Application of nuclear transport receptor importin-β as a target in the preparation of drugs for the treatment of radiation-induced liver fibrosis

Abstract

The present invention discloses the application of nuclear transport receptor importin-β as a target in the preparation of a drug for treating radiation-induced liver fibrosis, and belongs to the field of biomedicine. The present invention plays a role in treating radiation-induced liver fibrosis by inhibiting the nuclear entry of protein mediated by the nuclear transport receptor importin-β. The present invention finds that importazole, an inhibitor of nuclear transport receptor importin-β, can inhibit the entry of EIF4A3 protein of irradiated hepatic stellate cells into the nucleus, inhibit cell proliferation and promote apoptosis, and can also inhibit the expression of inflammatory factors and fibrosis factors of irradiated hepatic stellate cells, proving that inhibitors of nuclear transport receptor importin-β have the potential to prepare drugs for treating radiation-induced liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of nuclear transport receptor importin-β as an action target in the preparation of a drug for treating radiation-induced liver fibrosis. Background Art

[0002] Radiation-induced liver injury (RILI) is a common complication of radiotherapy for liver cancer. One of the main manifestations is radiation-induced liver fibrosis (RILF), which can lead to liver failure and death in severe cases. RILF is mainly caused by the activation of hepatic stellate cells. Screening for drugs that can effectively block the activation of hepatic stellate cells is of great significance for the effective prevention and treatment of radiation-induced liver fibrosis.

[0003] Activation of hepatic stellate cells (HSCs) plays a key role in the pathogenesis of RILF. Once liver damage occurs, quiescent HSCs are activated, transform into myofibroblasts, and produce a large amount of extracellular matrix, while excessive extracellular matrix deposition promotes the occurrence of liver fibrosis.

[0004] The nuclear transport receptor importin-β is a nuclear import receptor that can recognize and bind to different types of nuclear import proteins and mediate protein import into the nucleus through interaction with nucleoporins. The N-terminus of importinβ contains a conserved Ran binding domain (IBN-N domain), which can bind to RanGTP; the middle part is a HEAT domain, which can bind to nucleoporins; and the C-terminus can bind to substrates or adapter proteins. The nuclear transport receptor importin-β binds to cargo proteins with the assistance of adapter proteins and transports the latter into the cell nucleus through the nuclear pore complex. In the cell nucleus, importin-β binds to RanGTP, releasing cargo proteins, thereby completing the nuclear import of proteins.

[0005] Currently, small molecule inhibitors of the nuclear transport receptor importin-β are still in short supply. Importazole is a small molecule inhibitor that can effectively inhibit importin-β-mediated protein nuclear transport. It inhibits the entry of proteins into the cell nucleus by interfering with the binding of importin-β to RanGTP.

[0006] However, whether importazole, a small molecule inhibitor of the nuclear transport receptor importin-β, can help in the treatment of radiation-induced liver fibrosis requires further study. Summary of the invention

[0007] The purpose of the present invention is to provide an application of nuclear transport receptor importin-β as a target in the preparation of a drug for treating radiation-induced liver fibrosis, so as to solve the problems existing in the above-mentioned prior art. The present invention finds that the nuclear transport receptor importin-β inhibitor importazole has the potential to prepare a drug for treating radiation-induced liver fibrosis, providing a new treatment approach for radiation-induced liver fibrosis diseases.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides the use of nuclear transport receptor importin-β as a target in the preparation of a drug for treating radiation-induced liver fibrosis, and exerts the effect of treating radiation-induced liver fibrosis by inhibiting the nuclear importation of proteins mediated by the nuclear transport receptor importin-β.

[0010] Furthermore, inhibitors are used to block the nuclear transport receptor importin-β-mediated protein importation into the nucleus, thereby playing a role in treating radiation-induced liver fibrosis.

[0011] Further, the inhibitor comprises importazole, and the chemical formula of importazole is as follows:

[0012]

[0013] Furthermore, the inhibitor plays a role in treating radiation-induced liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells and the expression of inflammatory factors and fibrosis factors.

