Application of lysosomal cathepsin L inhibitor in medicine for treating or relieving allergic airway inflammation

Abstract

The invention discloses application of a lysosome cathepsin L inhibitor in a medicine for treating or relieving allergic airway inflammation, and relates to the field of allergic airway inflammation. It is found through research that inflammatory response of asthma can be relieved by inhibiting expression of lysosome CTSL in macrophages and animal models; the inflammatory response of asthma can be relieved by inhibiting expression of lysosome CTSL in macrophages by using a small-molecule inhibitor and myeloid specific knockout CTSL, so that the lysosome cathepsin L inhibitor can effectively relieve allergic airway inflammation, and a new direction can be provided for research and development of drugs for treating and relieving allergic airway inflammation in the future.

Description

Technical Field

[0001] The present invention relates to the field of allergic airway inflammation, and particularly to the application of lysosomal cathepsin L inhibitors in drugs for treating or alleviating allergic airway inflammation. Background Art

[0002] Bronchial asthma (asthma for short) is a heterogeneous chronic inflammatory lung disease, which is mainly manifested as allergic airway inflammation, airway remodeling, and airway hyperresponsiveness. According to epidemiological data, approximately 334 million people worldwide suffer from asthma. In China, the number of patients among people aged 20 and above reaches 45.7 million, with a prevalence rate of 4.2%. As a heterogeneous disease affected by genetic and environmental factors, a better understanding of the immune mechanism of asthma is crucial for improving the treatment of asthma. Asthma is an airway inflammatory response involving multiple cells and cell components, including various immune and inflammatory cells such as eosinophils, neutrophils, mast cells, dendritic cells, and macrophages, as well as structural cells such as epithelial cells, fibroblasts, and smooth muscle cells, all of which are involved in the occurrence and development of the disease. In the past, asthma research mainly focused on the role of adaptive immunity mediated by the Th2 cell-eosinophil axis in the pathogenesis of asthma. In recent years, studies have gradually revealed that asthma is not only the result of adaptive immune dysregulation, but also the dynamic regulation of innate immune cells is equally important. Among them, macrophages, as the most widely distributed innate immune cells in lung tissue, are important sentinels of host immune defense, participating in maintaining immune regulation, pathogen clearance, and homeostasis, and playing multiple roles in the initiation, progression, and regression of asthma. Macrophages can not only mediate the presentation of allergic antigens in asthma, but also undergo M2 polarization phenotype conversion under the action of IL-4 and IL-13, producing various chemokines such as CCL3, CCL5, CCL17, CCL22, CCL24, and GM-CSF, mediating the recruitment, retention, degranulation, and cytokine production of inflammatory cells in the inflammatory site, thereby promoting the occurrence and development of the disease. Therefore, studying the role of macrophages in asthma has important clinical value for enriching the study of disease immune mechanisms and targets.

[0003] Lysosomes in macrophages are very important organelles in macrophages by absorbing and degrading extracellular substances phagocytosed by macrophages. When macrophages sense the presence of pathogens, they will phagocytose the pathogens into phagocytic vesicles through phagocytosis, and then fuse with lysosomes to form phagosomes. Inside the phagosomes, acidic hydrolases and proteases in lysosomes will be activated, and these enzymes will decompose the pathogens inside the phagosomes, thereby eliminating the pathogens and removing harmful substances inside and outside the cells. Previous studies on macrophage lysosomes in asthma have basically focused on autophagy, believing that lysosomal autophagy plays a role in the occurrence and development of allergic airway inflammation in asthma. However, as an important organelle of macrophages, lysosomes have other functions in addition to their function of degrading as autophagolysosomes in cell autophagy. The function of lysosomes depends on their acidic internal environment and the activities of lysosomal membrane proteins (LMPs) and soluble lysosomal hydrolases. Lysosomal cathepsins are key hydrolases in lysosomes. Currently, the more studied lysosomal cathepsins mainly include Cathepsin B (CTSB), Cathepsin D (CTSD), Cathepsin S (CTSS), and Cathepsin L (CTSL), etc. They are almost involved in all lysosome-related processes, such as protein and lipid metabolism, cell autophagy, antigen presentation, inflammasome signal transduction, blood vessel remodeling, neuropeptide and hormone processing, cell death, etc. Further research found that lysosomal cathepsins are highly expressed in antigen-presenting cells such as dendritic cells and macrophages, and they have been reported to have a key regulatory role in chronic airway diseases. The European Respiratory Journal (ERJ) reported in 2019 that increased expression of CTSS was observed in the BALF supernatant and plasma of patients with chronic obstructive pulmonary disease, and knockout mice of CTSS were resistant to cigarette-induced inflammation, airway hyperresponsiveness, and lung function impairment. Another study reported by ERJ in March of the same year found that the expression of CTSS was upregulated in the lung tissues of patients with cystic fibrosis, and the use of drugs to inhibit CTSS in a mouse model could significantly reduce lung inflammation and injury. In an ovalbumin (OVA)-induced mouse asthma model, prophylactic use of a CTSS inhibitor before induction with ovalbumin could reduce the number of eosinophils in the bronchoalveolar lavage fluid of mice. Therefore, lysosomes and cathepsins play an important regulatory role in macrophages and asthma airway inflammation. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides the use of lysosomal cathepsin L inhibitors in the treatment or alleviation of allergic airway inflammation drugs. The technical solution adopted by the present invention to solve its technical problems is: the use of the lysosomal cathepsin L inhibitor in the treatment or alleviation of allergic airway inflammation drugs, and the lysosomal cathepsin L inhibitor is an inhibitor that targets and inhibits lysosomal activity.

[0005] Further, the lysosomal cathepsin L inhibitor is an inhibitor that targets and inhibits the activity of cathepsin L in lysosomes.

[0006] Further, using small molecule inhibitors and myeloid-specific knockout of CTSL to inhibit the expression of lysosomal CTSL in macrophages can alleviate the inflammatory response of asthma.

[0007] Further, the lysosomal cathepsin L inhibitor is Z-FY-CHO.

[0008] Further, a composite reagent formed by the lysosomal cathepsin L inhibitor and other drugs is used for the treatment of asthma.

[0009] The beneficial effects of the present invention are as follows:

[0010] The present invention studies and finds that inhibiting the expression of lysosomal CTSL in macrophages and animal models can alleviate the inflammatory response of asthma. Therefore, the lysosomal cathepsin L inhibitor can effectively alleviate allergic airway inflammation, and can provide a new direction for the research and development of future drugs for the treatment and alleviation of allergic airway inflammation. Description of the Drawings

[0011] Figure 1 In it, A is the enrichment of the lysosome pathway by Kegg analysis in the GEO public database; B is the fluorescence co-localization of macrophages (CD68) and lysosome-associated membrane protein 1 (LAMP1) representing the lysosome level observed by immunofluorescence in the BALF lavage fluid of asthma patients; C, D, and E are the single-cell sequencing data of the GEO database showing the number and acidification level of lysosomes in asthma patients.

[0012] Figure 2 In it, A is the HDM-induced mouse asthma model, and the treatment is carried out 24, 48, and 72 hours after the last airway instillation of HDM; B is to take the BALF lavage fluid of mice 24, 48, and 72 hours after the last airway instillation of HDM stimulation, and observe the co-localization of macrophages (CD68) and lysosome-associated membrane protein 1 (LAMP1) representing lysosomes by immunofluorescence; C is the proportion of lysosome-associated membrane protein 1 (LAMP1) positive macrophages in macrophages after quantification of the immunofluorescence results.

