Application of methyl gallate in preparation of medicine for treating silicosis

Abstract

The invention relates to application of methyl gallate in preparation of a medicine for treating silicosis. Tests prove that the methyl gallate can relieve fibrotic nodules of mouse lung tissues, improve pulmonary alveolitis and pulmonary fibrosis degree of the lung tissues, reduce the level of TGF-beta1, and reduce mRNA expression of FN and COLI and expression of COLI, FN and alpha-SMA proteins in a silicosis mouse model, so that the methyl gallate is expected to be used for treating silicosis and has relatively high practical value.

Description

Technical Field

[0001] The invention belongs to the technical field of silicosis treatment drugs and relates to the application of methyl gallate in the preparation of silicosis treatment drugs. Background Art

[0002] Silicosis is a lung disease caused by long-term excessive exposure to SiO2 dust, characterized by chronic lung inflammation and nodular fibrosis of lung tissue. Studies have shown that SiO2 dust inhaled into the lungs is recognized and phagocytosed by alveolar macrophages, stimulating the activation of alveolar macrophages and the release of a large number of cytokines, such as interleukin-1β, tumor necrosis factor α, transforming growth factor β1, etc., causing an inflammatory response. Since SiO2 particles cannot be cleared, macrophages repeatedly phagocytose and die, leading to an inflammatory cascade and abnormal repair of tissue damage. Continuous inflammatory response and tissue damage stimulate lung fibroblasts to proliferate and activate into myofibroblasts, which over-synthesize and secrete a large amount of type I collagen, α-smooth actin, fibronectin, and a variety of extracellular matrix components, resulting in excessive deposition of extracellular matrix and unbalanced degradation, causing fibrotic lesions in lung tissue.

[0003] Methyl gallate (MG), with a structure shown in Formula I, is a polyphenol compound that is widely found in natural plants and has been shown to have a variety of biological functions, including anti-tumor, anti-inflammatory, antioxidant, neuroprotective, hepatoprotective and antibacterial activities. Studies have found that methyl gallate can scavenge ROS and inhibit oxidative stress, protecting tissues such as the liver, nervous system, skin and blood vessels. Methyl gallate regulates TLR4 / NF-κB and MAPK signaling pathways to exert anti-inflammatory activity.

[0004]

[0005] CN114028376A discloses the use of MG in the preparation of NLRP3 pathway inhibitors for hyperuricemia nephropathy and / or gouty arthritis. CN108530314A discloses a methyl gallate analog and its use in the preparation of anti-inflammatory drugs. CN114931571A discloses the use of methyl gallate in the preparation of osteoarthritis therapeutic drugs.

[0006] However, there are no reports on the use of methyl gallate in the treatment of silicosis. Summary of the invention

[0007] The inventor team of the present application studied the effects of a series of anti-inflammatory active ingredients of traditional Chinese medicine on fibroblasts at the cellular level, and unexpectedly found that methyl gallate has the potential to significantly inhibit the activation of fibroblasts. Then, the silicon dioxide-induced silicosis mouse model was used as the research object to study the intervention effect of methyl gallate on silicosis. It was found that the administration of methyl gallate can reduce the alveolitis and fibrosis scores of the mouse lung tissue, and thus can be used to treat silicosis, thereby completing the present invention.

[0008] In one aspect, the present invention provides the use of methyl gallate in preparing a medicine for preventing or treating silicosis.

[0009] In the present invention, methyl gallate reduces fibrous nodules in lung tissue.

[0010] In the present invention, methyl gallate reduces the level of TGF-β1, the expression of FN and COLⅠ mRNA in lung tissue, and the expression of COLⅠ, FN and α-SMA proteins in lung tissue.

[0011] In another aspect, the present invention provides a drug for preventing or treating silicosis, wherein the drug comprises methyl gallate.

[0012] In another aspect, the present invention provides a method for preventing or treating silicosis, comprising administering methyl gallate to a subject in need thereof.

[0013] The object here can be a human or an animal, such as a mammal, such as a human, anthropoid, monkey, pig, horse, cow, sheep, dog, cat, lion, tiger, etc., but is not limited thereto.

