Application and verification method of small molecule for targeted inhibition of hyaluronic acid synthase 2

Abstract

The invention relates to application and a verification method of a small molecule for targeted inhibition of hyaluronic acid synthetase 2, the small molecule is moss phenol glucoside, and the small molecule is applied to targeted hyaluronic acid synthetase 2 protein to inhibit synthesis of hyaluronic acid; through molecular docking, a surface plasmon resonance experiment and a cell thermal migration technology experiment, the inventor finds that the moss phenol glucoside shows relatively high binding force and affinity to the hyaluronic acid synthetase 2 and has the potential of targeting the hyaluronic acid synthetase 2, and a subsequent enzyme-linked immunosorbent assay result shows that the moss phenol glucoside has relatively high affinity to the hyaluronic acid synthetase 2. The moss phenol glucoside can inhibit the expression increase of hyaluronic acid in fibroblasts induced by transforming growth factor-beta1. Through an animal model, the anti-pulmonary fibrosis effect and the inhibition effect on hyaluronic acid of mice with pulmonary fibrosis of the moss phenol glucoside are proved. The moss phenol glucoside can specifically target hyaluronic acid synthetase 2, so that the secretion of hyaluronic acid is inhibited, and the pulmonary fibrosis is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a small molecule that targets hyaluronic acid synthase 2 (HAS2) protein to inhibit hyaluronic acid (HA) synthesis and its application, specifically a small molecule that inhibits HA levels by targeting HAS2 protein, and its application in treating pulmonary fibrosis by regulating HA levels. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia. It is a chronic, progressive, fibrotic, unexplained interstitial lung disease that usually has characteristic imaging and histological manifestations and mainly occurs in middle-aged and elderly people. IPF is characterized by dry cough and exertional shortness of breath, and in the late stage it often causes heart and lung failure. It has a high mortality rate and is called a "tumor-like disease", with an average of 3-4 years from diagnosis to death. The main pathological feature of this disease is the imbalance of extracellular matrix homeostasis, resulting in excessive collagen deposition. The pathological process of pulmonary fibrosis is mainly driven by the differentiation of fibroblasts into myofibroblasts, which produce large amounts of collagen and extracellular matrix proteins, leading to impaired tissue and organ function.

[0003] Currently, only two anti-pulmonary fibrosis drugs, pirfenidone and nintedanib, are available on the market. Although they can slow down the progression of the disease, they are still unable to cure the disease. Therefore, effective pharmacological methods are still in great need of treatment for pulmonary fibrosis, especially preventing fibroblasts from differentiating into myofibroblasts and the resulting excessive deposition of extracellular matrix proteins.

[0004] HA (Hyaluronic Acid) is a glycosaminoglycan composed of disaccharides (D-glucuronic acid and N-acetylglucosamine) as basic units, also known as uronic acid and hyaluronic acid. HA is a ubiquitous natural glycosaminoglycan and the main component of the extracellular matrix. It is involved in biological processes such as water homeostasis, cell matrix signal transduction, tissue healing, inflammation, angiogenesis, cell proliferation and migration. HA can be secreted by type II lung fibroblasts, lung epithelial cells and endothelial cells in the lungs, among which fibroblasts can be stimulated by pathogenic factors to synthesize a large amount of HA. In IPF tissue samples and animal models, the HA content is significantly higher than that of normal tissues, and is positively correlated with the severity of IPF disease. In IPF animal models, increased HA levels can widely induce inflammatory responses in lung tissues. HA activates mouse fibroblasts through the CD44 receptor, promotes the proliferation of fibroblasts, and participates in the process of pulmonary fibrosis. In addition, HA can also activate the CD44 / S100A4 axis, affect the differentiation of mesenchymal stem cells into fibroblasts, and promote pulmonary fibrosis. Studies have found that HA is also involved in the occurrence of pulmonary fibrosis and emphysema.

[0005] HA synthase (Hyaluronic Acid Synthase, HAS) controls the synthesis of HA, mainly including three isomers HAS1, HAS2 and HAS3. Among them, HAS2 is the main subtype responsible for the production of HA in mesenchymal cells. The inventor's previous studies found that HAS2 was highly expressed in the lungs of IPF patients, the lungs of pulmonary fibrosis mouse models, and myofibroblasts induced by transforming growth factor β1 (Transforminggrowth factor-β1, TGF-β1). Existing studies have shown that overexpression of HAS2 in myofibroblasts promotes an aggressive phenotype, leading to severe fibrosis. Therefore, HAS2 may be a key determinant of the fate of fibrosis, and the synthesis of HA can be reduced by inhibiting the expression of HAS2, thereby treating pulmonary fibrosis. Therefore, inhibiting the generation and release of HA by intervening in the HAS2 target is a potential means of treating IPF. In order to solve the problem of intervening in the HAS2 target to inhibit the generation and release of HA, the applicant proposed an application and verification method of a small molecule that targets and inhibits hyaluronan synthase 2. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an application of a small molecule for targeted inhibition of hyaluronan synthase 2 in view of the above-mentioned defects of the prior art, and also provide a verification method for the application of the small molecule for targeted inhibition of hyaluronan synthase 2.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] An application of a small molecule for targeted inhibition of hyaluronan synthase 2 is constructed, wherein the small molecule is orcinol glucoside (OG), and the small molecule is used to target HAS2 protein to inhibit the synthesis of HA.

[0009] A method for verifying the application of a small molecule for targeted inhibition of hyaluronan synthase 2, wherein the method comprises the following steps:

[0010] Protein preparation: Download the predicted three-dimensional structure of Human HAS2, use the Protein Preparation Wizard module to hydrogenate the protein, then perform energy optimization, and use the Receptor Grid Generation module to create a grid file centered on amino acid residues CYS279 / SER310 / LYS190;

[0011] Compound preparation: 2D format of OG is passed through The software LigPrep Module performs hydrogenation and energy optimization and outputs 3D structures for molecular docking.

[0012] Molecular docking: The prepared compounds are imported and molecular docking is performed using the Glide module, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching, and the docking score is calculated.

