Application and validation methods of small molecules that target and inhibit hyaluronic acid synthase 2

Abstract

This invention relates to the application and verification method of a small molecule that targets and inhibits hyaluronic acid synthase 2. The small molecule is phytohexene glucoside, which is used to target the hyaluronic acid synthase 2 protein and inhibit hyaluronic acid synthesis. The inventors discovered through molecular docking, surface plasmon resonance experiments, and cell thermal migration experiments that phytohexene glucoside exhibits high binding force and affinity for hyaluronic acid synthase 2, demonstrating its potential to target hyaluronic acid synthase 2. Subsequent enzyme-linked immunosorbent assay (ELISA) results showed that phytohexene glucoside can inhibit the increase in hyaluronic acid expression in fibroblasts induced by transforming growth factor-β1. Animal models were used to confirm the anti-pulmonary fibrosis effect of phytohexene glucoside and its inhibitory effect on hyaluronic acid in pulmonary fibrosis mice. Phytohexene glucoside can specifically target hyaluronic acid synthase 2, thereby inhibiting hyaluronic acid secretion and improving pulmonary fibrosis.

Description

Technical Field

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

[0002] Idiopathic pulmonary fibrosis (IPF) is the most common type of idiopathic interstitial pneumonia. It is a chronic, progressive, fibrotic, and unexplained interstitial lung disease, typically characterized by distinctive imaging and histological features, and primarily affects middle-aged and elderly individuals. IPF is mainly characterized by dry cough and exertional dyspnea, and in its later stages, it often leads to heart and lung failure. It has a high mortality rate and is often referred to as a "neoplastic disease," with an average lifespan 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 progression of pulmonary fibrosis is primarily driven by the differentiation of fibroblasts into myofibroblasts, which produce large amounts of collagen and extracellular matrix proteins, leading to tissue and organ dysfunction.

[0003] Currently, only two drugs for treating pulmonary fibrosis are available: pirfenidone and nintedanib. Although both can slow disease progression, they cannot cure the disease. Therefore, effective pharmacological approaches are still needed for the treatment of pulmonary fibrosis, particularly to prevent fibroblasts from differentiating into myofibroblasts and the resulting excessive deposition of extracellular matrix proteins.

[0004] Hyaluronic acid (HA) is a glycosaminoglycan composed of disaccharides (D-glucuronic acid and N-acetylglucosamine) as its basic units. It is also known as hyaluronic acid. HA is a ubiquitous natural glycosaminoglycan and a major component of the extracellular matrix, participating in biological processes such as water homeostasis, matrix signaling, tissue healing, inflammation, angiogenesis, and cell proliferation and migration. In the lungs, HA is secreted by type II lung fibroblasts, lung epithelial cells, and endothelial cells. Fibroblasts can be stimulated by pathogenic factors to synthesize large amounts of HA. In IPF tissue samples and animal models, HA levels are significantly higher than in normal tissues and are positively correlated with the severity of IPF. In IPF animal models, elevated HA levels can widely induce inflammatory responses in lung tissue. HA activates mouse fibroblasts through the CD44 receptor, promoting fibroblast proliferation and participating in the process of pulmonary fibrosis. Furthermore, HA can activate the CD44 / S100A4 axis, affecting the differentiation of mesenchymal stem cells into fibroblasts and promoting pulmonary fibrosis. Studies have found that HA is also involved in the development of pulmonary fibrosis and emphysema.

