Use of nlrx1 protein inhibitors in the preparation of products having a protective effect on pulmonary fibrosis

Abstract

This invention discloses the application of NLRX1 protein inhibitors in the preparation of products with protective effects against pulmonary fibrosis, belonging to the field of biomedical technology. This invention, through the use of an NLRX1 gene knockout mouse model and siRNA silencing technology, demonstrates that inhibiting NLRX1 can significantly alleviate bleomycin-induced pulmonary fibrosis pathological damage. Its protective mechanisms include promoting the proliferation of type II alveolar epithelial cells, inhibiting their damage and apoptosis, and reducing myofibroblast infiltration and extracellular matrix deposition. Based on this, this invention provides for the first time a therapeutic strategy targeting the inhibition of the NLRX1 protein. Related products include recombinant vectors containing RNA molecules encoding NLRX1 expression inhibitors or other drugs capable of inhibiting NLRX1 expression. This invention provides new targets and drug development directions for the treatment of fibrotic diseases, especially pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of NLRX1 protein inhibitors in the preparation of drugs for the treatment or prevention of fibroproliferative diseases. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease of unknown etiology with a very high mortality rate, primarily affecting the elderly. Its main clinicopathological features include diffuse interstitial fibrosis, scarring, and progressive, irreversible alveolar structural destruction, culminating in diffuse fibrosis in later stages. Currently approved antifibrotic drugs such as pirfenidone and nintedanib can only slow the decline in lung function, but cannot reverse the continuously progressing pathological process of pulmonary fibrosis. The average survival time after diagnosis is only 3-5 years, even shorter than many malignant tumors, leading respiratory experts to call it "a cancer that isn't cancer." Currently, lung transplantation remains the most effective treatment for IPF; however, due to donor limitations, high costs, and post-transplant immune rejection, its widespread clinical application is limited. Therefore, in-depth exploration of the upstream mechanisms of lung injury and the development of more effective strategies to control the initiation of pulmonary fibrosis are imperative.

[0003] Type II alveolar epithelial cells (AT2 cells) are a type of metabolically highly active lung epithelial cell that plays a crucial role in the synthesis of alveolar surfactant and the maintenance of alveolar homeostasis. With a deeper understanding of AT2 cells, it has been found that damage, apoptosis, or dysfunction of AT2 cells are important drivers of the occurrence and development of interstitial lung disease (IPF). AT2 cells possess stem cell function; under injury conditions, they can self-renew through mitosis and transdifferentiate into AT1 cells, maintaining normal lung function and promoting damage repair. Another important function of AT2 cells is to maintain alveolar function by secreting surfactant, reducing alveolar surface tension, ensuring alveolar compliance, and thus preventing alveolar collapse at end-expiratory time. Continuous damage stimulation can lead to abnormal activation of AT2 cells and secretion of various profibrotic cytokines; on the other hand, damaged and / or apoptotic AT2 cells can synthesize and release large amounts of transforming growth factor (TGF)-β to synergistically promote the activation of fibroblasts and their transdifferentiation into myofibroblasts (MFBs), resulting in excessive deposition of extracellular matrix (ECM), which in turn promotes alveolar structural deformation and destruction.

[0004] The NOD-like receptor (NLR) family is a novel class of intracellular pattern recognition receptors, with 23 members in the human body. NOD-like receptor X1 (NLRX1, Genebank ID: 79671), located in mitochondria and cytoplasm, is the only member of the NOD-like receptor family with a mitochondrial targeting sequence. It plays a crucial regulatory role in life processes such as autoimmunity, embryonic development, apoptosis, and differentiation by modulating multiple downstream signal transduction pathways. Recent studies have found a close association between NLRX1 and respiratory diseases: NLRX1 mRNA and protein expression levels are significantly elevated in animal models of hyperoxia-induced acute lung injury, and NLRX1 gene knockout significantly inhibits lung tissue inflammation and apoptosis in mouse models; NLRX1 expression is suppressed in the lung tissue of COPD patients, and this suppression is strongly correlated with the degree of airflow limitation in COPD patients.

[0005] However, the function and mechanism of NLRX1 in IPF remain unclear. Therefore, it is necessary to provide more effective methods and means to treat or prevent pulmonary fibrosis. Summary of the Invention

[0006] Addressing the problems existing in the prior art, this invention, using an NLRX1 gene knockout mouse model and siRNA silencing technology, demonstrates that inhibiting NLRX1 can significantly alleviate BLM-induced pulmonary fibrosis pathological damage. Its protective mechanisms include promoting type II alveolar epithelial cell proliferation, inhibiting their damage and apoptosis, and reducing myofibroblast infiltration and extracellular matrix deposition. Based on this, this invention provides for the first time a therapeutic strategy targeting the inhibition of the NLRX1 protein. Related products include recombinant vectors containing RNA molecules encoding NLRX1 expression inhibitors or other drugs capable of inhibiting NLRX1 expression. This invention provides new targets and drug development directions for the treatment of fibrotic diseases, especially pulmonary fibrosis.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide the application of substances that inhibit NLRX1 protein expression in the preparation of medicaments for the prevention and treatment of fibroproliferative diseases.

