Use of snx3 inhibitors for the preparation of a medicament for the prevention or treatment of lung injury

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of an SNX3 inhibitor in preparation of a drug for preventing and treating lung injury. The application is researched by using a lung injury mouse model induced by tracheal intubation and oral tracheal instillation of bleomycin solution (2 mg / mL), and it is found that the expression of SNX3 protein and gene in the lung of the mouse increases with the increase of the degree of lung injury. The application is researched by using a lung SNX3 gene knockout mouse, and it is found that the deletion of the lung SNX3 gene can improve lung injury, which indicates that SNX3 can be used as a therapeutic target for lung injury. Meanwhile, the application finds that si-SNX3 capable of inhibiting the expression of SNX3 gene or protein can inhibit the expression of lung injury related proteins. The application provides a new means and a drug target for the treatment of lung injury, and has important value for the development of a clinical drug for lung injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, it relates to the application of SNX3 inhibitors in the preparation of drugs for the prevention and treatment of lung injury. Background Technology

[0002] Lung injury is one of the most common and critical illnesses in clinical practice, with extremely high morbidity and mortality rates. Severe cases can lead to acute respiratory failure, seriously threatening human life and health. Current research indicates that multiple factors can induce lung injury, including environmental factors such as inhalation of harmful substances and smoking; drug-induced injury such as improper use of medications like amiodarone and bleomycin; systemic immune diseases such as systemic lupus erythematosus and rheumatoid arthritis; and genetic factors, such as abnormalities in the MUC5B gene affecting normal lung repair, thus leading to lung injury. Due to the complex and unclear pathogenesis of lung injury, progress in drug development targeting lung injury has been slow.

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

[0004] SNX3 (sorting nexin 3) is a sorting connexin protein that plays a crucial role in maintaining intracellular protein homeostasis. Sorting connexins are a highly conserved class of eukaryotic proteins, not only playing important regulatory roles in several key cell signaling pathways but also influencing the occurrence and development of diseases. Previous research by the inventors' team (CN117883578A) showed that SNX3 gene deletion can alleviate bile duct ligation-induced liver fibrosis, suggesting that SNX3 could serve as a therapeutic target for liver fibrosis; si-SNX3, which inhibits SNX3 protein and gene expression, can effectively inhibit liver fibrosis; thus, SNX3 inhibitors are effective therapeutic drugs for liver fibrosis. However, because lung injury differs from pulmonary fibrosis, and lung tissue differs from liver tissue, the role of SNX3 in lung injury remains uncertain. Summary of the Invention

[0005] This invention aims to overcome the problem of limited clinical treatment methods and insufficient drugs for lung injury. Through research and exploration, it provides the application of SNX3 inhibitors in the prevention and treatment of lung injury, providing new targets and drug strategies for the prevention and treatment of lung injury.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention utilizes a mouse model of severe lung injury induced by endotracheal infusion of bleomycin solution (2 mg / mL) via endotracheal intubation. The study showed that the expression of SNX3 protein and gene in the mouse lungs increased with the severity of lung injury. Further research using lung SNX3 gene knockout mice showed that lung SNX3 gene deletion improved lung injury, indicating that SNX3 can serve as a therapeutic target for lung injury. Simultaneously, this invention discovered that si-SNX3, which inhibits SNX3 gene or protein expression, can inhibit the expression of lung injury-related proteins, potentially representing a novel therapeutic approach and drug target for lung injury. Therefore, this 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 the prevention and treatment of lung injury.

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

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

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

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

[0014] Gene knockdown technology, which inactivates or silences the SNX3 gene at the post-transcriptional or translational level, is well known to those skilled in the art. Such gene knockdown techniques include, but are not limited to, RNA interference, Morpholino interference, antisense nucleic acids, ribozymes, or dominant-negative repressive mutations.

[0015] Using shRNA or siRNA expressed by viruses (such as lentiviruses or adeno-associated viruses) to suppress the expression of the SNX3 gene and thus silence the SNX3 gene is a conventional existing technique well known to those skilled in the art.

[0016] In addition, the present invention also provides other options, wherein the SNX3 inhibitor is a siRNA, dsRNA, shRNA, miRNA, or antisense nucleic acid that targets SNX3 or its transcript as a target sequence and is capable of inhibiting SNX3 protein expression or gene transcription; or a vector construct that can express or form the siRNA, dsRNA, shRNA, miRNA, or antisense nucleic acid.