[0005] Hyaluronic acid synthase (HAS) controls HA synthesis and mainly includes three isoforms: HAS1, HAS2, and HAS3. HAS2 is the major isoform responsible for HA production in mesenchymal cells. Previous research by the inventors found high expression of HAS2 in the lungs of IPF patients, the lungs of mouse models of pulmonary fibrosis, and myofibroblasts induced by transforming growth factor-β1 (TGF-β1). Existing research indicates that overexpression of HAS2 in myofibroblasts promotes an aggressive phenotype, leading to severe fibrosis. Therefore, HAS2 may be a key determinant of fibrosis fate, and inhibiting HAS2 expression can reduce HA synthesis, thereby treating pulmonary fibrosis. Therefore, intervening in the HAS2 target to inhibit HA production and release is a potential treatment for IPF. To address the issue of intervening in the HAS2 target to inhibit HA production and release, the applicant has proposed an application and validation method for a small molecule that targets and inhibits hyaluronic acid 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 that targets and inhibits hyaluronic acid synthase 2, and to provide a method for verifying the application of the small molecule that targets and inhibits hyaluronic acid synthase 2, in view of the above-mentioned defects of the prior art.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] An application of a small molecule that targets and inhibits hyaluronic acid synthase 2 is described, wherein the small molecule is orcinol glucoside (OG), and the small molecule is used to target HAS2 protein to inhibit HA synthesis.

[0009] A method for validating the application of a small molecule that targets and inhibits hyaluronic acid synthase 2, wherein the method includes 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: The 2D format of the OG was processed via... The LigPrep Module software performs hydrogenation and energy optimization processing, and outputs 3D structures for molecular docking.

[0012] Molecular docking: The prepared compound is imported, and molecular docking is performed using the Glide module. This means that the acceptor and ligand molecules dock with each other through geometric matching and energy matching, and the docking score is calculated.

[0013] A method for validating the application of a small molecule that targets and inhibits hyaluronic acid synthase 2, wherein the method includes the following steps:

[0014] Chip preparation: Before injection, an activator was prepared by mixing 400 mM ethylenediaminetetraacetic acid (EDTA) 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 at a flow rate of 10 μL / min for 300 s to fix the level at approximately 10993 RU; the chip was deactivated by continuously injecting 1 M ethanolamine hydrochloride into the Fc1 and Fc2 sample channels at a flow rate of 10 μL / min for 800 s.

[0015] Run the analyte by kinematics / affinity: Dilute OG with run buffer to seven concentrations: 1250, 625, 312.5, 156.3, 78.2, 39.1, 19.6, and 0 μM; inject OG into channels Fc1-Fc2 at a flow rate of 30 μL / min for a 60-second binding phase followed by a 90-second dissociation phase; both binding and dissociation processes are handled in run buffer.

[0016] A method for validating the application of a small molecule that targets and inhibits hyaluronic acid synthase 2, wherein the method includes the following steps:

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

[0018] Protein extraction: NIH / 3T3 cells were collected after induction using a cell scraper and washed once with cold PBS. The cells were resuspended in cold PBS containing protease inhibitors and the cell suspension was repeatedly frozen and thawed three times using liquid nitrogen. The supernatant was collected by centrifugation to separate soluble lysates from cell debris. The protein concentration was determined by the BCA method, and the quantified protein was then diluted to 4 μg / μl.

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

[0020] Heat treatment: The lysate after drug administration was resuspended by pipetting, and then each lysate was divided into 12 equal portions and placed in PCR tubes; each tube was individually heated for 3 minutes at different temperatures using a PCR thermal cycler: 37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67°C, and 70°C, for a total of 12 temperatures, with each group spaced 3°C apart. The tubes were then cooled at room temperature for 3 minutes and then temporarily stored on ice.

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

[0022] Western blot: Load 10 μl of protein sample in order of increasing processing temperature.

[0023] A method for validating the application of a small molecule that targets and inhibits hyaluronic acid synthase 2, wherein the method includes the following steps:

[0024] The effects of OG on the proliferation of mouse embryonic fibroblast line NIH / 3T3 cells and human fibroblast HFL-1 cells were detected using the CCK-8 assay.

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

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

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

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

[0029] Continue incubation in a cell culture incubator for 1-4 hours, and then use a microplate reader to detect the absorbance of the 96-well plate at 450 nm.

[0030] A method for validating the application of a small molecule that targets and inhibits hyaluronic acid synthase 2, wherein the method includes the following steps:

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

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

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

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

[0035] Washing buffer 300 μl per well for 4 washes, blot dry, then incubate at room temperature for 1 hour with 100 μl of biotinylated antibody working solution per well.