[0013] A method for verifying the application of a small molecule for targeted inhibition of hyaluronan synthase 2, wherein the method comprises the following steps:

[0014] Chip preparation: Prior to injection, the activator was prepared by mixing 400 mM ethylenediaminetetraacetic acid with 100 mM N-hydroxysuccinimide; the mixture was injected into the Fc1 and Fc2 sample channels at a flow rate of 10 μL / min for 800 s; then, 10 mM sodium acetate (pH 4.0) was added to HAS2 at a concentration of 100 μg / mL and injected into the Fc2 sample channel for 300 s at a flow rate of 10 μL / min, with a fixed level of approximately 10993 RU; the chip was deactivated by injecting 1 M ethanolamine hydrochloride into the Fc1 and Fc2 sample channels at a flow rate of 10 μL / min for 800 s;

[0015] Analytes were run by kinetics / affinity: OG was diluted with running buffer to seven concentrations: 1250, 625, 312.5, 156.3, 78.2, 39.1, 19.6, and 0 μM; OG was injected into the Fc1-Fc2 of the channel at a flow rate of 30 μL / min for a 60 s association phase followed by a 90 s dissociation phase; both association and dissociation processes were processed in running buffer.

[0016] A method for verifying the application of a small molecule for targeted inhibition of hyaluronan synthase 2, wherein the method comprises the following steps:

[0017] Induced cells: NIH / 3T3 cells were stimulated with 5 ng / ml TGF-β1 for 24 h;

[0018] Protein extraction: Use cell scraper to collect induced NIH / 3T3 cells, wash once with cold PBS, add cold PBS containing protease inhibitors to resuspend the cells, and freeze and thaw the cell suspension three times with liquid nitrogen; centrifuge to obtain the supernatant to separate the soluble lysate from the cell debris; determine the protein concentration by BCA method, and then dilute the quantified protein to 4μg / μl;

[0019] Drug treatment: The quantified protein lysate was divided into two equal parts and placed in 1.5 ml centrifuge tubes, divided into a drug treatment group and a negative control group; the drug treatment group treated NIH / 3T3 cells with OG at a final concentration of 100 μM, and the negative control group was treated with DMSO and incubated at room temperature for 1 hour;

[0020] Heating treatment: resuspend the lysate after administration by pipetting, and then divide the lysate into 12 equal parts and place them in PCR tubes; use a PCR thermal cycler to heat at different temperatures for 3 minutes: 37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67°C, 70°C, a total of 12 temperatures, with an interval of 3°C for each group, and then cool at room temperature for 3 minutes, and then temporarily store on ice;

[0021] Preparation of loading protein: After centrifugation of the above samples, take the supernatant, add 5× loading buffer, mix well, and heat at 100°C for 10 minutes to denature the protein;

[0022] Western blot: Take 10 μl of protein sample and load it in the order of treatment temperature from low to high.

[0023] A method for verifying the application of a small molecule for targeted inhibition of hyaluronan synthase 2, wherein the method comprises the steps of:

[0024] The CCK-8 method was used to detect the effect of OG on the proliferation of mouse embryonic fibroblast cell line NIH / 3T3 cells and human fibroblast HFL-1 cells.

[0025] The steps of detecting the effect of OG on the proliferation of mouse embryonic fibroblast cell line NIH / 3T3 cells and human fibroblast HFL-1 cells by CCK-8 method are as follows:

[0026] Seeding: Seed the corresponding number of cells in a 96-well plate at 3000 cells per well;

[0027] Drug addition and incubation: Add drugs of corresponding concentration to each well and incubate in a cell culture incubator for 48 hours;

[0028] Add 10ul CCK-8 solution to each well;

[0029] Continue incubating in the cell culture incubator for 1-4 hours and use an ELISA reader to detect the absorbance of the 96-well plate at 450 nm.

[0030] A method for verifying the application of a small molecule for targeted inhibition of hyaluronan synthase 2, wherein the method comprises the steps of:

[0031] The effect of OG on the production of HA in NIH / 3T3 cells and HFL-1 cells induced by TGF-β1 was detected by enzyme-linked immunosorbent assay (ELISA).

[0032] The steps of detecting the effect of OG on the production of HA by TGF-β1-induced NIH / 3T3 cells and HFL-1 cells by ELISA are as follows:

[0033] Cell administration and sample collection: NIH / 3T3 cells or HFL-1 cells were seeded in culture plates, NIH / 3T3 cells and HFL-1 cells were stimulated with 5 ng / ml TGF-β1, and different concentrations of OG were given for intervention. After 48 hours, the cell culture medium was collected and the HA content was detected by ELISA.

[0034] Wash the plate and incubate the samples: wash the plate 3 times with 300 μl of washing buffer per well, pat dry, add 100 μl of sample to each well, and incubate at room temperature for 2 hours;

[0035] Wash the plate and incubate with biotinylated antibody working solution: wash the plate 4 times with 300 μl of washing buffer per well, pat dry, and incubate with 100 μl of biotinylated antibody working solution per well at room temperature for 1 hour;

[0036] Wash the plate and incubate the enzyme conjugate working solution: wash the plate 4 times with 300μl of washing buffer per well, pat dry, and incubate each well with 100μl of enzyme conjugate working solution at room temperature for 30 minutes;

[0037] Wash the plate and incubate the color substrate: wash the plate 5 times with 300μl washing buffer per well, pat dry, and incubate the plate with 100μl color substrate per well at room temperature for 30 minutes;

[0038] Stop color development: Add 50 μl of stop solution to each well and detect the absorbance at 450 nm using an ELISA reader.

[0039] A method for verifying the application of a small molecule for targeted inhibition of hyaluronan synthase 2, wherein the method comprises the steps of:

[0040] Animal modeling and drug administration;

[0041] The experimental animals were divided into 5 groups, with 6 mice in each group. Groups 1 to 5 were: normal control group, model group, OG low concentration group, OG medium concentration group and OG high concentration group respectively; Group 6 was the pirfenidone positive drug group; after anesthesia, groups 2-6 were subjected to bleomycin modeling, first anesthetized with isoflurane, and 2.5 mg / kg of bleomycin was injected through the trachea to establish a pulmonary fibrosis mouse model; the normal control group mice were injected with an equal amount of normal saline as a control; on the second day of modeling, mice in groups 3-6 began to receive oral gavage of corresponding drugs once a day for 2 consecutive weeks; groups 1-2 were gavaged with an equal amount of normal saline; after 2 weeks, samples were collected;

[0042] HE and Masson's methods were used to observe the pathological changes and fibrosis degree of mouse lung tissue;

[0043] Lung tissues of mice in each group were collected, and HE and Masson methods were used to observe the pathological changes and fibrosis degree of the lungs of mice in each group. The steps are as follows:

[0044] HE part:

[0045] Fixation and dehydration: The lung tissues removed from the dissected mice were fully immersed in 4% paraformaldehyde for 24 hours, immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for dehydration for 24 hours;

[0046] Embedding: trim the tissue, preheat the embedding machine to melt the paraffin, place the tissue on the mold, fully infiltrate the liquid paraffin, wait for it to cool, and store the embedded tissue at -20℃ to harden for subsequent sectioning;

[0047] Sectioning: Fix the sample in a semi-automatic paraffin slicer, install the blade, and start slicing. The slice thickness is 4μm. Quickly pick up the cut wax slice with tweezers and spread it in the sink. Use a glass slide to pick up the slice and lay it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and place it on the baking machine to dry the water.

