Pharmaceutical composition for treating silicosis

Abstract

The invention discloses a pharmaceutical composition for treating silicosis. The pharmaceutical composition is prepared from isoliquiritin, glycyrrhizic acid and gallic acid in a mass ratio of 180: 180: 3.75. Pharmacological data of cell and animal levels show that the pharmaceutical composition has an obvious treatment effect on silicosis, and the components of the pharmaceutical composition have a synergistic effect.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to a pharmaceutical composition, in particular to a pharmaceutical composition for treating silicosis. Background Art

[0002] Silicosis is a general term for a group of occupational lung diseases caused by long-term inhalation of dust composed mainly of free silica and its retention in the lungs during occupational activities, with diffuse fibrosis of lung tissue as the main feature. Its clinical manifestations are mainly respiratory symptoms, including cough, sputum, chest pain and dyspnea. Silicosis is not only a lung disease, but also a systemic disease. Even if the patient is out of the dust contact environment, the disease will still progress and worsen, and in severe cases it will cause respiratory failure and heart failure. Silicosis is characterized by a high prevalence and heavy economic burden. There is a lack of effective drugs and treatments for silicosis in clinical practice.

[0003] Currently, silicosis is mainly treated with oxygen therapy, lung rehabilitation and lung transplantation, but the treatment effect is often unsatisfactory. Drug treatments include codeine phosphate tablets, aminophylline tablets, bromhexine hydrochloride tablets, ipratropium bromide aerosol, tetrandrine tablets, etc., but these drugs can only relieve symptoms. Treatments such as large-volume whole lung lavage and lung transplantation are expensive and have a small range of indications, which limits their widespread clinical application.

[0004] Traditional Chinese medicine preparations are also used in the treatment of silicosis, including Feining Granules and Bailing Capsules, but these preparations have low active ingredient content, especially the complex ingredients of traditional Chinese medicine compound, no quality control indicators, and their safety is difficult to control. These shortcomings limit the promotion and use of traditional Chinese medicine preparations.

[0005] In order to develop a pharmaceutical composition for treating silicosis with obvious efficacy and clear ingredients, the present invention is specially proposed. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a pharmaceutical composition for treating silicosis.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A pharmaceutical composition for treating silicosis, comprising isoliquiritigenin, glycyrrhizic acid and gallic acid in a mass ratio of 180:180:3.75.

[0009] Application of the above-mentioned pharmaceutical composition in preparing medicine for treating silicosis.

[0010] Preferably, the drug uses the drug composition as an active ingredient for treating silicosis and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0011] More preferably, the auxiliary material is a solid, liquid or semi-solid auxiliary material.

[0012] More preferably, the dosage form is a tablet, capsule, injection or inhalant.

[0013] Beneficial effects:

[0014] The present invention has discovered a pharmaceutical composition for treating silicosis, which is composed of isoliquiritigenin, glycyrrhizic acid and gallic acid in a mass ratio of 180:180:3.75. Pharmacological data at the cell and animal levels show that the pharmaceutical composition has a significant therapeutic effect on silicosis and there is a synergistic effect between the components of the pharmaceutical composition, specifically:

[0015] The comprehensive efficacy values ​​of glycyrrhizic acid alone, gallic acid alone, and glycyrrhizic acid and gallic acid combined are as follows Figure 6 As shown, the comprehensive efficacy value of glycyrrhizic acid and gallic acid combined is significantly higher than the simple sum of the comprehensive efficacy values ​​of glycyrrhizic acid and gallic acid administered alone, which indicates that glycyrrhizic acid and gallic acid have a synergistic effect when they are administered together. From another perspective, the combined use of gallic acid with a negative comprehensive efficacy value and glycyrrhizic acid not only does not lower the comprehensive efficacy value of the composition, but significantly increases the comprehensive efficacy value of the composition, which also indicates that glycyrrhizic acid and gallic acid have a synergistic effect when they are administered together.

