Application of substance for promoting conversion from mM to AM in preparation of medicine for treating airway epithelial cell injury

Abstract

The invention discloses application of a substance for promoting conversion from mM to AM in preparation of a medicine for treating airway epithelial cell injury, and relates to the technical field of treatment of airway epithelial cell injury. It is found that activation of a PPAR-gamma signal channel can promote conversion from mM to AM, Club cell proliferation and migration are driven, and the airway epithelial injury repair process after streptococcus pneumoniae infection is accelerated. The discovery not only reveals the core regulation effect of the PPAR-gamma signal in the post-infection airway repair, but also provides a new theoretical basis for targeting macrophage phenotypic transformation and Plet1 function intervention of chronic airway diseases (such as asthma), and has important theoretical and potential clinical application values.

Description

Technical Field

[0001] The present invention relates to the technical field of treating airway epithelial cell damage, and more particularly to the use of a substance that promotes the conversion of mM to AM in the preparation of a drug for treating airway epithelial cell damage. Background Art

[0002] Streptococcus pneumoniae (S. pneumoniae) is the main pathogen of bacterial pneumonia, meningitis, bleeding infections, sinusitis and other diseases in children under 5 years old. S. pneumoniae usually colonizes the nasopharynx of healthy people. When the host's immunity is reduced (such as colds, fatigue or chronic diseases), the bacteria can invade the lower respiratory tract through the respiratory tract and cause infection. It is worth noting that S. pneumoniae infection not only causes acute inflammation, but may also become a key cause of chronic airway diseases. Epidemiological studies have found that the cumulative incidence of asthma before the age of 18 is significantly increased in newborns carrying S. pneumoniae; and preventing S. pneumoniae infection through vaccination in infancy can significantly reduce the risk of asthma in children at the age of 4. This suggests that there may be a potential causal relationship between early S. pneumoniae colonization / infection and the development of asthma.

[0003] Adhesins on the surface of Streptococcus pneumoniae (such as choline-binding protein and pneumococcal surface protein A) bind to receptors on host airway epithelial cells (such as platelet-activating factor receptor and intercellular adhesion molecule), promoting bacterial adhesion and colonization. The released pneumolysin (PLY) directly damages epithelial cells by forming membrane pores, activating the Toll-like receptor (TLR) 2 / 4 signaling pathway, inducing NF-κB-mediated production of proinflammatory cytokines (such as TNF-α and IL-6), recruiting neutrophils and macrophages to the site of infection, and inducing the release of reactive oxygen species (ROS) and proteases, such as neutrophil elastase (NE) and matrix metalloproteinases (MMPs), further disrupting the airway epithelial barrier function. Among them, goblet cells (formerly known as Clara cells), key secretory epithelial cells in the terminal bronchioles, exert anti-inflammatory and antioxidant effects and maintain mucosal integrity by secreting CC16 (Clara cell secretory protein). Impaired CC16 function is closely associated with airway hyperresponsiveness and remodeling. Studies have shown that reduced CC16 expression is not only a biomarker for childhood asthma but also a predictor of its continued progression into adulthood. Impaired club cell regeneration can lead to progressive decline in lung function. Therefore, elucidating the mechanisms of club cell repair after infection is key to intervening in chronic airway diseases.

[0004] Alveolar macrophages (AMs), resident immune cells in the lung, play a central role in pathogen clearance, inflammation regulation, and epithelial repair. Under physiological conditions, AMs maintain homeostasis through self-renewal. However, during infection or inflammatory damage, monocyte-derived macrophages (mMs) can migrate to the alveoli and differentiate into AMs to replenish the depleted cell pool. However, whether pneumococcal infection interferes with the conversion of mMs to AMs, thereby affecting airway epithelial repair, and whether promoting the conversion of mMs to AMs can reverse airway epithelial cell damage remains unclear.