[0014] Furthermore, by inhibiting the entry of EIF4A3 protein of hepatic stellate cells into the cell nucleus, the activation of hepatic stellate cells is inhibited.

[0015] Furthermore, the inflammatory factors include TNF-α, IL-6 and IL-1β; the fibrosis factors include α-SMA and collagen1.

[0016] The present invention also provides the use of an inhibitor of nuclear transport receptor importin-β in the preparation of a drug for treating radiation-induced liver fibrosis, wherein the inhibitor comprises importazole, and the chemical formula of importazole is as follows:

[0017]

[0018] The present invention also provides a medicine for treating radiation-induced liver fibrosis, which contains a nuclear transport receptor importin-β inhibitor.

[0019] Furthermore, the nuclear transport receptor importin-β inhibitor comprises importazole, and the chemical formula of importazole is as follows:

[0020]

[0021] Furthermore, the drug also includes pharmaceutically acceptable excipients; the excipients include any one or more of fillers, disintegrants, binders, lubricants and flavoring agents.

[0022] The present invention discloses the following technical effects:

[0023] The present invention finds that importazole, an inhibitor of nuclear transport receptor importin-β, can inhibit the entry of EIF4A3 protein of irradiated hepatic stellate cells into the cell nucleus, inhibit cell proliferation and promote apoptosis, and can also inhibit the expression of inflammatory factors and fibrosis factors of irradiated hepatic stellate cells, proving that the inhibitor has the potential to prepare drugs for treating radiation-induced liver fibrosis.

[0024] The importazole, an inhibitor of nuclear transport receptor importin-β of the present invention, has the following advantages: (1) It has no effect or little effect on other members of the nuclear transport family, greatly improving the targeting and specificity of the drug and reducing the incidence of its adverse reactions. (2) It is different from the principle of the radioactive liver fibrosis treatment drugs currently used in clinical practice, and can be used alone to inhibit the activation of hepatic stellate cells, or it can be used in combination with other drugs to achieve better therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 Radiation does not affect the expression of EIF4A3 mRNA and protein levels in hepatic stellate cells; A: qRT-PCR detection of EIF4A3 mRNA expression in hepatic stellate cells after 8Gy X-ray irradiation; B: Western blot detection of EIF4A3 expression in hepatic stellate cells after 8Gy X-ray irradiation; ns: no significant difference;

[0027] Figure 2Radiation promotes the nuclear translocation of EIF4A3 protein in hepatic stellate cells; A: Western blot detection of the nuclear and cytoplasmic expression of EIF4A3 in hepatic stellate cells after 8Gy X-ray irradiation; B: Immunofluorescence detection of the nuclear and cytoplasmic expression of EIF4A3 in hepatic stellate cells after 8Gy X-ray irradiation;

[0028] Figure 3 Importazole, an inhibitor of nuclear transport receptor importin-β, inhibited the nuclear transport of EIF4A3 protein in irradiated hepatic stellate cells; 24 hours after hepatic stellate cells were transfected with EIF4A3 overexpression plasmid, they were irradiated with 8Gy X-rays, and fresh culture medium containing or not containing 5μM importazole was replaced after irradiation. Detection was performed 48 hours later. A: Western blot detection of nuclear and cytoplasmic expression of EIF4A3 in hepatic stellate cells; B: Immunofluorescence detection of nuclear and cytoplasmic expression of EIF4A3 in hepatic stellate cells;

[0029] Figure 4 Importazole, an inhibitor of nuclear transport receptor importin-β, inhibited the proliferation of irradiated hepatic stellate cells and promoted their apoptosis; 24 hours after hepatic stellate cells were transfected with EIF4A3 overexpression plasmid, they were irradiated with 8Gy X-rays, and after irradiation, fresh culture medium containing or not containing 5μM importazole was replaced and the following tests were performed: A: CCK-8 assay to detect the proliferation of irradiated hepatic stellate cells; B: Flow cytometry to detect the apoptosis of irradiated hepatic stellate cells; *P<0.05; **P<0.01;