[0036] Washing buffer 300 μl per well for 4 washes, pat dry, then incubate with enzyme conjugate working solution 100 μl per well at room temperature for 30 minutes.

[0037] Washing buffer 300 μl per well for 5 washes, then blot dry and incubate with 100 μl of chromogenic substrate per well at room temperature for 30 minutes.

[0038] Stop color development: 50 μl of stop solution per well, and measure absorbance using a microplate reader at 450 nm.

[0039] A method for validating the application of a small molecule that targets and inhibits hyaluronic acid synthase 2, wherein the method includes the following steps:

[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, low concentration OG group, medium concentration OG group, and high concentration OG group, respectively; Group 6 was the pirfenidone positive control group. After anesthesia, bleomycin was used to establish the pulmonary fibrosis mouse model in groups 2-6. Mice were first anesthetized with isoflurane, and 2.5 mg / kg of bleomycin was injected intratracheally to establish the pulmonary fibrosis mouse model. Mice in the normal control group were injected with an equal volume of physiological saline as a control. On the second day after modeling, mice in groups 3-6 began to receive the corresponding drug treatment orally by gavage once a day for 2 weeks; mice in groups 1-2 were gavaged with an equal volume of physiological saline. After 2 weeks, samples were collected.

[0042] Observe the pathological changes and degree of fibrosis in mouse lung tissue using HE and Masson methods;

[0043] Lung tissues were collected from each group of mice, and pathological changes and the degree of fibrosis in the lungs of each group of mice were observed by HE and Masson's method. The steps are as follows:

[0044] HE section:

[0045] Fixation and dehydration: Lung tissues taken from dissected mice were fully immersed in 4% paraformaldehyde for 24 hours for fixation, then immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for 24 hours for dehydration.

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

[0047] Sectioning: Fix the sample in a semi-automatic paraffin microtome, load the blade, and start slicing. The section thickness is 4μm. Quickly pick up the cut paraffin section with tweezers and spread it in the water tank. Use a glass slide to pick up the section and spread it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and then place it on a slide dryer to dry the water.

[0048] Dewaxing: After drying the moisture, place the slices in an oven at 60℃ for 2-3 hours, then remove them for dewaxing. First, soak the slices in xylene for 10 minutes, then remove them 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 and violet staining: Immerse the sections in hematoxylin and violet for 5-8 minutes, rinse with tap water, then soak in 1% hydrochloric acid ethanol for 5-10 seconds, rinse with tap water to restore blue color.

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

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

[0052] Mounting: Air dry the sections in xylene at room temperature, add a drop of neutral resin, cover with a coverslip, spread the neutral resin evenly, ensuring there are no air bubbles on the tissue, and air dry in a ventilated place;

[0053] Masson section:

[0054] Fixation and dehydration: Lung tissues taken from dissected mice were fully immersed in 4% paraformaldehyde for 24 hours for fixation, then immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for 24 hours for dehydration.

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

[0056] Sectioning: Fix the sample in a semi-automatic paraffin microtome, load the blade, and start slicing. The section thickness is 4μm. Quickly pick up the cut paraffin section with tweezers and spread it in the water tank. Use a glass slide to pick up the section and spread it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and then place it on a slide dryer to dry the water.

[0057] Dewaxing: After drying the moisture, place the slices in an oven at 60℃ for 2-3 hours, then remove them for dewaxing. First, soak the slices in xylene for 10 minutes, then remove them 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] Chromating treatment or removal of mercury salt precipitation, followed by washing with tap water and distilled water in sequence;

[0059] Stain the nucleus with Regaud hematoxylin staining solution or Weigert iron hematoxylin staining solution for 5-10 minutes;

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

[0061] Add 0.1-1% lithium carbonate for 5 minutes to increase the blueing effect, then rinse with water;

[0062] Use Masson's Ponceau Acid Flavescent Solution for 5-10 minutes;

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

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

[0065] Without rinsing with water, directly stain with aniline blue solution or 1% light green solution for 1-2 minutes; then wash with 0.2% glacial acetic acid solution for 1 minute.

