody was recovered and washed 3 times with PBS buffer, each time for 5 minutes.

[0093] 2.4.6 Incubation with secondary antibody:

[0094] According to the instructions of the secondary antibody, the secondary antibody was diluted with 0.5% bovine serum albumin BSA. After dilution, 200 μL was added to each well and incubated at room temperature for 1 hour. The secondary antibody was recovered and washed 3 times with PBS buffer, each time for 5 minutes.

[0095] 2.4.7 Nuclear staining:

[0096] Nuclear staining was performed with 200 μL DAPI staining solution, and the cells were incubated at room temperature in the dark for 10 min. After incubation, the DAPI staining solution was discarded and the cells were washed three times with PBS buffer, each time for 5 min.

[0097] 2.4.8DAPI mounting:

[0098] After taking out the cell slide and drying it, drop a drop of anti-fluorescence quenching mounting medium on the slide, stick the cell slide with the cell side on the mounting medium, and fix the cell slide with neutral resin.

[0099] 2.4.9 Microscopic examination:

[0100] After sealing, the slides were protected from light and stored at 4°C, and then examined under a microscope after the neutral resin solidified.

[0101] 3. Results

[0102] 3.1 Safe concentration of citrus fruit components in blood

[0103] This example uses CCK-8 experiment to explore the effect of different concentrations of drugs on RAW264.7 cell viability. The experimental results are as follows Figure 1 As shown, the results show that N-trans-p-coumaryl tyramide significantly inhibits cell proliferation at a concentration of 50 μM or above. In order to effectively carry out subsequent cell experiments, this example selects a concentration of 25 μM for subsequent cell experiments.

[0104] 3.2 Blood components of citronella inhibit the expression of TLR4 / MyD88 / NF-κB inflammatory signaling pathway

[0105] The TLR4 / MyD88 / NF-κB inflammatory signaling pathway is a classic signaling pathway for the production of inflammatory factors. In this example, the expression of TLR4 and MyD88 proteins in the TLR4 / MyD88 / NF-κB inflammatory signaling pathway was detected by IF technology. The experimental results are shown in Figure 2As shown, the results showed that N-trans-p-coumaryltyramide could reduce the expression of TLR4 and MyD88 proteins.

[0106] NF-κB nuclear translocation is a key process in activating the TLR4 / MyD88 / NF-κB inflammatory signaling pathway. Using IF technology to examine its translocation can reflect the activation of the TLR4 / MyD88 / NF-κB inflammatory signaling pathway. Figure 3 As shown, compared with the LPS group, N-trans-p-coumaryltyramide could inhibit the translocation of NF-κB protein to the nucleus, thereby inhibiting the release of inflammatory factors.

[0107] 4. Conclusion

[0108] This example found that N-trans-p-coumaryl tyramide can inhibit the expression of TLR4 / MyD88 / NF-κB inflammatory signaling pathway, suggesting that N-trans-p-coumaryl tyramide can be used as an NF-κB inhibitor to prevent or treat inflammatory responses in the body.

[0109] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any technician familiar with the field can make many possible changes and modifications to the technical solution of the present invention by using the technical means and technical contents disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, all equivalent changes made according to the shape, structure and principle of the present invention without departing from the content of the technical solution of the present invention should be covered by the protection scope of the present invention.

Claims

1. An NF-κB inhibitor, characterized in that Such inhibitors include N-trans-p-coumaryl tyramide.

2. The NF-κB inhibitor according to claim 1, characterized in that The inhibitor inhibits the transfer of NF-κB protein to the cell nucleus to inhibit the release of inflammatory factors, thereby preventing or treating diseases caused by inflammation in the body.

3. A method for preparing the NF-κB inhibitor according to claim 1 or 2, characterized in that: The preparation method comprises: Step 1, macrophage recovery and culture; Step 2, macrophage cryopreservation; Step 3, using CCK-8 method to detect cell viability and screen the maximum safe concentration of NF-κB inhibitor; Step 4: Detect the expression of TLR4 / MyD88 / NF-κB in cells under the intervention of NF-κB inhibitor by immunofluorescence method.

4. The method for preparing the NF-κB inhibitor according to claim 3, characterized in that: The specific steps of step 1 include: The macrophages were taken out from liquid nitrogen, thawed in a 37°C water bath, and taken out when nearly thawed. The water was absorbed with cotton balls, and the macrophages in step 1-1 were disinfected with 75% alcohol, and transferred to an ultra-clean workbench. The cap of the cryopreservation tube was opened, 1 mL of complete culture medium was added, and the mixture was transferred to a centrifuge tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded and resuspended with complete culture medium, and the mixture was transferred to a 100 mm culture dish and cultured in a 37°C, 5% CO2 incubator, and the culture was observed and the medium was changed.