[0030] Figure 5 Importazole, an inhibitor of nuclear transport receptor importin-β, inhibited the secretion of inflammatory factors in irradiated hepatic stellate cells; 24 hours after hepatic stellate cells were transfected with EIF4A3 overexpression plasmid, they were irradiated with 8Gy X-rays, and fresh culture medium containing or not containing 5μM importazole was replaced after irradiation. After 48 hours, the cell culture supernatant was collected and the expression of inflammatory factors TNF-α, IL-6 and IL-1β in the supernatant was detected by ELISA; *P<0.05; **P<0.01;

[0031] Figure 6Importazole, an inhibitor of nuclear transport receptor importin-β, inhibited the expression of fibrotic factors in irradiated hepatic stellate cells. 24 hours after hepatic stellate cells were transfected with EIF4A3 overexpression plasmid, they were irradiated with 8 Gy X-rays. After irradiation, fresh culture medium containing or not containing 5 μM importazole was replaced. After 48 hours, the following tests were performed: A: Western Blot detection of the expression of inflammatory factors α-SMA and collagen1 in irradiated hepatic stellate cells; B: Immunofluorescence detection of the expression of inflammatory factors α-SMA and collagen1 in irradiated hepatic stellate cells. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Unless otherwise specified, the materials, instruments and reagents used in the present invention can be obtained from commercial channels; the experimental methods used are conventional experimental methods in the art unless otherwise specified.

[0038] The nuclear transport receptor importin-β inhibitor importazole used in the following examples was purchased from KKL Med, and its molecular formula is as follows:

[0039]

[0040] Example 1 Radiation promotes nuclear translocation of EIF4A3 protein in hepatic stellate cells

[0041] Human hepatic stellate cells LX2 were divided into irradiated and non-irradiated groups. The irradiated group was irradiated with 8Gy X-rays, and then qRT-PCR and Western blot were used to detect the expression of EIF4A3 mRNA and EIF4A3 protein, respectively. The results showed that compared with the non-irradiated group, radiation did not affect the expression of EIF4A3 mRNA and EIF4A3 protein in hepatic stellate cells ( Figure 1 ). However, when hepatic stellate cells LX2 were irradiated with 8Gy X-rays, Western blot and cell immunofluorescence were used to detect the nuclear and cytoplasmic distribution of EIF4A3 protein 48 hours later. The results showed that radiation promoted the translocation of EIF4A3 protein into the nucleus of hepatic stellate cells ( Figure 2 ), suggesting that radiation-mediated HSC activation may be associated with the nuclear translocation of EIF4A3.

[0042] The above-mentioned qRT-PCR, Western blot, cell nucleoplasmic protein separation and cell immunofluorescence detection methods are as follows:

[0043] (1) qRT-PCR

[0044] ① Extract total RNA from cells: 1) Discard the supernatant, wash once with PBS, add 1mL Trizol reagent to the cells in the 6-well plate, let stand on ice for 5 minutes, and then collect into a 1.5mL EP tube. 2) Add 200μL of chloroform (1 / 5 the amount of Trizol reagent) to the EP tube, flip the EP tube upside down 5-6 times to mix it thoroughly, let stand at room temperature for 5 minutes, and after the liquid in the tube is separated, put it into a 4℃ centrifuge for centrifugation at 12000rpm / min for 15min. 3) After the centrifugation, the mixed solution can be seen to be separated into layers, the upper layer is a colorless aqueous phase containing total RNA, and the lower layer is a light red phenol-chloroform phase. Collect the upper layer of the EP tube into a new EP tube, add an equal volume of isopropanol, flip the EP tube upside down 5-6 times, and let it stand at room temperature for 10min. Then centrifuge at 4℃, 12000rpm / min, 10min. 4) Remove the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to the tube to wash the RNA precipitate, and then place it in a 4°C centrifuge at 12,000 rpm / min for 5 minutes. Repeat this step twice. 5) Discard the supernatant, place it in a clean bench to dry thoroughly, and finally add 20-30 μL of pre-cooled DEPC water to dissolve it.