[0014] The drug of the present invention can be in various dosage forms conventional in the art, such as solid, semisolid or liquid forms, and can be aqueous solutions, non-aqueous solutions, suspensions, lozenges, capsules, tablets, granules, pills, powders and inhalants, etc. The administration route of the drug can be oral administration, injection administration or inhalation administration, especially oral administration. The injection administration can include intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

[0015] The drug may contain methyl gallate as an active ingredient for treating silicosis in any amount, as long as the expected preventive or therapeutic benefits can be obtained. For example, based on mass percentage, the drug may include 0.01-99.99%, such as 1% to 90%, such as 2%, 5%, 10%, 15%, 20%, 30%, 50% of methyl gallate, and 99.99-0.01%, such as 99% to 10% of pharmaceutically acceptable excipients.

[0016] The pharmaceutically acceptable conventional excipients may be selected from excipients, fillers, diluents, preservatives, colorants, flavoring agents, flavoring agents, sustained-release agents, coating agents, penetration enhancers, adhesives, wetting agents, disintegrants, surfactants, lubricants, solubilizers, fragrances, extenders, buffers, soothing agents, etc., but are not limited thereto.

[0017] In some embodiments, the medicament comprises methyl gallate as the only active ingredient for treating silicosis.

[0018] In some embodiments, the drug of the present invention may further comprise other active ingredients according to the needs of treatment or complications, etc. The other active ingredients may be other active ingredients for preventing or treating silicosis, or active ingredients of drugs for treating other diseases, disorders, or symptoms.

[0019] The dosage of the medicine according to the present invention can be selected according to the use instructions, the patient's age, sex and other conditions and the severity of the disease. For example, the daily dose of the medicine can be, based on the methyl gallate active ingredient, 0.1 mg-5000 mg / kg / day, for example 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000, 2000, 3000, 4000 mg / kg / day.

[0020] The present invention has been described in detail above, but the above embodiments are only illustrative in nature and are not intended to limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples.

[0021] Unless otherwise expressly stated, the numerical ranges throughout the application documents include any subranges therein and any numerical values ​​incremented by the smallest subunit of a given value therein. Unless otherwise expressly stated, the numerical values ​​throughout the application documents represent approximate measurements or limitations of the range of embodiments including slight deviations from the given values ​​and having approximately the values ​​mentioned and having the exact values ​​mentioned. Except for the working examples provided at the end of the detailed description, all numerical values ​​of parameters (e.g., quantities or conditions) in this application document (including the appended claims) should be understood in all cases as modified by the term "approximately", regardless of whether "approximately" actually appears before the numerical value. "Approximately" means that the numerical value described allows for slight imprecision (some close to precision in the value; approximately or reasonably close to the value; approximately). If the imprecision provided by "approximately" is not understood in this ordinary sense in the art, the "approximately" used herein at least represents the variation that can be produced by ordinary methods of measuring and using these parameters. For example, "approximately" can include a variation of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1% or less than or equal to 0.5%.

[0022] Beneficial Effects

[0023] The inventors have verified through experiments that methyl gallate can reduce fibrotic nodules in the lung tissue of mice with silicosis, improve the degree of alveolitis and pulmonary fibrosis in the lung tissue, reduce the level of TGF-β1, and reduce the expression of FN and COLⅠmRNA and the expression of COLⅠ, FN and α-SMA proteins. Therefore, methyl gallate is expected to be used to treat silicosis and has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1-3 The results showed that methyl gallate, ethyl gallate, gallic acid, apigenin and ginkgolide B inhibited the expression of fibroblast activation-related genes ACTA2 and FN1 mRNA and α-SMA protein expression.

[0025] Figure 4-7 The results showed that methyl gallate, ethyl gallate, gallic acid, apigenin and ginkgolide B inhibited the expression of TNF-α, IL-6 and IL-β mRNA and IL-6 protein in macrophages.

[0026] Figure 8 HE staining images (photographed under a 200x microscope) and Masson staining images (photographed under a 200x microscope) of mouse lung tissue are shown, where Control: normal group; Model: model group; 20mg / kg, 40mg / kg, 80mg / kg: methyl gallate groups; TET: tetrandrine group.

