Application of SNX3 inhibitor in preparation of medicine for preventing and treating lung injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an SNX3 inhibitor in preparation of a medicine for preventing and treating lung injury. A lung injury mouse model induced by dripping a bleomycin solution (2mg / mL) through a tracheal intubation and an oral tracheal tube is studied and shows that SNX3 protein and gene expression in mouse lungs is increased along with the lung injury degree. A research on a lung SNX3 gene knockout mouse shows that lung injury can be improved by lung SNX3 gene deletion, which indicates that SNX3 can be used as a treatment target of lung injury; meanwhile, the si-SNX3 for inhibiting the expression of the SNX3 gene or protein is found to be capable of inhibiting the expression of lung injury related protein. The invention provides a new means and drug target for treatment of lung injury, and has important value for development of clinical drugs for lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of SNX3 inhibitors in the preparation of drugs for preventing and treating lung injury. Background Art

[0002] Lung injury is one of the most common and critical diseases in clinical practice, with extremely high morbidity and mortality. In severe cases, acute respiratory failure may occur, which seriously threatens human life and health. Existing studies have shown that a variety of factors can induce lung injury, including environmental factors: inhalation of harmful substances and smoking; drug injury: improper use of drugs, such as amiodarone, bleomycin, etc.; systemic immune diseases: diseases such as systemic lupus erythematosus and rheumatoid arthritis may induce lung injury; genetic factors, such as MUC5B gene abnormalities, affect normal lung repair, thereby leading to the occurrence of lung injury. Due to the complex and unclear pathogenesis of lung injury, the development of drugs for lung injury has been slow.

[0003] At present, clinical treatment of lung injury mainly focuses on controlling inflammatory response and improving clinical symptoms. Lung transplantation is almost the only intervention for patients with severe lung injury. Therefore, studying the mechanism of lung injury is of great significance for disease treatment and drug development.

[0004] SNX3 (sorting nexin 3) is a sorting nexin protein that plays an important role in maintaining the homeostasis of intracellular proteins. Sorting nexins are a class of highly conserved eukaryotic proteins that not only play an important regulatory role in some important cell signaling pathways, but also affect the occurrence and development of diseases. The inventor's team's preliminary study CN117883578A showed that SNX3 gene deletion can alleviate bile duct ligation-induced liver fibrosis, suggesting that SNX3 can be used as a therapeutic target for liver fibrosis; si-SNX3 that inhibits SNX3 protein and gene expression can effectively inhibit liver fibrosis; it can be seen that SNX3 inhibitors are effective drugs for the treatment of liver fibrosis. However, since lung injury is different from pulmonary fibrosis, and lung and liver tissues are different, the role of SNX3 in lung injury cannot be determined. Summary of the invention

[0005] The present invention aims to overcome the problem of relatively single clinical treatment methods and insufficient drugs for lung injury. After research and exploration, the application of SNX3 inhibitors in drugs for the prevention and treatment of lung injury is provided, providing new targets and drug strategies for the prevention and treatment of lung injury.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention uses a mouse model of severe lung injury induced by oral instillation of bleomycin solution (2 mg / mL) through tracheal intubation to study and show that the expression of SNX3 protein and gene in the mouse lungs increases with the degree of lung injury. The present invention uses lung SNX3 gene knockout mice to study and show that lung SNX3 gene deletion can improve lung injury, indicating that SNX3 can be used as a therapeutic target for lung injury; at the same time, the present invention finds that si-SNX3 that inhibits the expression of SNX3 gene or protein can inhibit the expression of lung injury-related proteins, and can become a new treatment approach and drug target for lung injury. Therefore, the present invention claims protection for:

[0008] Application of SNX3 as a target in the preparation of drugs for the prevention and treatment of lung injury.

[0009] Application of SNX3 inhibitors in the preparation of drugs for preventing and treating lung injury.

[0010] The SNX3 inhibitor reduces the expression of SNX3 protein or gene.