[0045] ② Determination of RNA concentration: Use Nanodrop 2000 software to determine the extracted RNA concentration, keep experimental records, and store the samples at -80℃ for later use or immediately proceed with subsequent processing.

[0046] ③Reverse transcription of mRNA:

[0047] HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (purchased from Nanjing Novozyme Biotechnology) was used to reverse transcribe mRNA into cDNA, and the reaction system was prepared according to Table 1 for mRNA reverse transcription.

[0048] Table 1 mRNA reverse transcription reaction loading system

[0049]

[0050] After the above system is mixed, it is centrifuged instantly and the reaction program on the machine is: 50℃15min, 85℃5s.

[0051] ④qRT-PCR

[0052] Primer sequences of relevant genes were designed (see Table 2), and the reaction system was prepared according to the instructions of Taq Pro Universal SYBR qPCR MasterMix Kit (purchased from Nanjing Novozyme Biotechnology) (see Table 3), and amplification was performed using a real-time fluorescence quantitative PCR instrument Q6.

[0053] Table 2 Primer sequences of related genes

[0054]

[0055] Table 3 mRNA qRT-PCR reaction sample addition system

[0056]

[0057] After the above system is mixed, centrifuge it instantly, and the reaction program on the machine is: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing and extension for 34s and data collection, 40 cycles; melting curve analysis is performed after the cycle. Melting curve acquisition program: 95℃15s, 60℃1min, 95℃30s, 60℃15s, and melting curve data are acquired.

[0058] ⑤Analysis of experimental results

[0059] After the reaction, confirm the amplification curve and melting curve of qRT-PCR, collect the Ct value of each template, and use 2 -△△Ct The mRNA was calculated using GAPDH as an internal reference.

[0060] (2) Western blot

[0061] ① Cell lysis: Add an appropriate amount of RIPA protein lysis buffer to the cells to be tested to fully lyse them.

[0062] ② Protein concentration determination and sample preparation: Use the BCA method to detect the protein concentration, mix by pipetting according to the ratio of protein volume: 5×loading buffer = 4:1, and place the mixed sample in a 100°C metal bath and heat for 5 minutes.

[0063] ③ Electrophoresis: prepare 10% SDS-PAGE separation gel and 5% SDS-PAGE concentration gel, take 20μg protein sample for sample loading, set the electrophoresis voltage, concentration gel (upper gel) stage 60V, 20min, separation gel (lower gel) stage, 90V, 80-90min, turn off the power when bromophenol blue is about to reach the bottom of the separation gel.

[0064] ④ Transfer: After electrophoresis, set a constant current of 200 mA for 2 h at low temperature (4°C) to transfer the protein to the PVDF membrane.

[0065] ⑤ Blocking: Immerse the PVDF membrane in 5% BSA for blocking, and place it on a slow shaker (40 rpm / min) at room temperature for 1 hour.

[0066] ⑥ Primary antibody incubation: After blocking, take out the PVDF membrane and immerse it in TBST (1% TBST = 1L TBS buffer + 1mL Tween-20) solution for 5 minutes. Then, place the PVDF membrane in the prepared primary antibody solution and incubate it on a slow shaker at 4°C overnight.

[0067] ⑦ Secondary antibody incubation: The next day, take out the PVDF membrane, place it on a fast (120r / min) shaker at room temperature, wash it with TBST solution, 10min each time, repeat 3 times. Place the washed PVDF membrane in the corresponding secondary antibody solution, and shake it on a slow (40rpm / min) shaker at room temperature for 1h.

