Application of TGF-beta1 and diclapamine in preparation of medicine for inhibiting silicon lung EMT formation caused by quartz

Abstract

The invention belongs to the technical field of biological medicine, and provides application of TGF-beta1 and dichropamine DMI in preparation of a medicine for inhibiting silicon EMT formation caused by quartz. TGF-beta1 and DMI are jointly intervened to inhibit alveolar macrophage sphingomyelin metabolic pathways and Th1 type immunoreactions caused by quartz; the expression of alveolar II-type epithelial cell LTBP1 protein is inhibited, the increase of ASM activity is inhibited, and the Cer content is reduced. The method comprises the following steps: establishing a rat EMT model, and observing sphingomyelin metabolism change, T cell immune response and LTBP1 protein change; through targeted intervention of alveolar macrophage sphingomyelin metabolism and alveolar II-type epithelial cell LTBP1 protein expression, the specific mechanism of T cells participating in sphingomyelin signal pathway regulation of LTBP1 protein in EMT is discussed, and a dynamic regulation network of EMT in the silicosis pathogenesis process is comprehensively analyzed. And a theoretical basis and a potential intervention target are provided for developing a precise treatment strategy of a targeted immunometabolism and fibrosis process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of TGF-beta1 and desipramine DMI in preparation of a drug for inhibiting formation of silicosis EMT caused by quartz. BACKGROUND

[0002] Occupational pneumoconiosis is a disease caused by long-term inhalation of production mineral dust in occupational activities and retention in the lung, which is mainly characterized by diffuse fibrosis of lung tissue. Occupational pneumoconiosis has become a major public health problem in society and has brought new challenges to prevention and control work. Silicosis is one of the main types of occupational pneumoconiosis. The long incubation period, irreversible progression and high risk of complications of silicosis result in heavy social burden.

[0003] Silicosis is an occupational pulmonary fibrosis disease caused by long-term inhalation of inorganic dust containing quartz (particles with a particle size of <5 μm can reach the alveoli) in the workplace. Although the cause is clear, the pathogenesis is complex and not yet understood. The pathological changes of silicosis are divided into early inflammatory response and late lung tissue fibrosis.

[0004] In addition, more and more evidence confirms that EMT plays an important regulatory role in the development of silicosis fibrosis. Alveolar epithelial cell transformation to mesenchymal cell (Epithelial-Mesenchymal Transition, EMT) is one of the important sources of local fibroblasts in pulmonary fibrosis, and plays an important role in the occurrence and development of silicosis pulmonary fibrosis. EMT is considered as a process of complete transformation of epithelial cells into fibroblasts, which is manifested as loss of polarity of epithelial cells and morphological changes into fibroblast-like cells. This process modifies the expression of adhesion molecules on the cell surface, thereby making it have migratory and invasive behavior. During the process of EMT, the expression of epithelial cell markers such as E-cadherin and keratin (Keratin19) is down-regulated, while the expression of mesenchymal phenotype markers such as vimentin and alpha-SMA is up-regulated; in addition, the process of EMT is also accompanied by an increase in the expression of important components of extracellular matrix (extracellular matrix, ECM) type I and type III collagen. Ultimately, it causes pulmonary fibrosis and further forms nodules, silica nodules and cell masses. Therefore, it is of great significance to study the mechanism of EMT in the development of silicosis pulmonary fibrosis.

[0005] Because of the complex mechanism of EMT formation, it is not the same at different stages of the disease, and the types and expression levels of the proteins involved are also different. Proteomics is an emerging technology that mainly studies the structure and function of all proteins expressed by cells, tissues, etc. Proteomics provides a new platform for EMT research. In the previous pre-experiment, the proteomics of lung tissue in control and experimental groups of rats in EMT model was carried out, and a total of 3856 proteins were identified, and a total of 21 proteins with significant differences were obtained, of which 12 proteins were up-regulated and 9 proteins were down-regulated. Through functional analysis and gene screening, the research focused on LTBP1 protein. EMT formation is regulated by multiple cytokines, among which transforming growth factor-beta (TGF-β) plays an important role in the regulation of EMT. LTBP1 is a key regulator of TGF-β (TGFB1, TGFB2 and TGFB3), which controls TGF-β activation by maintaining it in a latent state during storage in the extracellular space; through specific binding with latent-associated peptide (LAP) by disulfide bond, LAP is a regulatory chain of TGF-β, and regulates the integrin-dependent activation of TGF-β. Studies have found that down-regulation of LTBP1 inhibits invasion and migration and metastatic ability in vitro and in vivo, not only reduces the mesenchymal phenotype, but also inhibits TGF-β-induced EMT in esophageal squamous cell carcinoma (ESCC) cells. Studies have found that up-regulation of LTBP1 in the lung is closely related to immune cell infiltration.

[0006] T cells are an important cell type in the immune system, playing a core role in lung immune response and inflammatory response, and the differentiation of T cell subtypes (Th1, Th2, etc.) will secrete different cytokines to regulate the EMT formation of alveolar epithelial cells, and the immune response enhanced by the differentiation of T cell subtypes is regulated by lung lipid metabolism. The extracellular environment in the lung is an important hub for communication between several cell types, including structural cells and immune cells. The extracellular environment of the distal lung is a complex system composed of multiple proteins, including fibrin, such as collagen, proteoglycans and glycosylated proteins. Lipids are part of the extracellular environment and play an important role in regulating the interaction between several cell types. Studies using mouse models have shown that the lipid composition found in this environment changes significantly at the onset of fibrosis. In addition to changes in lipid composition, immune cells also play an important role in the extracellular environment of the distal lung in silicosis. By using a non-targeted metabolomics method, a large number of lipid metabolites were identified in the lung tissue of control and experimental rats. Through manual screening and literature review, it was found that sphingomyelin metabolism has potential role in lung fibrosis, and sphingomyelin metabolism is closely related to quartz dust-induced lung fibrosis.

[0007] The pathological process of silicosis is closely related to the abnormal activation of sphingomyelin metabolic pathway, and sphingomyelin and its metabolites run through the early and late stages of silicosis development. SM is the main component of membrane vesicles, including early / late endosomes and lysosomes. SM is widely required for cell secretion activity; in the early stage of silicosis, after quartz particles are phagocytosed by alveolar macrophages, ASMase in lysosomes is activated to catalyze SM hydrolysis to generate Cer. Cer is an essential component of the plasma membrane, which regulates cell signaling, proliferation, differentiation, and apoptosis, and Cer can drive the release of pro-inflammatory factors by promoting mitochondrial ROS and NLRP3 inflammasome assembly. At the same time, Cer can induce alveolar type I epithelial cell apoptosis and release damage-associated molecular patterns (DAMPs), further amplifying the inflammatory response. In the late stage, sphingomyelin metabolism tilts towards S1P synthesis. S1P belongs to lysophospholipids and is a bioactive metabolite of ceramide, an important bioactive mediator, a molecule related to EMT and fibrosis progression, involved in multiple signal transduction pathways, and regulates many different cell functions. Studies have shown that S1P enhances the reprogramming process of alveolar type II epithelial cells induced by TGF-β or bleomycin, and S1P accumulates in the extracellular space, promoting the EMT of alveolar type II epithelial cells. SUMMARY

[0008] The application provides applications of TGF-β1 and Desipramine (DMI) in the preparation of drugs for inhibiting the formation of EMT of silicosis caused by quartz.

[0009] The application is implemented by the following technical solutions: applications of TGF-β1 and Desipramine (DMI) in the preparation of drugs for inhibiting the formation of EMT of silicosis caused by quartz.

[0010] Further, TGF-β1 and DMI joint intervention inhibit the sphingomyelin metabolic pathway of alveolar macrophages and Th1 type immune response caused by quartz.

[0011] Further, TGF-β1 and DMI joint intervention inhibit the expression of LTBPI protein of alveolar type II epithelial cells, inhibit the increase of ASM activity of alveolar macrophages caused by quartz, and reduce the content of Cer.