[0027] Fig. 9 The expression of TGF-β1 in mouse lung tissue is shown, among which, Control: normal group; Model: model group; MG: methyl gallate group; TET: tetrandrine group, n=6, compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01.

[0028] Fig.10 The expression of FN and COLⅠ mRNA in mouse lung tissues is shown, where Control: normal group; Model: model group; MG: methyl gallate group; TET: tetrandrine group, n=6, compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01.

[0029] Fig.11 The WB bands and quantitative results of COLⅠ, FN and α-SMA protein expression in mouse lung tissues are shown, where Control: normal group; Model: model group; MG: methyl gallate group; TET: tetrandrine group, n=6, compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01, # P<0.05. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described in detail below in conjunction with the examples. However, the following examples are provided only for easier understanding of the present invention, and the scope of the present invention is not limited thereto.

[0031] Example 1: Screening of active ingredients

[0032] Methyl gallate, ethyl gallate, gallic acid, apigenin and Ginkgolide B were used for screening.

[0033] 1.1 Materials and methods

[0034] 1.1.1 Reagents and materials

[0035] Lipopolysaccharide (Cat. No. L2880, Sigma, USA), 1640 culture medium (Cat. No. 31800, Solebio, Shanghai), PBS (Cat. No. G4202-500ML, Servicebio, Wuhan), fetal bovine serum (Cat. No. S711-001S, Lonsera, Uruguay); Ham's F-12K basal medium (Cat. No. PM150910, Procell, Wuhan), penicillin-streptomycin solution (Cat. No. PB180120, Procell, Wuhan), Human TGF-β1 (Cat. No. 100-21-10UG, PEPROTECH); reverse transcription reagent ( IIQ RT SuperMix for qPCR (Cat. No. R223, Novozymes, Nanjing), amplification reagent (ChamQUniversal SYBR qPCR Master Mix, Cat. No. Q711, Novozymes, Nanjing), mouse IL-6 ELISA kit (Cat. No. 555240, BD Bioscience), human α-SMA ELISA kit (catalog number: EH1509, Fine Biotechnology, Wuhan), methyl gallate (catalog number: B20853, Yuanye, Shanghai, CAS number 99-24-1, specification: analytical standard HPLC ≥ 98%), ethyl gallate (catalog number: B20855, Yuanye, Shanghai, CAS number 831-61-8, specification: analytical standard HPLC ≥ 98%), gallic acid (catalog number: A0110, Mansite Biotechnology, Chengdu, CAS number 149-91-7, specification: analytical standard HPLC ≥ 98%), apigenin (catalog number: A0113, Mansite Biotechnology, Chengdu, CAS number 520-36-5, specification: analytical standard HPLC ≥ 98%), ginkgolide B (catalog number: A0164, Mansite Biotechnology, Chengdu, CAS number 15291-77-7, specification: analytical standard HPLC ≥ 98%).

[0036] 1.1.2 Fibroblast culture

[0037] Human embryonic lung fibroblasts were purchased from Procell Life Science & Technology Co., Ltd., Wuhan. Fibroblasts were placed in Ham's F-12K (containing double antibody) complete medium containing 10% Lonsera fetal bovine serum, cultured in a 37°C, 5% CO2 incubator, and the medium was changed once every two days. When the density reaches more than 70% of the culture dish area, the cells can be subcultured at a 1:2 subculture ratio. Cells of passages 2 to 4 were used for experiments.

[0038] 1.1.3 Fibroblast grouping and treatment

[0039] Fibroblasts were collected at 2×10 5 Cells were inoculated in 35 mm dishes, and fibroblast activation was induced by TGF-β1 (5 ng / mL). Methyl gallate (30, 10, 3 μM), ethyl gallate (30, 10, 3 μM), gallic acid (60, 20, 6.7 μM), apigenin (50, 25, 12.5 μM), and ginkgolide B (50, 25, 12.5 μM) were given for 3-4 hours, and TGF-β1 was added to induce 24 hours except for the control group. Cells were collected by QIAzol, and the expression of ACTA2 and FN1 mRNA was detected by qPCR. The collected cells were resuspended in PBS, placed in -80℃, and the cells were repeatedly frozen and thawed for detection of α-SMA ELISA kit.