[0048] Dewaxing: After drying the slices, place them in an oven at 60°C for 2-3 hours, take them out for dewaxing, first soak them in xylene for 10 minutes, then take them out and soak them in new xylene for 10 minutes, then soak them in anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes in sequence, and finally soak them in ultrapure water for 5 minutes twice;

[0049] Hematoxylin nuclear staining: Place the sections in hematoxylin for 5-8 minutes, wash with tap water, and then soak in 1% hydrochloric acid ethanol for 5-10 seconds, and wash with tap water to turn blue;

[0050] Eosin staining: Place the sections in eosin staining solution for 1-3 minutes;

[0051] Dehydration: Then soak in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 3 minutes in sequence, then soak the slices in xylene for 10 minutes, take them out and soak them in new xylene for 10 minutes;

[0052] Sealing: air-dry xylene in room temperature slices, add neutral gum, cover with coverslip, spread the neutral gum evenly, make sure there are no bubbles on the tissue, and dry in a ventilated place;

[0053] Masson part:

[0054] Fixation and dehydration: The lung tissues removed from the dissected mice were fully immersed in 4% paraformaldehyde for 24 hours, immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for dehydration for 24 hours;

[0055] Embedding: trim the tissue, preheat the embedding machine to melt the paraffin, place the tissue on the mold, fully infiltrate the liquid paraffin, wait for it to cool, and store the embedded tissue at -20℃ to harden for subsequent sectioning;

[0056] Sectioning: Fix the sample in a semi-automatic paraffin slicer, install the blade, and start slicing. The slice thickness is 4μm. Quickly pick up the cut wax slice with tweezers and spread it in the sink. Use a glass slide to pick up the slice and lay it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and place it on the baking machine to dry the water.

[0057] Dewaxing: After drying the slices, place them in an oven at 60°C for 2-3 hours, take them out for dewaxing, first soak them in xylene for 10 minutes, then take them out and soak them in new xylene for 10 minutes, then soak them in anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes in sequence, and finally soak them in ultrapure water for 5 minutes twice;

[0058] Chromization treatment or demercuration salt precipitation, washing with tap water and distilled water in turn;

[0059] Stain the nuclei with Regaud's hematoxylin stain or Weigert's iron hematoxylin stain for 5 to 10 minutes;

[0060] Differentiate with 1% hydrochloric acid alcohol for 5-15 seconds, and then wash thoroughly with water;

[0061] Add 0.1-1% lithium carbonate dropwise for 5 minutes to increase the degree of blueing, and wash with water;

[0062] Use Masson Ponceau acid red solution for 5 to 10 minutes;

[0063] Wash with 2% glacial acetic acid aqueous solution for 1 minute;

[0064] Differentiate with 1% phosphomolybdic acid aqueous solution for 3-5 minutes, and wash with 2% glacial acetic acid aqueous solution for 1 minute;

[0065] Without washing, dye with aniline blue aqueous solution or 1% light green aqueous solution for 1 to 2 minutes; wash with 0.2% glacial acetic acid aqueous solution for 1 minute;

[0066] Dehydration: Then soak in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 3 minutes in sequence, then soak the slices in xylene for 10 minutes, take them out and soak them in new xylene for 10 minutes;

[0067] Sealing: air-dry xylene in room temperature slices, add neutral gum, cover with coverslip, spread the neutral gum evenly, make sure there are no bubbles on the tissue, and dry in a ventilated place;

[0068] Western Blot was used to detect the expression of α-SMA and COL1A1 in mouse lung tissue:

[0069] Protein extraction and denaturation: 50 g lung tissue of each mouse was weighed and immersed in 400 μl lysis buffer, three zircons were added, and the mixture was ground in a grinder for 3 minutes 3 times. The grinding solution was placed on ice for 30 minutes and then centrifuged at 14000 rpm for 30 minutes. The supernatant was taken and the protein concentration was measured. Then, loading buffer was added and the mixture was placed in a metal bath at 85°C for 10 minutes.

[0070] Electrophoresis: Prepare electrophoresis gel, add 30 μg protein to each well, and run electrophoresis in a 100 V constant voltage electric field for 90 minutes;

[0071] Transfer: Take out the gel and put it close to the PVDF membrane in the transfer clip, and transfer the membrane in a 300mA constant current electric field for 2 hours;

[0072] Blocking: Take out the PVDF membrane and soak it in 5% skim milk, and place it on a shaker for 1 hour;

[0073] Incubation with antibodies: wash the membrane with TBST for 5 minutes 3 times, add primary antibody and incubate at 4°C overnight, recover primary antibody, wash the membrane with TBST for 5 minutes 3 times, add secondary antibody and incubate at room temperature for 1 hour;

[0074] Chemiluminescence: Place the membrane in an automatic chemiluminescence instrument, evenly add luminescent liquid, and avoid light to emit light;

[0075] ELISA method to determine the content of HA in mouse serum:

[0076] The serum of mice in each group was collected, and ELISA experiment was performed to determine the HA content in the serum.

[0077] The beneficial effects of the present invention are as follows: the inventors found through molecular docking, surface plasmon resonance experiments, and cell thermal migration technology experiments that OG exhibits high binding force and affinity to HAS2, and has the potential to target HAS2, and subsequent ELISA results show that OG can inhibit the increased expression of HA in fibroblasts induced by TGF-β1. And through animal models, the anti-pulmonary fibrosis effect of OG and the inhibitory effect on HA in mice with pulmonary fibrosis were confirmed. Therefore, OG can specifically target HAS2, thereby inhibiting the secretion of HA and improving pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. The drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work:

[0079] Figure 1 Schematic diagram of the molecular formula of OG.