[0012] Further, TGF-β1 and DMI joint intervention promote the increase of E-Cadherin protein expression of alveolar type II epithelial cells and inhibit the expression of α-SMA protein.

[0013] The application establishes a rat EMT model through whole animal experiments, observes the changes of sphingomyelin metabolism and T cell immune response and the changes of LTBP1 protein, simultaneously establishes a cell EMT model by using rat alveolar macrophages, rat primary T cells and rat alveolar type II epithelial cells, and deeply explores the specific mechanism of T cells participating in the sphingomyelin signal pathway to regulate LTBP1 protein in EMT by targeted intervention on the sphingomyelin metabolism of alveolar macrophages and the expression of LTBP1 protein of alveolar type II epithelial cells, so as to provide key theoretical basis and potential intervention target for integrating multi-omics data to comprehensively analyze the dynamic regulation network of EMT in the pathogenesis of silicosis and developing precise treatment strategies targeting immune metabolism and fibrosis process. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a heat map of differential metabolites related to silicosis and fibrosis; Figure 2 is the body weight and lung organ coefficient changes of rats in each group; Figure 3 is the lung function index of rats in each group; Figure 4 is the HE staining of rat lung tissue (×200); Figure 5 is the Masson staining of rat lung tissue (×200); Figure 6 is the HYP content of rat lung tissue; Figure 7 is the activity of sphingomyelin and metabolites in rat bronchoalveolar lavage fluid; Figure 8 is the cytokine content in the bronchoalveolar lavage fluid of rats in each group; Figure 9 is the expression of LTBP1 protein in the lung tissue of rats in each group; in the figure: * indicates that compared with the one-month control group, P <0.05; # indicates that compared with the three-month control group, P <0.05; & indicates that compared with the six-month control group, P <0.05; Figure 10 is the expression of EMT marker E-Cadherin protein in the lung tissue of rats in each group; note: # indicates that compared with the three-month control group, P <0.05; & indicates that compared with the six-month control group, P <0.05; Figure 11 is the expression of EMT marker α-SMA protein in the lung tissue of rats in each group; note: * indicates that compared with the one-month control group, P <0.05; # indicates that compared with the three-month control group, P <0.05; & indicates that compared with the six-month control group, P <0.05; Figure 12 is the expression of cell EMT marker E-Cadherin protein; note: * indicates that compared with the control group, P <0.05; Figure 13Expression of cell EMT marker α-SMA protein; Note: * indicates compared with control group, P <0.05; Figure 14 Cell EMT alveolar macrophage supernatant sphingomyelin and metabolite activity; Note: * indicates compared with control group, P <0.05; Figure 15 Cell EMT model T cell supernatant cytokine content; Note: * indicates compared with control group, P <0.05; Figure 16 Cell EMT model alveolar type II epithelial cell LTBP1 expression; Note: * indicates compared with control group, P <0.05; Figure 17 Sphingomyelin pathway metabolite index after DMI intervention; Figure 18 T cell factor content after DMI intervention; Figure 19 LTBP1 protein expression after DMI intervention; Figure 20 EMT formation marker protein content after DMI intervention; Note: * indicates compared with control group, P <0.05; # indicates compared with Quartz group, P <0.05; & indicates compared with DMI group, P <0.05; Figure 21 EMT formation marker protein content after DMI intervention; Note: * indicates compared with control group, P <0.05; # indicates compared with Quartz group, P <0.05; ns indicates no statistical difference; Figure 22 TGF-β1 intervention alveolar type II epithelial cell LTBP1 protein expression; Note: * indicates compared with control group, P <0.05; # indicates compared with Quartz group, P <0.05; Figure 23 E-Cadherin protein expression after TGF-β1 intervention; Note: * indicates compared with control group, P <0.05; # indicates compared with Quartz group, P <0.05; & indicates compared with DMI group, P <0.05; Figure 24 α-SMA protein expression after TGF-β1 intervention; Note: * indicates compared with control group, P<0.05; # indicates DMI group compared to P <0.05; Figure 25 DMI and TGF-β1 co-intervention after E-Cadherin protein expression; Note: * indicates compared to control group, P <0.05; ns indicates DMI group compared to P >0.05; Figure 26 DMI and TGF-β1 co-intervention after E-Cadherin protein expression; Note: * indicates compared to control group, P <0.05; # indicates DMI group compared to P <0.05; Figure 27 DMI and TGF-β1 co-intervention after E-Cadherin protein expression; Note: * indicates compared to control group, P <0.05; # indicates DMI group compared to P <0.05. DETAILED DESCRIPTION

[0015] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work, fall within the protection scope of the present application.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials referred to in this disclosure are incorporated by reference.

[0017] The equivalent techniques of the described specific embodiments, which can be realized by a person skilled in the art through routine experiments, are included in the present application.

[0018] The experimental methods in the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The experimental materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.

[0019] Experimental reagents: 1% sodium pentobarbital, Tianjin Kaition; physiological saline (0.9% NaCl, sterile), Boster Bioengineering Co., Ltd., China; 75% alcohol, Hebei Ruikang Pharmaceutical Technology Co., Ltd., China; 4% paraformaldehyde solution, Beijing Regen Bioengineering Co., Ltd., China; BCA method protein concentration determination kit, ammonium persulfate (AP), ACRY, 5x loading buffer, Anti-GAPDH antibody, Anti-beta Actin antibody, ECL high-sensitivity chemiluminescence solution, horseradish peroxidase (HRP) labeled goat anti-mouse IgG, RIPA Lysis Buffer (Strong), Protease Inhibitor Cocktail (100x), Kangwei Century Biological Technology Co., Ltd., China; tetramethyl ethylene diamine (TEMED), Sigma, USA; E-Cadherin antibody, Sanying, China; alpha-Smooth Muscle Actin antibody, Bioo Sun, China; LTBP1 antibody, Abeam, USA; horseradish peroxidase (HRP) labeled goat anti-rabbit IgG, rat IL-4 ELISA kit, rat IFN-γ ELISA kit, Dr. De BOSTER Bioengineering, China; rat SM ELISA kit, rat ASM ELISA kit, rat Cer ELISA kit, rat S1P ELISA kit, Jingmei Bioengineering (Shanghai) Co., Ltd., China; tissue section HE staining, tissue section Masson staining, Wuhan Saivier Technology Co., Ltd., China; hydroxyproline (HYP) determination kit, Nanjing Jiancheng Bioengineering Institute, China; DMEM high-sugar culture medium, RPMI-1640, Beijing Haikelon Biochemical Co.; fetal bovine serum, Zhejiang Sijiqing Co.; penicillin mixture, EDTA-trypsin digestion solution, D-Hank's solution, Beijing Solabio Co.; CCK-8 kit, Shanghai Dongren Chemical Technology Co.; PVDF membrane, Millipore, USA; protein marker, Thermo Fisher Scientific (China); EasySep™ Rat CD4 + T Cell Isolation Kit, RoboSep™ Buffer, STEMCELL, Canada; DMI, MCE, USA.

[0020] Preparation: Quartz suspension preparation: First, the quartz dust is thoroughly ground, then dissolved with sterile normal saline, the final mass concentration is adjusted to 50 g / L, and sterilized by high pressure. According to the instructions, prepare 4x electrophoresis solution, 10x transfer solution, 10x TBS. Prepare 1x electrophoresis solution, 1x transfer solution, 1x TBST by equal dilution. Prepare blocking solution: weigh an appropriate amount of skimmed milk powder, dissolve with 1x TBST to prepare 5% blocking solution, mix thoroughly, and use immediately. Protein extraction solution: mix RIPA lysis buffer, phosphatase inhibitor and protease inhibitor at a ratio of 98:1:1 to prepare the protein extraction solution.