[0013] Figure 3In A and B, the lysosomal acidification levels of BMDM were detected by LysoTracker at the time points of HDM (100 ng / μl) intervention for 2, 4, 8, and 12 h, and were quantitatively analyzed; in C and D, the lysosomal acidification levels of BMDM were detected by LysoSensor at the time points of HDM (100 ng / μl) intervention for 2, 4, 8, and 12 h and after combined BafA1 intervention for 12 h, and were quantitatively analyzed; in E, the transcriptional levels of genes related to the acidification of V-ATPase-dependent organelles (ATP6V1H, ATP6V0E1, ATP6V0B) were detected by qPCR.

[0014] Figure 4 In A, differential gene analysis of cathepsin related to PBMC data of asthma patients was performed; in B, the transcriptional level of CTSL in PBMC of healthy controls and mild and severe asthma patients was detected by qPCR.

[0015] Figure 5 In C, the expression of CTSL in PBMC of asthma patients was shown by immunofluorescence.

[0016] Figure 6 In A and B, the protein level and activity level of CTSL in the supernatant of BALF lavage fluid of the HDM mouse asthma model were measured; in C and D, the results of immunofluorescence staining of macrophages (CD68) and CTSL in the BALF lavage fluid of the HDM mouse asthma model and the quantitative analysis of CTSL-positive macrophages were performed; in E and F, the immunohistochemical results of CTSL in the lung tissue pathological sections of the HDM mouse asthma model and the quantitative analysis of CTSL-positive macrophages were performed.

[0017] Figure 7 In A, the total number of cells in BALF of each group of models (NS group, NS-CTSL MKO group, HDM group, HDM-CTSL MKO group) was counted; in B, the number of eosinophils in the cell differential count in BALF of each group of models was counted; in C, D, and E, the pathological sections of lung tissue in each group of models were examined: HE staining, PAS staining, and the corresponding inflammation scores and the proportion of PAS-positive cells.

[0018] Figure 8 In F, G, H, I, and J, the transcriptional expression levels of asthma-related inflammatory factors in the lung tissue of each group of models were measured.

[0019] Figure 9 In A, the schematic diagram of the construction of the HDM-induced mouse asthma model and the intervention time and dose of the Z-FY-CHO inhibitor were shown; in B, the expression of CTSL protein in each group of models (NS group, Z-FY-CHO group, HDM group, HDM+Z-FY-CHO group) with or without Z-FY-CHO intervention was detected.

[0020] Figure 10In this study, A represents the total number of BALF cells in each group of models (NS group, Z-FY-CHO group, HDM group, HDM+Z-FY-CHO group); B represents the number of eosinophils in the cell differential count of BALF in each group of models; C, D, and E represent the pathological sections of lung tissues in each group of models: HE staining, PAS staining, and the corresponding inflammation scores.

[0021] Figure 11 In this study, A, B, and C represent the expression levels of macrophage M2 polarization-related genes Fizz1, Arg1, and Ccl24 in each group of models (NS group, NS-CTSL MKO group, HDM group, HDM-CTSL MKO group).

[0022] Figure 12 In this study, D represents the heatmap of macrophage M2 polarization-related differential genes in the HDM group and the HDM CTSL MKO group; E represents the expression level of Arg1 protein in the lung tissues of each group of models.

[0023] Figure 13 In this study, A represents the expression levels of CTSL and Arg1 proteins in BMDM cells of WT mice and CTSL MKO mice under the intervention of 100 ng / ml IL4; B, C, D, and E represent the expression levels of macrophage M2 polarization-related genes Arg1, Fizz1, Ccl22, and Cd206 in BMDM cells of WT mice and CTSL MKO mice under the intervention of 100 ng / ml IL4. Detailed implementation manners

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners. The embodiments of the present invention are given for the purpose of illustration and description, and are not exhaustive or limit the present invention to the disclosed form. Many modifications and variations are obvious to those of ordinary skill in the art. The embodiments are selected and described to better illustrate the principles and practical applications of the present invention, and enable those of ordinary skill in the art to understand the present invention and thus design various embodiments with various modifications suitable for specific purposes.

[0025] Example 1: By detecting the expression level of lysosomes in lung macrophages during asthma attack.

[0026] First, the RNA-seq data of asthma patients in the GEO public database were analyzed, and it was found that compared with healthy controls, the expression of lysosome-related pathways was upregulated in asthma patients ( Figure 1, A). Lysosome-associated membrane protein 1 (LAMP1) is one of the main proteins on the lysosomal membrane, which participates in maintaining the integrity and function of lysosomes. As a classical lysosomal membrane marker, it can be used to detect changes in the number of lysosomes. Subsequently, bronchoalveolar lavage fluid (BALF) from clinical asthma patients was collected and immunofluorescence experiments were performed. The results of immunofluorescence showed that the expression level of macrophage LAMP1 in BALF was up-regulated in asthma patients ( Figure 1 , B). In addition, by analyzing the single-cell sequencing data of asthma patients in public databases, the same conclusion of increased lysosome expression was also obtained. Moreover, the single-cell data of asthma patients also showed enhanced lysosome acidification levels that were beneficial to promoting lysosome function and internal enzyme activity ( Figure 1 , C, D, E).

[0027] Example 2: Increased lysosome expression in macrophages of a mouse asthma model induced by HDM.

[0028] An asthma mouse model was constructed using house dust mite extract (HDM). Healthy wild-type C57 / BL6J mice aged 6 - 8 weeks were selected. On days 0, 7, and 14, each mouse was given 100 μg of HDM by intratracheal instillation, and the same dose of normal saline (Saline) by intratracheal instillation was used as a control (NS). The mice were treated 24 h, 48 h, and 72 h after the last HDM challenge to observe the lysosome expression at different treatment times ( Figure 2 , A), and the BALF of the mice was collected for immunofluorescence experiments to detect the lysosome expression level of macrophages in the BALF.

[0029] 1. Animal model treatment and specimen collection

[0030] 1.1 Collection of mouse BALF and lung tissue samples:

[0031] Prepare the operating instruments. Anesthetize the mice with 0.2 ml of 2% sodium pentobarbital. After the mice show no response, fix them on the operating board. Disinfect the operating site with 75% ethanol, bluntly separate the skin, and cut along the sternum to fully expose the chest cavity. Use a 1 ml syringe to draw heart blood, and choose whether to collect blood samples as needed. Then ligate the three lobes of the right lung together with the upper lobe of the left lung. After fully exposing the trachea, make a small incision in the trachea with scissors, insert a BLAF lavage needle for alveolar lavage, inject 0.4 ml of PBS and aspirate repeatedly 3 times, and repeat this step 3 times to obtain approximately 1 ml of BALF lavage fluid, which is placed on ice for preservation. Subsequently, cut the ligated four-lobe lung tissue and place it in liquid nitrogen for preservation. After the treatment is completed, store the lung tissue in a -80°C refrigerator. Then perfuse 0.4 ml of 4% formaldehyde solution into the left lung for fixation, ligate the trachea, cut the left lung and soak it in 4% formaldehyde solution for external fixation, pending further histological examination. Aspirate 50 μl of the previously ice-cold BALF lavage fluid for cell counting. Centrifuge the remaining lavage fluid at 6000 g for 10 minutes, collect the supernatant and store it at -80°C. Perform cytocentrifugation on the cell pellet obtained by centrifugation, and then perform Giemsa staining for differential cell counting;