[0008] Furthermore, the substance is selected from any one or more of the following: (a) Compounds that inhibit the activity of NLRX1 protein; (b) A polynucleotide that inhibits NLRX1 gene expression or a recombinant vector containing said polynucleotide.

[0009] Furthermore, the polynucleotide is a DNA, RNA, and / or DNA-RNA hybrid that inhibits NLRX1 gene expression.

[0010] Furthermore, the RNA that inhibits NLRX1 gene expression includes NLRX1 siRNA.

[0011] Furthermore, the sequence of the NLRX1 siRNA is shown in SEQ ID NO.7-SEQ ID NO.8.

[0012] Furthermore, the fibroproliferative disease is pulmonary fibroproliferative disease.

[0013] Furthermore, the pulmonary fibrotic disease is idiopathic pulmonary fibrosis.

[0014] The second objective of this invention is to provide a drug for the prevention and treatment of fibroproliferative diseases, which contains NLRX1siRNA, the sequence of which is shown in SEQ ID NO.7-SEQ ID NO.8.

[0015] The drug in question is an NLRX1 protein expression inhibitor that has a protective effect against pulmonary fibrosis. It refers to a vector in the field of gene therapy containing Nlrx1 knockdown. The vector includes viral vectors, liposomes, and may also include one or more pharmaceutically acceptable vectors, such as absorption enhancers and adsorbents, and can be prepared into inhaled or injectable formulations.

[0016] The pulmonary fibrosis includes improvements in imaging and pathology, a reduction in myofibroblast infiltration and extracellular matrix deposition, and inhibition of alveolar epithelial cell damage and apoptosis.

[0017] Furthermore, this invention utilizes Nlrx1 gene knockout mice to establish a BLM-induced pulmonary fibrosis model, and experiments have shown that compared to Nlrx1... + / + (WT) + BLM group mice, Nlrx1 - / - The (KO) + BLM group mice showed significant improvement in imaging and pathology, reduced myofibroblast infiltration and extracellular matrix deposition, and reduced alveolar epithelial cell damage and apoptosis, ultimately alleviating pulmonary fibrosis. Therefore, this invention also provides for the application of this mouse model in the development or screening of any of the following products: (1) Products for the severity of pulmonary fibrosis; (2) Products that reduce myofibroblast infiltration and extracellular matrix deposition; (3) Products that reduce AT2 cell apoptosis and promote their proliferation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: Currently, there are no studies worldwide on the relationship between NLRX1 and pulmonary fibrosis. This invention, using an NLRX1 gene knockout mouse model and siRNA silencing technology, demonstrates that inhibiting NLRX1 can significantly alleviate BLM-induced pulmonary fibrosis pathological damage. Its protective mechanisms include promoting type II alveolar epithelial cell proliferation, inhibiting apoptosis and damage, and reducing myofibroblast infiltration and extracellular matrix deposition. Based on this, this invention provides for the first time a therapeutic strategy targeting the inhibition of NLRX1 protein. Related products include recombinant vectors containing RNA molecules encoding NLRX1 expression inhibitors or other drugs capable of inhibiting NLRX1 expression. This invention provides a new target and drug development direction for the treatment of fibrotic diseases, especially pulmonary fibrosis. Attached Figure Description

[0019] Figure 1 This figure shows the results of NLRX1 expression changes in lung tissue and AT2 cells of IPF patients and BLM-induced pulmonary fibrosis animal models in Example 1 of the present invention.

[0020] Figure 2 This is a graph showing the effect of adeno-associated virus (AAV2 / 9) mediated NLRX1 overexpression on the severity of BLM-induced pulmonary fibrosis in mice in Example 2 of this invention.

[0021] Figure 3 This is a graph showing the results of Nlrx1 gene knockout improving the severity of pulmonary fibrosis in Example 3 of the present invention.

[0022] Figure 4 This is a diagram showing the results of BLM-induced apoptosis and inhibited cell proliferation in AT2 cells cultured in Example 4 of this invention after overexpression of NLRX1.

[0023] Figure 5 This is a diagram showing the results of BLM-induced apoptosis reduction and enhanced cell proliferation after NLRX1 knockdown in AT2 cells in Example 5 of this invention. Detailed Implementation

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0025] The Nlrx1 gene knockout mice involved in the embodiments of the present invention were purchased from Cyagen Biosciences (product number: S-KO-08765), BLM was purchased from MedChemExpress (MCE) (Cat#: HY-17565), and human NLRX1 siRNA was purchased from Wuhan Jinkairui Biotechnology Co., Ltd.