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

[0067] Mounting: Air dry the sections in xylene at room temperature, add a drop of neutral resin, cover with a coverslip, spread the neutral resin evenly, ensuring there are no air bubbles on the tissue, and air dry in a ventilated place;

[0068] Western blot analysis of α-SMA and COL1A1 expression in mouse lung tissue:

[0069] Protein extraction and denaturation: Weigh 50g of lung tissue from each mouse and soak it in 400μl of lysis buffer. Add three zircon grains and grind in a grinder for 3 minutes three times. After the lysis buffer is placed on ice for 30 minutes, centrifuge at 14000rpm for 30 minutes at low temperature. Take the supernatant, measure the protein concentration, add loading buffer, and incubate in a metal bath at 85℃ for 10 minutes.

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

[0071] Transfer: Take out the gel and place it in close contact with the PVDF membrane in the transfer clamp, and transfer the membrane in a constant current electric field of 300mA for 2 hours;

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

[0073] Antibody incubation: Wash the membrane with TBST for 5 minutes 3 times, add primary antibody and incubate overnight at 4°C, recover the 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: The membrane is placed in an automated chemiluminescence analyzer, and the luminescent liquid is added evenly and emitted in the dark.

[0075] ELISA method for determining serum HA content in mice:

[0076] Serum from each group of mice was collected, and the HA content in the serum was determined by ELISA.

[0077] The beneficial effects of this invention are as follows: Through molecular docking, surface plasmon resonance experiments, and cell thermal migration experiments, the inventors discovered that OG exhibits high binding and affinity for HAS2, demonstrating its potential to target HAS2. Furthermore, subsequent ELISA results showed that OG can inhibit the increase in HA expression in fibroblasts induced by TGF-β1. Animal models were used to confirm the anti-pulmonary fibrosis effect of OG and its inhibitory effect on HA in pulmonary fibrosis mice. Therefore, OG can specifically target HAS2, thereby inhibiting HA secretion and improving pulmonary fibrosis. Attached Figure Description

[0078] To more clearly illustrate the technical solutions in the embodiments of the present invention or 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 skilled in the art, other drawings can be obtained based on these drawings without creative effort:

[0079] Figure 1 This is a schematic diagram of the molecular formula of OG.

[0080] Figure 2 This is a schematic diagram showing the results of determining the molecular docking fraction between 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 in one embodiment of the present invention, confirming the binding effect between OG and HAS2.

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

[0083] Figure 5 This is a diagram showing the results of finding that OG can inhibit the production of HA by myofibroblasts in one embodiment of the present invention.

[0084] Figure 6 This is a graph showing the results of ELISA assay to determine the inhibitory effect of oral OG on serum HA levels in mice with bleomycin-induced pulmonary fibrosis, according to one embodiment of the present invention. Detailed Implementation

[0085] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, a clear and complete description will be provided below in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0086] The preferred embodiment of the present invention relates to the application of a small molecule that targets and inhibits hyaluronic acid synthase 2. The small molecule is OG and is used to target HAS2 protein to inhibit HA synthesis.

[0087] OG is a phenolic glycoside isolated from the traditional Chinese medicine *Curculigo orchioides*, possessing various pharmacological effects including anti-inflammatory, antioxidant, and antidepressant properties; a molecular formula diagram of OG can be found here. Figure 1 ;

[0088] The inventors discovered through molecular docking, surface plasmon resonance experiments, and cell thermal migration experiments that OG exhibits high binding affinity and activity to HAS2, demonstrating its potential to target HAS2. Subsequent enzyme-linked immunosorbent assay (ELISA) results showed that OG can inhibit TGF-β1-induced increases in HA expression in fibroblasts. Animal models were used to confirm the anti-pulmonary fibrosis effect of OG and its inhibitory effect on HA in pulmonary fibrosis mice. Therefore, OG can specifically target HAS2, thereby inhibiting HA secretion and improving pulmonary fibrosis.