[0011] Specifically, the technical means for reducing the expression of SNX3 protein or gene can be achieved by conventional existing technologies such as gene silencing, gene knockout, gene editing or gene knockdown known to those skilled in the art. For example, RNA interference (RNAi) technology can be used to specifically eliminate or shut down the expression of specific genes; the tools using gene editing technology can be CRISPR / Cas9 technology, zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) technology, but are not limited thereto.

[0012] As an optional embodiment, the SNX3 inhibitor is si-SNX3.

[0013] More preferably, the sequence of the si-SNX3 is GCAACTTCCTCGAGATCGA.

[0014] The technology of inactivating SNX3 gene expression or silencing the gene at the post-transcriptional level or translational level by using gene knock-down technology is well known to those skilled in the art. The gene knock-down technology includes RNA interference, Morpholino interference, antisense nucleic acid, ribozyme or dominant negative inhibitory mutation, but is not limited thereto.

[0015] It is also a conventional prior art known to those skilled in the art to inhibit the expression of the SNX3 gene by using shRNA or siRNA expressed by a virus (such as a lentivirus or adeno-associated virus) to silence the SNX3 gene.

[0016] At the same time, the present invention also provides other options, the SNX3 inhibitor is a siRNA, dsRNA, shRNA, miRNA, antisense nucleic acid that uses SNX3 or its transcript as a target sequence and can inhibit SNX3 protein expression or gene transcription; or a vector construct that can express or form the siRNA, dsRNA, shRNA, miRNA, antisense nucleic acid.

[0017] Furthermore, the vector construct is at least one of a bacterial plasmid, a bacteriophage, a recombinant viral vector or a eukaryotic recombinant expression vector.

[0018] In addition, the SNX3 inhibitor may also be a small molecule inhibitor.

[0019] Furthermore, the drug may further include a pharmaceutically acceptable carrier.

[0020] Furthermore, the pharmaceutically acceptable carrier includes any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, suspending agents, surfactants, and stabilizers.

[0021] Furthermore, the administration routes of the drug include intravenous injection, intramuscular injection, subcutaneous injection or oral administration.

[0022] Furthermore, the dosage form of the drug is tablets, capsules, pills, powders, granules or oral liquids.

[0023] A drug for preventing and treating lung injury, comprising a SNX3 inhibitor.

[0024] Furthermore, the SNX3 inhibitor is si-SNX3.

[0025] Furthermore, the si-SNX3 sequence is GCAACTTCCTCGAGATCGA.

[0026] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0027] The present invention has the following beneficial effects:

[0028] The present application has been studied in the laboratory and has shown that after normal mice were induced with bleomycin-induced lung injury, the expression of SNX3 protein and gene in the mouse lungs increased with the lung injury; further, the present invention used SNX3 gene knockout mice as experimental subjects to study the role of SNX3 gene in lung injury, and the results showed that the lack of SNX3 gene in the lungs can reduce lung injury, suggesting that SNX3 can be used as a target for the prevention and treatment of lung injury, and SNX3 inhibitors are effective drugs for the prevention and treatment of lung injury; at the same time, the present invention found that si-SNX3, which inhibits the expression of SNX3 gene or protein, can significantly reduce the expression of fibronectin, collagen-Ⅰ (COL-Ⅰ) and α-actin (α-SMA) in human embryonic lung cells MRC5. The experimental results of the present invention show that SNX3 is expected to target the multiple pathogenesis of lung injury, providing a new means for the prevention and treatment of lung injury, and has the potential to become a target for the prevention and treatment of various lung diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Pulmonary function test and lung tissue pathology test of mouse lung injury model (PIF: maximum inspiratory flow rate, Penh: forced respiratory interval, MV: minute ventilation, Saline: normal saline group, BLM: model group, α-SMA: α-actin, COL-Ⅰ: collagen-Ⅰ);