[0026] The experimental animals used in this invention were: SPF-grade wild-type C57BL / 6 mice purchased from Guangdong Sijiajingda Biotechnology Co., Ltd., and C57BL / 6 background Nlrx1 gene knockout mice purchased from Cyagen Biosciences. All animals were bred and housed in the SPF-grade animal facility of the Experimental Animal Center of the First Affiliated Hospital of Guangzhou Medical University. Mice were randomly assigned to groups for experiments at 8-10 weeks of age. All animal use and handling were reviewed and approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University.

[0027] The statistical processing methods involved in this embodiment of the invention are as follows: GraphPad Prism 10.0 software is used for statistical analysis and software graphing. The experimental results are expressed as mean ± standard error (Mean ± SEM). The t-test is used to compare the means between two groups of samples, and the two-way ANOVA test is used to compare the means of multiple groups. p < 0.05 is considered statistically significant.

[0028] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0029] Example 1: The expression levels of NLRX1 in lung tissues and AT2 cells of IPF patients and BLM animal models were significantly increased. 1. Lung tissue specimens from IPF patients were all obtained from lung tissues of lung transplant patients at the First Affiliated Hospital of Guangzhou Medical University, and were clinically diagnosed as IPF; normal control lung tissues were obtained from lung transplant donor lung tissues at the First Affiliated Hospital of Guangzhou Medical University.

[0030] 2. Establishment of a mouse pulmonary fibrosis model: Bleomycin (BLM) was administered intratracheally via tracheotracheal intubation at a dose of 1.3 mg / kg. The control group received a corresponding dose of physiological saline. Mice were anesthetized by intraperitoneal injection of 1.2% aphthylamine (0.2 ml / 10 g), ensuring the anesthesia was not too deep. The mice were fixed to the surgical table, perpendicular to the surface. The laryngoscope was inserted into the mouse's mouth, and the entire laryngoscope was lifted upwards to expose the glottis. As the glottis opened, a 22 g intravenous catheter was inserted into the trachea, and approximately 0.1 ml of gas was injected into the trachea using a syringe tip. The mouse was observed to ensure it could still breathe normally and evenly. If the mouse experienced brief respiratory arrest, the tracheal intubation was considered successful. Diluted BLM was injected into the trachea, and the mouse's tail was gently pinched to stimulate breathing, further drawing the fluid from the catheter into the lungs. The mouse's body was gently shaken to promote the even distribution of BLM in the lungs.

[0031] 3. Western blot was used to detect the expression levels of relevant target proteins in lung tissue. The detection method is as follows: (1) Total protein extraction from lung tissue: The frozen lung tissue was taken out of the -80℃ freezer, washed with physiological saline, and dried. The weight of the lung tissue was weighed, and approximately 1 ml of protein lysis buffer was added for every 100 mg of lung tissue. The buffer was then placed in a homogenizing tube and homogenized using a high-speed homogenizer until the lysate became a thick soup-like consistency. The mixture was centrifuged in a high-speed refrigerated centrifuge at 4℃ and 12000g for 25 minutes. The supernatant was transferred to a new EP tube for subsequent protein concentration measurement, and the precipitate was discarded.

[0032] (2) Protein concentration determination: Follow the instructions for the Beyotime BCA protein concentration assay kit. Add 25 μl of serially diluted standards. Dilute 1.25 μl of sample 20 times with water to a total volume of 25 μl and add it to a 96-well plate. Mix reagent A and reagent B at a ratio of 50:1, and add 200 μl of the mixture to the 96-well plate containing the standards and sample. Incubate at 37°C in the dark for 30 minutes. Measure the OD value of each well at 562 nm using a full-wavelength microplate reader, construct a standard curve, and calculate the protein concentration of each sample.

[0033] (3) Preparation of loading protein: The total loading volume is 20 ng. Calculate the loading volume of each sample based on the measured protein concentration. Dilute the sample with protein lysis buffer to ensure a consistent final loading volume for each sample. Add 5× loading buffer according to the ratio. Boil the sample in 100℃ water for 5 minutes to denature the protein. After boiling, cool the sample on ice and store it at -20℃.

[0034] (4) Protein immunoblotting: Prepare 10% separating gel and 5% stacking gel in advance. Add electrophoresis buffer to the electrophoresis tank, pull out the sample comb vertically upward, and add the sample to the sample well. First, perform electrophoresis at a constant voltage of 70 V until the sample is pressed into a straight line by the stacking gel and the marker begins to separate. Increase the voltage to 120 V and continue electrophoresis at a constant voltage until the bromophenol blue reaches about 0.5 cm at the bottom. Soak the PVDF membrane in methanol, take it out and place it in a dish containing electrotransfer buffer. Discard the electrophoresis buffer, take the gel out of the electrophoresis tank, and prepare an electrotransfer "sandwich" in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge". Place the sandwich clip in the electrotransfer tank, pour the pre-cooled electrotransfer buffer into the electrotransfer apparatus, and place ice packs to keep the electrotransfer buffer at a low temperature. Perform electrotransfer at 300 mA for 120 minutes and cover the electrotransfer tank with ice. After electroporation, remove the PVDF membrane, add the corresponding primary antibody to the target protein band, and incubate overnight at 4°C with gentle shaking. The next day, wash the membrane three times with TBST for 10 minutes each time, add the corresponding secondary antibody, and incubate at room temperature for 1 to 2 hours. Wash the membrane three times with TBST for 10 minutes each time. Develop using a fully automated chemiluminescence imaging system, analyze the grayscale values ​​of the target band using ImageJ image analysis software, and perform statistical analysis using GraphPad software.