[0021] I. Experimental method 1. Animal experiment: healthy male SD rats were selected as experimental animals, which were purchased from Beijing Huafukang Biotechnology Co., Ltd. (License No. SCXK (Jing) 2019-0008). The experimental animals were raised in a special animal house, the indoor temperature was maintained at about 25℃, the relative humidity was controlled at 40% to 60%, and the light and dark alternation mode of 12 hours was used. Before the formal experiment, the rats were adaptively fed for 1 week. The experimental animal use plan has been reviewed and approved by the Shanxi Medical University Experimental Animal Management Committee (Approval No. SYDL2023023).

[0022] 2. Experimental animal exposure grouping and treatment: 48 healthy male SD rats (6W age, body weight 180-200g) were randomly divided into control group and quartz group according to the random number table method, 1, 3, 6 months of exposure, 8 rats in each group. After adaptive feeding for 1 week, the rats in each group were treated by non-exposed tracheal perfusion. The specific operation is one-time intratracheal perfusion, and the perfusion volume of each rat is 1mL. Among them, the rats in the quartz group were perfused with quartz suspension with a mass concentration of 50g / L, while the rats in the control group were perfused with the same volume (1mL) of 0.9% sodium chloride solution. At the 1st month, 3rd month and 6th month after perfusion, the laboratory indicators of rats in each group were detected.

[0023] The non-exposed tracheal perfusion method is used to treat the rats. The specific operation steps are as follows: first, put the gauze soaked with ether in a closed glass jar with a lid, put the rat into the jar, and make it inhale ether for anesthesia, which lasts about 1 minute. When it is observed that the skeletal muscle of the rat is completely relaxed, it is confirmed that it has reached a completely anesthetized state. Then, fix the limbs of the rat with a needle, adjust its body position, and make the head of the rat face the operator, and keep the body in a supine position. Then, gently pull out the rat's tongue with sterile gauze and place it on the left side of the oral cavity. At the same time, try to insert the small end of the small horn-shaped catheter (small horn) into the bottom of the oral cavity of the rat. With the right hand, accurately insert the tracheal tube into the trachea and connect the syringe. Quickly inject the liquid (1 mL of dust suspension or normal saline solution). Before injecting the liquid, the syringe needs to be withdrawn. If it can be easily withdrawn and bubbles are observed, it can be determined that the tracheal tube has been correctly inserted into the trachea, and then the perfusion operation is continued. After the liquid injection is completed, the tracheal tube and the small horn are carefully pulled out, the rat is kept in an upright position, and the rat is rotated left and right alternately while massaging the lungs on both sides. Repeat this operation 10 times to ensure that the perfusion liquid can completely enter the lungs and be evenly distributed.

[0024] 3. Lung function detection: At the first month, the third month and the sixth month after quartz exposure, the rats were intraperitoneally injected with 1% sodium pentobarbital for anesthesia. Then, the skin of the rat's neck was disinfected with 75.0% ethanol. Incision operation was performed along the median line of the neck, and the trachea was exposed by blunt separation technique. A small opening was cut on the trachea, a tracheal tube was inserted, and it was connected to a small animal lung function instrument for detecting lung function related indexes. The indexes detected include forced vital capacity (FVC), 0.2 second forced expiratory volume (FEV 0.2 ) and maximum minute ventilation (MVV).

[0025] 4. Sample collection: After completing the lung function detection of the rat, the limbs of the rat were fixed, and the trachea was fully exposed. A 5 mL syringe was used to extract normal saline, inserted into the trachea and directly injected to perform whole lung lavage. By massaging the lungs with both hands, it is ensured that the lavage fluid is in full contact with the lung tissue. After 1 minute, the lavage fluid is withdrawn using a syringe. After repeating the suction process 3 times, the bronchoalveolar lavage fluid (BALF) is collected. Then, the collected BALF is placed in a centrifuge and centrifuged at 4℃, 1500 rpm (centrifugal radius 10 cm) for 15 min. The supernatant is taken for standby. 7 rats in each group are selected for the above lavage treatment. After the treatment is completed, the rat's chest is cut open with scissors, the lung tissue is separated, washed with normal saline, dried with filter paper, and stored at -80℃ for standby. In addition, 1 rat in each group is not subjected to lavage treatment, and the whole lung tissue is directly immersed in a 4% paraformaldehyde solution for subsequent pathological detection.

[0026] 5. Lung tissue pathological staining: (1) Hematoxylin-eosin staining (HE staining): Hematoxylin dye is alkaline, which can make the cell nucleus and ribosome in the cell show blue-violet color; and eosin is an acid dye, which is mainly used to dye the cytoplasm and extracellular matrix into red.

[0027] Specific steps: Take out the fresh lung tissue soaked in paraformaldehyde in advance, and perform steps such as wax immersion, embedding, sectioning, and drying. The lung tissue paraffin section is subjected to xylene dewaxing treatment, repeated 2 times, each for 15 minutes; the section is treated with ethanol gradient of 100%, 95%, 90%, 85%, 75%, and 50% in turn, each for 5 minutes; the section is dyed with hematoxylin staining reagent for 5 minutes. Then, the residual hematoxylin solution is removed by flushing with running water for 30 seconds, followed by 1% hydrochloric acid alcohol treatment for 30 seconds, and then 75% alcohol solution for color separation treatment for 1 minute, and finally distilled water washing for 10 minutes; the section is dyed with 0.5% eosin solution for 2 minutes, and then treated with 95% ethanol for color removal for 30 seconds, and then treated with 100% ethanol for color removal for 2 times, each for 5 minutes; the section is treated with xylene solution for transparency treatment, repeated 3 times, each for 2 minutes; neutral gum is added on the paraffin section, and a cover glass is covered for fixation. Finally, the section is observed and photographed under an Olympus optical microscope.

[0028] (2) Masson staining: Masson staining is improved on the basis of Mallory tri-color staining, which is completed by the mixing or sequential action of two or three anionic dyes, and the staining effect is closely related to the molecular size of the anionic dye and the permeability of the tissue. Specific steps: The paraffin section is subjected to conventional dewaxing treatment until it is dewaxed to water phase; drop the Masson composite staining solution on the section, and dye for 5 minutes, and then wash with distilled water to remove excess dye; drop the phosphomolybdic acid solution, and dye for 5 minutes, and then dry the section (note that do not wash); drop the aniline blue staining solution, and dye for 5 minutes, and then wash gently with distilled water; drop the differentiation solution for differentiation treatment, repeated 2 times, each for 30 to 60 seconds; perform alcohol dehydration treatment according to the conventional method, and finally fix the glass with neutral gum. Observe and photograph under an Olympus optical microscope.

[0029] 6. Lung tissue hydroxyproline (HYP) content detection: The hydroxyproline determination kit is purchased from Nanjing Jiancheng Biological Company, and the sample processing and operation method is carried out according to the requirements of the hydroxyproline determination kit. The analysis is carried out according to the detection steps described in Table 1.

[0030] Table 1: Operation flow table 7. ELISA detection: SM content detection in bronchoalveolar lavage fluid: sample equilibration: take the bronchoalveolar lavage fluid sample from the-80℃ refrigerator and place it in a 36℃ water bath for equilibration for 60 minutes; add 50μL of standard solution of different concentrations to the standard well; in the sample well, first add 40μL of sample diluent, then add 10μL of the sample to be tested; add the sample to the bottom of the enzyme-labeled plate well, taking care not to touch the wall of the well, and gently shake the enzyme-labeled plate to mix the sample; add 100μL of enzyme-labeled reagent to each well (except for the blank well), seal the enzyme-labeled plate with a sealing film, and incubate it in a 37℃ shaking incubator for 1 hour; dilute the 20-fold concentrated washing solution with distilled water 20 times for standby use; after incubation, discard the liquid in the well and shake dry, add enough washing solution to each well, stand for 30 seconds, then discard, repeat this operation 5 times, and finally use a water-absorbing paper to dry the enzyme-labeled plate; add the reagent to each well according to the ELISA kit instructions, gently shake the enzyme-labeled plate to mix, then place the enzyme-labeled plate in an aluminum lunch box to avoid light, and place it in a 37℃ incubator for color development for 15 minutes; after color development, add 50μL of stop solution to each well to stop the reaction; use an enzyme-labeled instrument to detect the absorbance (OD value) of each well at 450nm wavelength, and calculate the sample concentration according to the standard curve.