[0032] 1.2 Total number and differential count of cells in BALF lavage fluid

[0033] Prepare a white blood cell lysate. Mix 50 μl of the aspirated BALF lavage fluid with 50 μl of the white blood cell lysate in an 8-well strip, then aspirate 20 μl and add it to a cell counting chamber, and place it in a Countstar cell counter for counting. After counting, according to the total number of cells obtained, aspirate a certain amount of PBS to resuspend the cell pellet obtained by centrifuging the BALF lavage fluid according to the total number of cells (100 μl of PBS is required for every 2.5x105 / ml cells) for differential counting. Take 50 μl of the resuspended cell suspension and place it on a cytocentrifuge, 850 rpm, for 2 minutes. Fix the cytospin on a glass slide, draw boundaries with paraffin 0.5 cm on each side of the cells, and perform Giemsa staining. First, stain with 150 μl of Giemsa solution A for 90 seconds, then add 300 μl of Giemsa solution B for decolorization, and gently blow and mix the solution B and solution A with an ear syringe for 10 minutes. Then gently rinse the slide with water for 10 seconds to remove excess dye. After the slide is dry, place the stained slide under an optical microscope for differential cell counting. Under the microscope, select about 200 cells to count in continuous fields of view, and classify different types of cells (such as eosinophils, macrophages, neutrophils, etc.) by observing the morphological characteristics of the cells;

[0034] 1.3 Extraction of lung tissue protein samples

[0035] Step 1: Rinse the mouse lung tissue thoroughly with PBS and use absorbent paper to absorb the residual PBS in the lung tissue as much as possible. Cut an appropriate amount of lung tissue, weigh it on a microbalance, record it, place it in a sterile homogenate tube, and store it on ice.

[0036] Step 2: After placing large and small ceramic beads in a homogenizer at a ratio of 1:2, add an appropriate amount of RIPA at a ratio of 1:15 according to the amount of lung tissue, and then grind the lung tissue in a homogenizer at a frequency of 60 Hz for 60 seconds. If there are still obvious tissue fragments after one grinding, grind it again at the same frequency and time.

[0037] Step 3: Place the homogenate tube in a small low-temperature centrifuge and centrifuge at 12000 rpm for 15 minutes.

[0038] Step 4: After centrifugation, transfer as much supernatant as possible from the homogenate tube to a new EP tube, take 25ul of the supernatant for BCA protein concentration detection, and store the remaining supernatant on ice. After the protein concentration test is completed, add 5x Loading Buffer, protease inhibitors, β-mercaptoethanol, etc. to the remaining supernatant to prepare a protein loading mixture.

[0039] Step 5: Store at -80℃.

[0040] 1.4 Preparation of lung tissue RNA samples

[0041] Place the lung tissue in a clean homogenizer tube and add 1 ml of Trizol. Place large and small porcelain beads in the homogenizer at a ratio of 1:2. Grind the lung tissue in a homogenizer at a frequency of 60 Hz for 60 seconds before using it to extract RNA for subsequent testing.

[0042] 1. Cell Culture and Sample Collection

[0043] 1.1 Cell recovery

[0044] Prepare items such as 10-cm dish culture dishes, 50-ml and 15-ml centrifuge tubes, and 1-ml pipette tip boxes in advance in a laminar flow hood, and perform ultraviolet disinfection for half an hour. At the same time, heat the water bath to 37 °C. After the preparation work is completed, quickly take out the cells from the -80 °C refrigerator, place the cells in a 37 °C water bath for heating, shake to accelerate thawing. After the cells are completely thawed into a cell suspension, use a pipette to transfer the cell suspension in the cryotube to a 15-ml centrifuge tube, add about 4 ml of complete medium, centrifuge at 400 rcf for 5 minutes to obtain a cell pellet, aspirate the supernatant, resuspend the cell pellet with a certain amount of complete medium according to the number of cell dishes to be resuscitated, and seed them in a 10-cm dish culture dish. After spraying 75% disinfected alcohol, place them in a 37 °C cell culture incubator for culture. The resuscitated cells need to be changed the medium after 24 hours, passaged after 48 hours, and passaged every 48 hours thereafter. During the whole operation process, attention should be paid to aseptic operation.

[0045] 2.2 Cell Passage

[0046] First, determine that the cell density has reached 90%-100%, and then perform cell passage operations. Prepare items in a laminar flow hood and perform ultraviolet disinfection for half an hour. For adherent cells, first wash them 1-2 times with PBS, then add 1 ml of trypsin and gently shake to make the trypsin contact the cell surface fully and evenly. Place the cells in a 37 °C cell culture incubator to make the trypsin in the best activity. After digesting the cells with trypsin for an appropriate time according to the cell type, add complete medium to terminate the digestion and collect them in a 15-ml centrifuge tube;

[0047] For suspension cells, they can be directly collected in a 15-ml centrifuge tube. Centrifuge at a speed of 400 rcf for 5 minutes, discard the supernatant to obtain a cell pellet, and then resuspend the cell pellet with complete medium according to the proportion required for the number of dishes to be passaged, and seed them in a 10-cm dish as needed, and passage once every 48 hours.

[0048] 2.3 Cell Cryopreservation

[0049] After collecting the cell pellet using the same method as the above cell passage, resuspend the cell pellet with 1 ml of serum-free cell cryopreservation solution, and aliquot the cell resuspension into cryotubes at 0.5 ml per tube. Seal them tightly with a sealing film, make marks such as the date, and cryopreserve the cells in liquid nitrogen or a -80 °C refrigerator.

[0050] 2.4 Collection of Cell Protein Samples

[0051] Adherent cells (taking 6-well plates as an example): Wash the adherent cells with PBS 2-3 times. Add 80-120 μl of protein extraction solution (RIPA or protein loading buffer) to each well according to the cell density. Ensure that the protein extraction solution is in full contact with the cell surface, and lyse the cells on ice for 5 minutes. Use a cell scraper to scrape and collect the cells on ice into a 1.5 ml EP tube. After ultrasonically lysing the cells thoroughly, heat them in a metal bath at 100 °C for 10 minutes and store them in a -80 °C refrigerator.

[0052] Suspension cells (taking 6-well plates as an example): Collect the cells as much as possible into a 15 ml centrifuge tube, centrifuge at 400 rcf for 5 minutes. Add 80-120 μl of protein extraction solution according to the cell pellet amount and transfer it to a 1.5 ml EP tube. Mix well with a pipette, sonicate on ice, then heat in a metal bath at 100 °C for 10 minutes and store in a -80 °C refrigerator.

[0053] 2.5 Collection of cell RNA samples

[0054] Adherent cells (taking 6-well plates as an example): Wash the adherent cells with PBS 2-3 times. Add 1 ml of Trizol to each well to lyse the cells thoroughly. After standing for 5 min, mix well with a 1 ml pipette and collect it into a 1.5 ml EP tube, then store it in a -80 °C refrigerator.

[0055] Suspension cells (taking 6-well plates as an example): Collect the suspension cells as much as possible with a sterile dropper into a 15 ml centrifuge tube, centrifuge at 400 rcf for 5 minutes. Add 1 ml of Trizol to each tube, mix well with a pipette, transfer it to a 1.5 ml EP tube, and store it in a -80 °C refrigerator.

[0056] 2. Fluorescent quantitative PCR (qPCR)

[0057] 3.1 RNA extraction

[0058] Step 1: Add 200 μl of chloroform solution to every 1 ml of Trizol, mix the sample by inverting it up and down, and let it stand

[0059] for 5 minutes.