[0035] 4. Immunofluorescence staining of lung tissue: (1) Baking, dewaxing, and gradient hydration of paraffin sections: Paraffin-embedded lung tissue sections were baked in a 65°C oven for 2 hours. After baking, the sections were quickly placed in a staining rack and dewaxed in xylene I and xylene II for 10 minutes each. Then, they were placed in 100% ethanol I and 100% ethanol II for 5 minutes each to remove residual xylene. The sections were then gradient hydrated in 95%, 85%, and 75% ethanol for 5 minutes each, and finally washed three times in PBS solution for 5 minutes each time.

[0036] (2) Antigen retrieval: The heat retrieval method was used. The slides were placed in a retrieval box containing pH 6.0 sodium citrate buffer and heated in a microwave oven on high for 30 minutes. After retrieval, the buffer was allowed to cool to room temperature naturally. The slides were then removed and washed three times with PBS for 5 minutes each time.

[0037] (3) Blocking and antibody incubation: Place the slides in a humidified chamber, gently absorb excess PBS with absorbent paper, and use an immunohistochemical pen to circle the tissue edges. Add 0.3% Triton X-100 solution for permeation, incubate at room temperature for 10 minutes, and wash with PBS 3 times, 5 minutes each time. Then add 3% hydrogen peroxide solution and incubate at room temperature for 15 minutes to block endogenous peroxidase activity, and wash with PBS 3 times, 5 minutes each time. After discarding the PBS, add 5% BSA blocking solution and incubate at room temperature for 30 minutes. After discarding the blocking solution, add SPC (1:500, abclonal, Cat#: A23181) directly and incubate overnight in a humidified chamber at 4°C in the dark. The next day, remove the slides, warm them to room temperature for 30 minutes, wash with PBS 3 times, 5 minutes each time, then add the corresponding fluorescently labeled secondary antibody and incubate at room temperature in the dark for 1 h. After incubation, wash with PBS 5 times, 5 minutes each time.

[0038] (4) TSA fluorescence staining and counterstaining: TSA fluorescent dye (TYR-520) was added and reacted for 10 minutes in the dark, followed by washing with PBS three times for 5 minutes each time. For multiple immunofluorescence staining, the antigen retrieval and staining steps were repeated; the primary antibody used was NLRX1 antibody (1:300, ImmunoWay, Cat# YT5802), and the signal was developed by reacting with TSA fluorescent dye (TYR-570) in the dark for 10 minutes. After staining, DAPI staining solution was added and incubated in the dark for 10 minutes to counterstain the cell nuclei. The cells were washed with PBS three times for 5 minutes each time, and finally, anti-fluorescence quenching mounting medium was added for mounting. After drying, the cells were observed under a microscope and images were acquired.

[0039] result: (1) Western blot results showed that the level of NLRX1 protein in the lung tissue of IPF patients was significantly increased ( Figure 1 A). Immunofluorescence staining results showed that NLRX1 expression was significantly increased in AT2 cells of lung tissue from IPF patients ( Figure 1 B).

[0040] (2) Western blot showed that compared with the control group, NLRX1 expression in the lung tissue of the BLM model was significantly upregulated. Figure 1 C); Immunofluorescence staining results showed that NLRX1 expression was significantly increased in AT2 cells of mouse lung tissue (C); Figure 1 D).

[0041] Example 2: Effect of NLRX1 overexpression on the degree of bleomycin-induced pulmonary fibrosis in mice 1. Establishment of a mouse model of pulmonary fibrosis To determine the role of NLRX1 in the development and progression of pulmonary fibrosis, we packaged adeno-associated virus (AAV2 / 9-NLRX1) overexpressing NLRX1 and a control virus (AAV2 / 9-NC). The adeno-associated virus was administered via endotracheal intubation to induce NLRX1 overexpression (5 × 10⁻⁶). 10 Two weeks later, pulmonary fibrosis models were established using the same method of intratracheal infusion of bleomycin (1.3 mg / kg), or a corresponding dose of saline was administered as a control. Subsequently, 14 days after bleomycin administration, mice in each group underwent micro-CT examination, and the harvested tissues were further analyzed to observe the effect of NLRX1 overexpression on bleomycin-induced pulmonary fibrosis in mice. The specific grouping of mice was as follows: AAV9-NC + Saline, AAV9-NC + BLM, AAV9-NLRX1 + Saline, and AAV9-NLRX1 + BLM. The method for preparing the pulmonary fibrosis mouse model was the same as above.

[0042] 2. In vivo CT scan of mice Mice were anesthetized with isoflurane and then placed in a Super Nova Micro-CT scanning chamber for chest CT imaging.