[0068] ⑧ Luminescence: After the secondary antibody incubation is completed, the PVDF membrane is washed three times with TBST solution as above, and the band image is collected using ECL chemiluminescence.

[0069] (3) Separation of nuclear and cytoplasmic proteins

[0070] The nuclear and cytoplasmic proteins of hepatic stellate cells were extracted using a nuclear protein and cytoplasmic protein extraction kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.). The specific steps are as follows:

[0071] ① Wash once with PBS, add an appropriate amount of PBS and scrape off the cells with a cell scraper, collect the cell suspension into a 1.5mL EP tube. Centrifuge at room temperature at 1000rpm for 5 minutes, discard the supernatant, and collect the cell pellet for later use;

[0072] ② Add 200 μL of cytoplasmic protein extraction reagent A with PMSF added to every 20 μL of cell pellet;

[0073] ③ Vortex vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet, and place on ice for 10 minutes;

[0074] ④ Add 10 μL of cytoplasmic protein extraction reagent B, vortex vigorously at the highest speed for 5 seconds, and place on ice for 1 minute;

[0075] ⑤ Centrifuge at the highest speed for 5 seconds and at 12,000 rpm for 5 minutes at 4°C;

[0076] ⑥Immediately aspirate the supernatant into a pre-cooled EP tube, which is the extracted cytoplasmic protein;

[0077] ⑦ For the precipitate, completely aspirate the remaining supernatant and add 50 μL of nuclear protein extraction reagent with PMSF added;

[0078] ⑧ Vortex vigorously at the highest speed for 30 seconds to completely suspend and disperse the cell pellet. Then return to the ice bath and vortex vigorously at high speed for 30 seconds every 2 minutes for a total of 30 minutes;

[0079] ⑨Centrifuge at 12,000 rpm for 10 min at 4°C;

[0080] ⑩Immediately pipette the supernatant into a pre-cooled EP tube, which is the extracted nuclear protein. Then use Western blot to detect the expression of nuclear and cytoplasmic related proteins.

[0081] (4) Cell Immunofluorescence

[0082] ① Plating: LX2 cells were inoculated in 24-well plates. After intervention, the culture medium was removed and the cells were washed once with PBS. ② Fixation: 0.5 mL of 4% paraformaldehyde fixative was added and fixed for 30 min.

[0083] ③ Washing: discard the fixative and wash with PBS three times, shaking slowly on a shaker (40 rpm / min), 5 min each time;

[0084] ④ Blocking: Add 0.5 mL 5% BSA for blocking and leave at room temperature for 1 hour;

[0085] ⑤ Apply primary antibody: discard the blocking solution, add 0.2mL of the diluted primary antibody, and shake slowly (40rpm / min) at 4℃ overnight;

[0086] ⑥ Apply secondary antibody: discard the primary antibody, wash with PBS 3 times, shake slowly on a shaker (40rpm / min), 5min each time; add 0.2mL of the secondary antibody diluted in proportion, shake slowly on a shaker (40rpm / min) at room temperature and away from light for 1h;

[0087] ⑦ Nuclear staining: Recover the secondary antibody; wash with PBS three times, shake slowly on a shaker (40 rpm / min), 5 min each time; add 0.2 mL of nuclear staining solution (DAPI), shake slowly on a shaker (40 rpm / min) for 5 min at room temperature and away from light;

[0088] ⑧Washing: discard the nuclear staining solution, wash with PBS three times, shake slowly on a shaker (40 rpm / min), 5 min each time;

[0089] ⑨ Taking photos: Use an inverted fluorescence microscope IX73 to observe and take fluorescent photos.