[0005] Peroxisome proliferator-activated receptor (PPAR)-γ (PPAR-γ) is a nuclear transcription factor that exhibits multiple biological activities upon ligand activation and is widely expressed in various tissues and organs throughout the body. Studies have shown that PPAR-γ plays multiple roles in regulating macrophage differentiation and function. Activation of PPAR-γ promotes polarization of monocytes into anti-inflammatory macrophages and enhances phagocytic and repair functions through molecules such as CD163 and CD36. However, the mechanism of action of PPAR-γ in AM transformation and airway epithelial regeneration following Streptococcus pneumoniae infection remains unclear. Summary of the Invention

[0006] In view of this, the present invention provides the use of a substance that promotes the conversion of mM to AM in the preparation of a drug for treating airway epithelial cell damage.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides use of a substance that promotes the conversion of mM to AM in the preparation of a drug for treating airway epithelial cell damage.

[0009] Furthermore, the substance that promotes the conversion of mM to AM is an agonist that activates the PPAR-γ signaling pathway; the agonist is rosiglitazone RSG.

[0010] Furthermore, the airway epithelial cell damage is airway epithelial cell damage caused by infection with Streptococcus pneumoniae;

[0011] Furthermore, the airway epithelial cells are Club cells;

[0012] Furthermore, the airway epithelial cell damage is manifested by a decrease in the mRNA level of Scgb1a1 and a decrease in the fluorescence intensity of Club cells CC16 in lung tissue.

[0013] Furthermore, the substance promoting the conversion of mM to AM increases the number of AM in BALF after S. pneumoniae infection; and increases the mRNA level of Scgb1a1 and the fluorescence intensity of Club cells CC16 in lung tissue.

[0014] Furthermore, AM cells promote the proliferation and migration of airway epithelial cells; the airway epithelial cells are 16HBE cells.

[0015] The second aspect of the present invention provides a drug for treating airway epithelial cell damage, the drug comprising a substance that promotes the conversion of mM to AM; the substance is an agonist that activates the PPAR-γ signaling pathway; the agonist is rosiglitazone RSG.

[0016] Through the above technical solutions, the present invention discovered that activating the PPAR-γ signaling pathway can promote the transformation of macrophages (mMs) to AMs, drive Club cell proliferation and migration, and accelerate the repair process of airway epithelial damage after Streptococcus pneumoniae infection. This discovery not only reveals the core regulatory role of PPAR-γ signaling in post-infection airway repair, but also provides a new theoretical basis for targeting macrophage phenotypic transformation and Plet1 function to intervene in chronic airway diseases (such as asthma), with important theoretical and potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0018] Attachment Figure 1 Figure 2. Flow cytometry analysis of AM and mM in BALF of mice on day 2 (A), day 4 (C), and day 7 (E) after S. pneumoniae infection. B. Quantitative analysis of AM and mM in BALF on day 2 (D), day 4 (F), and day 7 (F). Data are expressed as mean ± SD, n = 3; *p < 0.05, **p < 0.01, ****p < 0.0001 vs. control group.

[0019] Attachment Figure 2 Figure 1: Club cells in the airway epithelium of mice damaged by Streptococcus pneumoniae infection. A. Representative immunofluorescence staining of CC16 (scale bar: 20 μm); B. Mean fluorescence intensity of CC16. Data are expressed as mean ± SD, n = 4; ****p < 0.0001 vs. Control group.

[0020] Attachment Figure 3 KEGG and GSEA enrichment analysis results of DEGs in the process of mM to AM conversion; A. KEGG enrichment analysis results; B. GSEA enrichment analysis results.

[0021] Attachment Figure 4 Figure 2 shows the significant upregulation of PPAR-γ expression during the transformation from mM to AM; A. Volcano plot of differentially expressed genes; B. PPAR-γ mRNA levels; C. Heat map of differentially expressed genes.

[0022] Attachment Figure 5Figure 1 shows the decreased expression of PPAR-γ in the micromillipores of BALF from mice infected with Streptococcus pneumoniae. A. Flow cytometry analysis of PPAR-γ expression in the micromillipores of BALF from mice. B. Mean fluorescence intensity of PPAR-γ. Data are expressed as mean ± SD, n = 3; **p < 0.01 vs. Control group.

[0023] Attachment Figure 6 Effects of PPAR-γ activation and inhibition on the number of AMs and mMs on day 2 after S. pneumoniae infection; A. Flow cytometry analysis of the number of AMs and mMs in each group; B. Quantitative analysis of AMs and mMs. Data are expressed as mean ± standard deviation, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance.