[0017] Furthermore, the vector construct is at least one of bacterial plasmids, bacteriophages, recombinant viral vectors, or eukaryotic recombinant expression vectors.

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

[0019] Furthermore, the drug may also 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 routes of administration 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 the prevention and treatment of lung injury, the drug comprising an 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] Laboratory studies have shown that in normal mice, bleomycin-induced lung injury leads to increased expression of SNX3 protein and gene in the lungs. Furthermore, using SNX3 gene knockout mice as experimental subjects, this invention investigated the role of the SNX3 gene in lung injury. Results showed that SNX3 gene deletion in the lungs alleviated lung injury, suggesting that SNX3 can serve as a target for the prevention and treatment of lung injury, and that SNX3 inhibitors are effective drugs for the prevention and treatment of lung injury. Simultaneously, this invention discovered that si-SNX3, which inhibits SNX3 gene or protein expression, significantly reduced the expression levels of fibronectin, collagen-I (COL-I), and α-actin (α-SMA) in human embryonic lung cells (MRC5). These experimental results indicate that SNX3 holds promise for targeting multiple pathogenic mechanisms of lung injury, providing a new approach 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. Attached Figure Description

[0029] Figure 1 For lung function testing and lung tissue pathological examination of mouse lung injury model (PIF: maximum inspiratory flow rate, Penh: forced breathing interval, MV: minute ventilation, Saline: 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 a mouse lung injury model (SNX3: sorting linker protein 3, GADPH: glyceraldehyde-3-phosphate dehydrogenase as an internal reference protein, Relative protein expression (fold of Saline): expression of related proteins (fold of histone expression in saline)).

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

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

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

[0034] Figure 6 The effect of SNX3 knockout on the expression of lung injury-related proteins in a cellular lung injury model (Fibronectin, COL-Ⅰ, α-SMA, and GADPH were internal reference proteins). Detailed Implementation

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

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

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

[0038] Example 1: Construction of a mouse lung injury model and investigation of its effects on lung injury

[0039] 1. Construction of a mouse lung injury model

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

[0041] Mice were weighed and anesthetized by intraperitoneal injection of 3.5% chloral hydrate solution (0.1 ml / 10 g). Anesthetized mice were fixed on a plate, and an animal experiment lamp was used to illuminate the neck area to locate the trachea. Approximately 20 μL of bleomycin solution (2 mg / mL) was instilled via endotracheal intubation. The mice were immediately upright and rotated several times to ensure even distribution of the drug in the lungs. The control group received an equal volume of physiological saline via endotracheal instillation. Various indicators were measured 21 days later.

[0042] 2. Lung function test

[0043] Lung function in mice was assessed using the EMKA animal lung function testing system. The device key was connected, and the software was run. After setting and calibrating the parameters, the file was run, and 1 mL of gas was rapidly injected (using a syringe) into the recording chamber to perform channel signal correction and check the device's seal. The mice were placed in the recording chamber, and the testing began once their breathing and movement stabilized. The system was set to automatically record the mouse's respiratory wave values ​​every 5 seconds, for a total of 5 minutes.

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

[0045] Lung tissue was dissected from mice. The left lung tissue was fixed and immersed in 4% paraformaldehyde for 48 hours. Paraffin sections were prepared by Wuhan Sewell Biotechnology Co., Ltd., and subjected to HE staining, Masson staining, tissue immunofluorescence, and immunohistochemistry. The tissue sections were observed and photographed using cell imaging to assess alveolar structure, pulmonary inflammatory infiltration, collagen deposition, and expression of related proteins.

[0046] 4. Western blots

[0047] Weigh an appropriate amount of animal tissue and wash it on ice with PBS and discard the PBS. Add an appropriate amount of RIPA lysis buffer (containing 1% PMSF), and use an sonicator to release the tissue proteins. Vortex once every 5 minutes, centrifuge at 12000 rcf, 4°C for 15 minutes, and transfer the supernatant (protein) to another clean 1.5 mL EP tube. It can be used for BCA quantitative aliquoting or frozen at -80°C.

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

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

[0050] (3) Sample loading: Fix the solidified gel in the electrophoresis tank, pour the electrophoresis solution (prepared according to Table 4) into the tank so that it covers the comb, slowly pull out the comb, blow out the residual gel in the lane with the gun, and load the samples into their respective lanes in sequence. Add 4 μL of protein marker to the adjacent lanes of the two samples.