[0031] The same operation procedure as SM content detection is used to determine ASM content; the detection methods of Cer content, S1P content, IFN-γ content, and IL-4 content are consistent with SM content detection.

[0032] 8. Animal protein extraction and quantification: mix phosphatase inhibitor, protease inhibitor, and RAPI lysis buffer in a ratio of 1:1:98 to prepare protein extraction solution, mix thoroughly, and store on ice for a few minutes for standby use. Take the frozen lung tissue from the-80℃ refrigerator, and cut about 50mg of rat lung tissue. According to the ratio of 1:5 of tissue to extraction solution, add the lung tissue to the pre-cooled protein extraction solution. Use an ultrasonic homogenizer for manual homogenization, operate up and down 2 times, each for 7 seconds. Pre-cool for 1-2 minutes on ice before each crushing, and strictly maintain low temperature throughout the operation process. Then incubate the homogenized tissue on ice for 30 minutes to ensure complete lysis of the tissue. Then centrifuge at 14000rpm, 4℃ for 10min. After centrifugation, transfer the supernatant to a new pre-cooled centrifuge tube to obtain the total protein extract. According to the BCA protein quantification kit, prepare BSA standard and BCA working solution for protein detection, and use an enzyme-labeled instrument to detect the absorbance at 595nm wavelength. First, plot the standard curve according to the absorbance value and known concentration of the standard. Then, calculate the protein concentration according to the OD value of the protein to be tested and the standard curve.

[0033] According to the calculation results, add sample buffer and lysis buffer to each protein sample to be tested to adjust the protein concentration to the minimum level. The final protein concentration should be adjusted to 0.8 times the minimum protein concentration. Mix the sample well by vortexing, then place it in a boiling water bath for 5 minutes. After the treatment is completed, cool the sample to room temperature and store it in a -20°C refrigerator.

[0034] 9. Western Blot detection: Gel preparation: According to the molecular weight range of the target protein, prepare the separation gel and stacking gel. Carefully install the prepared gel into the electrophoresis tank. Make sure that the inner tank is completely filled with electrophoresis liquid, while the outer tank is filled with 2-3 cm high electrophoresis liquid to ensure uniform conduction of the electric current during electrophoresis. Take the protein sample from the storage condition, allow it to melt naturally at room temperature, and mix it evenly by vortexing. Add 1 μL and 1.5 μL of protein maker as molecular weight markers to the first and last sample wells of the electrophoresis gel, respectively, and add an equal amount of protein sample to the middle sample wells in turn. Connect the electrophoresis tank to the power supply and set the voltage to 60 V for 2 hours to ensure that the protein sample is fully separated in the gel. Membrane transfer: Cut the PVDF membrane. Before membrane transfer, soak the PVDF membrane in methanol for 1 to 2 minutes in advance. At the same time, soak the sponge pad and filter paper with recovered membrane transfer solution. Then, place the sponge pad, filter paper, gel, activated PVDF membrane, filter paper, and sponge pad in turn on the black plate of the membrane transfer clamp, and clamp the membrane transfer clamp. Pour the membrane transfer solution into the membrane transfer tank, half of which is recovered liquid and the other half is fresh membrane transfer solution, and adjust the liquid level to just overflow. Set the current to 300 mA and the membrane transfer time to 50 minutes. Blocking: Prepare a 5% blocking solution using skimmed milk or bovine serum albumin (BSA). After completing the membrane transfer operation, quickly remove the PVDF membrane and place it in the blocking solution, then use a shaker to perform blocking treatment at room temperature for 3 hours. Incubate the primary antibody: Dilute the primary antibody according to the ratio in Table 2 using 1 × TBST solution. Place the diluted antibody solution in a 4°C refrigerator on a shaker for overnight incubation. Wash the membrane: Wash the strip with 1 × TBST solution for 10 minutes each time, repeating 3 times. Incubate the secondary antibody: Dilute the secondary antibody with 1 × TBST according to the ratio in Table 3, then place it in a 37°C incubator for 1 hour of incubation. Wash the membrane: Wash the strip with 1 × TBST solution for 10 minutes each time, repeating 3 times. Develop: Mix the luminescent solution A and B in a ratio of 1:1 to prepare the luminescent solution. Immerse the strip in the prepared luminescent solution and allow it to soak for 1 to 2 minutes. Then remove the strip and use a developing instrument to develop it.

[0035] Table 2: Primary antibody dilution ratio Table 3: Secondary Antibody Dilution Ratio 10. Cell Experiments: NR8383 cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.; RLE-6TN cells, also known as rat type II alveolar epithelial cells, were purchased from Wuhan Shangen Biotechnology Co., Ltd.; primary rat CD4T cells were also used. + Cells were taken from the spleen of normal rats.

[0036] Grouping and processing of cells in experiments: (1) NR8383 cell culture: Cell revival: Take the frozen cells out of the -80℃ freezer and quickly place them in a 37℃ water bath until they are completely thawed. Transfer them to 5mL of 1640 medium that has been prepared in advance, centrifuge at 1000r / min for 5min, discard the old culture medium, add 1mL of new complete culture medium, gently mix the cell suspension with a pipette, and finally transfer it to a culture flask containing 5mL of 1640 medium. Place it in a 37℃, 5% CO2 incubator and observe the cell state to see if it can be passaged.

[0037] Cell passage: When cell ligation reaches 80%-90%, wash the culture medium with D-Hanks solution until the wash solution is clear. Add 1 mL of trypsin containing EDTA until white cell membranes are visible to the naked eye. Immediately add 3 mL of complete culture medium to stop digestion. Aspirate the cell suspension and centrifuge at 1000 r / min for 5 min. Discard the old culture medium and add 3 mL of fresh culture medium. Gently mix the cell suspension with a pipette and dispense into three culture flasks. Incubate in a 37℃, 5% CO2 incubator and observe the cell status to determine if the cells are ready for treatment with a toxin or intervention.

[0038] Cell cryopreservation: After 5-6 passages, cells need to be cryopreserved to maintain cell stability. Digest well-grown logarithmic phase cells with trypsin, collect them in EP tubes, centrifuge at 1000 r / min for 5 min, discard the supernatant, add 1 mL of cryopreservation solution, transfer to cryopreservation tubes, seal with sealing film, and freeze at -80℃.

[0039] Original CD4 + T cell culture: Adult SD rats were fasted and deprived of water for 12 hours prior to death. The rats were then sacrificed, and the spleens were harvested. The spleens were minced using forceps and scissors and passed through a pre-wetted 70μm mesh nylon sieve to remove aggregates and fragments. The cells were centrifuged at 120×g for 10 minutes. If aggregates remained, the cell suspension was passed through the nylon sieve again and centrifuged for another 10 minutes. The cell suspension was maintained at 2-8℃ until separation began. 0.5-1 ml of 5×10⁶ cells was collected. 7Cells / ml, add 50 μl / ml Isolation Cocktail in round bottom tube, mix at room temperature for 10 minutes (without bubbles), vortex for 30 seconds, add 25 μl / ml RapidSpheres™ without incubation, directly add the sample into the culture medium, pipette up and down gently for 2-3 times, supplement to 2.5 ml, put into the magnet for 3 minutes at room temperature, invert the magnet and the tube for 2-3 seconds, then return to vertical position without shaking, until the last drop drops, aspirate the supernatant into a new tube, and then put it into the magnet for 5 minutes, collect the supernatant, which is the CD4+ T cell suspension.