[0090] Example 2 Importazole, an inhibitor of nuclear transport receptor importin-β, inhibits the entry of EIF4A3 protein from irradiated hepatic stellate cells into the nucleus

[0091] Hepatic stellate cells were transfected with EIF4A3 overexpression plasmid or control plasmid (both purchased from Shandong Weizhen Biotechnology). 24 hours after transfection, they were irradiated with 8Gy X-rays. After irradiation, the complete culture medium containing or not containing 5μM importazole was replaced, and the following groups were set: ① Overexpression control group (NC-OE); ② Overexpression EIF4A3 group (EIF4A3-OE); ③ Overexpression EIF4A3+importazole treatment group (EIF4A3-OE+importazole). Western blot detection was performed after 48 hours of importazole treatment. The results showed that overexpression of EIF4A3 increased the expression of EIF4A3 protein in the nucleus of hepatic stellate cells LX2, while the addition of importazole significantly inhibited the entry of EIF4A3 protein into the nucleus ( Figure 3 Similarly, cell immunofluorescence assays also showed that importazole could significantly inhibit the entry of EIF4A3 protein into the nucleus of hepatic stellate cells ( Figure 3 (B).

[0092] Example 3 Importazole, an inhibitor of nuclear transport receptor importin-β, inhibits proliferation and promotes apoptosis of irradiated hepatic stellate cells

[0093] Hepatic stellate cells were transfected with EIF4A3 overexpression plasmid or control plasmid. 24 hours after transfection, they were irradiated with 8Gy X-rays. After irradiation, the complete culture medium containing or not containing 5μM importazole was replaced, and the following groups were set: ① overexpression control group (NC-OE); ② overexpression EIF4A3 group (EIF4A3-OE); ③ overexpression EIF4A3+importazole treatment group (EIF4A3-OE+importazole). The proliferation of irradiated hepatic stellate cells was detected by CCK-8 assay. The results showed that overexpression of EIF4A3 significantly enhanced the proliferation ability of irradiated hepatic stellate cells, while the addition of importazole could significantly inhibit the proliferation ability of irradiated hepatic stellate cells ( Figure 4 In addition, after 48 hours of importazole treatment, flow cytometry was used to detect the apoptosis of irradiated hepatic stellate cells. The results showed that overexpression of EIF4A3 significantly inhibited the apoptosis of irradiated hepatic stellate cells, while the addition of importazole could significantly promote the apoptosis of irradiated hepatic stellate cells ( Figure 4 (B).

[0094] The detection methods of the above CCK-8 experiment and flow cytometry experiment are as follows:

[0095] (1) CCK-8 experiment

[0096] ① The hepatic stellate cells transfected with plasmids were inoculated into 96-well plates (3000 cells / well) and given corresponding treatments;

[0097] ② The cell proliferation activity was detected using CCK-8 kit (purchased from Japan Tongren) at 6, 24, 48, 72, 96, and 120 h after cell irradiation;

[0098] ③ At the set time point, remove the culture medium in the wells, wash once with PBS, then add 100 μL of freshly prepared solution (containing 10 μL CCK-8 reagent and 90 μL DMEM culture medium) to each well and continue culturing for 2 hours;

[0099] ④ Use an ELISA reader to read the absorbance at 450nm and draw a cell proliferation curve.

[0100] (2) Flow cytometry to detect cell apoptosis

[0101] The cell apoptosis detection kit (purchased from Jiangsu Keygene Biotechnology) was used for detection.

[0102] ① Hepatic stellate cells were seeded into 6-well plates and given corresponding treatments;

[0103] ② After 48 hours of cell irradiation, discard the supernatant, wash once with PBS, digest the cells with trypsin and collect them into 1.5 mL EP tubes;

[0104] ③Discard the supernatant and add 500 μL Binding Buffer to suspend the cells;

[0105] ④ After adding 5μL AnnexinV-APC and mixing, add 5μL Propidium Iodide and mix;

[0106] ⑤React at room temperature in the dark for 10 minutes;

[0107] ⑥Perform flow cytometry testing within 1 hour.