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

[0052] (5) Electrospinning: Pre-cut a PVDF membrane (0.22μm) to a suitable size and immerse it in methanol for 5 minutes to activate it. After activation, immerse the membrane, along with the sponge pad and filter paper, in the electrospinning solution. At the beginning of the transfer, place the membranes in the sandwich structure, ensuring there are no air bubbles between the SDS gel and the PVDF membrane. Clamp the membranes in the electrospinning clamp and place them in the electrospinning tank. Pour in the pre-prepared and cooled electrospinning solution. Place two ice blocks in the electrospinning tank and place the entire tank in an ice box. Set the current to 220mA and electrospin for 2 hours (observe the voltage at approximately 100V to 120V).

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

[0054] (7) Incubation with primary antibody: Discard the blocking solution, wash with TBST buffer for 5 min, 3 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 with primary antibody dilution solution), and incubate overnight at 4°C by rotation to allow the primary antibody to fully bind to the protein on the membrane.

[0055] (8) Incubation with secondary antibody: Take out the membrane after incubation with primary antibody, add TBST buffer, place it on a decolorizing shaker and wash it three times for 10 minutes each time. Select a secondary antibody with the corresponding attribute according to the source of the primary antibody, and incubate the membrane with the secondary antibody with the corresponding attribute (prepared with 5% skim milk) at room temperature for 1 hour to allow the secondary antibody to fully bind with the primary antibody.

[0056] (9) Development: Take out the membrane after incubation with the secondary antibody, add TBST buffer, and wash it three times by shaking on a decolorizing shaker for 10 minutes each time. Prepare the developing solution (A solution: B solution = 1:1) under light-protected conditions, incubate the membrane in the luminescent solution for 2 minutes, expose the incubated membrane in a developing instrument, and save the image after exposure.

[0057] 5. Results

[0058] Combination Figure 1In the AC plot, blue dots represent the saline group (Saline) and red dots represent the model group (BLM). Experimental results showed a decrease in peak inspiratory flow (PIF), an increase in forced breathing interval (Penh), and a decrease in minute ventilation volume (MV). These results indicate significant lung function impairment in mice after bleomycin-induced modeling. Further HE staining... Figure 1 (D diagram) and Masson staining ( Figure 1 Figure E shows that, compared with the saline group, the model group exhibited disordered cell arrangement, massive proliferation of fibroblasts, inflammatory cell infiltration, and abnormal collagen deposition. During lung injury, a large amount of extracellular matrix is ​​released; immunohistochemical results indicate that α-SMA ( ) is present in the lung tissue of the model group mice. Figure 1 (Figure F) and COL-1 ( Figure 1 The level of bleomycin-induced lung injury in mice was significantly elevated (see Figure G). All these results indicate that the bleomycin-induced mouse lung injury model was successfully replicated.

[0059] Combination Figure 2 The AC figure shows that the expression changes of SNX3 in a mouse lung injury model were detected by immunohistochemistry, Western blots and immunofluorescence experiments. The results showed that SNX3 was significantly upregulated in the lung injury model.

[0060] Example 2: Construction of SNX3 gene knockout mice and investigation of their effects on lung injury

[0061] 1. Construction of SNX3 gene knockout mice

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

[0063] 2. Investigation into the effects on lung injury

[0064] The transgenic mice with SNX3 gene knockout and the 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 tested.

[0067] N-cKO+BLM group: 10-month-old N-cKO mice were given 2 mg / mL bleomycin solution via intratracheal instillation to induce a fibrosis model. Lung function was then assessed 21 days later.

[0068] SNX3-cKO+BLM group: 10-month-old SNX3-cKO knockout mice were given 2 mg / mL bleomycin solution via intratracheal infusion to induce a fibrosis model. Lung function was tested 21 days later.

[0069] After the grouped experiments were completed as described above, the tissues were dissected, and the histopathological examination procedures of Example 1 were performed.

[0070] 3. Results

[0071] Combination Figure 3 The AC plot showed that lung function in 10-month-old SNX3-cKO mice was not significantly different from that in N-cKO mice. Furthermore, by observing lung tissue cell arrangement, collagen deposition, and lung injury-related proteins in N-cKO and Snx3-cKO mice, we found no significant differences between the two groups. Figure 4 (DG map).

[0072] Combination Figure 4 The AC diagram shows a lung injury model induced by simultaneous intratracheal instillation of bleomycin in N-cKO and SNX3-cKO mice. Lung function was assessed 21 days later. The results indicated that SNX3-cKO mice significantly downregulated PIF and upregulated Penh and MV. Further histological observation showed that bleomycin-induced cell disorganization, inflammatory infiltration, collagen deposition, and abnormal expression of lung injury-related proteins were all improved in SNX3-cKO mice. Figure 5 (DG map).