[0030] Figure 2 The expression level of SNX3 was significantly increased in the mouse lung injury model (SNX3: sorting junction protein 3, GADPH: glyceraldehyde-3-phosphate dehydrogenase as the internal reference protein, Relative protein expression (fold of Saline): the expression of related proteins (divided by the fold of histone expression in normal saline));

[0031] Figure 3 Comparison of lung function and histopathology between mice in the N-cKO group and the SNX3-cKO group (PIF: maximum inspiratory flow rate, Penh: forced respiratory interval, MV: minute ventilation);

[0032] Figure 4 Comparison of lung function and histopathology between mice in the N-cKO+BLM group and mice in the SNX3-cKO+BLM group (PIF: maximum inspiratory flow rate, Penh: forced respiratory interval, MV: minute ventilation);

[0033] Figure 5To construct a lung injury model at the cellular level and the expression changes of SNX3 (FN: fibronectin, COL-Ⅰ: collagen-Ⅰ, α-SMA: α-actin, TGFβ1: transforming growth factor β1, Relative protein expression (foldof GADPH): expression of related proteins (divided by the fold of GADPH protein expression));

[0034] Figure 6 This is the effect of knocking out SNX3 on the expression of lung injury-related proteins in a cellular lung injury model (Fibronectin: fibronectin, COL-Ⅰ: collagen-Ⅰ, α-SMA: α-actin, GADPH: glyceraldehyde-3-phosphate dehydrogenase as internal reference proteins). DETAILED DESCRIPTION

[0035] The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.

[0036] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field.

[0037] Unless otherwise specified, all reagents and materials used in the examples are commercially available.

[0038] Example 1 Construction of mouse lung injury model and study of the effects on lung injury

[0039] 1. Construction of mouse lung injury model

[0040] Twenty male C57BL / 6 mice (SPF grade, weight 25±2g) were randomly divided into two groups: control group and model group, with 10 mice in each group.

[0041] The mice were weighed and anesthetized by intraperitoneal injection of 3.5% chloral hydrate solution (0.1 ml / 10 g). The anesthetized mice were fixed on a flat plate, and the neck of the mice was illuminated by a lamp for animal experiments. The mouse trachea was found with the help of the light. About 20 μL of bleomycin solution (2 mg / mL) was instilled through the trachea through the mouth using a tracheal cannula. The mice were immediately stood upright and rotated several times to make the drug solution evenly distributed in the lungs. The control group was instilled with an equal amount of normal saline through the trachea, and various indicators were tested 21 days later.

[0042] 2. Pulmonary function test

[0043] Use the EMKA animal pulmonary function testing system to test the lung function of mice. Connect the device key and run the software. After setting the parameter calibration, run the file and quickly inject (syringe) 1mL of gas into the recording box to calibrate the signal of the channel and test the sealing of the equipment. Place the mouse in the recording box, and start the test after the mouse's breathing action is stable. Set it to automatically record the mouse's respiratory wave value every 5s, and the total time is 5min.

[0044] 3. HE staining, Masson staining, tissue immunofluorescence and immunohistochemistry

[0045] The lung tissue of mice was dissected, and the left lung was fixed in 4% paraformaldehyde for 48 hours. Wuhan Sewell Biotechnology Co., Ltd. was commissioned to prepare paraffin sections and perform HE staining, Masson staining, tissue immunofluorescence and immunohistochemistry. Tissue sections were observed and photographed by cell imaging to evaluate alveolar structure, lung inflammatory infiltration, collagen deposition or expression of related proteins.

[0046] 4. Western blots

[0047] Weigh an appropriate amount of animal tissue, wash it with PBS on ice and discard it. Add an appropriate amount of RIPA lysis buffer (containing 1% PMSF), use an ultrasonic device to release tissue protein, vortex once every 5 minutes, centrifuge at 12000rcf, 4℃ for 15 minutes, transfer the supernatant (protein) to another clean 1.5mL EP tube, and perform BCA quantitative packaging or freeze at -80℃.