[0024] Attachment Figure 7 Effects of PPAR-γ activation and inhibition on the number of AMs and mMs on day 4 after S. pneumoniae infection. A. Flow cytometry analysis of the number of AMs and mMs in each group. B. Quantitative analysis of AMs and mMs. Data are expressed as mean ± SD, n = 3; **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0025] Attachment Figure 8 Figure 7: Effects of PPAR-γ activation and inhibition on the number of AMs and mMs after infection with Streptococcus pneumoniae. A. Flow cytometry analysis of the number of AMs and mMs in each group of mice. B. Quantitative analysis of AMs and mMs. Data are presented as mean ± SD, n = 3; ns indicates not significant.

[0026] Attachment Figure 9 PPAR-γ activation promotes Scgb1a1 mRNA expression.

[0027] Attachment Figure 10 PPAR-γ activation promotes the expression of CC16 in airway epithelium.

[0028] Attachment Figure 11 AMs were isolated and purified from mouse BALF for flow cytometry.

[0029] Attachment Figure 12 Effects of AM from mice with different treatments on epithelial cell proliferation.

[0030] Attachment Figure 13 Effects of AM from mice with different treatments on epithelial cell migration. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1 Animal Model Preparation

[0033] Female Balb / c mice, 3 weeks old (10 ± 1 g), were purchased from Enswell Biotechnology Co., Ltd. All animal experiments were conducted in accordance with standard animal research guidelines. Mice were housed in an independent ventilation cage (IVC) system with a 12-h light / 12-h dark cycle at appropriate temperature and humidity, with free access to standard chow and tap water. After acclimating to the feeding regimen for 1 week, 48 juvenile mice were randomly divided into four groups, each containing 12 mice: control (Con), Streptococcus pneumoniae infection group (Sp), Streptococcus pneumoniae infection plus a PPAR-γ agonist group (Sp+RSG), and Streptococcus pneumoniae infection plus a PPAR-γ inhibitor group (Sp+T007). RSG (3 mg / kg / d) and T007 (1 mg / kg / d) were intraperitoneally injected three days before infection and continued until 7 days after infection. The Con and Sp groups received equal volumes of normal saline intraperitoneally for 14 consecutive days.

[0034] The infection method of Streptococcus pneumoniae was as follows: 200 μl of the standard strain D39 stored at -80°C was inoculated into Todd-Hewitt broth yeast extract (THY) medium and cultured in a 5% CO2, 37°C incubator until the logarithmic growth phase. The bacteria were scraped off and centrifuged (3000 rpm / min, 5 min), washed twice with sterile PBS, and resuspended. Mice in the Sp group, S.p + RSG group, and S.p + T007 group were instilled into the nasal cavity with 10 μl (approximately 2×10 6 The control group was instilled with an equal volume of PBS. All experimental operations were performed in a clean bench.

[0035] The animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

[0036] Example 2 Verification of Streptococcus pneumoniae damage to airway epithelial cells

[0037] 1. Collection of serum, BALF, and lung tissue samples from model mice and normal mice

[0038] (1) Serum Collection: The model mice described in Example 1 and normal mice were anesthetized with 1.25% avertin and the right eyeballs were enucleated. Eyeball blood was collected in 1.5 ml EP tubes. After standing at room temperature for 3 h, the blood was centrifuged at 2500 rpm for 20 min. The upper serum layer was aspirated into a new 1.5 ml EP tube and stored at -80°C.

[0039] (2) BALF collection: The mouse was fixed in a supine position on the operating table. The hair was wetted and disinfected with 75% alcohol. The neck skin was cut open with ophthalmic scissors, and the muscle tissue was bluntly separated to fully expose the trachea. After tracheal intubation, the tracheal tube was fixed with surgical sutures. The mouse chest cavity was exposed, and the hilum of the left lung of the mouse was clamped with hemostatic forceps. 0.5 ml of pre-cooled PBS was taken from the tracheal tube to lavage the right lung three times to obtain bronchoalveolar lavage fluid (BALF).