[0080] Figure 2 This is a schematic diagram of the results of determining the molecular docking scores of OG and HAS2 using the molecular docking method in one embodiment of the present invention.

[0081] Figure 3 This is a diagram showing the results of a surface plasmon resonance experiment confirming the binding effect between OG and HAS2 in one embodiment of the present invention.

[0082] Figure 4 This is a diagram showing the results of using cell thermal migration technology to confirm the binding effect between OG and HAS2 in one embodiment of the present invention.

[0083] Figure 5 This is a graph showing the result of finding that OG can inhibit myofibroblasts from producing HA in one embodiment of the present invention.

[0084] Figure 6 This is a graph showing the results of determining the inhibitory effect of oral administration of OG on serum HA content in mice with bleomycin-induced pulmonary fibrosis by ELISA in one embodiment of the present invention. DETAILED DESCRIPTION

[0085] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the following will be described clearly and completely in combination with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0086] The application of a small molecule for targeted inhibition of hyaluronan synthase 2 in a preferred embodiment of the present invention, the small molecule is OG, and the small molecule is used to target HAS2 protein to inhibit the synthesis of HA;

[0087] OG is a phenolic glycoside isolated from the Chinese medicinal plant Curculigo orchioides, which has multiple pharmacological effects such as anti-inflammatory, antioxidant, and antidepressant. Figure 1 ;

[0088] The inventors found through molecular docking, surface plasmon resonance experiments, and cell thermal migration technology experiments that OG showed high binding force and affinity to HAS2, and had the potential to target HAS2. The subsequent enzyme-linked immunosorbent assay (ELISA) results showed that OG could inhibit the increased expression of HA in fibroblasts induced by TGF-β1. And through animal models, OG's anti-pulmonary fibrosis effect and its inhibitory effect on HA in mice with pulmonary fibrosis were confirmed. Therefore, OG can specifically target HAS2, thereby inhibiting the secretion of HA and improving pulmonary fibrosis.

[0089] The specific verification method implementation instructions are as follows:

[0090] Example 1: Molecular docking of OG and HAS2

[0091] In this example, the molecular docking method was used to determine the molecular docking between the OG small molecule and the HAS2 protein. The principle steps are briefly described as follows:

[0092] 1. Protein preparation: Download the predicted three-dimensional structure of Human HAS2 from the AlphaFold website (AlphaFoldID: AF-Q92819-F1). Use the Protein Preparation Wizard module to hydrogenate the protein, followed by energy optimization (OPLS2005 force field, RMSD ). The grid file was created using the Receptor Grid Generation module with amino acid residues CYS279 / SER310 / LYS190 as the center. The box sizes were set to

[0093] 2. Compound preparation: 2D format of OG is prepared by The software LigPrep Module performs hydrogenation, energy optimization and other processing, and outputs 3D structures for molecular docking.

[0094] 3. Molecular docking: Import the prepared compounds and use the Glide module to perform molecular docking, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching, and the docking score is calculated.

[0095] Experimental results: see Figure 2 The molecular docking score of OG and HAS2 is -7.325, indicating that OG and HAS2 have a good degree of binding. OG can form 5 hydrogen bonds and 2 π-π interactions with the target protein: the three hydroxyl groups on the sugar ring can form four hydrogen bonds with GLU339, GLN350, ARG353, and ASP212, and the distances are shown in the 3D interaction diagram; the phenolic hydroxyl group, as a hydrogen bond donor, forms a hydrogen bond with ASP314, and the distance is In addition, the benzene ring can form two π-π interactions with TRP354.

[0096] Example 2: Surface plasmon resonance experiments confirm the direct binding of OG to HAS2 protein

[0097] In this example, the molecular affinity between the OG small molecule and the HAS2 protein was determined by surface plasmon resonance experiment. The steps are briefly described as follows:

[0098] 1. Chip preparation (HAS2): Before injection, the activator was prepared by mixing 400mM ethylenediaminetetraacetic acid with 100mM N-hydroxysuccinimide. The mixture was injected into the Fc1 and Fc2 sample channels at a flow rate of 10μL / min for 800s. Then, 10mM sodium acetate (pH 4.0) was added to HAS2 at a concentration of 100μg / mL and injected into the Fc2 sample channel for 300s at a flow rate of 10μL / min, with a fixed level of approximately 10993RU. The chip was deactivated by injecting 1M ethanolamine hydrochloride into the Fc1 and Fc2 sample channels at a flow rate of 10μL / min for 800s.

[0099] 2. Run analytes by kinetics / affinity: OG was diluted to 7 concentrations (1250, 625, 312.5, 156.3, 78.2, 39.1, 19.6 and 0 μM) with running buffer (1*PBS-P, 5% DMSO buffer, ethanolamine hydrochloride). OG was injected into the Fc1-Fc2 of the channel at a flow rate of 30 μL / min for a 60-second association phase, followed by a 90-second dissociation. Both the association and dissociation processes were processed in running buffer.

[0100] Experimental results: see Figure 3 The dissociation constant KD (M) between OG small molecule and HAS2 protein is 3.039*10-4, which has good molecular affinity.

[0101] Example 3: Cell thermal migration technology confirms the direct binding of OG to HAS2 protein.

[0102] 1. Induced cells: Add 5ng / ml TGF-β1 to stimulate NIH / 3T3 cells for 24 hours;

[0103] 2. Protein extraction: Use a cell scraper to collect the induced NIH / 3T3 cells, wash them once with cold PBS, add cold PBS containing protease inhibitors (protease inhibitors: PBS = 1:100) to resuspend the cells, and use liquid nitrogen to freeze and thaw the cell suspension three times. Centrifuge (4°C, 20,000×g, 20 minutes) to take the supernatant to separate the soluble lysate from the cell debris. Determine the protein concentration by the BCA method, and then dilute the quantified protein to 4μg / μl;

[0104] 3. Drug treatment: The quantified protein lysate was divided into two equal parts and placed in 1.5 ml centrifuge tubes, which were divided into a drug treatment group and a negative control group. The drug treatment group treated NIH / 3T3 cells with OG at a final concentration of 100 μM, and the negative control group was treated with DMSO and incubated at room temperature for 1 hour;

[0105] 4. Heating treatment: resuspend the lysate after administration by blowing, and then divide the lysate into 12 equal parts and place them in PCR tubes. Use a PCR thermal cycler to heat at different temperatures for 3 minutes (37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67°C, 70°C, a total of 12 temperatures, each group with an interval of 3°C), then cool at room temperature for 3 minutes, and then temporarily store on ice;

[0106] 5. Prepare the loading protein: After centrifugation of the above samples (4°C, 20,000×g, 20 minutes), take the supernatant, add 5× loading buffer, mix well, and heat at 100°C for 10 minutes to denature the protein;

[0107] 6.Western blot: Take 10μl protein sample and load it in the order of treatment temperature from low to high.