[0089] The specific verification method is explained in the following instructions:

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

[0091] This embodiment uses molecular docking to determine the molecular docking between OG small molecules and HAS2 protein. The principle and steps are briefly described below:

[0092] 1. Protein Preparation: The predicted 3D structure of Human HAS2 was downloaded from the AlphaFold website (AlphaFoldID: AF-Q92819-F1). Hydrogenation of the protein was performed using the Protein Preparation Wizard module, followed by energy optimization (OPLS2005 force field, RMSD: ). A grid file was created using the Receptor Grid Generation module, centered on amino acid residues CYS279 / SER310 / LYS190, with all boxes set to a specific size.

[0093] 2. Compound preparation: Convert the OG 2D format through... The LigPrep Module software performs hydrogenation, energy optimization, and other processing to output a 3D structure for molecular docking.

[0094] 3. Molecular docking: The prepared compound is imported, and molecular docking is performed using the Glide module. This means that the acceptor and ligand molecules dock with each other through geometric matching and energy matching, and the docking score is calculated.

[0095] Experimental results: see Figure 2 The molecular docking fraction between OG and HAS2 was -7.325, indicating a good binding affinity between OG and HAS2. 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 (distances shown in the 3D interaction diagram); the phenolic hydroxyl group acts as a hydrogen bond donor, forming one hydrogen bond with ASP314 (distance is...). In addition, the benzene ring can form two π-π interactions with TRP354.

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

[0097] In this embodiment, the molecular affinity between OG small molecules and HAS2 protein was determined using a surface plasmon resonance experiment. The steps are briefly described below:

[0098] 1. Chip Preparation (HAS2): Before injection, an activator was prepared by mixing 400 mM ethylenediaminetetraacetic acid (EDTA) 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 at a flow rate of 10 μL / min for 300 s to fix the level at approximately 10993 RU. The chip was deactivated by continuously injecting 1 M ethanolamine hydrochloride into the Fc1 and Fc2 sample channels at a flow rate of 10 μL / min for 800 s.

[0099] 2. Running the analyte by kinematics / affinity: Dilute the OG with running buffer (1*PBS-P, 5% DMSO buffer, ethanolamine hydrochloride) to seven concentrations (1250, 625, 312.5, 156.3, 78.2, 39.1, 19.6, and 0 μM). Inject the OG into channels Fc1-Fc2 at a flow rate of 30 μL / min for a 60-second binding phase, followed by a 90-second dissociation phase. Both binding and dissociation processes are handled in running buffer.

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

[0101] Example 3: The results of cell thermal migration technology confirming the direct binding of OG to HAS2 protein.

[0102] 1. Cell induction: NIH / 3T3 cells were stimulated with 5 ng / ml TGF-β1 for 24 hours;

[0103] 2. Protein Extraction: NIH / 3T3 cells were collected using a cell scraper and washed once with cold PBS. The cells were resuspended in cold PBS containing a protease inhibitor (protease inhibitor:PBS = 1:100). The cell suspension was repeatedly frozen and thawed three times using liquid nitrogen. The cells were centrifuged (4℃, 20000×g, 20 min) to separate soluble lysates from cell debris. Protein concentration was determined using the BCA method, and the quantified protein was then diluted to 4 μg / μl.

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

[0105] 4. Heat treatment: Resuspend the lysate after drug administration by pipetting, and then divide the lysate into 12 equal portions and place them in PCR tubes. Heat each tube individually at different temperatures for 3 minutes using a PCR thermal cycler (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 a 3°C interval between each group), then cool at room temperature for 3 minutes, and then temporarily store on ice.

[0106] 5. Preparation of loading protein: After centrifuging the above sample (4℃, 20000×g, 20 minutes), take the supernatant, add 5× loading buffer, mix well, and heat at 100℃ for 10 minutes to denature the protein.

[0107] 6. Western blot: Load 10 μl of protein sample in order of increasing processing temperature.