[0040] (3) Lung tissue collection: After BALF collection, release the hemostatic clamp that clamps the left lung hilum, cut the left atrial appendage with ophthalmic scissors, and perfuse the heart with 10 ml of pre-chilled PBS to flush the blood from the pulmonary vessels. Remove the left lung and place it in 4% paraformaldehyde. Fix it at room temperature in the dark for at least 24 hours. Subsequently, dehydrate the lung tissue and embed it in paraffin. Remove the right lung and place it in a 1.5 ml EP tube at -80°C for subsequent experiments.

[0041] 2. Flow cytometry was used to detect the number of AM and mM cells in BALF.

[0042] (1) Cell collection: Centrifuge the collected BALF at 700 g for 5 min at 4°C and remove the supernatant; add 1 ml of red blood cell lysis buffer to the pellet, resuspend and let stand for 2 min, add PBS to terminate the reaction, centrifuge again at 700 g for 5 min at 4°C and remove the supernatant, retaining the cell pellet.

[0043] (2) Washing: The collected cell pellet was washed twice with PBS, centrifuged at 700 g for 5 min at 4°C, and the supernatant was removed. The cell suspension was resuspended in HBSS buffer containing 2% FBS.

[0044] (3) Closure: Count Star counter, collect about 1×10 5 Each tube of cell suspension was centrifuged at 700 g at 4°C for 5 min, the supernatant was removed and the serum collected above was added, and the mixture was blocked at room temperature for 20 min.

[0045] (5) Antibody incubation: Centrifuge at 700g for 5 min at 4°C and discard the supernatant. Add the mixed flow cytometry antibody and stain at 4°C for 30 min in the dark, flicking the tube every 15 min.

[0046] (6) Washing: Add 1 ml of PBS buffer and wash once, centrifuge at 700g, 4℃ for 5 min, and remove the supernatant.

[0047] (7) Fixation: Add 300 μl of paraformaldehyde to fix the cells and store at 4°C in the dark.

[0048] (8) Flow cytometry: After filtering with a 300-mesh filter, flow cytometry was performed. The gate strategy for lung tissue cells is as follows: mononuclear macrophages (F4 / 80 int CD11b + ) and alveolar macrophages (F4 / 80 high CD11c high Siglec-F high ).

[0049] See the results Figure 1 Compared with the Con group, on the second day after pneumococcal infection, the number of AM in the BA LF of the infected mice was significantly reduced (33213467.8 vs. 38253±1126, p<0.01), and the number of mM was significantly increased (23701±388.1 vs. 2677±321.2, p<0.0001) ( Figure 1 A and B in Figure 4). On the 4th day after infection, the AM of the infected group mice gradually increased, but was still significantly lower than that of the control group (29007±2382 vs 39086±3475, p<0.05) ( Figure 1 C and D). On day 7 after infection, the number of AMs in the BALF of the infected mice returned to the baseline level (41285±1949 vs. 38315±1730, ns) ( Figure 1 E and F), suggesting that S. pneumoniae infection can lead to a decrease in AM.

[0050] 3. Detect whether the airway epithelial cell CLUB in the lung tissue of model mice is damaged

[0051] Immunofluorescence staining was used to detect the expression of CC16 in mouse airway epithelium. The specific method is as follows.

[0052] (1) Baking: Place the paraffin sections in a 60°C constant temperature oven and bake for at least 24 hours.

[0053] (2) Dewaxing and hydration: Place the paraffin sections in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min, and then rinse in tap water for 1 min.

[0054] (3) Antigen retrieval: Add the pre-prepared 1 mM Tris / EDTA solution (pH 9.0) to a beaker and heat the solution to a boil in a microwave oven over medium-high heat. Place the paraffin sections in the beaker and heat over medium-low heat for 10 min. Transfer the beaker from the microwave oven to room temperature and allow it to cool naturally. Place the paraffin sections that have cooled to room temperature in a wash box and wash them three times with PBS on a shaker for 5 min each.

[0055] (4) Blocking: Wipe the PBS around the slices with filter paper, cover the tissue with 5% BSA, and block in a wet box at room temperature for 1 hour.