[0043] 3. HE staining of mouse lung tissue The isolated left mouse lung was fixed with 10% neutral formalin, and then dehydrated, embedded in paraffin, and sectioned sequentially. The sections were placed in a 56℃ oven for 1 hour to fully melt the paraffin. Dewaxing was performed using xylene (10 minutes x 2 times), anhydrous ethanol (5 minutes x 2 times), 95% ethanol (3 minutes x 1 time), 85% ethanol (2 minutes x 1 time), and 75% ethanol (1 minute x 1 time), followed by rinsing with running water for 5 minutes. Hematoxylin staining of the nucleus was performed for 10 minutes, followed by rinsing with running water for 4 minutes. Differentiation with 2% hydrochloric acid alcohol was performed for 15 seconds, followed by rinsing with running water for 2 minutes, and the staining was observed under a microscope. Eosin staining was performed for 1 minute, followed by rinsing with running water for 4 minutes, and the staining was observed under a microscope. Dehydration and clearing were performed using 95% ethanol (5 minutes x 2 times), anhydrous ethanol (5 minutes x 2 times), and xylene twice. After the slides dried, they were mounted with neutral resin. After the neutral resin dried, a panoramic scanner was used to observe the lung lesions.

[0044] 4. Masson staining of mouse lung tissue After fixation in 10% neutral formalin, the left lung of mice underwent routine dehydration, embedding, sectioning, and dewaxing. The specific steps after dewaxing were as follows: overnight in fixative at room temperature, followed by rinsing with running water; staining with azurite blue for 2 minutes; staining with hematoxylin and eosin for 2 minutes; differentiation with 1% hydrochloric acid ethanol, followed by rinsing with running water for 10 minutes; treatment with fuchsin solution for 10 minutes; treatment with 1% phosphoaluminate solution for 10 minutes; staining with 2% aniline blue solution for 5 minutes; treatment with 0.2% glacial acetic acid solution for 2 minutes; dehydration with graded alcohols, clearing with xylene, and mounting with neutral resin; after drying, panoramic scanning was used to observe collagen deposition in the lungs.

[0045] 5. Detection of the expression level of the target protein in mouse lung tissue by immunoblotting (method as above) result: (1) Micro-CT results showed that, compared with the control group (AAV9-NC + Saline), the model group (AAV9-NC + BLM) mice had significantly more pulmonary consolidation and fibrosis. Furthermore, compared with the model group (AAV9-NC + BLM), the NLRX1 overexpression (AAV9-NLRX1 + BLM) mice showed further aggravation of pulmonary consolidation and fibrosis. Figure 2 A).

[0046] (2) HE and Masson staining results showed that the alveolar structure of the control group mice was intact, with no inflammatory cell infiltration in the airways and alveoli, and no fibrous tissue hyperplasia or collagen deposition. The alveolar structure of the model group (AAV9-NC + BLM) mice was destroyed, the alveolar walls and alveolar septa were thickened, and inflammatory cell infiltration in the airways and alveoli was visible. Fibrous tissue hyperplasia was observed in the alveolar septa. The lung tissue destruction and collagen deposition of the NLRX1 overexpressing (AAV9-NLRX1 + BLM) mice were significantly increased compared with the model group. Figure 2 B, 2C).

[0047] (3) Western blot results showed that the protein expression of extracellular matrix components Fibronectin (FN) and Collagen 1 (Col 1) in the lung tissue of model group mice was significantly increased, and NLRX1 overexpression further promoted the protein expression of FN and Col1. Figure 2 D, E).

[0048] Example 3: Effect of NLRX1 gene knockout on bleomycin-induced pulmonary fibrosis in mice 1. Nlrx1 gene knockout (abbreviated as Nlrx1) - / - Identification of Nlrx1 KO mice (1) Cut off and lyse the mouse tail: Number the mice, cut off the mouse tail with clean scissors and place it in a 1.5ml EP tube. Add 204 μl of mouse tail lysis buffer to each EP tube, mix and centrifuge, incubate at 56℃ for 35 minutes or overnight at 56℃, and then boil at 95℃ for 5 minutes to inactivate proteinase K (the specimen can be stored at 4℃ for one week).

[0049] (2) Primer sequences are shown in Table 1 below: Table 1 Nlrx1 gene knockout mice (Nlrx1 - / - ) Primer sequences for genotype identification

[0050] Note: NEO primers are used to amplify the NLRX1 mutant gene; WT primers are used to amplify the NLRX1 wild-type gene.

[0051] (3) Prepare the PCR reaction system (total volume 25 μl) according to Table 2 below: Table 2 Nlrx1 - / - Mouse gene identification reaction system

[0052] Note: F: upstream primer; R: downstream primer.