[0108] Example 4: Importazole, a nuclear transport receptor importin-β inhibitor, inhibits the secretion of inflammatory factors in irradiated hepatic stellate cells

[0109] Hepatic stellate cells were transfected with EIF4A3 overexpression plasmid or control plasmid. 24 hours after transfection, they were irradiated with 8Gy X-rays. After irradiation, the complete culture medium containing or not containing 5μM importazole was replaced, and the following groups were set: ① Overexpression control group (NC-OE); ② EIF4A3 overexpression group (EIF4A3-OE); ③ EIF4A3 overexpression + importazole treatment group (EIF4A3-OE + importazole). After 48 hours of importazole treatment, the supernatant of cell culture was collected, and the expression of inflammatory factors TNF-α, IL-6 and IL-1β in the supernatant was detected by ELISA. The results showed that overexpression of EIF4A3 significantly enhanced the secretion of inflammatory factors TNF-α, IL-6 and IL-1β in irradiated hepatic stellate cells, while the addition of importazole could significantly inhibit the secretion of the above inflammatory factors ( Figure 5 ).

[0110] The detection method of the above ELISA is as follows:

[0111] TNF-α, IL-6 and IL-1β cytokine ELISA detection kits (purchased from Shanghai ELISA Biologicals) were used to detect the content of TNF-α, IL-6 and IL-1β in the cell supernatant. The specific steps are as follows:

[0112] ① Sample collection: Collect the cell supernatant, centrifuge at 2000 rpm for 10 min at room temperature, and collect the supernatant in a 1.5 mL centrifuge tube;

[0113] ② Standard sample addition: set standard sample wells and sample wells, and add 50μL of standard samples of different concentrations to each standard sample well;

[0114] ③ Sample addition: Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40 μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10 μL of the test sample (the final sample dilution is 5 times);

[0115] ④ Add enzyme: add 100 μL of enzyme-labeled reagent to each well, except for the blank well;

[0116] ⑤ Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour;

[0117] ⑥ Liquid preparation: dilute the 20 times concentrated washing solution with 20 times distilled water for later use;

[0118] ⑦ Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with detergent, let it stand for 30 seconds and then discard, repeat 5 times;

[0119] ⑧ Color development: first add 50 μL of color development solution A to each well, then add 50 μL of color development solution B, gently shake and mix, and develop the color at 37℃ in the dark for 15 min;

[0120] ⑨ Determination: Set the blank well to zero and measure the absorbance (OD value) of each well in turn at a wavelength of 450nm;

[0121] ⑩Calculation: Collect data, draw standard curves for each cytokine and generate corresponding formulas to calculate the content of each cytokine.

[0122] Example 5 Importazole, an inhibitor of nuclear transport receptor importin-β, inhibits the fibrotic phenotype of irradiated hepatic stellate cells

[0123] Hepatic stellate cells were transfected with EIF4A3 overexpression plasmid or control plasmid. 24 hours after transfection, they were irradiated with 8Gy X-rays. After irradiation, the complete culture medium containing or not containing 5μM importazole was replaced, and the following groups were set: ① overexpression control group (NC-OE); ② EIF4A3 overexpression group (EIF4A3-OE); ③ EIF4A3 overexpression + importazole treatment group (EIF4A3-OE + importazole). After 48 hours of importazole treatment, the expression of α-SMA and collagen1 proteins in irradiated hepatic stellate cells was detected by Western blot and immunofluorescence. The results showed that overexpression of EIF4A3 significantly promoted the expression of fibrotic factors α-SMA and collagen1 in irradiated hepatic stellate cells, while the addition of importazole could significantly inhibit the expression of the above fibrotic factors ( Figure 6 ).

[0124] In summary, importazole, an inhibitor of nuclear transport receptor importin-β, can inhibit the activation, inflammation and fibrotic phenotypes of irradiated hepatic stellate cells, which proves that the inhibitor can inhibit radiation-induced liver fibrosis and has the potential to treat radiation-induced liver fibrosis.

[0125] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an inhibitor of nuclear transport receptor importin-β in the preparation of a drug for treating radiation-induced liver fibrosis, characterized in that: The inhibitor is importazole, and the chemical formula of importazole is as follows: 。

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