[0016] The comprehensive efficacy values ​​of isoliquiritigenin alone, gallic acid alone, and isoliquiritigenin combined with gallic acid are as follows Figure 7 As shown, the comprehensive efficacy value of the combined administration of isoliquiritigenin and gallic acid is significantly higher than the simple sum of the comprehensive efficacy values ​​of isoliquiritigenin and gallic acid administered alone, which indicates that there is a synergistic effect between isoliquiritigenin and gallic acid when they are administered together. From another perspective, the combined use of gallic acid with a negative comprehensive efficacy value and isoliquiritigenin not only does not lower the comprehensive efficacy value of the composition, but significantly increases the comprehensive efficacy value of the composition, which also indicates that there is a synergistic effect when isoliquiritigenin and gallic acid are administered together.

[0017] The comprehensive efficacy of isoliquiritigenin alone, glycyrrhizic acid alone, gallic acid alone, and the combination of the three is as follows Figure 8 As shown, the comprehensive efficacy value of the three combined administration is significantly higher than the simple sum of the comprehensive efficacy values ​​of isoliquiritigenin, glycyrrhizic acid, and gallic acid administered separately, which indicates that there is a synergistic effect when the three are administered in combination. From another perspective, the combined use of gallic acid with a negative comprehensive efficacy value and the combination of isoliquiritigenin + glycyrrhizic acid not only did not lower the comprehensive efficacy value of the three-combination combination, but significantly increased the comprehensive efficacy value of the three-combination combination, which also shows that there is a synergistic effect when isoliquiritigenin, glycyrrhizic acid and gallic acid are administered in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The effect of different concentrations of isoliquiritigenin, glycyrrhizic acid and gallic acid monomers on the viability of MH-S cells;

[0019] Figure 2 The effect of different concentrations of isoliquiritigenin monomer on the expression level of polarization markers of MH-S cells;

[0020] Figure 3 The effect of different concentrations of glycyrrhizic acid monomer on the expression level of polarization markers of MH-S cells;

[0021] Figure 4 The effect of different concentrations of gallic acid monomer on the expression level of polarization markers of MH-S cells;

[0022] Figure 5 is the comprehensive efficacy value of the composition with different proportions;

[0023] Figure 6 The comprehensive efficacy values ​​of glycyrrhizic acid alone, gallic acid alone, and glycyrrhizic acid and gallic acid combined were compared;

[0024] Figure 7 The comprehensive efficacy values ​​of isoliquiritigenin alone, gallic acid alone, and isoliquiritigenin and gallic acid combined were compared.

[0025] Figure 8 The comprehensive efficacy values ​​of isoliquiritigenin alone, glycyrrhizic acid alone, gallic acid alone, and the combination of the three were compared;

[0026] Fig. 9 A is a schematic diagram of the modeling and drug administration process of the silica-induced silicosis model; B is the HE staining result of the lung tissue of silicosis mice; C is the Masson staining result of the lung tissue of silicosis mice;

[0027] Fig.10 The expression levels of marker proteins IL-6, IL-1β, TNF-α, CD206 and ARG-1 in lung tissues of silicotic mice. DETAILED DESCRIPTION

[0028] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0029] 1. Experimental Materials

[0030] 1. Chemicals: Isoliquiritigenin, glycyrrhizic acid and gallic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd., lipopolysaccharide (LPS) was purchased from Beijing Solebao Company, and interleukin 4 (IL-4) was purchased from Peprotech Company, USA.

[0031] 2. Cell experiment reagents: Mouse alveolar macrophages (MH-S cells) were purchased from the Cell Resource Center of Shanghai Institutes for Life Sciences, Chinese Academy of Sciences, RPMI 1640 culture medium was purchased from Beijing Solebow Technology Co., Ltd., Beijing, fetal bovine serum was purchased from Lonsera, Uruguay, PBS buffer and 0.25% trypsin solution (containing EDTA, dissolved in PBS) were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0032] 3. PCR-related reagents: QIAzol Lysis Reagent was purchased from Qiagen, USA; DEPC-Treated Water was purchased from Ambion, USA; isopropanol was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; anhydrous ethanol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; II 1st Strand cDNA Synthesis Kit, ChamQTM Universal qPCR Master Mix was purchased from Nanjing Novozymes Biotech Co., Ltd., and mouse primers were synthesized by Suzhou Genewise Biotech Co., Ltd.