[0060] Step 2: Centrifuge at 12000 g at 4 °C for 15 minutes. Meanwhile, prepare clean and sterilized 1.5 ml EP tubes corresponding to the number of sample tubes.

[0061] Step 3: After centrifugation, the sample liquid can be seen to be divided into three layers. The lower layer is a red organic layer (containing chloroform, protein, etc.), the middle layer is a white thin layer (containing protein, DNA, etc.), and the upper layer is a clear, transparent and colorless aqueous layer (containing RNA). Use a pipette to aspirate 400 μl of the liquid from the upper aqueous layer and transfer it to a prepared 1.5 ml EP tube. Note that during the aspiration process, do not stick to the tube wall and do not get close to the middle white thin layer. Add 500 μl of isopropanol to each tube, gently invert and mix up and down, and let it stand for five minutes.

[0062] Step 4: Centrifuge at 12000 g for 15 minutes at 4 °C. At the same time, prepare 75% ethanol (dilute absolute ethanol to 75% with DEPC water) and pre-cool it on ice.

[0063] Step 5: After centrifugation, a white RNA precipitate adhering to the wall can be seen in the EP tube. At this time, discard the supernatant. If there is some liquid residue, it can be aspirated clean with a pipette after quickly spinning the EP tube in the centrifuge. Do not touch the precipitate during the aspiration process. Whether the supernatant is completely aspirated will directly affect the quality of the subsequent RNA.

[0064] Step 6: Add 1 ml of pre-cooled 75% ethanol to each EP tube, gently invert and rotate to make the precipitate float and come into full contact with the ethanol.

[0065] Step 7: Centrifuge at 9000 g for 5 minutes at 4 °C.

[0066] Step 8: After centrifugation, the white precipitate can be seen to adhere to the wall again. Discard the supernatant, and use a pipette to aspirate as much residual liquid as possible. Open the lid and let it stand at room temperature to dry (but do not dry completely, otherwise the precipitate will become transparent and affect dissolution).

[0067] Step 9: Add an appropriate amount of DEPC water according to the amount of precipitate, pipette and mix well, place on ice, and measure the concentration with a nanodrop. Generally, the concentration should be preferably 200 - 300 ng / μl, and the A260 / A280 ratio should be 1.8 - 2.0 to ensure qualified purity. If no subsequent operations are to be carried out, the RNA needs to be stored in a -80 °C refrigerator, or continue with reverse transcription and other operations.

[0068] 3.2 Reverse Transcription (RT)

[0069] (1) RT System:

[0070] 5x Evo M-MLV RT Master Mix 2 μl

[0071] RNA 500 ng

[0072] DPEC water up to 10 μl

[0073] (2) Thaw the required RNA enzyme in advance and place it on ice. Since the enzyme may precipitate at the bottom, gently flick it to mix evenly and add 2 μl of 5x RT Mix to each well in the eight-well strip according to the number of samples.

[0074] (3) According to the RNA concentration of the samples measured previously, pipette 500 ng of RNA into each sample and add it to the eight-well strip.

[0075] (4) According to the amount of 5x RT Mix and RNA added, supplement with DEPC water to 10 μl.

[0076] (5) Gently flick to mix evenly. RNA is unstable, so be careful and gently spin the liquid to the bottom in a centrifuge.

[0077] (6) Perform the reaction according to the reverse transcription program set on the PCR instrument. The specific program is as follows:

[0078] 37°C for 15 min

[0079] 85°C for 5 sec

[0080] (7) After the reverse transcription is completed, the obtained cDNA product can be directly used for subsequent qPCR or stored in a -80°C refrigerator.

[0081] 3.3 qPCR

[0082] (1) Real-time fluorescence quantitative PCR (qPCR) system

[0083] 2x SYBR Green Pro Taq Premix

[0084]

[0085]

[0086] (2) Take out the enzyme and upstream and downstream primers required for qPCR and thaw them in advance. Do not wear rubber gloves during subsequent operations to avoid affecting the PCR results.

[0087] (3) According to the amount required for the samples, mix the SYBR Green enzyme and upstream and downstream primers in the system ratio and prepare one more portion for every 10 portions to prevent intermediate losses. Mix the cDNA product obtained from RT with ddH2O in a ratio of 1:3.6.

[0088] (4) Add 5.4 μl of the SYBR Green enzyme and upstream and downstream primer mixture and 3.6 μl of the cDNA and ddH2O mixture to each well in the PCR plate, for a total of 10 μl. After adding the samples, quickly spin the PCR plate in a horizontal centrifuge to make the liquid reach the bottom.

[0089] (5) Operate on the real-time fluorescence quantitative PCR instrument according to the SYBR Green enzyme instruction manual or the pre-set program. Select whether to run the melting curve as needed.

[0090] (6) Obtain the qPCR data and export it for analysis.

[0091] 4 Western Blot

[0092] 4.1 Preparation of PAGE gel

[0093] (1) Select a PAGE gel rapid preparation kit with the appropriate concentration according to the specific molecular weight of the protein to be run.

[0094] (2) Prepare the corresponding number of gels according to the number of proteins to be run. One thin and one thick glass slide are required for each gel preparation. Check whether the glass slides are damaged before use, and place the glass slides under running water to clean them, avoiding debris residue. Prepare the gel-making rack and gel strips. Stack the thin and thick glass slides on the gel-making rack, pay attention to clamping. Add ddH2O to the gap between the thin and thick glass slides, and let it stand for 5 minutes. Check whether the liquid level drops and there is water leakage.

[0095] (3) During the standing period, prepare the corresponding lower-layer separating gel mixture according to the required PAGE gel concentration using the preparation kit, and place it on a shaker to mix well quickly.

[0096] (4) After checking for water leakage, pour out the ddH2O in the gap of the glass slides, invert the glass slides together with the gel-making rack to drain the ddH2O in the gap as much as possible. After sucking the water clean with absorbent paper, add the lower-layer separating gel mixture pre-mixed on the shaker to the gap between the thin and thick glass slides at a ratio of 100:1 with the rapid coagulant promoter, and add 8 ml per gel-making glass slide. Use 500 μl of isopropanol to flatten the liquid surface of the mixture, and wait for 10 minutes for the lower-layer gel to solidify.

[0097] (5) During the waiting period for the lower-layer gel to solidify, prepare the corresponding upper-layer stacking gel mixture using the gel preparation kit, and place it on a shaker to mix well quickly.

[0098] (6) Rinse the isopropanol for pressing the gel with ddH2O 3 - 5 times. After the isopropanol is rinsed clean, suck out the liquid as much as possible with absorbent paper. Add the upper-layer stacking gel mixture pre-mixed on the shaker to the gap between the glass slides at a ratio of 100:1 with the rapid coagulant promoter, and add 4 ml per gel-making glass slide. Insert a 10 / 15-well gel comb according to the number of sample loading wells, and let it stand for 15 minutes. After the upper-layer gel solidifies, take out the gel for sample loading experiment.

[0099] 4.2 Western blot sample preparation

[0100] If it is a freshly collected sample, load the sample directly after heating in a 100°C metal bath.

[0101] If it is a sample frozen at -80°C, it needs to be thawed first, then placed on ice and sonicated, and then heated in a 100°C metal bath for 8 minutes before loading.