[0073] Example 3: Construction of a cell-based lung injury model and its impact on lung injury.

[0074] 1. Construction of a cell-mediated lung injury model

[0075] (1) Cell culture and passage: 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%, passage was started. Taking a large dish as an example, before passage, PBS and culture medium need to be preheated in a 37°C water bath; discard the old culture medium, add 2mL of PBS and wash twice; discard the PBS, add 1mL of trypsin to wet the entire bottom of the dish, incubate at 37°C for about 30 seconds, observe under a microscope, and when the cells become wrinkled or rounded, immediately add 2mL of complete culture medium (containing 10% FBS) to stop digestion; use a pipette to slowly and repeatedly pipette to detach the cells growing at the bottom of the dish to form a cell suspension, transfer the cell suspension to a 15mL autoclaved centrifuge tube; centrifuge at 1000rpm for 5min, discard the supernatant, add 4mL of fresh complete culture medium to resuspend the cells; according to the experimental needs, seed the cells at an appropriate density into culture plates or dishes (passage ratio of 1:3).

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

[0077] (3) Inoculation: Five well-grown human embryonic lung cells (MRC5) were inoculated into a 6-well plate containing culture medium and allowed to adhere to the plate for 24 hours.

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

[0079] 2. Western blots

[0080] Cell culture dishes were washed with PBS on ice and discarded. An appropriate amount of RIPA lysis buffer (containing 1% PMSF) was added, and the dishes were placed on ice for 30 minutes for lysis, vortexing every 5 minutes. The dishes were then centrifuged at 12000 rcf at 4°C for 15 minutes. The supernatant (protein) was transferred to another clean 1.5 mL EP tube for BCA aliquoting or freezing at -80°C. Subsequent processing was the same as in Experiment 4 of Example 1.

[0081] 3. Immunofluorescence (IF)

[0082] (1) The cells were seeded in confocal dishes (approximately 1 × 10⁴ cells) and allowed to adhere to the dish for 12 hours before proceeding with the subsequent experimental treatments.

[0083] (2) After the experimental treatment, discard the culture medium and rinse once with PBS. Add 100 μL of 4% paraformaldehyde preheated in a 37℃ water bath and fix at room temperature for 15 min.

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

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

[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 of goat serum and block at room temperature for 1 h (or overnight at 4°C). Discard the goat serum. No washing is required.

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

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

[0090] (9) Add 100 μL of fluorescent secondary antibody (appropriate ratio) prepared with goat serum corresponding to the primary antibody 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, decolorize in the dark and shake for 10 min, repeat three times.

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

[0093] (12) Add 1 mL of PBS, place on a decolorizing shaker and wash 3 times for 5 min each time. After washing, add 1 mL of PBS. Observe and take pictures under a fluorescence microscope.

[0094] 4. Results

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

[0096] Example 4: Investigation into the effect of cellular knockdown of SNX3 on lung injury

[0097] The culture medium used was a complete culture medium: low-glucose DMEM containing 10% fetal bovine serum and 1% antibiotics; culture conditions: 5% carbon dioxide, 37℃.

[0098] siRNA solution: After silicification of si-SNX3 lyophilized powder, add an appropriate volume of DEPC water to dilute the siRNA to a 20 μM storage solution, aliquot and store at -20℃.

[0099] (1) Five well-grown human embryonic lung cells (MRC5) from Experiment 1 of this embodiment were seeded into 6-well plates containing culture medium. After 24 hours of cell adhesion, 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 incubate for 24 h in a cell culture incubator containing 5% carbon dioxide at 37°C;

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

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

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

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

[0105] Combination Figure 6Figures A and B show the effects of SNX3 on lung injury-related proteins in a TGF-β1-induced lung injury model, which was induced by knocking down SNX3. Western blot analysis was used to examine the effects of SNX3 on these proteins. Compared to the control group, the TGF-β1-induced model showed a significant upregulation of SNX3 expression. Figure 6 Figure A shows that knocking down SNX3 inhibited the expression of lung injury-related proteins induced by TGF-β1. Figure 6 (Figure B in the middle)

[0106] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of SNX3 inhibitors in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, characterized in that, The SNX3 inhibitor is si-SNX3, and the si-SNX3 sequence is GCAACTTCCTCGAGATCGA.

2. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

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