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

[0109] Example 4: Results of OG inhibiting HA production by myofibroblasts.

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

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

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

[0113] 2. Drug incubation: Add the corresponding concentration of drug 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 10 μL of CCK-8 solution to each well.

[0115] 4. Continue incubation in a cell culture incubator for 1-4 hours, and then use a microplate reader to detect the absorbance of the 96-well plate at 450 nm.

[0116] Experimental results: Figure 4 A and Figure 4 B showed that OG at various concentrations had no significant cytotoxic effect on NIH / 3T3 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] This embodiment uses the ELISA method (OG) to investigate the effect of TGF-β1-induced HA production in NIH / 3T3 and HFL-1 cells. The steps are briefly described below:

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

[0120] 2. Washing and incubating samples: Wash the plate 3 times per well with 300 μl of washing buffer, pat dry, add 100 μl of sample to each well, and incubate at room temperature for 2 hours.

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

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

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

[0124] 6. Stop color development: 50 μl of stop solution per well, and measure the absorbance with a microplate reader at 450 nm.

[0125] Experimental results: Figure 5 C and Figure 5 D showed that OG could dose-dependently inhibit HA secretion 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: Experimental animals were divided into 5 groups, with 6 mice in each group. 1. Normal control group; 2. Model group; 3. Low concentration OG group (25 mg / kg); 4. Medium concentration OG group (50 mg / kg); 5. High concentration OG group (100 mg / kg); 6. Pirfenidone positive control group. After anesthesia, groups 2-6 underwent bleomycin modeling. Mice were first anesthetized with isoflurane, and 2.5 mg / kg of bleomycin was injected intratracheally to establish a mouse model of pulmonary fibrosis. Mice in the normal control group were injected with an equal volume of physiological saline as a control. On the second day after modeling, mice in groups 3-6 began to receive the corresponding drug treatment by oral gavage once a day for 2 consecutive weeks; mice in groups 1-2 were given an equal volume of physiological saline by gavage. After 2 weeks, samples were collected.

[0128] (2) HE and Masson's method were used to observe pathological changes and degree of fibrosis in mouse lung tissue.

[0129] Lung tissues were collected from each group of mice, and pathological changes and the degree of fibrosis in the lungs of each group of mice were observed by HE and Masson's method. The steps are briefly described below:

[0130] HE section:

[0131] 1. Fixation and dehydration: Lung tissues taken from dissected mice were fully immersed in 4% paraformaldehyde for 24 hours for fixation, then immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for 24 hours for dehydration.

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

[0133] 3. Sectioning: Fix the sample in a semi-automatic paraffin microtome, attach the blade, and start slicing. The section thickness is 4μm. Quickly pick up the cut paraffin section with tweezers and spread it in the water tank. Use a glass slide to pick up the section and spread it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and then place it on a slide dryer to dry.

[0134] 4. Dewaxing: After drying the slices, bake them in an oven at 60℃ for 2-3 hours. Then, remove them and dewax them. First, soak the slices in xylene for 10 minutes, then remove them and soak them in fresh xylene for 10 minutes. Then, soak them in anhydrous ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 minutes in sequence. Finally, soak them in ultrapure water for 5 minutes twice.

[0135] 5. Hematoxylin and violet staining: Immerse the sections in hematoxylin and violet for 5-8 minutes, rinse with tap water, then soak in 1% hydrochloric acid ethanol for 5-10 seconds, rinse with tap water to restore blue color.

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

[0137] 7. Dehydration: Then soak the slices in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 3 minutes in sequence. Then soak the slices in xylene for 10 minutes. Remove them and soak them in fresh xylene for 10 minutes.

[0138] 8. Mounting: Air dry the sections in xylene at room temperature, add neutral resin, cover with a coverslip, spread the neutral resin evenly, ensuring there are no air bubbles on the tissue, and air dry in a ventilated place.

[0139] Masson section:

[0140] 1. Fixation and dehydration: Lung tissues taken from dissected mice were fully immersed in 4% paraformaldehyde for 24 hours for fixation, then immersed in 75% ethanol for 24 hours, and then placed in a dehydrator for 24 hours for dehydration.