[0053] (4) The reaction conditions for PCR amplification are shown in Table 3 below: Table 3 Nlrx1 - / - Mouse genotype identification reaction conditions

[0054] (5) Agarose gel electrophoresis Place the sample comb in the appropriate position on the sample loading tank, weigh 2.6 g of agarose powder into the conical flask, add 130 ml of 1xTAE solution, and heat in a microwave oven until the agarose is completely dissolved. After it cools slightly, add the EB substitute (10 μl of EB substitute per 100 ml of agarose gel), mix well, and pour into the prepared sample loading tank. After the agarose gel has completely solidified, remove the sample comb and place the agarose gel in the agarose electrophoresis tank. Add 1xTAE until the agarose gel is completely submerged, then add the DL1000 marker, sample, and negative control sample in sequence. Perform electrophoresis at 100–130 V for 40 minutes. After electrophoresis, photograph the agarose gel using a gel imaging system, save and analyze the results.

[0055] 2. Establishment of a mouse model of pulmonary fibrosis C57 mice were randomly divided into four groups: Nlrx1 + / + Mouse saline group (Nlrx1)+ / + +saline), Nlrx1 - / - Mouse control group (Nlrx1) - / - +saline), wild-type mice treated with BLM (Nlrx1) + / + +BLM), Nlrx1 - / - Mouse BLM treatment group (Nlrx1) - / - +BLM). The dosage of BLM was 1.3 mg / kg, administered via a single intratracheal infusion. The control group received the corresponding dose of physiological saline. The method for preparing the pulmonary fibrosis mouse model was the same as above.

[0056] 3. The in vivo CT scan method for mice is the same as above.

[0057] 4. The HE staining method for mouse lung tissue is the same as above.

[0058] 5. The Masson staining method for mouse lung tissue is the same as above.

[0059] 6. Immunoblotting was used to detect the expression level of the target protein in mouse lung tissue, using the same method as above.

[0060] result: Micro-CT results showed that, compared with the WT control group (Nlrx1) + / + Compared to +saline mice, the WT model group (Nlrx1) + / + +BLM) mice showed significant lung consolidation and fibrosis. Compared to the WT model group mice, Nlrx1 - / - The lung consolidation and fibrosis in the model group mice were significantly improved. Figure 3 A). HE and Masson staining results showed that Nlrx1 gene knockout significantly suppressed the severity of pulmonary fibrosis pathological changes in pulmonary fibrosis mice. Figure 3 B, C). Western blot results showed that Nlrx1 gene knockout inhibited bleomycin-induced deposition of mouse ECM proteins (B, C). Figure 3 D, E).

[0061] Example 4: NLRX1 overexpression promotes BLM-induced apoptosis and inhibits proliferation of human AT2 cells (HPAEpiCs). 1. Cell lines All in vitro experiments were performed using HPAEpiC cells (human type II alveolar epithelial cells), which were purchased from Guangzhou Genio Biotechnology Co., Ltd.

[0062] 2. Cell Culture and Processing Plating: Add 2 ml of 0.25% trypsin to a 10 cm dish and digest for approximately 2 minutes in a cell culture incubator, then stop digestion; centrifuge at 800 rpm for 5 minutes and resuspend in 2 ml of DMEM high-glucose culture medium containing 10% FBS; add 2 ml of DMEM high-glucose culture medium containing 10% FBS to each well of a six-well plate, and add 200 μl of cell suspension; gently shake the plate in a "X" pattern to distribute the cells evenly. After even distribution, incubate at 37°C in a 5% CO2 cell culture incubator.

[0063] 3. Lentiviral transfection (taking one well of a six-well plate as an example) (1) Melt the NLRX1 overexpressing lentivirus at 4℃.

[0064] (2) Lentiviral dilution: Based on the MOI value of Hpaepic cells and the titer of NLRX1 overexpressing virus, calculate the required virus volume of 4 μl, and then add 4 μl of virus to 2 ml of prepared DMEM high glucose culture medium containing 10% FBS.

[0065] (3) After culturing the cells for about 24 hours, discard the culture medium containing lentivirus and replace it with 10% FBS DMEM high glucose complete culture medium. Continue culturing until the 72nd hour, and then carry out subsequent experiments, such as Western blot, apoptosis detection, EdU detection, etc.

[0066] 4. EdU assay for cell proliferation capacity Based on the incorporation of the thymidine deoxynucleoside analog EdU (5-ethynyl-2'-deoxyuridine) during DNA synthesis, EdU is labeled by Alexa Fluor 555 through a subsequent Click reaction, which can be observed directly through a fluorescence microscope.

[0067] (1) Prepare 2× EdU working solution: rewarm EdU stock solution and dilute 10 mM EdU stock solution to 20 μM with complete culture medium (10% FBS + 1% antibiotics + 89% DMEM) at a ratio of 1:500.

[0068] (2) Culture the cells in a 6-well plate to an appropriate number (density of about 80%). First, remove 1 ml of culture medium, then add 1 ml of 2X EdU working solution (20 μM) preheated at 37℃ to the 6-well plate to make the final concentration of EdU in the 6-well plate 1X. Then, put the plate back into the CO2 incubator and incubate for 2 h (the incubation time of EdU depends on the growth rate of the primary fibroblasts, generally about 10% of the cell cycle).