[0040] RLE-6TN cell culture: cell recovery: take the frozen cells from the -80°C refrigerator, quickly place them in a 37°C water bath until they are completely melted, transfer them to 5 mL of complete medium in advance, centrifuge at 1000 r / min for 5 min, discard the old culture medium, add 1 mL of new complete culture medium, gently blow the cell suspension with a gun, and finally transfer it to 7 mL of complete culture medium, and incubate in a 37°C, 5% CO2 incubator. Observe the cell state to determine whether it can be passaged.

[0041] Cell passage: when the cell connection reaches 80%-90%, wash the medium with D-Hanks solution until the wash is transparent, add 1 mL of trypsin containing EDTA, and immediately add 2 mL of complete medium to terminate digestion when the white cell membrane is visible to the naked eye. Centrifuge the cell suspension at 1000 r / min for 5 min, discard the old culture medium, add 7 mL of new culture medium, gently blow the cell suspension with a gun, and divide it into three culture dishes, and incubate in a 37°C, 5% CO2 incubator. Observe the cell state to determine whether it can be treated or intervened.

[0042] Cell freezing: after 5-6 passages, freezing is required to maintain cell stability. Well-grown logarithmic phase cells are trypsinized and collected in an appropriate EP tube. After centrifugation at 1000 r / min for 5 min, discard the supernatant, add 1 mL of freezing medium to the freezing tube, seal with a sealing film, and freeze at -80°C.

[0043] Cell infection and intervention: Establishment of cell EMT model: NR8383 was treated with 100 μg / ml quartz solution for 48 h, and the supernatant of NR8383 was collected to stimulate primary T cells for 48 h. The supernatant of T cells was collected to stimulate RLE-6TN for 72 h to establish the cell EMT model.

[0044] Intervention with DMI: NR8383 was intervened with 5 mM DMI for 1 h before quartz stimulation, and then the above operation was performed. Experimental groups: control group, quartz group, DMI group, DMI + quartz group, DMSO group.

[0045] Intervention with TGF-β1: NR8383 was stimulated with quartz for 48 h, and then the supernatant of NR8383 was used to stimulate T cells for 48 h. The supernatant of T cells was collected, and RLE-6TN cells were intervened with 5 ng / mL TGF-β1 for 1 h. Then the supernatant of T cells was used to stimulate RLE-6TN for 72 h. Experimental groups: control group, quartz group, TGF-β1 group, TGF-β1 + quartz group, DMSO group.

[0046] Intervention with DMI and TGF-β1: NR8383 was intervened with 5 mM DMI for 1 h before quartz stimulation, and then the supernatant was collected after NR8383 was stimulated with quartz for 48 h. The supernatant was used to stimulate T cells for 48 h, and then the supernatant of T cells was collected. RLE-6TN cells were intervened with 5 ng / mL TGF-β1 for 1 h, and then the supernatant of T cells was used to stimulate RLE-6TN for 72 h. Experimental groups: control group, quartz group, DMI + TGF-β1 group, DMI + TGF-β1 + quartz group, DMSO group.

[0047] Specific operation: (1) The quartz suspension was mixed with 1640 medium to prepare a 100 ug / ml toxicant solution. DMI and TGF-β1 were dissolved in DMSO. When NR8383 cells grew to 50%-60%, the cells were treated with the toxicant, and then cultured in a 37°C, 5% CO2 incubator for 48 h. After that, the cell supernatant was collected for use. Before adding the toxicant, DMI was added for intervention for 1 h, and then the toxicant was added. (2) The collected NR8383 cell supernatant was mixed with 1640 medium at a ratio of 1:1 and added to the extracted T cells after 24 h. The cells were cultured in a 37°C, 5% CO2 incubator for 48 h, and then the cell supernatant was collected for use. (3) The collected T cell supernatant was mixed with complete medium at a ratio of 1:1 and added to RLE-6TN cells with a growth rate of 50%-60%. The cells were cultured in a 37°C, 5% CO2 incubator for 72 h, and then the cells and cell supernatant were collected for the next experiment. Before intervention, LTBP1 was added for intervention for 1 h before the toxicant was added, and then the toxicant was added. Finally, the cells and supernatant were collected for the next detection.

[0048] Cell supernatant ELISA detection was the same as the previous method. Cell protein extraction and quantification: after the cell treatment or intervention was completed, the cells were collected, 80-100 µL cell protein extraction agent was added to each tube, and the cell protein was extracted. The rest was the same as the previous method. Western Blot detection was the same as the previous method.

[0049] 11、Statistical analysis: SPSS 26.0 software was used for data processing. For measurement data conforming to normal distribution, mean ± standard deviation was used. When comparing multiple groups, one-way ANOVA was used for overall test; pairwise comparison: LSD test was used for equal variance, and Dunnett test was used for unequal variance. The influence of animal experiment design group treatment and treatment time on related indicators was analyzed by two-factor (group treatment factor (2 levels), observation time factor (3 levels)) 2x3 factorial design ANOVA. The influence of quartz treatment and DMI intervention, TGF-β1 intervention on experimental indicators was analyzed by two-factor 2x2 factorial analysis, with test level α=0.05 (two-sided).

[0050] II. Experimental results 1. Animal experiment (1) Screening of silicosis differential metabolites: The differential proteins and metabolites of the control group and the quartz group were screened, and the screening standard was P <0.05, and the proteins and metabolites related to silicosis fibrosis were selected. LTBP1 was the main differential protein, as shown in Table 4. Due to too many differential metabolites, the heat map showed the top 16 differential metabolites with adjusted P value, as shown in Figure 1 Sphingomyelin ranked first among the differential metabolites.

[0051] Table 4: Analysis of significantly different protein expression (2) General condition of each group: The control group rats were agile and active, with smooth and full-colored fur, normal mood, diet, water intake, defecation and urination, and obvious body weight gain. The general physiological condition was good. Some rats in the quartz group showed irritability, jumping, dry and sparse fur, lack of luster, irritability, and slight weight loss.

[0052] (3) Changes in body weight and lung organ coefficient of each group: The body weight and lung organ coefficient of rats in each group showed significant differences in the main effects and interaction effects of group treatment and time (body weight: F value was 10.191, 0.219, 5.128, organ coefficient: F value was 6.587, 7.747, 1.883) P <0.05; organ coefficient: F value was 6.587, 7.747, 1.883, P <0.05). From Figure 2As shown, quartz led to an increase in body weight and lung organ coefficient in rats. With increasing time, both body weight and lung organ coefficient increased, indicating a negative interaction between the two. This resulted in increased lung wet weight, a slight initial increase followed by a decrease in body weight, and an initial decrease followed by an increase in lung organ coefficient in the quartz group over time. Compared to the 1-month control group, there was no difference in lung weight, body weight, or lung organ coefficient in the quartz group. P >0.05); compared with the 3-month control group, the lung organ coefficient of the quartz group rats decreased ( P <0.05); compared with the 6-month control group, the quartz group rats had decreased body weight and increased lung organ coefficient ( P <0.05).

[0053] (4) Changes in lung function in each group: FVC and FEV in rats in each group 0.2 The differences in main effect and interaction effect of MVV were statistically significant (FVC: F values ​​were 45.264, 25.146, and 8.883, respectively). P All values ​​are <0.001; FEV 0.2 The F-values ​​were 21.432, 36.399, and 4.752, respectively. P All values ​​were <0.05, indicating that lung function impairment worsens with prolonged dust exposure.