[0048] (1) Sample pretreatment: According to the BCA method, the protein is divided into 1.5 mL centrifuge tubes according to the appropriate system. Place on ice, add sample, PBS and 5× Loading buffer in sequence, centrifuge, place in a dry thermostat at 100°C for 5 min, centrifuge, and perform subsequent operations or store in a -80°C refrigerator.

[0049] (2) Gel preparation: Select appropriate concentrations of separation gel and concentrated gel according to the molecular weight of the protein. Fix the glass plate, add reagents in order to prepare the separation gel, and add about 7.2 mL of separation gel to the fixed glass plate. Quickly seal it with isopropanol. After the separation gel solidifies (a clear dividing line appears between the isopropanol and the separation gel), recover the isopropanol, add the prepared concentrated gel, immediately insert the comb (no bubbles are generated), and let it stand until the concentrated gel solidifies.

[0050] (3) Sample loading: Fix the solidified gel in the electrophoresis tank, pour the electrophoresis solution (configured according to Table 4) into the tank until it covers the comb, slowly pull out the comb, blow out the residual gel remaining in the lane with a gun, and load the samples into their respective lanes in order. Add 4 μL of protein marker to the adjacent lanes on both sides of the samples.

[0051] (4) Electrophoresis: Set the voltage to 70 V and perform pre-electrophoresis for about 30 min. When the proteins reach the same horizontal line, set the voltage to 120 V and continue electrophoresis until the desired bands are completely separated (about 60 min).

[0052] (5) Electrotransfer: Pre-cut a PVDF membrane (0.22 μm) of appropriate size and soak the membrane in methanol for 5 min to activate it. After activation, soak the membrane, sponge pad and filter paper in electrotransfer solution. At the beginning of the transfer, place them in the order of sandwich structure. Make sure there are no bubbles between SDS glue and PVDF membrane. Clamp the electrotransfer clip and place it in the electrotransfer tank. Pour the pre-prepared and cooled electrotransfer solution. Put two ice bricks in the electrotransfer tank. Place the entire electrotransfer tank in an ice box. Set the current to 220 mA for 2 h (observe the voltage to be about 100 V to 120 V).

[0053] (6) Blocking: After electroporation, place the membrane in 5% skim milk (prepared in fresh TBST) and block at room temperature for 1 h with slow shaking on a decolorizing shaker.

[0054] (7) Incubation with primary antibody: discard the blocking solution, wash with TBST buffer for 5 min, three times, cut the membrane according to the molecular weight of the target protein and the protein marker, incubate the cut membrane in the corresponding primary antibody (prepared in primary antibody diluent), and incubate in a refrigerator at 4°C with rotation overnight to allow the primary antibody to fully bind to the protein on the membrane.

[0055] (8) Incubation with secondary antibody: Remove the membrane after incubation with primary antibody, add TBST buffer, place on a decolorizing shaker and shake quickly for 3 times, 10 min each time, select a secondary antibody with corresponding properties based on the source of the primary antibody, and incubate the membrane in a secondary antibody with corresponding properties (prepared in 5% skim milk) at room temperature for 1 h to allow the secondary antibody to fully bind to the primary antibody.

[0056] (9) Development: Remove the membrane after incubation with secondary antibody, add TBST buffer, and wash it on a decolorizing shaker for 3 times, 10 min each time. Prepare the developer (A solution: B solution = 1:1) under light-proof conditions, incubate the membrane in the luminescent solution for 2 min, expose the incubated membrane in a developer, and save the exposed image.