[0040] 1.1.4 Macrophage culture

[0041] Macrophage MHS cells were cultured in 1640 medium containing 10% fetal bovine serum, and passaged every 2 days at a ratio of 1:3. After passage, they were cultured in a 37°C, 5% CO2 incubator for 24 hours for induction.

[0042] 1.1.5 Macrophage grouping and treatment

[0043] Macrophages were counted at 1×10 6 The cells were inoculated in 35 mm dishes, and LPS (100 ng / mL) was used to induce macrophage inflammatory response. Methyl gallate (30, 10, 3 μM), gallic acid (60, 20, 6.7 μM), apigenin (50, 25, 12.5 μM), and ginkgolide B (50, 25, 12.5 μM) were given for 3-4 hours, and LPS was added for 12 hours. The cells were collected with QIAzol to extract cell RNA, and qPCR technology was used to detect TNF-α, IL-6, and IL-β mRNA expression. The cell supernatant was collected and the IL-6 expression level was detected by ELISA.

[0044] 1.1.6 ELISA for detection of IL-6 and α-SMA levels

[0045] After collecting the cell supernatant, centrifuge at 1000×g and take the supernatant. Follow the instructions of the ELISA kit for IL-6, read the absorbance value at 450nm wavelength using an enzyme reader, and calculate the concentration based on the standard curve. Centrifuge the cell lysate at 1500×g to remove cell debris and collect the supernatant. Follow the instructions of the ELISA kit for α-SMA, read the absorbance value at 450nm wavelength using an enzyme reader, and calculate the concentration based on the standard curve.

[0046] 1.1.7 QIAzol method for total RNA extraction and qPCR reaction technology

[0047] Remove the supernatant from the cells, add 2mL PBS to each dish and wash once; add 1mL QIAzol lysis buffer to each dish, let stand at room temperature for 10 minutes, collect the lysate in a 1mL enzyme-free tube; add 200μL chloroform to each tube, vortex to mix, let stand at room temperature for 5 minutes; centrifuge at 4℃, 12000g for 15 minutes, and keep 400μL of supernatant in a new enzyme-free tube; add an equal volume of isopropanol to each tube, vortex to mix; centrifuge at 4℃, 12000g for 10 minutes, and remove the supernatant; add 1mL 75% alcohol (diluted with DEPC water) to each tube, vortex for 5s; centrifuge at 4℃, 12000g for 5 minutes, and remove the supernatant; add 30μL DEPC water to each tube to dissolve RNA; detect and adjust the RNA concentration to 100-200μg / mL; reverse transcription: 10μL sample + 4μL suPermixⅡ + 6μL DEPC water, reverse transcription according to the conditions of 50℃, 15min→85℃, 10s; fluorescence quantitative analysis: 2μL DEPC water + 0.3μL primers (front and back chains) + 2.5μL SYBR green + 0.2μL reverse transcribed RNA sample, amplification according to the conditions of 50℃, 2min→90℃, 2min→90℃, 15s→60℃, 1min→95℃, 15s→60℃, 1min→95℃, 15s. The primers used for human fibroblasts are shown in Table 1, and the primers used for mouse macrophages are shown in Table 2.

[0048] Table 1 Primer sequences used for human fibroblasts

[0049]

[0050] Table 2 Primer sequences used for mouse macrophages

[0051]

[0052] 1.1.8 Statistical methods

[0053] The experimental data were statistically analyzed using IBM SPSS22.0 statistical software. One-way ANOVA was used to compare the quantitative data between groups. If the variance was equal, the least significant difference method (LSD) was used for pairwise comparison; if the variance was unequal, Dunnett's T3 method was used. The significance level was α = 0.05, and the data were expressed as mean ± standard deviation (s) for statistical description.

[0054] 1.2 Results

[0055] 1.2.1 Effects of methyl gallate, ethyl gallate, gallic acid, apigenin, and ginkgolide B on inhibiting the expression of genes and proteins related to fibroblast activation

[0056] Figure 1-3 The results showed that methyl gallate, ethyl gallate, gallic acid, apigenin and ginkgolide B inhibited the expression of fibroblast activation-related genes ACTA2 and FN1 mRNA and α-SMA protein expression. Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ** P<0.01, * P<0.05.