5. The method for preparing the NF-κB inhibitor according to claim 4, characterized in that: The complete culture medium is prepared by a penicillin-streptomycin mixed solution, fetal bovine serum and α-MEM liquid culture medium in a volume ratio of 1:10:

89.

6. The method for preparing the NF-κB inhibitor according to claim 4, characterized in that: The specific steps of step 2 include: Take the macrophages in step 1, and when the macrophages are cultured to 80%, digest the cells with trypsin, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fetal bovine serum and dimethyl sulfoxide in a volume ratio of 9:1 to resuspend, transfer to a cryopreservation tube, mark it, transfer to -80°C after overnight at -20°C, and then transfer to a liquid nitrogen tank for long-term cryopreservation.

7. The method for preparing the NF-κB inhibitor according to claim 6, characterized in that: The specific steps of step 3 include: The macrophages from step 2 were plated at 1×10 4 100 μL was inoculated into each well at a density of 10 cells / mL. When the macrophages grew to 50-60%, 100 μL of NF-κB inhibitor was added into each well at a series of concentrations of 200, 100, 50, 25, and 12.5 μM to treat the macrophages for 24 hours, in parallel for 3 times. Then 10 μL of CCK-8 solution was added to each well, and cell viability was measured after 2 h; The absorbance was measured at 450 nm using a microplate reader; The in vitro safety of NF-κB inhibitors was evaluated according to cell survival rate = (OD experimental group / OD control group) × 100%, and the maximum safe concentration of NF-κB inhibitors was screened as the drug concentration for subsequent experiments.

8. The method for preparing the NF-κB inhibitor according to claim 7, characterized in that: In step 3, the method for preparing the NF-κB inhibitor comprises: Accurately weigh an appropriate amount of N-trans-p-coumaryl tyramide and place it in a brown sterile 1.5 mL EP tube. Add a certain amount of DMSO to prepare a 200 mM NF-κB inhibitor and store it at -20°C for later use.

9. The method for preparing the NF-κB inhibitor according to claim 8, characterized in that: The specific steps of step 4 include: Step 4-1, seeding board: After sterilizing the cell slides on flame, place them in a 24-well plate and add 75% alcohol for sterilization for 30 min. After sterilization, wash them three times with PBS buffer. HepG2 cells were plated in 24-well plates at a density of 2 × 10 4 500 μL was inoculated into each well at a density of 10 cells / mL, and cell growth was observed; Step 4-2, drug administration: When the cells grow to about 50%, add NF-κB inhibitor and continue culturing; Step 4-3; Fixation: After 24 h of NF-κB inhibitor intervention, the 24-well plate was removed, the cell culture medium was discarded, 200 μL of PBS buffer was added to each well, and the cells were washed after standing for 5 min. After repeating 3 times, 200 μL of 4% paraformaldehyde was added to each well and fixed for 30 min at room temperature. Step 4-4, closure: After fixation, 200 μL of PBS buffer was added again for washing three times, each time for 5 min; After washing, the cells were treated with 200 μL 0.5% Triton X-100 for 5 min; After the treatment, the cells were washed three times with PBS buffer, each time for 5 min; 1% bovine serum albumin was added to the 24-well plate and blocked at room temperature for 30 min; After blocking, wash with PBS buffer three times, 5 min each time; Step 4-5, incubate with primary antibody: TLR4\MyD88\NF-κB were diluted with 5% bovine serum albumin, and the diluted primary antibody was added to each well, 200 μL, and incubated at 4°C overnight. The primary antibody was recovered and washed three times with PBS buffer, each time for 5 min; Steps 4-6, incubation with secondary antibody: The secondary antibody was diluted with 0.5% bovine serum albumin, and 200 μL was added to each well. The cells were incubated at room temperature for 1 h, and the secondary antibody was recovered and washed three times with PBS buffer for 5 min each time. Steps 4-7, nuclear staining: Nuclear staining was performed with 200 μL DAPI staining solution, and the cells were incubated at room temperature in the dark for 10 min. After the incubation, the DAPI staining solution was discarded and the cells were washed three times with PBS buffer, each time for 5 min. Steps 4-8, DAPI mounting: After taking out the cell slide and drying it, drop a drop of anti-fluorescence quenching mounting medium on the slide, stick the cell slide with the cell side on the mounting medium, and fix the cell slide with neutral resin; Steps 4-9, microscopic examination: After sealing, the slides were protected from light and stored at 4°C, and then examined under a microscope after the neutral resin solidified.

10. Use of the NF-κB inhibitor according to claim 1 or 2 in a drug for preventing or treating inflammation-related diseases.

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