[0033] 4. Animal experimental reagents: 70 male C57BL6J mice, weighing 20-25 g, were purchased from Beijing SPF Co., Ltd., with animal license number SYXK(Yu)2021-0015. Silica was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0034] 5. Main experimental instruments:

[0035] Cell CO2 incubator: Thermo Fisher, USA;

[0036] 1300 Series Class II Type A2 Biological Safety Cabinet: Thermo Fisher Scientific, USA;

[0037] ECLIPSE TS100 inverted biological microscope: Nikon, Japan;

[0038] Applied Biosystems Veriti PCR instrument: ABI, United States;

[0039] QuantStudio 6Flex real-time fluorescence quantitative PCR instrument: ABI, United States;

[0040] Kylin-bell vortex mixer VORTEX-5: Kylin-bell, Haimen;

[0041] Hiclave HVE-50 autoclave: Hirayama, Japan.

[0042] 2. Experimental Methods

[0043] 1. MH-S cell culture and passaging

[0044] Mouse alveolar macrophages MH-S were cultured in 1640 complete medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The cell growth status was observed daily. When the cell coverage rate was 80-90%, the complete medium was preheated in a 37°C water bath, the medium in the culture dish was removed, 1 mL of fresh medium was added, the cells were dispersed by blowing and counted, and the cells were passaged at a ratio of 1:4.

[0045] 2. Determination of the effect of drugs on the survival rate of MH-S cells

[0046] Using DMSO as solvent, prepare the drug stock solution under a sterile operating table and store it at -80℃ for future use. Dilute it with complete culture medium to a certain concentration of working solution to treat cells according to experimental requirements. 6 / mL MHS cell suspension was inoculated in a 96-well plate, 100μL per well. Set up a blank group and a drug group, with 3-6 replicates in each group, cultured in a cell culture incubator for 24 hours, and observed the cell adhesion under a microscope. After the cells are completely attached, remove the original culture medium, add 100μL of culture medium containing different concentrations of drugs, and treat for 24 hours. The CCK8 method was used to detect cell viability. First, remove the drug-containing culture medium, add 100μL of 1640 complete culture medium containing 10% CCK8, and place it in a 37°C, 5% CO2 incubator for 1-2 hours. Use an enzyme marker to detect the OD value at a wavelength of 450nm. And use Graph PadPrism 6 to draw a graph to draw its cell activity reaction graph.

[0047] 3. Determination of the inhibitory effect of drugs on MH-S cell polarization

[0048] MH-S cells were cultured at 0.5×10 6 The cells were inoculated in 35 mm cell culture dishes and divided into blank group, model group and 6 different concentrations of drug administration groups, a total of 8 groups. Different concentrations of the test compound were added to the drug administration group. After 3 hours, except for the blank group, LPS (20 ng / mL) was added to the model group and different concentrations of drug administration groups of the M1 type group, and IL-4 (20 ng / mL) was added to the M2 type group. They were induced for 12 hours respectively, and then the mRNA expression levels of M1 markers IL-6, IL-1β, TNF-α and M2 markers CD206 and ARG-1 were detected by qPCR method, as follows:

[0049] The cells were washed with 1 mL PBS, lysed with 1 mL QIAzol for 10 minutes, the lysate was transferred to a 1.5 mL enzyme-free EP tube, 200 μL chloroform was added and shaken for 15 seconds, the tube was placed at room temperature for 15 minutes, and centrifuged at 4°C and 12000 g for 15 minutes. 490 μL supernatant was transferred to a new 1.5 mL enzyme-free EP tube, an equal volume of isopropanol was added, the tube was shaken for 10 minutes at room temperature, and centrifuged at 4°C and 12000 g for 10 minutes, and the supernatant was discarded; 1 mL 75% ethanol was added, the tube was inverted 3 times, and centrifuged at 4°C and 7500 g for 5 minutes, and the supernatant was discarded; the tube was dried in a fume hood for 5 minutes, 31 μL DEPC water was added to dissolve the precipitate, the total RNA concentration was measured by nanodrop, and the purity A260 / A230 and A260 / A280 were recorded. 1 μg of total RNA was added to eight consecutive EP tubes for reverse transcription synthesis of cDNA, and 4 μL 5×HiScriptⅡqRTSuperMixⅡ, 6μL DEPC water and 10μL RNA were centrifuged instantly and placed in a PCR instrument for reverse transcription to synthesize cDNA. The reaction conditions were 50℃ for 15 minutes, 85℃ for 10s, and 4℃ to +∞. The cDNA after reverse transcription was amplified using a fluorescent quantitative PCR instrument. The relative expression of mRNA was calculated according to the formula, which is F=2 -ΔΔCT ΔΔCT = (average CT of target gene in test group - average CT of reference gene in test group) - (average CT of target gene in blank group - average CT of reference gene in blank group). The qPCR reaction system and reaction conditions are shown in Tables 1 to 3.

[0050] Table 1 Gene primer sequences and amplification lengths

[0051]

[0052] Table 2 qPCR 384-well plate reaction system

[0053]

[0054] Table 3 qPCR reaction conditions and cycle numbers

[0055]

[0056] 4. Calculation of comprehensive efficacy index

[0057] In order to comprehensively evaluate the inhibitory effect of the synergistic components on MH-S cell polarization, we used a multi-index evaluation and gave each indicator the same weight coefficient. The comprehensive efficacy OutputP of each combination was obtained by evaluating the effects on macrophage-related M1 markers IL-6, IL-1β, TNF-α and M2 markers CD206 and ARG-1.

[0058] Calculation formula: V = 0.2*(M IL-6-S IL-6 ) / M IL-6 +0.2*(M IL-1β -S IL-1β ) / M IL-1β +0.2*(M TNF-α -S TNF-α ) / M TNF-α +0.2*(M CD206 -S CD206 ) / M CD206 +0.2*(M ARG-1 -S ARG-1 ) / M ARG-1 .

[0059] Among them: V represents the comprehensive efficacy of each combination, M represents the measured value of each indicator model group, and S represents the measured value of each indicator after each combination of drugs is given.

[0060] 5. Animal Models and Treatments

[0061] Animal experiments were conducted on 70 male C57BL6J mice. A silicosis mouse model was established by a single intratracheal instillation of silica (25 mg silica suspension) and maintained for 2 weeks. The mice were then divided into six groups (normal, model, positive drug, low, medium, and high dose drug combinations), with 10 animals in each group. From the 3rd to 6th week, 10 mice in the normal group and the model group were gavaged with 0.5 mL of normal saline every day; 10 mice in the positive drug group were gavaged with hanfangji A (15 mg kg -1 ·d -1 ); the same number of mice in the drug combination group were gavaged with the drug combination (low, medium, and high doses were 4.04, 20.21, and 50.52 mg·kg -1 ·d -1 All mice were sacrificed at the end of 6 weeks, and lung tissues were taken for pathological examination and immunohistochemistry. The dosage was adjusted every 3 days according to the body weight of each mouse.

[0062] 6. Histopathological examination

[0063] Lung tissues were fixed with formalin, embedded in paraffin, and cut into 4 μm thick sections. These sections were stained with hematoxylin and eosin (HE) or Masson's trichrome. Histopathological changes were observed under an optical microscope. Quantitative evaluation was performed by three independent researchers in a blinded manner. HE-stained sections were scored using the Szapiel score, while Masson's trichrome-stained sections were used to assess the severity of pulmonary fibrosis using the Ashcroft score.