[0054] Depend on Figure 3 As shown, quartz caused rats to initially increase and then decrease FVC and FEV0.2 levels, while MVV showed a gradually decreasing trend. FVC results showed no statistically significant difference among the groups after 1 month. P >0.05); compared with the 3-month and 6-month control groups, the quartz group showed reductions of 63% and 64%, respectively ( P <0.05). FEV0.2 results showed that, compared with the 1-, 3-, and 6-month control groups, the quartz group showed reductions of 26%, 33%, and 64%, respectively (…). P <0.05). MVV results showed no statistically significant difference between the 1-month control group and the quartz group ( P >0.05); compared with the 3-month and 6-month control groups, the quartz group showed reductions of 40% and 73.82%, respectively ( P <0.05). This suggests that dust exposure significantly reduces vital capacity, deteriorates airway condition and lung reserve function in rats. Early lung function impairment is mainly characterized by restrictive ventilatory dysfunction, while mixed ventilatory dysfunction appears in the fibrotic stage.

[0055] (5) Pathological morphological staining of lung tissue in each group: HE staining results are as follows Figure 4As shown, the results indicated that the lung tissue structure of the control group rats was clear, the alveolar and capillary walls were thin, and there was no inflammatory cell infiltration in the alveolar cavity. With the increase of dust exposure time, the quartz group showed a large number of inflammatory cells and erythrocyte infiltration, and the alveolar septa widened. After 1 month, the alveolar cavity of the quartz group was significantly enlarged and inflammatory cell foci appeared. After 3 and 6 months, the alveolar cavity disappeared or merged, normal alveolar structure was rare, and small clump-like lung nodules appeared.

[0056] Masson staining results are as follows: Figure 5 As shown, the results indicated that the lung tissue of the control group had a small amount of normal blue-stained areas without fibrous tissue proliferation. With increasing exposure time, the amount of blue collagen fibers in the quartz group gradually increased. This suggests that quartz induces early inflammatory responses in the lungs of rats, with fibrotic lesions appearing at 3 and 6 months.

[0057] (6) Hydroxyproline (HYP) content in lung tissue of each group: The degree of pulmonary fibrosis in the lung tissue of rats was reflected by measuring the HYP content. The results showed that the HYP content of each group of rats differed significantly in terms of the main effect and interaction effect of treatment and treatment time (F values ​​were 8.090, 35.301, and 40.874, respectively). P The value was <0.05. This indicates that the degree of fibrosis in the lung tissue of the quartz group worsened with prolonged exposure to dust.

[0058] Depend on Figure 6 As shown, dust exposure led to an increase in HYP content in lung tissue, and there was a positive interaction between dust exposure and time. The HYP content in the quartz group increased with time; compared to the 3- and 6-month control groups, the HYP content in the quartz group increased by 47% and 59%, respectively. P <0.05). This suggests that the collagen fiber content in rat lung tissue gradually increases with increasing dust exposure time.

[0059] (7) Activity of sphingomyelin and metabolites in bronchoalveolar lavage fluid of each group: There were statistically significant differences in the main effect and interaction effect of SM and S1P in each group of rats. P <0.05), and it was a positive interaction. The difference in treatment between groups for Cer was statistically significant ( P <0.05), indicating that the activities of SM and S1P increase with increasing dust contamination time. Figure 7 As shown, the contents of SM, Cer, and S1P generally increased with increasing dust accumulation time. The SM results showed no statistically significant difference in the quartz group compared to the 1-month control group. P >0.05); compared with the 3-month and 6-month control groups, the quartz group increased by 10% and 13%, respectively ( P <0.05). The Cer results showed no statistically significant difference in the quartz group compared to the 1-month control group ( P>0.05); compared with the 3-month and 6-month control groups, the quartz group increased by 5% and 4%, respectively ( P <0.05). S1P results showed no statistically significant difference in the quartz group compared with the 1-month control group ( P >0.05); compared with the 3-month and 6-month control groups, the quartz group increased by 13% and 30%, respectively ( P <0.05). The differences in ASM levels among the rat groups were statistically significant ( ). P <0.05), compared with the control group, the quartz content increased by 15% ( P <0.05).

[0060] (8) Cytokine content in bronchoalveolar lavage fluid of each group: Figure 8 As shown, the main effects and interaction responses of IFN-γ and IL-4 in rats from each group were statistically significant in terms of treatment and time. P <0.05). IFN-γ results showed that in January, March, and June, the quartz content in the group increased by 13%, 12%, and 14% compared to the control group, respectively. P <0.05); IL-4 results showed that compared with the 1-month and 6-month control groups, the quartz group had increased levels by 38% and 19%, respectively ( P <0.05).

[0061] (9) LTBP1 protein expression in lung tissue of each group: Western blot was used to detect LTBP1 protein expression in rat lung tissue at each time point to verify the protein sequencing results. Figure 9 As shown, the results indicated that the expression of LTBP1 protein in the lung tissue of rats in each group showed statistically significant differences in both the main effect and the interaction effect between treatments and time treatments (F values ​​were 47.01, 53.54, and 20.97, respectively). P All values ​​were <0.05. With increasing exposure time, the expression of LTBP1 protein in rat lung tissue showed a trend of first increasing and then decreasing. Compared with the control groups at 1, 3, and 6 months, the content in the quartz group increased by 20%, 80%, and 65%, respectively. P <0.05). The above results suggest that LTBP1 protein is activated in the early stages of silicosis, which may activate subsequent fibrotic responses.

[0062] (10) Expression of EMT marker proteins in lung tissue of each group: Western blot was used to detect the protein expression of EMT in rat lung tissue at each time point. The differences in E-cadherin content in lung tissue of each group of rats were statistically significant in terms of both main effect and interaction effect (F values ​​were 103.3, 396.11, 128.3, respectively). P (All values ​​< 0.05). Figure 10As shown in Fig. 4, compared with the control group in January, the E-cadherin content in the quartz group did not change significantly, and compared with the control groups in March and June, the E-cadherin protein expression in the quartz group decreased by 20% and 35% respectively (P<0.05). P The α-SMA protein expression in the lung tissue of rats in each group had statistical significance in the main effect and interaction effect of treatment and time (F values were 110.9, 288.1 and 153.1 respectively, P<0.05). P As shown in Fig. 5, compared with the control groups in January, March and June, the α-SMA expression in the quartz group increased by 35%, 50% and 60% respectively (P<0.05). Figure 11 The results of protein expression of EMT in the lung tissue of rats, HE staining and HYP content in the lung tissue suggested that the epithelial mesenchymal transition process occurred in the lung tissue of rats at 3 months, and entered the fibrosis stage of silicosis.

[0063] 2. Cell experiment (1) Expression of marker proteins of EMT model: The cell EMT model was established by using rat NR8383, rat primary T cells and rat RLE-6TN alveolar type II epithelial cells. Figure 12 , 13 As shown in Fig. 6, compared with the control group, the expression of E-Cadherin in the quartz group decreased by 40%, and the expression of α-SMA increased by 14% (P<0.05). P The results suggested that the cell EMT modeling was successful.

[0064] (2) Sphingomyelin and metabolite activity in the supernatant of alveolar macrophages in EMT model: The sphingomyelin and metabolite activity in the supernatant of alveolar macrophages was detected. Figure 14 As shown in Fig. 7, compared with the control group, the activities of SM, ASM and Cer in the supernatant of alveolar macrophages in the quartz group increased by 23%, 17% and 20% respectively (P<0.05). P The activity of S1P had no statistical significance (P>0.05). P The results suggested that the activities of SM, ASM and Cer in alveolar macrophages increased in the EMT process.

[0065] (3) Cytokine content in the supernatant of T cells in EMT model: The detection results of T cell supernatant are shown in Fig. 8. Figure 15 As shown in Fig. 8, compared with the control group, the content of IFN-γ in the quartz group increased by 62%, and the content of IL-4 decreased by 31% (P<0.05). P It is suggested that T cells participate in the formation of EMT, and quartz activates the immune response of T cells, mainly in Th1 type.

[0066] (4) LTBP1 expression in type II alveolar epithelial cells of the EMT model: The results of LTBP1 protein expression in type II alveolar epithelial cells are as follows: Figure 16 As shown, compared with the control group, the expression of LTBP1 in the quartz group increased by 18% ( P <0.05). This suggests that LTBP1 protein expression is increased in type II alveolar epithelial cells during EMT.