[0102] 4.3 Sample Loading and Electrophoresis

[0103] (1) While waiting for the gel, the 10x electrophoresis buffer can be diluted to 1x with ddH2O and vortexed well. Prepare the electrophoresis tank, electrophoresis apparatus, electrophoresis core, three-color prestained protein marker, etc. in advance.

[0104] (2) Clamp the prepared gel with the electrophoresis core, being careful not to skew it, and place it in the electrophoresis tank. Pay attention to the positive and negative electrodes. If the positive and negative electrodes are reversed, the sample will float out of the loading wells during electrophoresis. Add 1x electrophoresis buffer. At least fill the inner part of the electrophoresis core with electrophoresis buffer, and then add an appropriate amount of electrophoresis buffer to the outer tank. Gently pull out the comb from the gel.

[0105] (3) First, add the three-color prestained protein marker to the outermost well of the loading wells as much as possible. Generally, add 5 μl. Then add a certain volume of the sample according to the protein expression level. If running a certain protein for the first time, generally a loading volume of 10 μl can be used for a preliminary experiment, and then adjust the loading volume according to the concentration of the band that runs out. If the loading volume of the marker and the sample is quite different, the loading well of the marker can be filled up with 1x Loading Buffer.

[0106] (4) After all the samples are loaded, cover the lid of the electrophoresis apparatus. First, perform electrophoresis at a voltage of 60 V. When the sample leaves the upper stacking gel and enters the lower separating gel, and the bands of the marker are clearly separated, the voltage can be adjusted to 90 V or 120 V and continue electrophoresis. When the loading band is almost at the bottom, determine the specific electrophoresis time according to the protein molecular weight.

[0107] 4.4 Blotting

[0108] (1) Stop electrophoresis, pour out the electrophoresis buffer, rinse the remaining electrophoresis buffer in the electrophoresis tank under running water, then place the gel in ddH2O. Prepare two liquid boxes, each containing a certain amount of methanol and 1x membrane equilibration buffer. The 1x membrane equilibration buffer can be diluted to 1x with 10x membrane equilibration concentrate and ddH2O. Prepare items such as scissors, gel cutting board, ballpoint pen, etc.

[0109] (2) Cut a PVDF membrane that matches the size of the cutting and transfer membrane instrument. Immerse it in methanol for 1 minute and then transfer it to 1x membrane equilibration solution for sufficient immersion. It is appropriate that the PVDF membrane is fully immersed in the membrane equilibration solution. During the immersion of the PVDF membrane, take out the gel plate, separate the thick and thin glass slides, and it is appropriate to leave the gel on the thin glass slide. Cut the gel with a gel cutting plate. Generally, retain the width of the corresponding marker near the target band and two markers above and below it. After cutting, place it in ddH2O. Take out the transfer membrane clamping plate in the transfer membrane instrument, with the positive electrode facing down. Pay attention to the positive and negative electrodes. If reversed, the protein bands on the gel will be transferred to the transfer membrane solution rather than the PVDF membrane. Place a square cotton gasket on the positive electrode, place the PVDF membrane on the gasket, and place the cut gel on the PVDF membrane. Note that there should be no gap between the PVDF membrane and the gel. Exhaust air bubbles, then place another gasket on the gel, press the negative transfer membrane clamping plate, and insert it into the transfer membrane instrument for membrane transfer.

[0110] (3) After the membrane transfer is completed, mark the membrane with a pen and appropriately cut the PVDF membrane in ddH2O with scissors. Note that the membrane should be kept moist and not dried.

[0111] 4.5 Blocking

[0112] (1) During the operation of the transfer membrane instrument, take 2.5 g of skim milk and place it in a 50 ml centrifuge tube. Add 1xTBST and vortex to preliminarily mix and make up the volume to 50 ml to prepare 5% skim milk, and place it on a shaker and shake quickly to mix evenly.

[0113] (2) Take out the cut membrane and place it in 5% skim milk, and shake it on a horizontal shaker at low speed for 1 hour to block. If the blocking time is too short, it is easy for non-target proteins to affect the band signal. If the blocking time is too long, it is easy for the target band signal to weaken.

[0114] (3) Use 1xTBST to rinse and wash off the 5% skim milk used for blocking. Do not shake and wash quickly on the shaker. The milk blocking is not very tight, and quick washing is easy for milk particles to detach from the PVDF membrane.

[0115] 4.6 Primary Antibody Incubation

[0116] (1) Mix the antibody in a 1:1000 ratio in the primary antibody diluent, place it in a 50 ml centrifuge tube, place the blocked membrane in the centrifuge tube, and make the membrane fully contact with the antibody. Incubate overnight at 4°C in a vertical mixer.

[0117] (2) Recover the primary antibody and quickly shake and wash it with 1xTBST on a horizontal shaker for 5 minutes, three times in total.

[0118] 4.7 Secondary Antibody Incubation

[0119] (1) Prepare 5% skim milk, add the corresponding secondary antibody to the milk at a ratio of 1:2000, mix well, immerse the membrane in the secondary antibody, and incubate it with gentle shaking on a horizontal shaker for 1 hour.

[0120] (2) Recover the secondary antibody, wash it quickly with 1xTBST on a horizontal shaker for 5 minutes, three times in total.

[0121] 4.8 Development

[0122] ChemiDoc TM Image development with a chemiluminescence imaging system and expose the bands.

[0123] 5 Mouse genotype identification

[0124] (1) Cut the tip of the tail of the mouse for genotype identification and place it in a 1.5 ml EP tube, and cut the mouse's toe for marking.

[0125] (2) Turn on the metal bath in advance and preheat it to 100°C. Add 200 μl of 40 mM 1xNaOH solution to each EP tube, place it in the 100°C metal bath and heat for 20 minutes, and pay attention to opening the lid to drain the steam halfway.

[0126] (3) Quickly centrifuge and flick the liquid to the bottom. Add 20 μl of Tris-HCl (pH = 6.8) to each EP tube to stop the reaction, place it on ice and flick it evenly quickly, and centrifuge at 12000 g for 5 minutes in a low-temperature centrifuge. The supernatant is the solution containing DNA.

[0127] (4) PCR system:

[0128]

[0129]

[0130] (5) After loading the samples in the eight-well strip according to the above PCR system, cover the lid and centrifuge quickly, and place it in the PCR instrument to perform PCR according to the editing program of the required gene.

[0131] (6) Rinse the conical flask under clean water, add an appropriate amount of TAE to rinse the inner wall of the conical flask. Weigh 2 g of agarose using an electronic balance, place it in the conical flask, add slightly more than 100 ml of 1x TAE, shake well, and place it in the microwave oven to heat at a temperature above medium heat for 3 min. Take out the conical flask and observe whether there is still agarose precipitation. Shake the conical flask and place it in the microwave oven again to heat for 2 minutes until the liquid is clear and transparent. Rinse the conical flask under clean water to cool it down to about 50 °C, generally when the palm touches the bottom of the conical flask without discomfort. Then add 10 μl of EB solution to the conical flask, shake well, prepare the gel mold and comb, pour the liquid in the conical flask into the mold, insert the comb, ensure there are no air bubbles, and let it stand for about half an hour until the gel cools and solidifies.

[0132] (7) Remove the comb from the solidified gel and place it on the horizontal electrophoresis apparatus. Pour in enough 1x TAE solution. Take out the PCR-amplified mouse samples to be genotyped, add them to the wells of the gel sample, and add DNA loading marker as needed. Electrophoresis is carried out at 160 V.

[0133] (8) Take an image using a gel imaging system, and determine the mouse genotype based on the position of the bands and whether the bands are developed.