[0057] 5. Results

[0058] Combination Figure 1In the AC figure, the blue dots represent the saline group (Saline) and the red dots represent the model group (BLM). The experimental results showed that the peak inspiratory flow (PIF) decreased, the forced respiratory interval (Penh) value increased, and the minute ventilation volume (MV) decreased. The above results indicate that the lung function of mice was significantly damaged after bleomycin modeling. Further HE staining ( Figure 1 D) and Masson staining ( Figure 1 Figure E in the middle shows that compared with the saline group, the model group had disordered cell arrangement, massive proliferation of fibroblasts, infiltration of inflammatory cells, and abnormal deposition of collagen. A large amount of extracellular matrix is ​​released during lung injury. Immunohistochemical results showed that α-SMA ( Figure 1 (Fig. F) and COL-1 ( Figure 1 The above results indicate that the bleomycin-induced mouse lung injury model was successfully replicated.

[0059] Combination Figure 2 In Figure AC, immunohistochemistry, Western blots and immunofluorescence experiments were used to detect the expression changes of SNX3 in the mouse lung injury model. The experimental results showed that SNX3 was significantly upregulated in the lung injury model.

[0060] Example 2 Construction of SNX3 gene knockout mice and study of its effect on lung injury

[0061] 1. Construction of SNX3 knockout mice

[0062] SNX3-cKO knockout mice and N-cKO mice with the same background were purchased from Shanghai Model Organisms Science Co., Ltd. and bred in the Experimental Animal Center of Sun Yat-sen University (SPF grade, 25±0.5°C, humidity 60-70%, 12h light / 12h dark).

[0063] 2. Study on the effect on lung injury

[0064] The above-mentioned SNX3 gene knockout transgenic mice and N-cKO mice with the same background were divided into 4 groups:

[0065] N-cKO group: 10-month-old N-cKO mice, lung function was tested.

[0066] SNX3-cKO group: 10-month-old SNX3-cKO knockout mice, lung function was detected.

[0067] N-cKO+BLM group: 10-month-old N-cKO mice were given 2 mg / mL bleomycin solution by tracheal instillation to induce fibrosis model, and lung function was tested 21 days later.

[0068] SNX3-cKO+BLM group: 10-month-old SNX3-cKO knockout mice were given 2 mg / mL bleomycin solution by tracheal instillation to induce fibrosis model, and lung function was tested 21 days later.

[0069] After the above grouping experiment was completed, the patient was dissected and the histopathological detection experiment operation of Example 1 was performed.

[0070] 3. Results

[0071] Combination Figure 3 Figure AC shows that the lung function of 10-month-old SNX3-cKO mice was tested, and there was no significant difference between them and N-cKO mice. Furthermore, we observed the arrangement of lung tissue cells, collagen deposition, and lung injury-related proteins in N-cKO and Snx3-cKO mice and found that there was no significant difference between N-cKO and Snx3-cKO mice ( Figure 4 (DG in Figure 5).

[0072] Combination Figure 4 Figure AC, N-cKO mice and SNX3-cKO mice were simultaneously instilled with bleomycin to induce a lung injury model. Lung function was tested 21 days later. The experimental results showed that SNX3-cKO mice could significantly downregulate PIF and upregulate Penh and MV. Further tissue morphological observation showed that the cell arrangement disorder, inflammatory infiltration, collagen deposition and abnormal expression of lung injury-related proteins caused by bleomycin could be improved by SNX3-cKO mice ( Figure 5 (DG in Figure 5).

[0073] Example 3 Construction of cell lung injury model and study of the effect on lung injury