[0067] (5) Sphingomyelin and metabolite activity after DMI intervention: There were statistically significant differences in the main effect and interaction effect of SM and ASM in the alveolar macrophage supernatant of each group (F values ​​14.608, 49.890, 3.646, respectively). P (All values ​​< 0.05). Figure 17 As shown, compared with the control group, the SM content in the quartz group increased by 14% and the ASM content increased by 17%. Compared with the quartz group, the SM content in the DMI+quartz group increased by 30% and the ASM content decreased by 14% (P<0.05). The differences in the main effect and interaction effect of Cer content in the alveolar macrophage supernatant among the groups were statistically significant. P <0.05), and there was a negative interaction; compared with the control group, the Cere activity in the quartz group increased by 18%, and compared with the quartz group, the Cere content in the DMI+quartz group decreased by 29% ( P <0.05). There were no statistically significant differences in the main effect and interaction effect of S1P activity in the supernatant of alveolar macrophages among the groups. P >0.05). The results suggest that DMI can inhibit the increase in ASM activity and Cer content induced by quartz.

[0068] (6) T cell cytokine levels after DMI intervention: CD4 levels in each group + The main effects and interaction effects of IFN-γ and IL-4 in T cell supernatant were statistically significant. P <0.05) and there is a negative interaction. (By...) Figure 18 As shown, compared with the control group, the quartz group showed a 65% increase in IFN-γ and a 22% decrease in IL-4; compared with the quartz group, the DMI+quartz group showed a 12.5% ​​decrease in IFN-γ and a 17% increase in IL-4. P <0.05). This suggests that DMI can suppress quartz-induced T-cell immune responses, primarily by suppressing Th1-type immune responses.

[0069] (7) LTBP1 protein expression after DMI intervention: The expression of LTBP1 in alveolar type II epithelial cells of each group showed statistically significant differences in both the main effect and the interaction effect (F values ​​were 49.7, 2.7, and 32.336, respectively). PThe values ​​are <0.05, 0.137, and <0.05), and there is a negative interaction. (By...) Figure 19 As shown, compared with the control group, the expression of LTBP1 in the quartz group increased by 17% ( P <0.05), compared with the quartz group, the expression of LTBP1 in the DMI+quartz group was reduced by 25% ( P <0.05). The results suggest that DMI inhibited the expression of LTBP1 protein in quartz-induced alveolar type II epithelial cells.

[0070] (8) Expression of EMT marker proteins after DMI intervention: The expression of E-cadherin and α-SMA, marker proteins for EMT formation in alveolar type II epithelial cells, showed statistically significant differences in both the main effect and the interaction effect among the groups (F values ​​were 61.3, 32.5, and 56.9, respectively). P The values ​​are all <0.05), and there is a negative interaction. (From...) Figure 20 , 21 As shown, compared with the control group, E-Cadherin expression decreased by 25% and α-SMA expression increased by 11% in the quartz group. Compared with the quartz group, E-Cadherin expression increased by 42% and α-SMA expression decreased by 22% in the DMI+quartz group. Compared with the control group, E-Cadherin expression increased by 43% and α-SMA expression decreased by 15% in the DMI+quartz group. The results suggest that DMI can inhibit quartz-induced EMT formation.

[0071] (9) LTBP1 protein expression after TGF-β1 intervention: The relative expression of LTBP1 protein in type II alveolar epithelial cells of each group showed statistically significant differences in both the main effect and the interaction effect, and a negative interaction existed (F values ​​were 204.5, 425.1, and 268.1, respectively). P (All values ​​< 0.05). Figure 22 As shown, compared with the control group, the expression of LTBP1 protein in the quartz group increased by 35% ( P <0.05). Compared with the quartz group, LTBP1 protein expression was reduced by 53% in the TGF-β1+ quartz group ( P <0.05). The results suggest that TGF-β1 significantly inhibits the expression of LTBP1 protein induced by quartz.

[0072] (10) Expression of EMT marker proteins after TGF-β1 intervention: The expression of E-Cadherin and α-SMA proteins in type II alveolar epithelial cells of each group showed statistically significant differences in both the main effect and the interaction effect, and there was a negative interaction (F values ​​were 49.4, 2.4, and 99.7, respectively). P (Value < 0.05). Figure 23 , Figure 24As shown in Figs. 6 and 7, compared with the control group, the expression of E-Cadherin protein in the quartz group decreased by 24%, and the expression of a-SMA protein increased by 24% (P<0.05); compared with the quartz group, the expression of E-Cadherin protein in the TGF-β1+quartz group increased by 128%, and the expression of a-SMA protein decreased by 40% (P<0.05). The results suggest that TGF-β1 can inhibit the EMT formation caused by quartz. P

[0073] (11) LTBP1 protein expression after DMI and TGF-β1 co-intervention: The expression of LTBP1 protein in alveolar type II epithelial cells in each group had statistical significance in main effect and interaction effect, and there was a negative interaction (F value was 6.8, 12.6, 89.8, P all P<0.05). There was a negative interaction. As shown in Figs. 8 and 9, compared with the control group, the expression of LTBP1 protein in the quartz group increased by 25% (P<0.05), and compared with the quartz group, the expression of LTBP1 protein in the TGF-β1+DMI+quartz group decreased by 40% (P<0.05). The results suggest that TGF-β1 and DMI combined inhibit the expression of LTBP1 protein caused by quartz. Figure 25 P P

[0074] (12) Marker protein expression of EMT formation after DMI and TGF-β1 co-intervention: The expression of E-Cadherin protein in alveolar type II epithelial cells in each group had statistical significance in main effect and interaction effect, and there was a negative interaction (F value was 5.4, 89.01, 1.7, P all P<0.05). As shown in Figs. 10 and 11, compared with the control group, the expression of E-Cadherin protein in the quartz group decreased by 60%, and the expression of a-SMA increased by 24% (P<0.05). Compared with the quartz group, the expression of E-Cadherin protein in the TGF-β1+DMI+quartz group increased by 20%, and the expression of a-SMA decreased by 40% (P<0.05). The results suggest that TGF-β1 and DMI combined inhibit the EMT formation caused by quartz. Figure 26 27 P P

[0075] ​​​​​​​​The present application takes SD rats as the research object, and induces lung tissue fibrosis of rats by non-exposure tracheal perfusion method to give dust suspension to establish a silicosis rat model. The rats are sacrificed at 1, 3 and 6 months after dusting, and the samples are collected for detection of relevant laboratory indexes. The main pathological process of silicosis is divided into two stages, the early inflammatory reaction stage and the progressive pulmonary fibrosis stage. Epidemiological investigation finds that the dust exposure cycle of silicosis workers is about 20 years, but generally, the disease occurs in 5-10 years. The dusting time of rats in the present application is set to 1, 3 and 6 months, corresponding to the early inflammatory reaction stage and the fibrosis stage of silicosis, which is helpful to find the changes of markers in different stages of silicosis.

[0076] The lung dysfunction mainly occurs in the progressive stage, and the lung compliance decreases, and restrictive ventilation dysfunction may occur, which is manifested as the decrease of lung capacity, total lung capacity, residual volume and maximum ventilation, and further deterioration in the fibrosis stage. In the present application, lung function detection is performed on rats at different time points to observe the change trend of the ventilation function of rat lungs, and indexes such as FVC (forced expiratory lung capacity), FEV0.2 (forced expiratory volume in 0.2 seconds), MVV (maximum non-invasive ventilation volume) are selected. FVC and FEV0.2 mainly reflect the expiratory volume and ventilation flow rate, which are indexes reflecting the lung ventilation function, and are commonly seen in the case of lung function impairment caused by obstruction of large and small airways. The size of MVV is related to the respiratory muscle strength, chest elasticity and lung tissue elasticity, and it is an important index that can comprehensively evaluate the lung ventilation function reserve, and can reflect the overall efficiency of the respiratory system. The lung function detection results of the present application show that quartz processing and time have separate effects and interaction on FVC, FEV0.2 and MVV, which indicates that the longer the dusting time, the more serious the changes of FVC, FEV0.2 and MVV of each quartz group. Among the above changes, the MVV index decreases earliest and most obviously, which indicates that the overall efficiency of the respiratory system of rats decreases in the later stage. It is inferred that the serious fibrosis lesion in the later stage of the rat model leads to poor lung tissue elasticity and obstructive lung ventilation dysfunction.