[0134] 6 Flow cytometry

[0135] 6.1 Sample acquisition (taking mouse lung tissue as an example)

[0136] (1) Anesthetize the mouse with 0.2 ml of 2% sodium pentobarbital. After the mouse is anesthetized and motionless, spray the chest with alcohol for disinfection, fully expose the chest cavity, take a 1 ml syringe to draw cardiac blood, and sever the abdominal aorta. (It is also possible to directly sever the abdominal aorta and quickly suck out the blood with a syringe, but blood clots are likely to form.)

[0137] (2) Use a 10 ml syringe to gently and uniformly inject sterile PBS from the coronary artery of the heart. Flush the pulmonary blood vessels to make the lungs turn porcelain white. If too much force is used, it will cause the pulmonary blood vessels to burst, allowing PBS to enter the alveolar cavity, resulting in pulmonary edema and making the lungs appear edematous.

[0138] (3) Cut off the bilateral lung tissues of the mouse, rinse them with PBS, put them into clean and sterile EP tubes according to the pre-arranged groups, add a little Hank's balanced salt solution (HBSS), and use blunt scissors to cut the lung tissues in the EP tubes into pieces as small as possible, making them into a paste-like tissue mass of 1 - 3 mm. The smaller the better.

[0139] 6.2 Single-cell sample acquisition

[0140] (1) Prepare 5% collagenase I diluted with HBSS (collagenase I can be used for the digestion and separation of epithelial tissue, lung tissue, adipose tissue, etc. The working concentration is 1-2 mg / ml, and a certain amount of CaCl2 can be added to enhance the activity). Transfer the cut lung tissue from the EP tube to the corresponding 15 ml centrifuge tube, wash the EP tube with collagenase I, and aspirate the residual lung tissue and collect it in the 15 ml centrifuge tube.

[0141] (2) Incubate in a 37 °C incubator (the temperature at which collagenase I has the strongest activity) for 60 minutes. Prepare 50 ml centrifuge tubes and 44 μm filters according to the number of samples.

[0142] (3) Open the 50 ml centrifuge tube, place the 44 μm filter on the 50 ml centrifuge tube, aspirate the digested cell debris from the previous step into the filter for filtration, and use the sterile 5 ml syringe plunger to grind the unfiltered lung tissue on the filter. At the same time, rinse the ground lung tissue debris with 1640 medium until it is finally ground into a slag-like state.

[0143] (4) Centrifuge at 400 g for 5 minutes at 4 °C in a horizontal centrifuge. The cell pellet is at the bottom of the 50 ml centrifuge tube. Discard the supernatant, add 3 ml of red blood cell lysate and lyse for 3 minutes, and terminate the lysis with 1640 medium at about 10 times the volume of the red blood cell lysate (about 30 ml).

[0144] (5) Filter the solution after terminating lysis through a 44 μm filter into a new 50 ml centrifuge tube, and centrifuge at 400 g for 5 minutes at 4 °C in a horizontal centrifuge.

[0145] 6.3 Cell surface marker staining

[0146] (1) Prepare the Fc block solution diluted with PBS (to block cell Fc receptors and prevent interference with subsequent flow cytometry results). Discard the supernatant in the 50 ml centrifuge tube, resuspend the cell pellet with 200 μl of Fc block solution per tube, and stain for 10 minutes. At the same time, prepare flow cytometry tubes (divided into blank tubes, single-label tubes, and all-positive tubes) and flow cytometry antibodies corresponding to the staining strategy. Generally, the flow cytometry antibody for surface markers is 0.5 μl / sample, and 50 μl of PBS / sample is added to resuspend to obtain the flow cytometry antibody solution.

[0147] (2) After staining, wash with sterile PBS, centrifuge at 400 g for 5 minutes at 4 °C, and then invert the flow cytometry tube to discard the supernatant (generally, 50 μl of liquid is left by default).

[0148] (3) Add 100 μl of PBS to the blank tube for later use. Add the prepared corresponding flow cytometry antibody solution to the single-label tubes and all-positive sample tubes to form a 100 μl system, mix well, and stain at 4 °C in the dark for 15 minutes.

[0149] (4) Wash with sterile PBS, centrifuge at 400 g for 5 minutes at 4 °C, and resuspend with FCS.

[0150] 6.4 Fixation, permeabilization and intracellular marker staining (if necessary)

[0151] (1) Add 100 μl of fixation and permeabilization solution to each sample tube and incubate in the dark at 4 °C for 15 minutes.

[0152] (2) Wash with sterile PBS, centrifuge at 400 g for 5 minutes at 4 °C, and the subsequent staining steps are similar to those for surface marker staining.

[0153] (3) Run on a flow cytometer, set parameters to adjust voltage and compensation, and record data for analysis.

[0154] 7 Immunofluorescence technique

[0155] 7.1 Cell seeding and intervention

[0156] (1) Use a suction device to hold the cell slide and gently shake it to place it at the bottom of a 12-well plate. Seed the cells at an appropriate density (not too dense to avoid affecting subsequent imaging and filming effects) in the 12-well plate with the slide at the bottom.

[0157] (2) Perform intervention on the cells as needed.

[0158] 7.2 Fixation, permeabilization and blocking

[0159] (1) Discard the culture medium supernatant in the 12-well plate, wash the cells 2 - 3 times with sterile PBS, and then fix with 4% formaldehyde solution for 5 minutes. If staining for membrane surface antigens, do not permeabilize; if for intracellular antigens, add 500 μl of permeabilization solution to each well and incubate at room temperature for 30 minutes.

[0160] (2) Prepare 5% BSA solution and use it immediately. Add 1 ml of 5% BSA to each well in the 12-well plate for blocking and incubate at room temperature for half an hour.

[0161] 7.3 Primary antibody incubation

[0162] (1) Wash 3 times with sterile PBS, dilute the selected antibody with 5% BSA at a ratio of 1:100, 50 μl / sample. Select a humid chamber with a lid, place a long strip of sealing film at the bottom of the humid chamber, and add 50 μl of antibody to each sample on the sealing film with a pipette according to the number of cell slides in the samples.

[0163] (2) Use forceps to pick out the cell slides in the 12-well plate and invert them onto the antibody on the sealing film, taking care to leave no gaps or bubbles; cover the lid of the humid chamber and incubate for 2 hours (note to keep both sides of the humid chamber moist to prevent the cells on the slides from drying).

[0164] 7.4 Secondary antibody incubation

[0165] (1) Use forceps to pick up the cell slide from the humid chamber, place it upright (with the cell side facing up, not inverted) back into the 12-well plate, and wash it 3 times with PBS.

[0166] (2) Dilute the fluorescent secondary antibody with 5% BSA in the dark, at a ratio of 1:100. Take a new long strip of sealing film and place it in the humid chamber. Use a pipette to add 50 μl of the fluorescent secondary antibody. Also, invert the cell slide onto the fluorescent secondary antibody, leaving no gaps or bubbles, and incubate in the dark for 1 hour.

[0167] 7.5 Nuclear counterstaining and fixation

[0168] (1) Pick up the slide from the humid chamber, place it upright in the 12-well plate, and wash it 3 times with PBS.

[0169] (2) Blot dry the PBS, add 500 μl of DAPI solution, and incubate in the dark for 10 minutes;

[0170] (3) After incubation, wash it 3 times with PBS. At this time, each PBS wash is for 5 minutes. After the last wash, leave an appropriate amount of PBS in the 12-well plate.