[0064] 7. Immunohistochemistry

[0065] Lung tissue sections (4 μm thick) were blocked with 5% bovine serum albumin for 30 min at 37° C. and then incubated with anti-IL-1, anti-IL-6, anti-TNF-α, anti-CD206 (Changsha Aifang Biotechnology Co., Ltd.) or anti-Arg-1 antibodies (Proteintech Group, Wuhan Co., Ltd.) for 12 h at 4° C. The integrated optical density was measured using IPP 6.0.

[0066] 8 Statistical methods

[0067] 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, the LSD method was used after data conversion. The significance level was α = 0.05, and the data were statistically described as mean (`x) ± standard deviation (SD), ***P < 0.001, **P < 0.01, *P < 0.05.

[0068] 3. Experimental Results

[0069] 1. Evaluation of Monomer Cytotoxicity

[0070] The results of the cytotoxic effect of isoliquiritigenin monomer on MH-S cells are shown in Figure 1 As shown in A, isoliquiritigenin had no effect on the viability of MH-S cells at 0-180 μg / mL.

[0071] The results of the cytotoxic effect of glycyrrhizic acid monomer on MH-S cells are as follows Figure 1 As shown in B, glycyrrhizic acid had no effect on the viability of MH-S cells at 0-180 μg / mL.

[0072] The results of the cytotoxic effect of gallic acid monomer on MH-S cells are as follows Figure 1 As shown in C, gallic acid has an effect on the viability of MH-S cells above 7.5 μg / mL, but has no significant effect at 0-3.75 μg / ml.

[0073] 2. Results of Monomer Efficacy Test

[0074] The results of the inhibitory effect of isoliquiritigenin monomer on MH-S cell polarization are shown in Figure 2 As shown, isoliquiritigenin is effective for IL-1, IL-6, TNF-α, and CD206 indicators, but ineffective for Arg-1 indicator.

[0075] The results of the inhibitory effect of glycyrrhizic acid monomer on MH-S cell polarization are shown in Figure 3 As shown, glycyrrhizic acid is effective on IL-1, IL-6, TNF-α, CD206, and Arg-1 indicators.

[0076] The results of the inhibitory effect of gallic acid monomer on MH-S cell polarization are as follows Figure 4 As shown in the results, gallic acid has a strong inhibitory effect on IL-1, IL-6, and TNF-α indicators, but has a strong promoting effect on CD206 and Arg-1 indicators, which may aggravate the progression of silicosis.

[0077] 2. Optimization of drug combination ratio

[0078] The efficacy of 12 drug combinations generated by the "RandomGen_Initiation" algorithm written in MATLAB was evaluated, and the comprehensive efficacy value of each combination ratio was calculated. It was found that combination 8, that is, the ratio of isoliquiritigenin, glycyrrhizic acid, and gallic acid was 180:180:3.75, had the best comprehensive efficacy. The drug combination ratio design is shown in Table 4, and the comprehensive efficacy results are shown in Figure 5 shown.

[0079] Table 4 Drug combination ratio design

[0080]

[0081] 3. Evaluation of synergistic effects

[0082] We evaluated whether there is a synergistic effect in the combination of isoliquiritigenin, glycyrrhizic acid, and gallic acid in a mass ratio of 180:180:3.75. The specific analysis and evaluation are as follows:

[0083] The comprehensive efficacy values ​​of glycyrrhizic acid alone, gallic acid alone, and glycyrrhizic acid and gallic acid combined are as follows Figure 6 As shown, the comprehensive efficacy value of glycyrrhizic acid and gallic acid combined is significantly higher than the simple sum of the comprehensive efficacy values ​​of glycyrrhizic acid and gallic acid administered alone, which indicates that glycyrrhizic acid and gallic acid have a synergistic effect when they are administered together. From another perspective, the combined use of gallic acid with a negative comprehensive efficacy value and glycyrrhizic acid not only does not lower the comprehensive efficacy value of the composition, but significantly increases the comprehensive efficacy value of the composition, which also indicates that glycyrrhizic acid and gallic acid have a synergistic effect when they are administered together.