[0069] (3) After EdU labeling cells, remove the EdU working solution, add 1 ml of 4% paraformaldehyde to each well, and fix at room temperature for 15 min.

[0070] (4) Remove the fixative and wash the cells three times with PBS containing 3% BSA, shaking at medium speed for 5 minutes each time.

[0071] (5) Remove the washing solution, add 1 ml of PBS containing 0.3% Triton X-100 to each well to permeate the cells, and incubate at room temperature for 10 min.

[0072] (6) Remove the permeabilizing solution, wash the cells three times with PBS containing 3% BSA, and shake on a medium speed for 5 min each time.

[0073] (7) Prepare the Click reaction solution according to the following ratio, 500 μl per well of a 6-well plate, calculate the required amount, and prepare the large system. Use the prepared Click reaction solution within 15 min.

[0074] Table 4 Click Reaction Solution Formulation

[0075] (8) Remove the washing buffer from the 6-well plate, add 500 μl of Click reaction solution to each well, and incubate on a shaker at low speed at room temperature in the dark for 30 min to ensure that the reaction solution covers all cells.

[0076] (9) Remove the Click reaction solution, wash the cells three times with PBS containing 3% BSA, and shake on medium speed for 5 min each time.

[0077] (10) Staining cell nuclei: Dilute Hoechst 33342 (1000X) with PBS at a ratio of 1:1000, add 500 μl to each well of a 6-well plate, and incubate on a shaker at low speed at room temperature in the dark for 10 min.

[0078] (11) Remove Hoechst 33342 diluent, wash cells three times with PBS containing 3% BSA, shake on medium speed for 5 min each time.

[0079] (12) Add 2 ml of PBS to each well of a 6-well plate, observe the cells and take pictures using an inverted fluorescence microscope.

[0080] 5. TUNEL assay for apoptosis During apoptosis, cells activate certain DNA endonucleases that cleave genomic DNA between nucleosomes. Under the action of terminal deoxynucleotidyl transferase (TdT), the exposed 3'-hydroxyl (3'-OH) ends of the genomic DNA fragments are incorporated with YSFluorTM 640-12-dUTP, allowing for detection using fluorescence microscopy or flow cytometry.

[0081] (1) Adherent cells (density of about 80%) were cultured on Lab-Tek chamber slides and washed twice with PBS after apoptosis induction treatment.

[0082] (2) Fix the cells by immersing the slide in a staining jar containing 4% fresh paraformaldehyde prepared in PBS and placing it at 4°C for 25 min.

[0083] (3) Wash the slides and immerse them in PBS, then let them stand at room temperature for 5 minutes. Repeat the washing with PBS once.

[0084] (4) Gently remove excess liquid and carefully blot away excess liquid around the sample on the slide with filter paper. At this point, you can use a hydrophobic pen to trace the outline of the sample distribution around the sample to facilitate downstream permeability processing and equilibration marking.

[0085] (5) Dilute the 2 mg / mL Proteinase K solution with PBS at a ratio of 1:100 to achieve a final concentration of 20 μg / mL.

[0086] (6) Add 100 μL of Proteinase K solution with a concentration of 20 μg / mL to each sample to completely cover it, and incubate at room temperature for 5 min.

[0087] (6) Rinse the sample three times with PBS. Gently remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper. Place the treated sample in a humidified chamber.

[0088] (7) Dilute 5×Equilibration Buffer with deionized water at a ratio of 1:5.

[0089] (8) Add 100 μL of 1×Equilibration Buffer to each sample to completely cover the sample area to be tested, and incubate at room temperature for 10-30 min.

[0090] (9) Prepare sufficient TdT incubation buffer for all experiments according to Table 5.

[0091] Table 5. TdT Incubation Buffer Formulation (10) After equilibration, wash away most of the 100 μL 1×Equilibration Buffer around the cells with absorbent paper, and then add 100 μL TdT incubation buffer to the cells. Place the slide in a humidified chamber and incubate at 37°C in the dark for 60 min.

[0092] (11) Place the slices in PBS solution and incubate at room temperature for 5 min, then repeat twice with PBS.

[0093] (12) Immerse the glass slide in a staining jar containing DAPI solution in the dark and leave it at room temperature for 10 min.

[0094] (13) Wash the sample, immerse the slide in deionized water, and leave it at room temperature for 5 min. Repeat twice.

[0095] (14) Wipe off any excess water from the slide and add 100 μL of PBS to the sample area to keep the sample moist. Analyze the sample immediately under a fluorescence microscope.

[0096] result: Compared with the control group, BLM-stimulated human AT2 cells showed significantly inhibited cell proliferation and significantly increased apoptosis; while NLRX1 overexpression further promoted apoptosis and inhibited cell proliferation. Figure 4 ).