[0057] Depend on Figure 1-3 It can be seen that compared with the control group, the ACTA2 and FN1 mRNA in the model group were significantly increased (P < 0.01), and the α-SMA protein expression was significantly increased, indicating that the modeling was successful.

[0058] Depend on Figure 1-3 It can also be seen that compared with the model group, 30 and 10 μM methyl gallate significantly inhibited the expression of ACTA2 and FN1 mRNA and α-SMA protein expression (P < 0.01), while ethyl gallate, gallic acid, apigenin, and ginkgolide B had no significant effect on the expression of ACTA2 and FN1 mRNA and α-SMA protein expression (P > 0.05).

[0059] These results indicate that methyl gallate can significantly inhibit fibroblast activation, while ethyl gallate, gallic acid, apigenin and ginkgolide B have no significant effect on fibroblast activation.

[0060] 1.2.2 Effects of methyl gallate, ethyl gallate, gallic acid, apigenin, and ginkgolide B on inhibiting the expression of inflammation-related genes and proteins in macrophages

[0061] Figure 4-7 The results showed that methyl gallate, ethyl gallate, gallic acid, apigenin and ginkgolide B inhibited the expression of TNF-α, IL-6 and IL-β mRNA and IL-6 protein in macrophages, respectively. Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ** P<0.01, * P<0.05.

[0062] Depend on Figure 4-7 It can be seen that compared with the control group, the expression of TNF-α, IL-6, IL-β mRNA and IL-6 protein in the model group were significantly increased (P < 0.01), indicating that the modeling was successful.

[0063] Depend on Figure 4-7 It can also be seen that compared with the model group, 30 and 10 μM methyl gallate, 30 μM ethyl gallate, 60 μM gallic acid, 50 μM apigenin and 50 μM ginkgolide B can significantly inhibit the expression of TNF-α, IL-6 and IL-β mRNA (P < 0.05, P < 0.01). In addition, 25 μM apigenin and 25 μM ginkgolide B significantly inhibited the expression of IL-β mRNA (P < 0.05). 30 and 10 μM methyl gallate, 30 μM ethyl gallate, 60 μM gallic acid, 50 and 25 μM apigenin and 50 μM ginkgolide B can all significantly inhibit the expression of IL-6 protein (P < 0.01).

[0064] These results indicate that methyl gallate, ethyl gallate, gallic acid, apigenin and ginkgolide B can effectively inhibit the inflammatory response of macrophages.

[0065] 1.3 Conclusion

[0066] Through the activity evaluation of fibroblast activation and macrophage inflammatory response models, it was found that methyl gallate can effectively inhibit the inflammatory response and lung fibroblast activation, and has the potential to improve pulmonary fibrosis; ethyl gallate, gallic acid, apigenin and ginkgolide B can all effectively inhibit the macrophage inflammatory response, but have no obvious effect on lung fibroblast activation.

[0067] Example 2: Animal Experiment

[0068] 2.1 Experimental Materials

[0069] 2.1.1 Experimental animals

[0070] SPF-grade C57BL / 6 mice, male (weight: 20 ± 1 g; animal license number: SYXK [Henan] 2021-0015), were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0071] 2.1.2 Experimental drugs

[0072] 2.1.2.1 Methyl gallate

[0073] Methyl gallate (Cat. No.: S30155) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. An appropriate amount of methyl gallate powder was weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare a 30 mM clear mother solution, which was stored at -20°C for use in cell experiments.

[0074] An appropriate amount of methyl gallate powder was weighed and dissolved in an aqueous solution containing 0.5 wt% sodium carboxymethyl cellulose (CMC-Na) to prepare 4 mg / ml, 2 mg / ml, and 1 mg / ml suspensions, which were stored at 4°C for use in animal experiments.

[0075] 2.1.2.2 Tetrandrine (positive control)

[0076] Jin Aikang (Fangfangjisu Tablets) was provided by Zhejiang Jinhua Kangenbei Biopharmaceutical Co., Ltd. (Specification: 20 mg / tablet, batch number: PH2003006). Before use, the drug was ground into powder and dissolved in pure water according to the dosage to prepare a 0.75 mg / ml suspension.