[0077] In the present application, lung tissue sections are subjected to HE staining to observe the degree of lung tissue damage, and Masson staining is performed to observe the collagen fiber proliferation of lung tissue. The results of HE show that inflammatory cell infiltration occurs in the lung tissue of rats in the 1-month and 3-month silicosis groups, and the alveolar structure integrity is destroyed at 6 months of dusting time, which indicates that the early stage of silicosis may be dominated by alveolitis. Blue collagen fibers are found by Masson staining at 6 months. It is indicated that the lung tissue fibroblasts begin to proliferate in large quantities, and the degree of pulmonary interstitial fibrosis gradually increases. It is indicated that the early stage of lung pathological changes caused by quartz is mainly dominated by inflammatory reaction, and the middle and late stages are characterized by lung fibrosis.

[0078] HYP is a key component of collagen, which can reflect the health status of the tissue at the metabolic level, and is also an indirect indicator of the degree of fibrosis in connective tissue, i.e. pulmonary fibrosis. The present application uses the alkali hydrolysis method to find that the HYP content of silicosis rats at each time point is significantly higher than that of the control group, which indicates that the collagen content of lung tissue gradually increases with the increase of dust exposure time. The study of the changes of lung function and hydroxyproline level can dynamically evaluate the progress of pulmonary fibrosis and the treatment effect, and provide key clinical and molecular biology basis for early intervention, anti-fibrosis drug development and efficacy monitoring of silicosis and other interstitial lung diseases. The results of the EMT marker proteins in each group show that, compared with the 1-month control group, the E-cadherin content in the quartz group does not change significantly, and compared with the 3-month and 6-month control groups, the E-Cdherin protein expression in the quartz group is reduced by 20% and 35% respectively (P<0.05). With the extension of dust exposure time, compared with the 1-month, 3-month and 6-month control groups, the expression of α-SMA in the quartz group increased by 35%, 50% and 60% respectively (P<0.05), which indicates that the EMT model is successfully constructed in the 3-month quartz exposure animal experiment. Compared with the control group, the E-Cadherin in the cell EMT model decreased by 40%, and the α-SMA increased by 14% (P<0.05). It is indicated that the cell EMT model is successfully constructed. P <0.05). It is indicated that the cell EMT model is successfully constructed.

[0079] Pulmonary fibrosis in silicosis can severely damage the alveolar structure, and fibroblasts can also proliferate, and the extracellular matrix is over-deposited, and sphingomyelin metabolism abnormalities may participate in this process by regulating inflammatory cell apoptosis and fibrosis. The present application collects the lung tissue of the rats after they are sacrificed and uses metabolomics to measure the activity of sphingomyelin, which is significantly increased in the silicosis group of rats, so it is found that sphingomyelin metabolites may play an important role in the inflammation and fibrosis stages of silicosis. The results of animal dust exposure show that the activities of SM, Cer and S1P in the lung lavage fluid of the quartz group gradually increase with the extension of time, which verifies the results of metabolomics, and further indicates that sphingomyelin metabolism plays an important role in silicosis fibrosis. ASM plays an important role in pulmonary fibrosis, and it is found that the activity of ASM in the lung lavage fluid of IPF patients is positively correlated with the severity of the disease, which may be related to the increase of ASM activity, leading to the accumulation of Cer in the lung.

[0080] The present application adopts alveolar macrophages, T cells and alveolar type II epithelial cells to establish a cell EMT model by quartz dust, and uses DMI to intervene in the ASM activity of alveolar macrophages, detects various indexes, and verifies the animal experiment. The cell EMT model result shows that quartz can cause the increase of the activities of SM, ASM and Cer in the supernatant of NR8383, and after the intervention of DMI, the ASM activity can be inhibited, and the content of Cer is reduced. The result shows that DMI can inhibit the increase of the ASM activity of alveolar macrophages caused by quartz, and reduce the activities and contents of downstream metabolites, thereby reducing the lung inflammation.

[0081] Therefore, the change of sphingomyelin metabolism in alveolar macrophages is the key to the early inflammatory response in the lung, and is the key driving factor of pulmonary fibrosis. By inhibiting the ASM activity in the sphingomyelin pathway, a new treatment direction for early pulmonary fibrosis can be provided.

[0082] The content of IFN-γ in the lung lavage fluid of the EMT model of rats gradually increases with the prolongation of quartz dusting time, and the content of IL-4 decreases with the prolongation of dusting time, indicating that the immune characteristics in the process of pulmonary fibrosis in silicosis are the continuous increase of IFN-γ and the increase and then decrease of IL-4. In the inflammatory response stage of the disease, Th1 reaction can play a protective role by inhibiting the past repair. Activated macrophages can act as antigen-presenting cells, recruit and stimulate T lymphocytes, initiate adaptive immune response, and interval inflammation and fibrosis reaction. Therefore, it is proved by the cell EMT model that quartz causes the increase of T cell factor IFN-γ and the decrease of IL-4 content, and after the inhibition of the ASM activity of macrophages by DMI, the content of T cell factor IFN-γ decreases, the content of IL-4 increases, the content of epithelial phenotype marker E-Cadherin of alveolar type II epithelial cells increases, and the content of mesenchymal phenotype marker alpha-SMA decreases, indicating that DMI inhibits the EMT formation process caused by quartz.

[0083] The present application selects human TGF-β1 as an inhibitor of LTBPl of alveolar type II epithelial cells, and the results show that after the inhibition of human TGF-β1, the expression of LTBPl protein is obviously inhibited, and E-Cadherin and alpha-SMA of alveolar epithelial cells are improved in opposite changes, which indicates that the EMT of alveolar type II epithelial cells is slowed down. When DMI and TGF-β1 are used to intervene in the cell model, it is found that the content of LTBPl protein decreases by 53%, and the decrease of TGF-β1 is only 40%, which is not much different. In the EMT process of silicosis, alveolar type II cells play a major role.

[0084] Both alveolar macrophage sphingomyelin metabolism and T cell immune response are involved in the process of EMT formation induced by quartz, which is manifested as the activation of SM and ASM activity by quartz, the increase of Cer synthesis, the activation of T cell immune response, the increase of alveolar type II epithelial cell LTBP1 protein, and the formation of EMT. The intervention measures for alveolar macrophage sphingomyelin metabolism and alveolar type II epithelial cell LTBP1 suggest that alveolar type II epithelial cells play a major role in the process of EMT formation.

[0085] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. The use of TGF-β1 and desipramine DMI in the preparation of a drug for inhibiting the formation of silicosis EMT caused by quartz.

2. Use according to claim 1, characterized in that: TGF-β1 and DMI joint intervention, inhibit the sphingomyelin metabolic pathway of alveolar macrophages and Th1 type immune response caused by quartz.

3. Use according to claim 2, characterized in that: TGF-β1 and DMI joint intervention, inhibit the expression of LTBPl protein in alveolar type II epithelial cells, inhibit the increase of ASM activity of alveolar macrophages caused by quartz, and reduce the content of Cer.

4. Use according to claim 2, characterized in that: TGF-β1 and DMI joint intervention, promote the expression of E-Cadherin protein in alveolar type II epithelial cells, and inhibit the expression of α-SMA protein.

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