[0097] Example 5: NLRX1 knockdown can inhibit BLM-induced apoptosis and promote the proliferation of human type II alveolar epithelial cells (HPAEpiCs). 1. Cell lines All in vitro experiments were performed using HPAEpiCs (human AT2 cells), which were purchased from the same source as above.

[0098] 2. Cell Culture and Processing Plating: Add 2 ml of 0.25% trypsin to a 10 cm dish and digest for approximately 2 minutes in a cell culture incubator, then stop digestion; centrifuge at 800 rpm for 5 minutes and resuspend in 4 ml of DMEM high-glucose culture medium containing 10% FBS; add 1.8 ml of DMEM high-glucose culture medium containing 10% FBS to each well of a six-well plate, and add 200 μl of cell suspension; gently shake the plate in a "X" pattern to distribute the cells evenly. After even distribution, incubate at 37°C in a 5% CO2 cell culture incubator.

[0099] 3. siRNA transfection: When the cell confluence reaches 40-50%, siRNA transfection can be performed. The specific steps are as follows: (1) Configure the transfection system (taking one well of a six-well plate as an example) System 1: ① Mix 100 μl of blank DMEM solution / well with 5 μl of SiMUC1 solution by gentle pipetting. ② Mix 100 μl of blank DMEM solution per well with 5 μl of Lipo8000™ solution by gentle pipetting. ③ = ① + ②, mix gently and thoroughly, and let stand at room temperature for 15 minutes to obtain system one; System Two: ① Mix 100 μl of blank DMEM solution / well with 5 μl of SiNC solution / well by gentle pipetting. ② Mix 100 μl of blank DMEM solution per well with 5 μl of Lipo8000™ solution by gentle pipetting. ③ = ① + ②, mix gently and thoroughly, and let stand at room temperature for 15 minutes to obtain system two; (2) Discard the supernatant, wash twice with PBS, add 200 μl of the prepared transfection system one or two to each well, let stand for 5 minutes, add 1800 μl of blank DMEM medium (without antibiotics and FBS), and mix gently. (3) 24 hours after transfection, add 20 μl of BLM solution (the initial concentration of BLM is 5 mg / ml). After 48 hours of BLM treatment, perform subsequent treatments such as Western blot, apoptosis detection, and EdU detection.

[0100] The siRNA sequence for the negative control is: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.5) 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.6) The siRNA sequence of NLRX1 is: 5'-GAGGAGGACUACUACAACGAU-3' (SEQ ID NO.7) 5'-AUCGUUGUAGUAGUCCUCCUC-3' (SEQ ID NO.8) 4. The experimental method for detecting cell proliferation capacity with EuU is the same as above.

[0101] 5. The experimental method for detecting cell apoptosis using TUNEL is the same as above.

[0102] 6. Extraction of total cellular protein and determination of the content of related target proteins. Discard the culture medium from the cell culture plate or dish, wash once with PBS, discard the PBS, add the prepared protein lysis buffer, and lyse on ice for 30 min. Scrape cells with a cell scraper, aspirate the protein lysis buffer and cell debris into a 1.5 ml EP tube, and centrifuge at 12000 rpm for 30 min at 4°C in a pre-chilled centrifuge. Transfer the supernatant to a new 1.5 ml EP tube, discard the precipitate, and store at -80°C for subsequent experiments. The subsequent protein immunoblotting assay is performed using the same method.

[0103] result: Compared with the control group, BLM-stimulated human AT2 cells showed significantly inhibited cell proliferation and significantly increased apoptosis; NLRX1 knockdown significantly inhibited AT2 cell apoptosis and promoted cell proliferation. Figure 5 ).

[0104] Note: Because in NIPO ST.26, "T" represents uracil in the RNA sequence and thymine in the DNA sequence, the "U" in the above sequences was converted to "T" during the manufacturing process.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of substances that inhibit NLRX1 protein expression in the preparation of drugs for the prevention and treatment of fibroproliferative diseases.

2. The application according to claim 1, characterized in that, The substance is selected from any one or more of the following: (a) Compounds that inhibit the activity of NLRX1 protein; (b) A polynucleotide that inhibits NLRX1 gene expression or a recombinant vector containing said polynucleotide.

3. The application according to claim 2, characterized in that, The polynucleotide is a DNA, RNA, and / or DNA-RNA hybrid that inhibits NLRX1 gene expression.

4. The application according to claim 2, characterized in that, The RNA that inhibits NLRX1 gene expression includes NLRX1siRNA.

5. The application according to claim 4, characterized in that, The sequence of the NLRX1 siRNA is shown in SEQ ID NO.7-SEQ ID NO.

8.

6. The application according to claim 5, characterized in that, The fibroproliferative disease mentioned is pulmonary fibroproliferative disease.

7. The application according to claim 6, characterized in that, The pulmonary fibrotic disease mentioned is idiopathic pulmonary fibrosis.

8. A drug for the prevention and treatment of fibrotic diseases, characterized in that, It contains NLRX1 siRNA, the sequence of which is shown in SEQ ID NO.7-SEQ ID NO.8.

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