[0108] Experimental results: see Figure 4 Compared with the DMSO control group, the OG small molecule can enhance the stability of the HAS2 protein at different temperatures, indicating that the OG small molecule can directly bind to the HAS2 protein.

[0109] Example 4: OG can inhibit the production of HA by myofibroblasts.

[0110] (1) CCK-8 assay to detect the effect of OG on the proliferation of mouse embryonic fibroblast cell line NIH / 3T3 cells and human fibroblast HFL-1 cells

[0111] In this example, the CCK-8 method was used to detect the effect of OG on the proliferation of NIH / 3T3 cells and HFL-1 cells. The steps are briefly described as follows:

[0112] 1. Seeding: Seed the corresponding number of cells in a 96-well plate at 3000 cells per well.

[0113] 2. Drug addition and incubation: Add drugs of corresponding concentration to each well (set 8 concentration gradients from 0 to 100 μM) and incubate in a cell culture incubator for 48 hours.

[0114] 3. Add 10ul CCK-8 solution to each well.

[0115] 4. Continue incubating in the cell culture incubator for 1-4 hours and use an ELISA reader to detect the absorbance of the 96-well plate at 450 nm.

[0116] Experimental results: Figure 4 A and Figure 4 B shows that OG at various concentrations had no significant cytotoxic effect on NIH / 3T3 cells and HFL-1 cells.

[0117] (2) ELISA method was used to determine the effect of OG on TGF-β1-induced HA production in NIH / 3T3 cells and HFL-1 cells

[0118] In this example, the ELISA method was used to determine the effect of OG on the production of HA by NIH / 3T3 cells and HFL-1 cells induced by TGF-β1. The steps are briefly described as follows:

[0119] 1. Cell administration and sampling: NIH / 3T3 cells or HFL-1 cells were seeded in culture plates, NIH / 3T3 cells and HFL-1 cells were stimulated with 5 ng / ml TGF-β1, and different concentrations of OG were given for intervention. After 48 hours, the cell culture medium was collected and the HA content was detected by ELISA.

[0120] 2. Wash the plate and incubate the samples: Wash the plate three times with 300 μl of washing buffer per well. After patting dry, add 100 μl of sample to each well and incubate at room temperature for 2 hours.

[0121] 3. Wash the plate and incubate with biotinylated antibody working solution: Wash the plate 4 times with 300 μl of washing buffer per well. After patting dry, incubate each well with 100 μl of biotinylated antibody working solution at room temperature for 1 hour.

[0122] 4. Wash the plate and incubate the enzyme conjugate working solution: Wash the plate 4 times with 300μl of washing buffer per well. After patting dry, incubate each well with 100μl of enzyme conjugate working solution at room temperature for 30 minutes.

[0123] 5. Wash the plate and incubate the chromogenic substrate: Wash the plate 5 times with 300 μl of washing buffer per well. After patting dry, incubate the plate with 100 μl of chromogenic substrate per well at room temperature for 30 minutes.

[0124] 6. Stop color development: Add 50 μl of stop solution to each well and detect the absorbance at 450 nm using an ELISA reader.

[0125] Experimental results: Figure 5 C and Figure 5 D shows that OG can dose-dependently inhibit the secretion of HA by myofibroblasts induced by TGF-β1 (*P<0.05, **P<0.01 VS TGF-β1 group).

[0126] Example 5: Animal experiments confirmed the anti-pulmonary fibrosis effect of OG and its inhibitory effect on serum HA in mice with pulmonary fibrosis.

[0127] (1) Animal modeling and drug administration: The experimental animals were divided into 5 groups, with 6 mice in each group. 1. Normal control group; 2. Model group; 3. OG low concentration group (25 mg / kg); 4. OG medium concentration group (50 mg / kg); 5. OG high concentration group (100 mg / kg). 6. Pirfenidone positive drug group. After anesthesia, groups 2-6 were subjected to bleomycin modeling. They were first anesthetized with isoflurane and 2.5 mg / kg of bleomycin was injected through the trachea to establish a pulmonary fibrosis mouse model. The normal control group mice were injected with an equal amount of normal saline as a control. On the second day of modeling, mice in groups 3-6 began to receive oral gavage of the corresponding drug treatment once a day for 2 consecutive weeks; groups 1-2 were gavaged with an equal amount of normal saline. After 2 weeks, the samples were collected.

[0128] (2) Observation of pathological changes and fibrosis degree of mouse lung tissue by HE and Masson method

[0129] Lung tissues of mice in each group were collected, and HE and Masson methods were used to observe the pathological changes and fibrosis degree of the lungs of mice in each group. The steps are briefly described as follows:

[0130] HE part:

[0131] 1. Fixation and dehydration: The lung tissue removed from the dissected mouse was fully immersed in 4% paraformaldehyde for 24 hours, immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for dehydration for 24 hours.

[0132] 2. Embedding: trim the tissue, preheat the embedding machine to melt the paraffin, place the tissue on the mold, fully infiltrate it with liquid paraffin, wait for it to cool, and store the embedded tissue at -20℃ to harden for subsequent sectioning.

[0133] 3. Slicing: Fix the sample in a semi-automatic paraffin slicer, install the blade, and start slicing. The slice thickness is 4μm. Quickly pick up the cut wax slice with tweezers and spread it in the sink. Use a glass slide to pick up the slice and spread it flat on the glass slide. Gently shake off the moisture, mark the information with a pencil, and place it on the slide dryer to dry the moisture.

[0134] 4. Dewaxing: After drying the slices, place them in an oven at 60°C for 2-3 hours, take them out for dewaxing. First, soak the slices in xylene for 10 minutes, then take them out and soak them in new xylene for 10 minutes, then soak them in anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes in sequence, and finally soak them in ultrapure water for 5 minutes twice.