[0077] 2.1.3 Experimental reagents

[0078] 2.1.3.1 Hematoxylin-eosin (H&E) staining, Masson's Trichrome staining related reagents

[0079] Paraformaldehyde (P804536) was provided by Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Paraffin (39601006) was provided by Leica Microsystems Trading Co., Ltd. (Illinois, USA). Anhydrous ethanol (500 ml) and xylene (500 ml) were provided by Tianjin Zhiyuan Chemical Reagent Co., Ltd. Hematoxylin staining solution (G1140), eosin staining solution (G1100), and neutral gum (G8590) were provided by Beijing Solebold Technology Co., Ltd. Masson staining solution (G1006) was provided by Wuhan Saiweier Biotechnology Co., Ltd.

[0080] 2.1.3.2 TGF-β1 detection related reagents

[0081] Transforming growth factor TGF-β1 enzyme-linked immunosorbent assay kit (E-EL-0162c-96T) was purchased from Wuhan Elaruite Biotechnology Co., Ltd.

[0082] 2.1.3.3 qPCR detection reagents

[0083] HiScript i Q RT SuperMix for qPCR (+gDNA wiper) was purchased from Nanjing Novozymes Biotech Co., Ltd., SYBR GREEN kit (Q711-02) was purchased from Nanjing Novozymes Biotech Co., Ltd., anhydrous ethanol (E809056-500ML) was purchased from MACKLIN Biological Co., Ltd., and DEPC water (AM9922) was purchased from Invitrogen Co., Ltd.

[0084] 2.1.3.4 Western Blot Detection Reagents

[0085] BCA protein quantification kit (ZJ101) was purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., PAGE gel rapid preparation reagent (PG112) was purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., GAPDH Rabbit Polyclonal Antibody (10494-1-AP), Fibronectin Polyclonal Antibody (22018-1-AP), Collagen Type I Polyclonal antibody (14695-1-AP), and Smooth Muscle Actin Polyclonal Antibody (14395-1-AP) were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0086] 2.1.4 Experimental instruments

[0087] The experimental instruments are shown in Table 3.

[0088] Table 3

[0089]

[0090]

[0091] 2.2 Research Methods

[0092] 2.2.1 Model preparation

[0093] C57BL / 6 / mice were adaptively raised for 7 days, and a silicosis mouse model was prepared by a one-time intratracheal injection of SiO2 suspension. After anesthesia, the mice were fixed on the operating table in a supine position. The fine wire of the hanging hemostatic forceps was placed on the upper incisors of the mice to fix the upper jaw. The left hand was padded with gauze to pull out the tongue to expose the throat. The oral mucus was wiped with a cotton swab. The skin of the mouse throat was illuminated with a cold light source. The bright spot that opened and closed with the mouse's breathing was seen by adjusting the angle of vision. This was the tracheal opening. The trocar was inserted into the tracheal cannula from the throat, the needle core was pulled out, and cotton wool was placed at the cannula mouth. The cotton wool was seen swinging back and forth with the mouse's breathing frequency, indicating that the tracheal intubation was successful. Then 25mg / mL SiO2 suspension was quickly injected into the trachea, and then 0.5mL of air was injected. The upright mice were shaken left and right and the lungs were patted to make the suspension evenly distributed in the lungs. The control group was dripped with sterile saline in the same way.

[0094] 2.2.2 Grouping and Dosing

[0095] SPF C57BL / 6J male mice were randomly divided into control group, model group, methyl gallate (MG) high, medium and low dose groups, and tetrandrine group (TET) according to the random number table method after one week of adaptive feeding. Silicosis mouse model was prepared by intratracheal instillation of 25 mg / mL SiO2 suspension. The modeling day was set as day 0. Methyl gallate and tetrandrine were used for intervention on the 14th day after modeling. The high, medium and low dose groups of methyl gallate were given high (80 mg / kg / d), medium (40 mg / kg / d) and low (20 mg / kg / d) methyl gallate by gavage, respectively. The tetrandrine group was given tetrandrine (15 mg / kg / d) by gavage. The control group and model group were given CMC-NA by gavage once a day. Samples were collected after 28 days of treatment.