[0056] (5) Incubation with primary antibody: Cover the sliced tissue with 20 μl of primary antibody (CC16 antibody: PBS = 1:100) and incubate overnight at 4°C in a humidified chamber. After recovering the primary antibody, wash the sections with PBS three times on a shaker for 5 minutes each time.

[0057] (6) Incubation with secondary antibody: Dry the sections with filter paper and cover the tissue with secondary antibody (Alexa Fluor 488-conjugated goat anti-mouse secondary antibody: 5% BSA = 1:100). Incubate in a humidified chamber at room temperature in the dark for 1 h. After recovering the secondary antibody, wash the sections three times with PBS on a shaker for 5 min each time.

[0058] (7) DAPI staining: Cover the tissue with DAPI and incubate at room temperature in the dark for 10 min. Wash with PBS three times, 5 min each time.

[0059] (8) Sealing: After sealing the slides with anti-fluorescence quencher, observe and take pictures under an upright laser confocal microscope.

[0060] See the results Figure 2 , found that the mean fluorescence intensity (MFI) of CC16 in the airway epithelium of mice infected with Streptococcus pneumoniae was significantly lower than that of the control group (Con) (MFI: 0.06450±0.01028 vs 0.1675±0.01258, p<0.0001) ( Figure 2 A and B), indicating that S. pneumoniae infection damages airway club cells.

[0061] Example 3: Clarifying the relationship between pneumococcal infection, mM to AM conversion, and PPAR-γ expression

[0062] To explore the mechanism by which Streptococcus pneumoniae infection inhibits the transformation from mM to AM, we analyzed the public database (G SE225406) and found that the differentially expressed genes in the process of mM to AM transformation were most significantly enriched in the PPAR signaling pathway ( Figure 3 A in Figure 1). Further GSEA enrichment analysis revealed that the PPAR pathway showed an up-regulation trend ( Figure 3Further analysis revealed that PPAR-γ was enriched in the PP AR pathway, and the expression of PPAR-γ was significantly upregulated ( Figure 4 AC in (Fig. 3A), suggesting that PPAR-γ plays a key role in the transformation of mM to AM.

[0063] In order to clarify the effect of pneumococcal infection on PPAR-γ, flow cytometry was used to detect the expression level of PPAR-γ in macrophages in BALF of the above model mice. The results showed that the level of PPAR-γ in macrophages of the pneumococcal infection group was significantly lower than that of the control group (MFI: 9751±277.8 vs 13179±754.6, p<0.01) ( Figure 5 A and B in Figure 5), suggesting that S. pneumoniae inhibits the expression of PPAR-γ in mM.

[0064] To further investigate the effect of PPAR-γ on the conversion of mM to AM, mice infected with Streptococcus pneumoniae were given the PPAR-γ agonist RSG and inhibitor T007, respectively. The number of mM and AM was measured on days 2, 4, and 7 after infection. The results showed that on day 2 after infection, compared with the control group, the number of AM in the BALF of the infected mice was significantly reduced (1.500±0.1575 vs. 3.272±0.4608, p<0.001), while the number of mM was significantly increased (3.698±0.7742 vs. 0.04546±0.007082, p<0.01). Compared with the mice infected with Streptococcus pneumoniae, the number of AM in the BALF of the RSG group mice was significantly increased (2.245±0.2137 vs 1.500±0.157, p < 0.05), and the number of AM in the T007 group mice was further decreased compared with the infection group (0.7253±0.2440 vs 1.500±0.157, p < 0.05) ( Figure 6 A and B in ).

[0065] On the 4th day after infection with Streptococcus pneumoniae, the number of AM in the infected group gradually increased, but was still significantly lower than that in the control group (1.859±0.2066 vs 3.315±0.06604, p<0.001). Compared with the infected group, after administration of RSG, the number of AM in mice increased significantly (3.045±0.4479 vs 1.859±0.2066, p<0.01), which was similar to the level of the control group. However, the number of AM in the T007 group was not significantly different from that in the infected group (2.157±0.3275 vs 1.859±0.2066, p<0.01). Figure 7 A and B in ).