[0135] 5. Hematoxylin nuclear staining: Place the sections in hematoxylin for staining for 5-8 minutes, wash with tap water, and then soak in 1% hydrochloric acid ethanol for 5-10 seconds, and wash with tap water to turn blue.

[0136] 6. Eosin staining: Place the sections in eosin staining solution for 1-3 minutes.

[0137] 7. Dehydration: Then soak the sections in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 3 minutes in turn, then soak the sections in xylene for 10 minutes, take them out and soak them in new xylene for 10 minutes.

[0138] 8. Sealing: Air-dry the slices with xylene at room temperature, add a drop of neutral gum, cover with a coverslip, spread the neutral gum evenly without any bubbles on the tissue, and dry in a ventilated place.

[0139] Masson part:

[0140] 1. Fixation and dehydration: The lung tissue removed from the dissected mouse was fully immersed in 4% paraformaldehyde for 24 hours, immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for dehydration for 24 hours.

[0141] 2. Embedding: trim the tissue, preheat the embedding machine to melt the paraffin, place the tissue on the mold, fully infiltrate it with liquid paraffin, wait for it to cool, and store the embedded tissue at -20℃ to harden for subsequent sectioning.

[0142] 3. Slicing: Fix the sample in a semi-automatic paraffin slicer, install the blade, and start slicing. The slice thickness is 4μm. Quickly pick up the cut wax slice with tweezers and spread it in the sink. Use a glass slide to pick up the slice and spread it flat on the glass slide. Gently shake off the moisture, mark the information with a pencil, and place it on the slide dryer to dry the moisture.

[0143] 4. Dewaxing: After drying the slices, place them in an oven at 60°C for 2-3 hours, take them out for dewaxing. First, soak the slices in xylene for 10 minutes, then take them out and soak them in new xylene for 10 minutes, then soak them in anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes in sequence, and finally soak them in ultrapure water for 5 minutes twice.

[0144] 5. After chromization or mercury salt precipitation, wash with tap water and distilled water in turn.

[0145] 6. Stain the nuclei with Regaud's hematoxylin stain or Weigert's iron hematoxylin stain for 5 to 10 minutes.

[0146] Differentiate with 7.1% hydrochloric acid and alcohol for 5 to 15 seconds and wash thoroughly with water.

[0147] 8. Add 0.1-1% lithium carbonate for 5 minutes to increase the degree of blueing, and wash with water.

[0148] 9. Use Masson Ponceau Acid Fuchsin Solution for 5 to 10 minutes.

[0149] 10. Wash with 2% glacial acetic acid aqueous solution for 1 minute.

[0150] Differentiate with 11.1% phosphomolybdic acid aqueous solution for 3-5 minutes and wash with 2% glacial acetic acid aqueous solution for 1 minute.

[0151] 12. Dye with aniline blue aqueous solution or 1% light green aqueous solution for 1 to 2 minutes without washing; wash with 0.2% glacial acetic acid aqueous solution for 1 minute.

[0152] 13. Dehydration: Then soak the sections in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 3 minutes in turn, then soak the sections in xylene for 10 minutes, take them out and soak them in new xylene for 10 minutes.

[0153] 14. Sealing: Air-dry the slices with xylene at room temperature, add a drop of neutral gum, cover with a coverslip, spread the neutral gum evenly, leave no bubbles on the tissue, and dry in a ventilated place.

[0154] Experimental results: Figure 6 As shown in A, the mice in the model group had obvious lung inflammation infiltration and lung fibrosis compared with the control group, while the inflammation and fibrosis levels of the lung tissues of the mice in the OG group were reduced compared with those in the model group.

[0155] (3) Western Blot analysis of the expression of α-SMA and COL1A1 in mouse lung tissue

[0156] α-SMA and COL1A1 are marker proteins of fibrosis. In this example, the expression of α-SMA and COL1A1 proteins in the lungs of each group of mice was determined by Western Blot method to study the effect of OG on pulmonary fibrosis. The steps are briefly described as follows:

[0157] 1. Protein extraction and denaturation: Weigh 50 g of lung tissue from each mouse, immerse it in 400 μl of lysis buffer, add three zircons, grind it in a grinder for 3 minutes 3 times, let the grinding solution stand on ice for 30 minutes, then centrifuge it at 14000 rpm for 30 minutes, take the supernatant, measure the protein concentration, add loading buffer, and place it in a metal bath at 85°C for 10 minutes.

[0158] 2. Electrophoresis: Prepare electrophoresis gel, add 30 μg protein to each well, and run electrophoresis in a 100 V constant voltage electric field for 90 minutes.

[0159] 3. Transfer: Take out the gel and place it close to the PVDF membrane in the transfer clip, and transfer the membrane in a 300mA constant current electric field for 2 hours.

[0160] 4. Blocking: Take out the PVDF membrane and soak it in 5% skim milk, and place it on a shaker for 1 hour.

[0161] 5. Incubation with antibodies: Wash the membrane with TBST for 5 minutes three times, add primary antibody and incubate at 4°C overnight, recover primary antibody, wash the membrane with TBST for 5 minutes three times, add secondary antibody and incubate at room temperature for 1 hour.

[0162] 6. Chemiluminescence: Place the membrane in an automatic chemiluminescence instrument, evenly add luminescent liquid, and avoid light.

[0163] Experimental results: Figure 6 B shows that the expression of pulmonary fibrosis marker proteins α-SMA and COL1A1 in the model group mice was significantly increased compared with the control group, while the OG group could inhibit the expression of α-SMA and COL1A1 in the lungs of model mice (*P<0.05, **P<0.01VS model group).

[0164] (3) ELISA method for determination of mouse serum HA content

[0165] The serum of mice in each group was collected and the HA content in the serum was determined by ELISA experiment. The operation steps were the same as Example 4.

[0166] Experimental results: Figure 6 C shows that the HA content in the serum of the model group mice was significantly higher than that of the control group, while the OG group was able to reduce the HA content in the serum of the model mice, with the medium and high dose groups showing a more obvious trend.

[0167] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. An application of a small molecule for targeted inhibition of hyaluronan synthase 2, characterized in that: The small molecule is orcinol glucoside, and the small molecule is used to target hyaluronan synthase 2 protein to inhibit the synthesis of hyaluronan.