[0084] The comprehensive efficacy values ​​of isoliquiritigenin alone, gallic acid alone, and isoliquiritigenin combined with gallic acid are as follows Figure 7 As shown, the comprehensive efficacy value of the combined administration of isoliquiritigenin and gallic acid is significantly higher than the simple sum of the comprehensive efficacy values ​​of isoliquiritigenin and gallic acid administered alone, which indicates that there is a synergistic effect between isoliquiritigenin and gallic acid when they are administered together. From another perspective, the combined use of gallic acid with a negative comprehensive efficacy value and isoliquiritigenin not only does not lower the comprehensive efficacy value of the composition, but significantly increases the comprehensive efficacy value of the composition, which also indicates that there is a synergistic effect when isoliquiritigenin and gallic acid are administered together.

[0085] The comprehensive efficacy of isoliquiritigenin alone, glycyrrhizic acid alone, gallic acid alone, and the combination of the three is as follows Figure 8 As shown, the comprehensive efficacy value of the three combined administration is significantly higher than the simple sum of the comprehensive efficacy values ​​of isoliquiritigenin, glycyrrhizic acid, and gallic acid administered separately, which indicates that there is a synergistic effect when the three are administered in combination. From another perspective, the combined use of gallic acid with a negative comprehensive efficacy value and the combination of isoliquiritigenin + glycyrrhizic acid not only did not lower the comprehensive efficacy value of the three-combination combination, but significantly increased the comprehensive efficacy value of the three-combination combination, which also shows that there is a synergistic effect when isoliquiritigenin, glycyrrhizic acid and gallic acid are administered in combination.

[0086] 4. Pharmacodynamic evaluation of drug combination on silicosis mouse model

[0087] We further established a silica-induced silicosis model to verify the effectiveness of the synergistic composition we identified (a combination of isoliquiritigenin, glycyrrhizic acid, and gallic acid in a mass ratio of 180:180:3.75). To evaluate its therapeutic effect on silicotic mice, the drug combination (e.g., Fig. 9 (A). Fig. 9 The HE and Masson staining results shown in B and C in the figure show that compared with the control group, the lungs of the silicotic mice in the model group showed alveolar collapse, inflammatory cell infiltration, fibroblast proliferation and accumulation of fibrous nodules, indicating that the model was successful. Compared with the model group, the alveolar collapse, inflammatory infiltration, fibroblast proliferation and accumulation of fibrous nodules in the silicotic mice in the drug composition administration groups with different concentrations were significantly alleviated, and there was an obvious dose effect. The positive drug group (hanfangjisu A) was able to significantly improve the above indicators, proving that the model and method are effective. In addition, we used immunohistochemical analysis to detect the protein expression levels of IL-6, IL-1β, TNF-α, CD206 and ARG-1 in lung tissues. The results are shown in Figure 2. Fig.10 As shown. Compared with the control group, the protein expression levels of IL-6, IL-1β, TNF-α, CD206 and ARG-1 in the lung tissue of silicotic mice in the model group were significantly upregulated, indicating that the model was successfully established. Compared with the model group, the protein expression levels of IL-6, IL-1β, TNF-α, CD206 and ARG-1 in the lung tissue of silicotic mice in the groups treated with different concentrations of the drug composition were significantly downregulated.

[0088] The results of animal experiments show that the composition provided by the present invention has an obvious effect in treating silicosis.

[0089] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A pharmaceutical composition for treating silicosis, characterized in that: The pharmaceutical composition consists of isoliquiritigenin, glycyrrhizic acid and gallic acid in a mass ratio of 180:180:3.

75.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating silicosis.

3. The use according to claim 2, characterized in that: The medicine uses the medicine composition as an active ingredient for treating silicosis and is prepared into a pharmaceutically acceptable dosage form through pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The auxiliary material is solid, liquid or semi-solid auxiliary material.

5. The use according to claim 3, characterized in that: The dosage form is tablet, capsule, injection or inhalant.

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