[0066] On day 7 after infection with Streptococcus pneumoniae, the number of AM in the BALF of the infected mice had completely recovered and was not significantly different from that of the control group (2.969±0.2199 vs 3.248±0.3639, ns). The number of AM in the RSG and T007 groups was also not significantly different from that of the control and infected groups ( Figure 8 A and B in ).

[0067] Example 4 RT-qPCR detection of the effect of PPAR-γ expression on airway epithelial cells

[0068] RNA extraction:

[0069] (1) Lysis: For lung tissue: Weigh 30 mg of lung tissue, dry the excess water on the tissue surface with filter paper, and place it in a 1.5 ml EP tube without enzyme. Add 300 μl of lysis buffer 2. Place 3 magnetic beads in the EP tube and grind it in a homogenizer. After grinding thoroughly, let it stand at room temperature for 5 minutes.

[0070] For cells cultured in vitro: After removing the cells from the incubator, discard the old culture medium, wash twice with PBS, aspirate the PBS, add 1 ml of lysis buffer 2, mix well with a pipette, and let it stand at room temperature for 5 minutes.

[0071] (2) Centrifugation: Centrifuge at 4°C, 12,000 rpm for 10 min. Carefully aspirate the supernatant and transfer it to the adsorption column. Centrifuge at 12,000 rpm for 30 s at room temperature. Keep the column in the purification column and discard the liquid in the collection tube.

[0072] (3) Washing: Add 600 μl of washing solution to the adsorption column and centrifuge at 12,000 rpm for 30 seconds at room temperature. Discard the liquid in the collection tube and retain the adsorption column. Repeat this step for a total of two washes.

[0073] (4) Empty column centrifugation: After discarding the liquid in the collection tube, the adsorption column was centrifuged at 10,000 rpm for 1 min at room temperature and left at room temperature for about 15 min to remove the residual washing solution.

[0074] (5) Redissolution: Connect the adsorption column to a new enzyme-free 1.5 ml EP tube, add 20 μl of DEPC water to the center of the adsorption column filter membrane, let it stand at room temperature for 5 minutes, and then centrifuge at room temperature and 12,000 rpm for 30 seconds to obtain the re-dissolved RNA.

[0075] (6) Concentration determination: The total RNA concentration was detected using Nanodrop.

[0076] Reverse transcription:

[0077] (1) Calculate the required RNA volume based on the measured RNA concentration, i.e., RNA volume (μl) = 1000 (ng) / RNA concentration (ng / ul).

[0078] (2) Remove genomic DNA and prepare the reaction system according to the following ingredients:

[0079]

[0080] (3) The reaction was carried out under the following conditions: heating at 42°C for 2 min, then at 4°C forever.

[0081] (4) Reverse transcribe RNA into cDNA and prepare the reaction system according to the following ingredients:

[0082]

[0083] (5) Incubate the reaction in a reaction machine according to the following conditions: 37°C for 15 min, 85°C for 5 s, and 4°C forever.

[0084] Add sample:

[0085] (1) Prepare the reaction system according to the following ingredients and add the liquid to each well of the eight-tube strip:

[0086]

[0087] (2) The primer sequences used are as follows:

[0088]

[0089] On board:

[0090] The reaction was carried out under the following conditions: 95°C for 2 min, 95°C for 10 s, and 57°C for 30 s, 40 cycles.

[0091] See the results Figure 9 and Figure 10 Club cells are an important component of epithelial cells. We evaluated the number of Club cells in the epithelium by detecting the Scgb1a1 level in the lung tissue of each group of mice and the immunofluorescence intensity of CC16 in the airway epithelium. The results showed that compared with the infection group, the mRNA level of Scgb1a1 in the lung tissue of the RSG-treated mice was significantly increased (1.066±0.06950 vs 0.6493±0.06073, p<0.001) ( Figure 9), indicating that PPAR-γ activation can reduce or reverse the damage of pneumococcal infection to airway epithelial cells. We further used immunofluorescence to detect the expression of CC16 in the airway epithelium of the lung tissue of each group of mice. The results showed that compared with the infection group, RSG treatment could significantly increase the immunofluorescence intensity of CC16 in the airway epithelium (MFI: 0.1429±0.03399 vs 0.03784±0.01755, p < 0.01) ( Figure 10 A and B in the figure indicate that RSG treatment activates PPAR-γ and promotes the increase of Club cells in the mouse airway epithelium.