2. A method for verifying the application of a small molecule that targets and inhibits hyaluronan synthase 2, characterized in that: The method comprises the following steps: Protein preparation: Download the predicted three-dimensional structure of Human hyaluronan synthase 2, use the Protein Preparation Wizard module to hydrogenate the protein, then perform energy optimization, and use the Receptor Grid Generation module to create a grid file centered on amino acid residues CYS279 / SER310 / LYS190; Compound preparation: The 2D format of orcinol glucoside was prepared by The software LigPrep Module performs hydrogenation and energy optimization and outputs 3D structures for molecular docking. Molecular docking: The prepared compounds are imported and molecular docking is performed using the Glide module, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching, and the docking score is calculated.

3. A method for verifying the application of a small molecule that targets and inhibits hyaluronan synthase 2, characterized in that: The method comprises the following steps: Chip preparation: Prior to injection, the activator was prepared by mixing 400 mM ethylenediaminetetraacetic acid with 100 mM N-hydroxysuccinimide; the mixture was injected into the Fc1 and Fc2 sample channels at a flow rate of 10 μL / min for 800 s; then, 10 mM sodium acetate (pH 4.0) was added to hyaluronic acid S2 at a concentration of 100 μg / mL and injected into the Fc2 sample channel for 300 s at a flow rate of 10 μL / min, with a fixed level of approximately 10993 RU; the chip was deactivated by injecting 1 M ethanolamine hydrochloride into the Fc1 and Fc2 sample channels at a flow rate of 10 μL / min for 800 s; Analytes were run by kinetics / affinity: Orcinol glucoside was diluted with running buffer to seven concentrations: 1250, 625, 312.5, 156.3, 78.2, 39.1, 19.6, and 0 μM; Orcinol glucoside was injected into the Fc1-Fc2 of the channel at a flow rate of 30 μL / min for a 60 s association phase followed by a 90 s dissociation phase; both association and dissociation processes were processed in running buffer.

4. A method for verifying the application of a small molecule that targets and inhibits hyaluronan synthase 2, characterized in that: The method comprises the following steps: Induced cells: NIH / 3T3 cells were stimulated with 5 ng / ml TGF-β1 for 24 h; Protein extraction: Use cell scraper to collect induced NIH / 3T3 cells, wash once with cold PBS, add cold PBS containing protease inhibitors to resuspend the cells, and freeze and thaw the cell suspension three times with liquid nitrogen; centrifuge to obtain the supernatant to separate the soluble lysate from the cell debris; determine the protein concentration by BCA method, and then dilute the quantified protein to 4μg / μl; Drug treatment: The quantified protein lysate was divided into two equal parts and placed in 1.5 ml centrifuge tubes, divided into a drug treatment group and a negative control group; the drug treatment group treated NIH / 3T3 cells with orcinol glucoside at a final concentration of 100 μM, and the negative control group treated with DMSO and incubated at room temperature for 1 hour; Heating treatment: resuspend the lysate after administration by pipetting, and then divide the lysate into 12 equal parts and place them in PCR tubes; use a PCR thermal cycler to heat at different temperatures for 3 minutes: 37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67°C, 70°C, a total of 12 temperatures, with an interval of 3°C for each group, and then cool at room temperature for 3 minutes, and then temporarily store on ice; Preparation of loading protein: After centrifugation of the above samples, take the supernatant, add 5× loading buffer, mix well, and heat at 100°C for 10 minutes to denature the protein; Western blot: Take 10 μl of protein sample and load it in the order of treatment temperature from low to high.

5. A method for verifying the application of a small molecule that targets and inhibits hyaluronan synthase 2, characterized in that: The method comprises the steps of: The CCK-8 method was used to detect the effect of orcinol glucoside on the proliferation of mouse embryonic fibroblast cell line NIH / 3T3 cells and human fibroblast HFL-1 cells.

6. The verification method for the application of a small molecule for targeted inhibition of hyaluronan synthase 2 according to claim 5, characterized in that: The steps of detecting the effect of orcinol glucoside on the proliferation of mouse embryonic fibroblast cell line NIH / 3T3 cells and human fibroblast HFL-1 cells by CCK-8 method are as follows: Seeding: Seed the corresponding number of cells in a 96-well plate at 3000 cells per well; Drug addition and incubation: Add drugs of corresponding concentration to each well and incubate in a cell culture incubator for 48 hours; Add 10ul CCK-8 solution to each well; Continue incubating in the cell culture incubator for 1-4 hours and use an ELISA reader to detect the absorbance of the 96-well plate at 450 nm.

7. A method for verifying the application of a small molecule that targets and inhibits hyaluronan synthase 2, characterized in that: The method comprises the steps of: Effect of orcinol glucoside on the production of hyaluronic acid in NIH / 3T3 cells and HFL-1 cells induced by TGF-β1 was evaluated by ELISA.

8. The verification method for the application of a small molecule for targeted inhibition of hyaluronan synthase 2 according to claim 7, characterized in that: The steps of affecting the production of hyaluronic acid by TGF-β1-induced NIH / 3T3 cells and HFL-1 cells by ELISA are as follows: Cell administration and sample collection: NIH / 3T3 cells or HFL-1 cells were seeded in culture plates, and NIH / 3T3 cells and HFL-1 cells were stimulated with 5 ng / ml TGF-β1, and different concentrations of orcinol glucoside were given for intervention. After 48 hours, the cell culture medium was collected and the hyaluronic acid content was detected by ELISA. Wash the plate and incubate the samples: wash the plate 3 times with 300 μl of washing buffer per well, pat dry, add 100 μl of sample to each well, and incubate at room temperature for 2 hours; Wash the plate and incubate with biotinylated antibody working solution: wash the plate 4 times with 300 μl of washing buffer per well, pat dry, and incubate with 100 μl of biotinylated antibody working solution per well at room temperature for 1 hour; Wash the plate and incubate the enzyme conjugate working solution: wash the plate 4 times with 300μl of washing buffer per well, pat dry, and incubate each well with 100μl of enzyme conjugate working solution at room temperature for 30 minutes; Wash the plate and incubate the color substrate: wash the plate 5 times with 300μl washing buffer per well, pat dry, and incubate the plate with 100μl color substrate per well at room temperature for 30 minutes; Stop color development: Add 50 μl of stop solution to each well and detect the absorbance at 450 nm using an ELISA reader.