[0171] (4) Take a glass slide and add 10 μl of fluorescent quenching protective agent onto the slide. Take out the cell slide from the 12-well plate, blot dry the liquid on the absorbent paper, invert it and place it on the fluorescent quenching protective agent, leaving no gaps or bubbles, and then fix it around the slide with nail polish. Store it in the dark at 4 °C.

[0172] (5) Observe and collect the cell fluorescence images under an inverted fluorescence microscope for subsequent analysis.

[0173] The experimental results showed that over time, the proportion of lysosome-associated membrane protein 1 (LAMP1)-positive macrophages in the BALF lavage fluid of mice gradually increased and reached a statistically significant difference 48 h after the last HDM challenge ( Figure 2 , B, C), indicating that there was also an increase in macrophage lysosome expression in the asthma mouse model.

[0174] In vitro experiments were conducted to extract mouse bone marrow-derived macrophages (BMDMs) for primary culture, and HDM (100 ng / μl) was used to intervene in BMDMs in a time-gradient manner for 2, 4, 8, and 12 hours respectively. Lysosomal fluorescent probes LysoTracker and LysoSensor can penetrate cell membranes and can be used for specific fluorescent staining of lysosomes in living cells, and can evaluate the degree of lysosomal acidification according to the fluorescence intensity. Moreover, LysoSensor is more sensitive than LysoTracker and has a stronger pH dependence. In BMDMs after HDM intervention, the LysoTracker probe was first used for preliminary detection, and it was found that as the intervention time increased, the lysosomal acidification level of BMDM cells gradually increased with the extension of the intervention time until it decreased at 12 hours ( Figure 3 , A, B). Using the same intervention conditions, an additional group with HDM combined with 1 mM bafilomycin A1 (BafA1) intervened for 12 h was set up. BafA1 has been proven to inhibit acidification and protein degradation in cell lysosomes, and then the LysoSensor probe was used for detection. Similar to the results obtained by the LysoTracker probe detection, the lysosomal acidification level of BMDM cells also gradually increased with the extension of the intervention time and decreased at 12 hours, and we found that BafA1 significantly inhibited the fluorescence intensity detected by the probe ( Figure 3 , C, D). Lysosomes use the energy of hydrolyzing ATP by V-type ATPase (vesicular or vacuolar-ATPase, V-ATPase) to pump protons into the lysosomal lumen to generate and maintain its pH gradient, maintaining its acidic environment so that lysosomes can function normally. When detecting the expression levels of V-ATPase-related genes (ATP6V1H, ATP6V0E1, ATP6V0B), it was found that they were all up-regulated in the early stage of intervention, namely at 2 h and 4 h, although the expression levels decreased again at 8 h and 12 h ( Figure 3 , E).

[0175] Under acidic conditions, lysosomes can cause an increase in the activity of cathepsin in lysosomes. By analyzing the RNA-seq peripheral blood mononuclear cell (PBMC) data of asthma patients in the previously retrieved public database and annotating various cathepsins, it was found that among all lysosomal cathepsins, only cathepsin L (CTSL) was increased in the PBMC of asthma patients ( Figure 4, A). Peripheral blood of asthmatic patients was collected clinically and PBMC was isolated for detection. The qPCR results showed that, compared with healthy controls, the expression level of CTSL in PBMC of asthmatic patients was indeed significantly increased, and this increasing trend might have a certain correlation with the severity of asthma. The expression level of CTSL in severe asthmatic patients was significantly higher than that in mild asthmatic patients. Although the expression level of CTSL in mild asthmatic patients did not reach a statistically significant difference compared with healthy controls ( Figure 4 , B). In addition, an immunofluorescence experiment was also performed on PBMC of asthmatic patients, and it was found that the immunofluorescence results were consistent with the aforementioned qPCR results, and the expression level of CTSL in PBMC increased to a certain extent. Figure 5 , C).

[0176] Meanwhile, in vivo experiments of animal models were carried out for verification. A batch of HDM-induced murine asthma models was constructed again, and the BALF lavage fluid and lung tissues of mice treated 24 h, 48 h, and 72 h after the last HDM challenge were collected. After centrifuging the BALF lavage fluid to collect the supernatant for detection, it was found that, compared with the control group, the CTSL protein in the supernatant of the 24 h treatment group after HDM challenge increased significantly, and then decreased in the 48 h and 72 h treatment groups, indicating that CTSL might be upregulated in the early stage of asthma onset. Figure 6 , A). Similarly, when detecting the activity of CTSL in the supernatant, it was also found that the activity of CTSL was enhanced to a certain extent in the 24 h treatment group, and then the activity decreased in the 48 h and 72 h treatment groups. Figure 6 , B). At the same time, the cells in the BALF lavage fluid collected from the above groups were immunofluorescently stained, and it was found that, compared with the control group, the proportion of CTSL-positive macrophages showed an obvious upward trend in the murine BALF lavage fluid collected at any time point after HDM challenge. Figure 6 , C, D). Then, the lung tissues collected from each group of mice were prepared into pathological sections for CTSL immunohistochemistry experiments, and similar results were also obtained in the immunohistochemistry. Figure 6 , E, F). These results all indicate that there is also an increase in the expression and enhancement of the activity of CTSL in the asthma animal model, which is consistent with the data obtained from asthmatic patients.

[0177] To further study the role of CTSL in macrophages in vivo in asthma, in this example, CTSL knockout CTSL flox / flox mice were bred with Lyz2-Cre mice specifically located in myeloid cells to obtain myeloid-specific CTSL knockout mice (Lyz2-Cre CTSL flox / floxMice, hereinafter referred to as CTSL MKO mice), in this example, wild-type C57 / BL6J mice of the same size and age (hereinafter referred to as WT mice) were used as controls, and HDM-induced mouse asthma models were constructed, which were divided into four groups: WT mice airway instilled with normal saline control group (NS group), CTSL MKO mice airway instilled with normal saline control group (NS-CTSL MKO group), WT mice airway instilled with HDM asthma model group (HDM group), CTSL MKO mice airway instilled with HDM asthma model group (HDM-CTSL MKO group). In this example, the models of each group were treated and the BALF lavage fluid and lung tissue of the mice were collected. By counting the cells in the BLAF lavage fluid, it was found that the total number of cells in the HDM group increased significantly compared with the two NS control groups, while in the HDM-CTSL MKO group, the total number of cells decreased significantly compared with the HDM group ( Figure 7 , A). At the same time, in this example, the BLAF lavage fluid was centrifuged, and the collected cell smear was stained with Giemsa and classified and counted. It was found that the number of eosinophils in the HDM group increased significantly, while the number of eosinophils in the HDM-CTSL MKO group decreased significantly compared with the HDM group ( Figure 7 , B).

[0178] In this example, the lung tissues of each group of mice were also prepared into pathological sections for HE staining and PAS staining, and the inflammation levels of the pathological sections of the lung tissues of each group of mice were detected and quantified, and similar conclusions were also obtained, that is, the inflammation level and the proportion of PAS-positive cells in the HDM group both increased significantly, while the inflammation level and the proportion of PAS-positive cells in the HDM-CTSL MKO group decreased significantly compared with the HDM group ( Figure 7 , C, D, E). In addition, in this example, the lung tissue RNA of each group of mice was extracted and the expression levels of asthma-related inflammatory factors (IL13, IL4, MUC5AC, IL25, IL33) were detected. When the expression levels of inflammatory factors in the HDM group all increased, the expression levels of inflammatory factors in the lung tissues of the HDM-CTSL MKO group were all down-regulated to varying degrees ( Figure 8 , F, G, H, I, J). These data all indicate that knocking out CTSL in macrophages will significantly inhibit the inflammation of asthma, suggesting the important regulatory role of macrophage CTSL in asthma inflammation.