[0074] 1. Construction of Cellular Lung Injury Model

[0075] (1) Cell culture and passaging: Human embryonic lung cells MRC5 were cultured in a low-glucose medium containing 10% fetal bovine serum (FBS) and placed in a 37°C constant temperature incubator with 5% CO2. When the cells grew to a density of about 80% to 90%, passaging began. Taking the large dish as an example, before subculturing, PBS and culture medium need to be preheated in a constant temperature 37℃ water bath in advance; discard the old culture medium, add 2mL PBS to wash twice; discard PBS, add 1mL trypsin to allow the trypsin to infiltrate the entire bottom of the dish, incubate in a 37℃ constant temperature incubator for about 30 seconds, observe under a microscope, and when the cells appear to shrink or become round, immediately add 2mL of complete culture medium (containing 10% FBS) to terminate digestion; use a pipette to slowly and repeatedly blow to make the cells growing on the bottom of the dish fall off to form a cell suspension, and transfer the cell suspension to a 15mL centrifuge tube that has been sterilized by high pressure; centrifuge at 1000rpm for 5min, discard the supernatant, and add 4mL fresh complete culture medium to resuspend the cells; inoculate the cells at an appropriate density into the culture plate or dish according to the experimental needs (the subculturing ratio is 1:3).

[0076] (2) Preparation of transforming growth factor-β1 (TGF-β1) solution: Weigh TGF-β1 powder and culture it in complete culture medium to a concentration of 5 ng / mL.

[0077] (3) Inoculation: Well-growing human embryonic lung cells MRC5 were seeded in a 6-well plate filled with culture medium and allowed to adhere to the plate for 24 hours.

[0078] (4) Induction: Remove the culture medium and add 2 mL of 5 ng / mL TGF-β1 solution (the solvent in the solution is complete culture medium) to each well for culture in a cell culture incubator containing 5% carbon dioxide and 37°C for 24 h.

[0079] 2. Western blots

[0080] The cell culture dish was washed with PBS on ice and discarded. An appropriate amount of RIPA lysis buffer (containing 1% PMSF) was added, and the cells were lysed on ice for half an hour, vortexed once every 5 minutes, centrifuged at 12000 rcf, 4°C for 15 minutes, and the supernatant (protein) was transferred to another clean 1.5 mL EP tube, which can be quantitatively packaged by BCA or frozen at -80°C. The subsequent treatment was consistent with Experiment 4 in Example 1.

[0081] 3. Immunofluorescence (IF)

[0082] (1) The cells were seeded into confocal dishes (about 1×104 cells) and after the cells adhered to the dish (12 h), the corresponding subsequent experimental treatments were performed.

[0083] (2) After the experimental treatment, the culture medium was discarded, and the cells were rinsed once with PBS. 100 μL of 4% paraformaldehyde preheated in a 37°C water bath was added and fixed at room temperature for 15 min.

[0084] (3) Discard the paraformaldehyde fixative, add 1 mL of PBS, place on a decolorizing shaker and wash for 5 min. Repeat twice.

[0085] (4) Add 100 μL of 0.3% Triton (prepared in PBS) and permeabilize the membrane for 15 min at room temperature.

[0086] (5) Discard 0.3% Triton, add 1 mL PBS, place on a decolorizing shaker and wash for 5 min. Repeat three times.

[0087] (6) Add 100 μL goat serum and block at room temperature for 1 h (or overnight at 4°C), discard the goat serum, and do not wash.

[0088] (7) Immediately add 100 μL of primary antibody (prepared with goat serum in proportion), cover the lid and place in a homemade wet box, and incubate at 4°C overnight.

[0089] (8) Recover the primary antibody, add PBS to wash away the unbound primary antibody, decolorize on a shaker for 10 min, and repeat three times.

[0090] (9) Add 100 μL of fluorescent secondary antibody prepared with goat serum of the same property as the primary antibody (in an appropriate ratio) and incubate at room temperature in the dark for 1 h.

[0091] (10) Discard the secondary antibody, add PBS to wash away the unbound secondary antibody, and decolorize on a shaker in the dark for 10 min. Repeat three times.

[0092] (11) Add 100 μL DAPI (prepared in PBS) to stain the cell nucleus for 10 min.

[0093] (12) Add 1 mL of PBS and wash on a decolorizing shaker three times, 5 min each time. After washing, add 1 mL of PBS. Observe and take photos under a fluorescence microscope.