[0096] 2.2.3 Lung tissue pathology

[0097] The mouse lung tissue was fixed with 4% formaldehyde solution for 24 hours, and then routinely dehydrated, paraffin-embedded, and sectioned for routine HE and Masson staining. The pathological changes of the lung tissue were observed under a light microscope.

[0098] HE staining was used to score the alveolitis pathologically according to the Szapiel method, and Masson staining was used to score the degree of fibrosis according to the Ashcroft scoring system.

[0099] The pathological changes of lung tissue were observed under an optical microscope. Six sections were selected from each group and six fields of view were selected from each section. The degree of alveolitis and pulmonary fibrosis was scored according to the standards shown in Table 4.

[0100] Table 4 Alveolitis and pulmonary fibrosis grading and scoring criteria

[0101]

[0102] 2.2.4 Detection of TGF-β1 in lung tissue:

[0103] 10 mg of lung tissue was weighed and ground into lung homogenate using a grinder. The supernatant was collected by centrifugation and tested according to the procedure of the mouse TGF-β1 ELISA kit.

[0104] 2.2.5 Detection of COLⅠ and FN mRNA expression in lung tissue by qPCR

[0105] The total RNA of lung tissue was extracted by QIAzol method, and the concentration and purity were determined. After reverse transcription into cDNA, the amplification reaction was carried out by real-time fluorescence quantitative PCR instrument. -ΔΔCT Calculate the relative expression of mRNA.

[0106] 2.2.6 Western Blot analysis of COLⅠ, FN and α-SMA protein expression in lung tissue

[0107] Mouse lung tissue was placed in RIPA lysis buffer, homogenized and centrifuged to collect the supernatant. The protein concentration was determined and quantified by the BCA method. The protein was denatured at 100°C for 10 min. After protein denaturation, gel preparation and SDS-PAGE electrophoresis were performed in sequence. The membrane was transferred to PVDF membrane by wet transfer method at 200 mA, blocked in 10% skim milk powder at room temperature for 1 h, and incubated with primary antibody in a refrigerator at 4°C overnight. After washing with TBST, the secondary antibody was incubated at room temperature for 1 h. After washing with TBST, ECL luminescent solution was added for development. GAPDH was used as the internal reference protein and analyzed by Image J software.

[0108] 2.2.7 Statistical methods

[0109] The experimental data were analyzed using Graphpad Prism 8.0.1 software. The statistical differences between the two groups were compared by t-test, and the statistical differences between multiple groups were compared by one-way analysis of variance. P<0.05 was considered statistically significant.

[0110] 2.3 Results

[0111] 2.3.1 Effects of methyl gallate on lung tissue pathology and fibrosis scores in silicotic mice

[0112] HE and Masson staining results are shown in Figure 8 The scoring results are shown in Table 5.

[0113] Table 5 Effects of methyl gallate on lung tissue pathological scores in silicotic mice

[0114]

[0115] Note: n = 6. Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01.

[0116] HE staining results showed that the lung tissue structure of mice in the normal group was clear, and no inflammatory cells were found in the alveolar cavity. In the model group, the lung tissue structure was obviously damaged, the alveolar wall was ruptured and fused, and obvious inflammatory cell infiltration was observed; the lung tissue of mice in the high, medium, and low dose groups of methyl gallate and the tetrandrine group was improved to varying degrees compared with the model group, with reduced lung tissue damage and a small number of inflammatory cells.

[0117] The results of MASSON staining showed that no fibrotic lesions were found in the lung tissues of the mice in the normal group; obvious fibrotic nodules were found in the lung tissues of the mice in the model group, and collagen deposition was significantly increased; fibrotic nodules were observed in the lung tissues of the mice in the high-, medium-, and low-dose methyl gallate groups and the tetrandrine group to varying degrees, but they were all alleviated compared with the model group.

[0118] Compared with the normal group, the alveolitis and fibrosis scores of the lung tissue of the mice in the model group were significantly increased (P<0.01); compared with the model group, the alveolitis and fibrosis scores of the lung tissue of the mice in the high-, medium-, and low-dose methyl gallate groups and the tetrandrine group were reduced (P<0.01).

[0119] 2.3.2 Effect of methyl gallate on TGF-β1 expression in lung tissue of silicotic mice

[0120] The expression of TGF-β1 in mouse lung tissue was detected by ELISA. The results are shown in Table 6. Fig. 9 .