[0179] Since this embodiment has shown that macrophage CTSL has a regulatory effect on asthma inflammation, during the construction of the HDM-induced mouse asthma model in this embodiment, a CTSL-specific inhibitor (Z-FY-CHO) was specifically administered, and it was proved that it could effectively and specifically inhibit the expression of CTSL without affecting the occurrence and development of the disease

[21] . In this embodiment, 15 mg / kg dose of Z-FY-CHO was intraperitoneally injected 2 hours before each HDM stimulation for intervention ( Figure 9 , A), and an equal dose of DMSO was intraperitoneally injected as a control without changing other modeling conditions. A total of four groups were divided: WT mouse airway instillation of normal saline combined with intraperitoneal injection of DMSO control group (NS group), WT mouse airway instillation of normal saline combined with intraperitoneal injection of Z-FY-CHO control group (Z-FY-CHO group), WT mouse airway instillation of HDM combined with intraperitoneal injection of DMSO control group (HDM group), WT mouse airway instillation of HDM combined with intraperitoneal injection of Z-FY-CHO control group (HDM+Z-FY-CHO group). Finally, the mouse models of each group were treated and BALF lavage fluid and lung tissue were collected. WB detection of the extracted mouse lung tissue protein found that Z-FY-CHO did inhibit the expression of CTSL in the asthma mouse model ( Figure 9 , B).

[0180] After determining that Z-FY-CHO inhibited the expression of CTSL, the total number of cells and eosinophils in the BALF lavage fluid of each group of mice collected in this embodiment were counted, and it was found that compared with the HDM group, under the action of Z-FY-CHO, both the total number of cells and the number of eosinophils in the HDM+Z-FY-CHO group decreased significantly ( Figure 10 , A, B), and the lung tissues of each group of mice were prepared into pathological sections for HE staining and PAS staining. Statistical analysis found that the inflammation level and score decreased significantly ( Figure 10 , C, D, E), indicating that inhibiting the expression of CTSL can effectively inhibit airway inflammation in asthma.

[0181] Previous research data have shown that the expression of macrophage CTSL is upregulated in asthma patients and murine asthma models, and the inhibition of CTSL can effectively alleviate asthma airway inflammation. Therefore, an investigation was conducted on how macrophage CTSL regulates asthma airway inflammation. It is known that during the asthma inflammatory response, macrophages can aggregate in the lung tissue and undergo M2 polarization. The number of CD206-positive macrophages increases in asthma patients, and there is a correlation between the percentage of M2 macrophages and the disease severity. Therefore, in this example, the expression of genes related to macrophage M2 polarization was detected in the lung tissue of the HDM asthma mouse model previously constructed in WT mice and CTSL MKO mice. It was found that the expression levels of genes related to macrophage M2 polarization (Fizz1, Arg1, Ccl24) were upregulated in the HDM group, while these genes decreased significantly in the HDM-CTSL MKO group( Figure 11 , A, B, C). Subsequently, in this example, a new batch of asthma mouse models was constructed again in WT mice and CTSL MKO mice respectively. Macrophages in the lungs of each group were sorted by flow cytometry, and RNA-seq was performed to obtain data. By analyzing and processing the RNA-seq data, a differential gene heat map of genes related to M2 polarization was made for the HDM group and the HDM-CTSL MKO group. It was found that compared with the control group, the genes related to macrophage M2 polarization (Arg1, Irf4, Ccl17, Ccl22, Ccl24) were basically upregulated in the HDM group. In contrast, the genes related to M2 polarization decreased to varying degrees in the HDM-CTSL MKO group( Figure 12 , D). By detecting the mouse lung tissue by WB, it was also found that the expression level of the protein Arg1 related to macrophage M2 polarization in the lung tissue of the HDM-CTSL MKO group of mice decreased compared with the HDM group( Figure 12 , E). In summary, the above experimental results all suggest that CTSL has a certain regulatory effect on macrophage M2 polarization during the pathogenesis of asthma.

[0182] In vitro experiments were conducted to verify the results obtained from in vivo models and RNA-seq. BMDM cells were isolated from WT mice and CTSL MKO mice for primary culture. Since macrophages undergo M2 polarization under the action of IL4 and IL13 during the pathogenesis of asthma, and both IL4 and IL13 are key cytokines in asthma development, IL4 was selected as the intervention factor for the experiment. BMDM cells were intervened with IL4 at a dose of 100 ng / ml, and cell proteins were collected for WB detection. After confirming that IL4 could induce an increase in CTSL in BMDM cells and that CTSL was effectively knocked out in BMDM cells of CTSL MKO mice, it was found that after IL4 intervention, the expression of the M2 polarization-related protein Arg1 increased in BMDM cells of WT mice, while the trend of increased Arg1 protein expression induced by IL4 in BMDM cells of CTSL MKO mice was inhibited( Figure 13 , A). Additionally, BMDM cells were intervened with 100 ng / ml IL4 at different time points of 0, 6, 12, and 24 h, and cell RNA was collected for qPCR detection. It was found that compared with BMDM cells of WT mice, the expression levels of M2 polarization-related genes (Arg1, Fizz1, Ccl22, Cd206) in BMDM cells of CTSL MKO mice decreased to varying degrees at almost every time point( Figure 13 , B, C, D, E).

[0183] Therefore, in this example, through means such as clinical specimens and in vitro and in vivo experiments, combined with techniques such as RNA-seq, the activation of macrophage lysosomes and the increase in cathepsin during the pathogenesis of asthma were studied. The results showed that there was lysosome activation in macrophages during the pathogenesis of asthma, and the expression of CTSL in the activated lysosomes increased. The increased CTSL could affect the development of asthma by promoting macrophage M2 polarization, and inhibiting CTSL could relieve asthma inflammation.

[0184] The following conclusions were drawn:

[0185] There is macrophage lysosome activation in asthma patients and asthma mouse models, and the expression of lysosomal cathepsin L (CTSL) increases;

[0186] The increased expression of CTSL can promote the occurrence and development of asthma, and small molecule inhibitors and knockout of CTSL to inhibit its expression can relieve the inflammatory response of asthma.

[0187] Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art and related fields based on the embodiments in the present invention without creative efforts shall fall within the scope of protection of the present invention. The structures, devices, and operation methods not specifically described and explained in the present invention shall be implemented by conventional means in the art unless otherwise specified and limited.

Claims

1. Use of lysosomal cathepsin L inhibitor in drugs for treating or alleviating allergic airway inflammation, characterized in that: The lysosomal cathepsin L inhibitor is an inhibitor that targets and inhibits lysosomal activity.

2. The application according to claim 1, wherein The lysosomal cathepsin L inhibitor is an inhibitor that targets and inhibits the activity of cathepsin L in lysosomes.

3. The application according to claim 1, wherein Using small molecule inhibitors and myeloid-specific knockout of CTSL to inhibit the expression of lysosomal CTSL in macrophages can alleviate the inflammatory response of asthma.

4. The application according to claim 1, characterized in that The lysosomal cathepsin L inhibitor is Z-FY-CHO.

5. The application according to claim 1, characterized in that, A composite reagent formed by a lysosomal cathepsin L inhibitor and other drugs is used for the treatment of asthma.

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