9. A method for verifying the application of a small molecule that targets and inhibits hyaluronan synthase 2, characterized in that: The method comprises the steps of: Animal modeling and drug administration; The experimental animals were divided into 5 groups, with 6 mice in each group. Groups 1 to 5 were: normal control group, model group, orcinol glucoside low concentration group, orcinol glucoside medium concentration group and orcinol glucoside high concentration group; Group 6 was pirfenidone positive drug group; after anesthesia, bleomycin modeling was performed on groups 2-6, first anesthetized with isoflurane, and 2.5 mg / kg bleomycin was injected through the trachea to establish a pulmonary fibrosis mouse model; the normal control group mice were injected with an equal amount of normal saline as a control; on the second day of modeling, mice in groups 3-6 began to receive oral gavage of corresponding drugs once a day for 2 consecutive weeks; groups 1-2 were gavaged with an equal amount of normal saline; after 2 weeks, samples were collected; HE and Masson's methods were used to observe the pathological changes and fibrosis degree of mouse lung tissue; Lung tissues of mice in each group were collected, and HE and Masson methods were used to observe the pathological changes and fibrosis degree of the lungs of mice in each group. The steps are as follows: HE part: Fixation and dehydration: The lung tissues removed from the dissected mice were fully immersed in 4% paraformaldehyde for 24 hours, immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for dehydration for 24 hours; Embedding: trim the tissue, preheat the embedding machine to melt the paraffin, place the tissue on the mold, fully infiltrate the liquid paraffin, wait for it to cool, and store the embedded tissue at -20℃ to harden for subsequent sectioning; Sectioning: Fix the sample in a semi-automatic paraffin slicer, install the blade, and start slicing. The slice thickness is 4μm. Quickly pick up the cut wax slice with tweezers and spread it in the sink. Use a glass slide to pick up the slice and lay it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and place it on the baking machine to dry the water. Dewaxing: After drying the slices, place them in an oven at 60°C for 2-3 hours, take them out for dewaxing, first soak them in xylene for 10 minutes, then take them out and soak them in new xylene for 10 minutes, then soak them in anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes in sequence, and finally soak them in ultrapure water for 5 minutes twice; Hematoxylin nuclear staining: Place the sections in hematoxylin for 5-8 minutes, wash with tap water, and then soak in 1% hydrochloric acid ethanol for 5-10 seconds, and wash with tap water to turn blue; Eosin staining: Place the sections in eosin staining solution for 1-3 minutes; Dehydration: Then soak in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 3 minutes in sequence, then soak the slices in xylene for 10 minutes, take them out and soak them in new xylene for 10 minutes; Sealing: air-dry xylene in room temperature slices, add neutral gum, cover with coverslip, spread the neutral gum evenly, make sure there are no bubbles on the tissue, and dry in a ventilated place; Masson part: Fixation and dehydration: The lung tissues removed from the dissected mice were fully immersed in 4% paraformaldehyde for 24 hours, immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for dehydration for 24 hours; Embedding: trim the tissue, preheat the embedding machine to melt the paraffin, place the tissue on the mold, fully infiltrate it with liquid paraffin, wait for it to cool, and store the embedded tissue at -20℃ to harden for subsequent sectioning;. Sectioning: Fix the sample in a semi-automatic paraffin slicer, install the blade, and start slicing. The slice thickness is 4μm. Quickly pick up the cut wax slice with tweezers and spread it in the sink. Use a glass slide to pick up the slice and lay it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and place it on the baking machine to dry the water. Dewaxing: After drying the slices, place them in an oven at 60°C for 2-3 hours, take them out for dewaxing, first soak them in xylene for 10 minutes, then take them out and soak them in new xylene for 10 minutes, then soak them in anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes in sequence, and finally soak them in ultrapure water for 5 minutes twice; Chromization treatment or removal of mercury salt precipitation, washing with tap water and distilled water in turn; Stain the nuclei with hematoxylin or iron hematoxylin stain for 5 to 10 minutes; Differentiate with 1% hydrochloric acid alcohol for 5-15 seconds, and then wash thoroughly with water; Add 0.1-1% lithium carbonate dropwise for 5 minutes to increase the degree of blueing, and wash with water; Use Masson Ponceau acid red solution for 5 to 10 minutes; Wash with 2% glacial acetic acid aqueous solution for 1 minute; Differentiate with 1% phosphomolybdic acid aqueous solution for 3-5 minutes, and wash with 2% glacial acetic acid aqueous solution for 1 minute; Without washing, dye directly with aniline blue aqueous solution or 1% light green aqueous solution for 1 to 2 minutes; Wash with 0.2% glacial acetic acid aqueous solution for 1 minute; Dehydration: Then soak in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 3 minutes in sequence, then soak the slices in xylene for 10 minutes, take them out and soak them in new xylene for 10 minutes; Sealing: air-dry xylene in room temperature slices, add neutral gum, cover with coverslip, spread the neutral gum evenly, make sure there are no bubbles on the tissue, and dry in a ventilated place; Western Blot was used to detect the expression of α-SMA and COL1A1 in mouse lung tissue: Protein extraction and denaturation: 50 g lung tissue of each mouse was weighed and immersed in 400 μl lysis buffer, three zircons were added, and the mixture was ground in a grinder for 3 minutes 3 times. The grinding solution was placed on ice for 30 minutes and then centrifuged at 14000 rpm for 30 minutes. The supernatant was taken and the protein concentration was measured. Then, loading buffer was added and the mixture was placed in a metal bath at 85°C for 10 minutes. Electrophoresis: Prepare electrophoresis gel, add 30 μg protein to each well, and run electrophoresis in a 100 V constant voltage electric field for 90 minutes; Transfer: Take out the gel and put it close to the PVDF membrane in the transfer clip, and transfer the membrane in a 300mA constant current electric field for 2 hours; Blocking: Take out the PVDF membrane and soak it in 5% skim milk, and place it on a shaker for 1 hour; Incubation with antibodies: wash the membrane with TBST for 5 minutes 3 times, add primary antibody and incubate at 4°C overnight, recover primary antibody, wash the membrane with TBST for 5 minutes 3 times, add secondary antibody and incubate at room temperature for 1 hour; Chemiluminescence: Place the membrane in an automatic chemiluminometer, evenly add luminescent liquid, and avoid light to emit light; ELISA method to determine the content of hyaluronic acid in mouse serum: The serum of mice in each group was collected, and the ELISA experiment was performed to determine the hyaluronic acid content in the serum.

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