[0121] Table 6 Effect of methyl gallate on TGF-β1 expression in lung tissue of silicotic mice

[0122]

[0123] Note: n = 6. Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01.

[0124] The results showed that compared with the normal group, the level of TGF-β1 in the lung tissue of mice in the model group was significantly increased (P<0.01); compared with the model group, the levels of TGF-β1 in the lung tissue of mice in the high-, medium-, and low-dose methyl gallate groups and the tetrandrine group were decreased (P<0.01).

[0125] 2.3.3 Effect of methyl gallate on the expression of COLⅠ and FN mRNA in lung tissue of silicotic mice

[0126] The expression of FN and COLⅠ mRNA in mouse lung tissue was detected by qPCR technology, as shown in Table 7, Fig.10 .

[0127] Table 7 Effects of methyl gallate on the expression of FN and COLⅠ mRNA in lung tissue of silicotic mice

[0128]

[0129] Note: n = 6. Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01.

[0130] The results showed that compared with the normal group, the expression of FN and COLⅠ mRNA in the lung tissue of mice in the model group was significantly increased (P<0.01); compared with the model group, the expression of FN and COLⅠ mRNA in the lung tissue of mice intervened by high, medium and low doses of methyl gallate and tetrandrine was significantly decreased (P<0.01).

[0131] 2.3.4 Effects of methyl gallate on the expression of COLⅠ, FN and α-SMA proteins in lung tissue of silicotic mice

[0132] The expression of COLⅠ, FN and α-SMA proteins in mouse lung tissue was detected by WB technology. The results are shown in Table 8. Fig.11 .

[0133] Table 8 Effects of methyl gallate on the expression of COLⅠ, FN and α-SMA proteins in lung tissue of silicotic mice

[0134]

[0135] Note: n = 6. Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01, # P<0.05.

[0136] The results showed that compared with the normal group, the expression of COLⅠ, FN and α-SMA proteins in the lung tissue of mice in the model group was significantly increased (P<0.01); compared with the model group, the expression of COLⅠ, FN and α-SMA proteins in the lung tissue of mice intervened by high, medium and low doses of methyl gallate and tetrandrine was significantly decreased (P<0.01, P<0.05).

[0137] 2.4. Conclusion

[0138] In summary, methyl gallate can reduce fibrotic nodules in the lung tissue of mice in the treatment of silicosis, relieve symptoms, improve alveolitis and pulmonary fibrosis in lung tissue, reduce TGF-β1 levels, reduce FN and COLⅠ mRNA expression and COLⅠ, FN and α-SMA protein expression. Therefore, methyl gallate is expected to be used in the treatment of silicosis and has high practical value.

[0139] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are within the scope of the technical solution of the present invention.

Claims

1. The use of methyl gallate in the preparation of drugs for preventing or treating silicosis.

2. The use according to claim 1, wherein: Methyl gallate prevents or treats silicosis by reducing the fibrous nodules in the lung tissue.

3. The use according to claim 1. Methyl gallate prevents or treats silicosis by reducing TGF-β1 levels, FN and COLⅠ mRNA expressions, and COLⅠ, FN, and α-SMA protein expressions in lung tissue.

4. The use according to claim 1, wherein: The medicament contains methyl gallate in an arbitrary content as an active ingredient for treating silicosis.

5. The use according to claim 1, wherein: Based on mass percentage, the drug comprises 0.01 to 99.99% of methyl gallate and 99.99 to 0.01% of pharmaceutically acceptable excipients.

6. The use according to claim 1, wherein: The medicament contains methyl gallate as the only active ingredient for the treatment of silicosis.

7. The use according to claim 1, wherein: The drug further comprises other active ingredients besides methyl gallate. The other active ingredients are other active ingredients for preventing or treating silicosis, or are active ingredients of drugs for treating other diseases, disorders, and symptoms besides silicosis.

8. The use according to claim 1, wherein: The administration route of the drug is oral administration.

9. The use according to claim 1, wherein: The daily dosage of the drug is 0.1 mg-5000 mg / kg / day based on the active ingredient of methyl gallate.

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