[0092] Example 5 Effects of Differently Treated Mouse-Derived AM on Epithelial Cell Proliferation and Migration

[0093] 1) Isolation and culture of primary mouse AM cells in vitro

[0094] After Sp infection and drug administration in mice, AMs were extracted from the BALF of mice in each group for in vitro studies.

[0095] (1) BALF collection: BALF from each group of mice was collected into sterile 1.5 ml EP tubes.

[0096] (2) Cell collection: Centrifuge at 700g, 4°C for 5 min, discard the supernatant, and retain the cell pellet. Add 1 ml of red blood cell lysis buffer to resuspend and let stand for 2 min. Wash twice with PBS.

[0097] (3) Culture: After centrifugation, high-glucose DMEM medium containing 20% FBS and 1% penicillin / streptomycin was added and cultured in a 5% CO2, 37°C incubator for subsequent experiments.

[0098] 2) Flow cytometry to identify AM purity

[0099] Take the primary mouse alveolar macrophages after separation and culture, discard the culture medium, wash the cells with PBS, centrifuge at 300g for 5 minutes, remove the supernatant and add rat serum, block with 5% BSA at room temperature for 20 minutes, centrifuge and remove the supernatant. Add the mixed flow cytometry antibodies (F4 / 80, CD11c and Siglec-F), stain at room temperature in the dark at 4℃ for 30 minutes, add 1ml PBS buffer to wash once, centrifuge at 1500rpm / min at room temperature for 30s, and discard the supernatant. Add 300μl paraformaldehyde to fix, filter with a 300-mesh filter, and detect the purity of AM on a flow cytometer. See Figure 11 AM was screened using F4 / 80high, Siglec-F+, and CD11c+ markers, and the purity of the purified AM was >80%.

[0100] 3) 16HBE cell recovery, passaging and cryopreservation

[0101] All operations were performed in an ultra-clean cell workbench after UV irradiation.

[0102] (1) Cell recovery

[0103] After removing the cells from the liquid nitrogen tank, quickly place them in a 37°C water bath and shake repeatedly to thaw them quickly. Transfer the completely thawed cells to a sterile 15ml centrifuge tube containing 3ml of complete culture medium (containing 10% FBS and 1% double-antibody) prepared in advance. Centrifuge at 1000rpm / min for 5min, discard the supernatant, retain the white precipitate at the bottom of the tube, add 4ml of complete culture medium, gently pipette to mix, transfer to a culture flask, mix thoroughly, and continue culturing in a cell culture incubator. Subculture when the cells grow to approximately 80% density.

[0104] (2) Cell passage

[0105] Remove the cells, discard the old culture medium, add 2 ml of sterile PBS to wash once, and aspirate the PBS; add 1 ml of trypsin, shake left and right to allow all cells to contact the trypsin, place in the incubator and digest for 1 min, remove and observe under a microscope to see if the cells have rounded, gently tap the bottle to suspend the cells in the liquid, and then quickly add 2 ml of complete culture medium to terminate digestion; mix well with a pipette and transfer to a 15 ml sterile centrifuge tube. Subsequent centrifugation and inoculation steps are the same as for cell recovery.

[0106] (3) Cell cryopreservation

[0107] When the cells grow to about 80% density, they can be frozen. After digesting and centrifuging the cells according to the passaging method, add 1 ml of serum-free cell freezing solution to the cells, pipette to mix, and transfer to cryopreservation tubes. Store at -80℃ and transfer to liquid nitrogen the next day for long-term storage.