[0094] 4. Results

[0095] Combination Figure 5 As shown in Figure AC, Western blots ( Figure 3 Figure A) shows that the lung injury cell model was successfully established. Western blots ( Figure 3 B) and immunofluorescence ( Figure 3 (C) The expression changes of SNX3 were detected, and the experimental results showed that the expression level of SNX3 was significantly upregulated.

[0096] Example 4 Study on the effect of knockdown of SNX3 at the cellular level on lung injury

[0097] The culture medium used was a complete culture medium: low-glucose DMEM containing 10% fetal bovine serum and 1% double antibody; the culture conditions were 5% carbon dioxide and 37°C.

[0098] siRNA solution: After the si-SNX3 lyophilized powder is centrifuged, an appropriate volume of DEPC water is added to dilute the siRNA to a 20 μM storage solution, which is then aliquoted and stored at -20°C.

[0099] (1) The well-grown human embryonic lung cells MRC5 in Experiment 1 of this Example were seeded in a 6-well plate filled with culture medium. After 24 hours of adherence, the cells were divided into 4 groups:

[0100] Control group: remove the culture medium, add 2 mL of complete culture medium to each well, and culture in a cell culture incubator containing 5% carbon dioxide and 37°C for 24 h;

[0101] TGF-β1 group: remove the culture medium, add 2 mL of TGF-β1 solution (the solvent in the solution is complete culture medium) with a concentration of 5 ng / mL to each well, and culture in a cell culture incubator containing 5% carbon dioxide and 37°C for 24 h;

[0102] si-SNX3 group: dilute Lipofectamine 2000 mixture (250μL OPTI-MEM I reduced serum medium + 5μL Lipofectamine 2000), incubate at room temperature for 5min; dilute siRNA (250μL OPTI-MEM I reduced serum medium + 5μL siRNA); mix the diluted siRNA with Lipofectamine 2000, incubate at room temperature for 25min to obtain siRNA-Lipofectamine 2000 mixture; discard the culture medium before transfection, wash once with PBS, and wash once with 1mL Eupetra; add 2mL siRNA-Lipofectamine 2000 mixture to each well, place the cells in a CO2 incubator at 37℃ and culture for 4-6h, then replace with DMED medium and continue to culture for 48h;

[0103] TGF-β1 (5 ng / mL) + si-SNX3 group: After 24 h of treatment as described above for the si-SNX3 group, TGF-β1 was added at a concentration of 5 ng / mL and culture was continued for 24 h.

[0104] (2) After the above grouping process is completed, Experiments 2 and 3 of Example 3 are carried out.

[0105] Combination Figure 6As shown in Figures A and B, by knocking down SNX3 and simultaneously administering TGF-β1 to induce a cell lung injury model, the effect of SNX3 on lung injury-related proteins was detected by Western blots. Compared with the control group, the expression level of SNX3 was significantly upregulated in the TGF-β1-induced model ( Figure 6 Figure A), and knocking down SNX3 at the same time found that knocking down SNX3 could inhibit the expression of lung injury-related proteins caused by TGF-β1 ( Figure 6 (Fig. B).

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of SNX3 as a target in the preparation of drugs for the prevention and treatment of lung injury.

2. Application of SNX3 inhibitors in the preparation of drugs for preventing and treating lung injury.

3. The application according to claim 2, characterized in that: The SNX3 inhibitor reduces the expression of SNX3 protein or gene.

4. The use according to claim 2 or 3, characterized in that: The SNX3 inhibitor is si-SNX3.

5. The application according to claim 4, characterized in that: The si-SNX3 sequence is GCAACTTCCTCGAGATCGA.

6. The use according to any one of claims 1 to 5, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

7. A drug for preventing and treating lung injury, characterized in that: The drugs include SNX3 inhibitors.

8. The drug according to claim 7, characterized in that: The SNX3 inhibitor is si-SNX3.

9. The drug according to claim 8, characterized in that: The si-SNX3 sequence is GCAACTTCCTCGAGATCGA.

10. The drug according to any one of claims 7 to 9, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

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