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

[0142] 3. Sectioning: Fix the sample in a semi-automatic paraffin microtome, attach the blade, and start slicing. The section thickness is 4μm. Quickly pick up the cut paraffin section with tweezers and spread it in the water tank. Use a glass slide to pick up the section and spread it flat on the glass slide. Gently shake off the water, mark the information with a pencil, and then place it on a slide dryer to dry.

[0143] 4. Dewaxing: After drying the slices, bake them in an oven at 60℃ for 2-3 hours. Then, remove them and dewax them. First, soak the slices in xylene for 10 minutes, then remove them and soak them in fresh xylene for 10 minutes. Then, soak them in anhydrous ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 minutes in sequence. Finally, soak them in ultrapure water for 5 minutes twice.

[0144] 5. Chromate treatment or remove mercury salt precipitation, then wash with tap water and distilled water in sequence.

[0145] 6. Stain the nucleus with Regaud hematoxylin staining solution or Weigert iron hematoxylin staining solution for 5-10 minutes.

[0146] Differentiate with 7.1% hydrochloric acid alcohol for 5-15 seconds, then rinse thoroughly with water.

[0147] 8. Add 0.1-1% lithium carbonate for 5 minutes to increase the blueing effect, then rinse with water.

[0148] 9. Apply Masson's Ponceau Acid Fructus for 5-10 minutes.

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

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

[0151] 12. Without rinsing with water, directly stain with aniline blue solution or 1% light green solution for 1-2 minutes; then wash with 0.2% glacial acetic acid solution for 1 minute.

[0152] 13. Dehydration: Then soak the slices in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 3 minutes in sequence. Then soak the slices in xylene for 10 minutes. Remove them and soak them in fresh xylene for 10 minutes.

[0153] 14. Mounting: Air dry the sections in xylene at room temperature, add neutral resin, cover with a coverslip, spread the neutral resin evenly, ensuring there are no air bubbles on the tissue, and air dry in a ventilated place.

[0154] Experimental results: such as Figure 6 As shown in Figure A, compared with the control group, the model group mice showed obvious pulmonary inflammatory infiltration and pulmonary fibrosis, while the OG group mice showed a decrease in the degree of inflammation and fibrosis in their lung tissue compared with the model group.

[0155] (3) Western blot analysis was used to detect the expression of α-SMA and COL1A1 in mouse lung tissue.

[0156] α-SMA and COL1A1 are marker proteins of fibrosis. In this example, Western blotting was used to determine the expression of α-SMA and COL1A1 proteins in the lungs of mice in each group to study the effect of oral globulin (OG) on pulmonary fibrosis. The steps are briefly described below:

[0157] 1. Protein extraction and denaturation: Weigh 50g of lung tissue from each mouse and soak it in 400μl of lysis buffer. Add three zircon grains and grind in a grinder for 3 minutes three times. After the lysis buffer is placed on ice for 30 minutes, centrifuge at 14000rpm for 30 minutes at low temperature. Take the supernatant, measure the protein concentration, add loading buffer, and incubate in a metal bath at 85℃ for 10 minutes.

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

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

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

[0161] 5. Antibody incubation: Wash the membrane with TBST for 5 minutes 3 times, add primary antibody and incubate overnight at 4°C, recover the primary antibody, wash the membrane with TBST for 5 minutes 3 times, add secondary antibody and incubate at room temperature for 1 hour.

[0162] 6. Chemiluminescence: Place the membrane in an automated chemiluminescence analyzer, add the luminescent liquid evenly, and emit light in the dark.

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

[0164] (3) ELISA method for determining serum HA content in mice

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

[0166] Experimental results: Figure 6 The results showed that the serum HA content in the model group mice was significantly higher than that in the control group, while the OG group could reduce the serum HA content in 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 modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of a phenol glucoside in the preparation of a drug for treating pulmonary fibrosis.

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