[0108] 4) Co-culture of primary AM cells and airway epithelial cells

[0109] Airway epithelial cells were co-cultured with AM cells using a co-culture system. Airway epithelial cell line 16HBE cells were seeded into the lower chamber of a 0.4 μm pore size transwell culture plate, and 8×10 5 The upper chamber was inoculated with primary AM cells of each group of mice, 3×10 5The cells were divided into the following groups: control group (16HBE), airway epithelial cells combined with control macrophages (16HBE+AM), airway epithelial cells combined with Sp macrophages (16HBE+AM+Sp), airway epithelial cells combined with RSG macrophages (16HBE+AM+Sp+RSG), and airway epithelial cells combined with T007 macrophages (16HBE+AM+Sp+T007). The cells were co-cultured in an incubator for 24 hours and used for subsequent CCK-8 experiments on 16HBE cells.

[0110] 5) CCK-8 assay to detect cell proliferation activity

[0111] For co-cultured 16HBE cells: After co-culture with each group of primary AM, the 16HBE cells were digested and seeded at 8,000 cells / well in a 96-well plate. 200 μl of DMEM containing 10% FBS was added to each well and cultured for 24 hours. 100 μl of serum-free DMEM containing 10% CCK-8 solution was added to each well and cultured in an incubator for 2 hours. After the cell culture medium changed color, the absorbance of the cells in each well was measured at 450 nm using a microplate reader. Figure 12 Compared with the control group (16HBE), the activity of 16HBE cells co-cultured with AM of mice in the Streptococcus pneumoniae infection group was significantly decreased (OD450: 0.3930±0.002449 vs 0.4803±0.01573, p < 0.01). Compared with the infection group, the activity of 16HBE cells co-cultured with AM of mice in the RSG supplementation group after infection was significantly increased (OD450: 0.7248±0.05726 vs 0.3930±0.002449, p < 0.0001), indicating that AM of mice treated with RSG can promote the proliferation of airway epithelial cells.

[0112] 6) Scratch assay to detect cell migration ability

[0113] Take 16HBE cells in the logarithmic growth phase, digest them with trypsin, centrifuge at 1000 rpm / min, retain the pellet, add complete medium to resuspend, and inoculate equal amounts into six-well plates. Add 2 ml of complete medium to each well and culture in a cell culture incubator. Observe the growth every day. Perform the scratch test when the cell density reaches more than 90%. Use a 200 μl pipette tip to draw a straight line in each well of the six-well plate, wash twice with PBS, and add the supernatant of primary AM culture from each group of mice to the 6-well plate respectively. After 24 hours of treatment, observe the changes in the scratch area. Figure 13Compared with the control group, the scratch area of 16HBE cells co-cultured with AM of mice in the infection group for 24 h was significantly increased (healing rate: 0.8338±0.1042 vs 1.000±0.05602, p < 0.01), and the scratch area of 16HBE cells co-cultured with AM of mice in the RSG supplementation group was significantly reduced compared with the infection group (healing rate: 1.457±0.02805 vs 0.8338±0.1042, p < 0.0001) ( Figure 13 A and B), indicating that AM from RSG-treated mice can promote airway epithelial cell migration.

[0114] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0115] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of substances that promote the conversion of mM to AM in the preparation of drugs for treating airway epithelial cell damage.

2. The use according to claim 1, characterized in that The substance that promotes the conversion of mM to AM is an agonist that activates the PPAR-γ signaling pathway; the agonist is rosiglitazone RSG.

3. The use according to claim 1, characterized in that The airway epithelial cell damage is airway epithelial cell damage caused by infection with Streptococcus pneumoniae; The airway epithelial cells are Club cells; The airway epithelial cell damage is manifested by a decrease in the mRNA level of Scgb1a1 in lung tissue and a decrease in the fluorescence intensity of Club cells CC16.

4. The use according to any one of claims 1 to 3, characterized in that The substance that promotes the conversion of mM to AM increases the number of AM in BALF after Streptococcus pneumoniae infection; and increases the mRNA level of Scgb1a1 in lung tissue and the fluorescence intensity of Club cell CC16.

5. The use according to claim 4, characterized in that AM cells promote the proliferation and migration of airway epithelial cells; the airway epithelial cells are 16HBE cells.

6. A drug for treating airway epithelial cell damage, characterized in that: The drug includes a substance that promotes the conversion of mM to AM; the substance is an agonist that activates the PPAR-γ signaling pathway; and the agonist is rosiglitazone RSG.

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