A pharmaceutical composition for treating silicosis

Abstract

The present invention discloses a pharmaceutical composition for treating silicosis. The composition comprises isoliquiritin, glycyrrhizic acid, and gallic acid in a mass ratio of 180:180:3.75. Pharmacological data at the cellular and animal levels demonstrate that the composition exhibits significant therapeutic effects on silicosis and exhibits synergistic effects among its components.

Description

Technical Field

[0001] The present 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, primarily composed of free silica, which accumulates in the lungs during occupational activities and is characterized by diffuse fibrosis of the lung tissue. Its clinical manifestations are primarily respiratory symptoms, including cough, sputum, chest pain, and dyspnea. Silicosis is not only a lung disease but also a systemic disease. Even after patients are removed from dust exposure, their condition can progress and worsen, leading to respiratory and heart failure in severe cases. Silicosis is characterized by a high prevalence and a heavy economic burden. There is a lack of effective medications and treatments for silicosis.

[0003] Currently, silicosis is primarily treated with oxygen therapy, pulmonary rehabilitation, and lung transplantation, but these treatments are often unsatisfactory. Drug treatments include codeine phosphate tablets, aminophylline tablets, bromhexine hydrochloride tablets, ipratropium bromide aerosol, and tetrandrine tablets, but these medications only alleviate symptoms. Treatments such as large-volume whole lung lavage and lung transplantation are expensive and have limited indications, limiting their widespread clinical application.

[0004] Traditional Chinese medicine preparations, including Feining Granules and Bailing Capsules, have also been used to treat silicosis. However, these preparations contain low levels of active ingredients, are complex in composition, lack quality control indicators, and are difficult to assess for safety. These limitations limit the promotion and use of these preparations.

[0005] In order to develop a pharmaceutical composition for treating silicosis with obvious efficacy and clear ingredients, the present invention is 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 consists of isoliquiritin, 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 pharmaceutical composition as the 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, comprising isoliquiritin, glycyrrhizic acid, and gallic acid in a mass ratio of 180:180:3.75. Pharmacological data at the cellular and animal levels demonstrate that the composition exhibits significant therapeutic effects against silicosis and exhibits synergistic effects among its components. 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 combined efficacy value of glycyrrhizic acid and gallic acid is significantly higher than the simple sum of the combined efficacy values ​​of glycyrrhizic acid and gallic acid when administered alone, indicating that glycyrrhizic acid and gallic acid have a synergistic effect when administered together. From another perspective, the combined use of gallic acid, which has 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 administered together.

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

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

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

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

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

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

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

[0023] Figure 6 To compare the comprehensive efficacy of glycyrrhizic acid alone, gallic acid alone, and glycyrrhizic acid and gallic acid combined;

[0024] Figure 7 To compare the comprehensive efficacy of isoliquiritin alone, gallic acid alone, and the combination of isoliquiritin and gallic acid;

[0025] Figure 8 To compare the comprehensive efficacy of isoliquiritin alone, glycyrrhizic acid alone, gallic acid alone, and the combination of the three;

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

[0027] Figure 10 is the expression level of marker proteins IL-6, IL-1β, TNF-α, CD206 and ARG-1 in the lung tissue of silicosis mice. DETAILED DESCRIPTION

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

[0029] 1. Experimental Materials

[0030] 1. Chemicals: Isoliquiritin, glycyrrhizic acid, and gallic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd., lipopolysaccharide (LPS) was purchased from Beijing Solaibao 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 Biological Studies, Chinese Academy of Sciences; RPMI 1640 medium was purchased from Beijing Solebold 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 Punosai 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 Biotechnology Co., Ltd., and mouse primers were synthesized by Suzhou Genewise Biotechnology 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 Scientific, 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, USA;

[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 complete medium 1640 supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Cell growth was monitored daily. When cell confluence reached 80-90%, complete medium was preheated in a 37°C water bath. The culture medium was removed from the dish and 1 mL of fresh medium was added to disperse the cells by pipetting. The cells were counted and passaged at a 1:4 ratio.

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

[0046] Use DMSO as solvent to prepare the drug stock solution under the sterile operating table, store it at -80℃ for future use, and dilute it with complete culture medium to a certain concentration of working solution to treat cells according to experimental requirements. 6 A suspension of MHS cells at a concentration of 100 μL / mL was inoculated into a 96-well plate, with 100 μL per well. A blank group and a drug-treated group were set up, with 3-6 replicates per group. The cells were cultured in a cell culture incubator for 24 hours. The cell adhesion was observed under a microscope. After the cells were completely attached, the original culture medium was removed and 100 μL of culture medium containing different concentrations of drugs was added for 24 hours. The cell viability was detected using the CCK8 method. The drug-containing culture medium was first removed, and 100 μL of 1640 complete culture medium containing 10% CCK8 was added. The cells were placed in a 37°C, 5% CO2 incubator and allowed to stand for 1-2 hours. The OD value was measured at a wavelength of 450 nm using a microplate reader. Graph PadPrism 6 was used to plot the cell activity response diagram.

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

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

[0049] Wash the cells with 1 mL of PBS, add 1 mL of QIAzol to lyse for 10 minutes, transfer the lysate to a 1.5 mL enzyme-free EP tube, add 200 μL of chloroform and shake thoroughly for 15 seconds, let it stand at room temperature for 15 minutes, centrifuge at 4°C and 12000g for 15 minutes, take 490 μL of supernatant and transfer it to a new 1.5 mL enzyme-free EP tube, add an equal volume of isopropanol, shake thoroughly and let it stand at room temperature for 10 minutes, centrifuge at 4°C and 12000g for 10 minutes, discard the supernatant; add 1 mL of 75% ethanol, turn it upside down 3 times, centrifuge at 4°C and 7500g for 5 minutes, discard the supernatant; dry it in a fume hood for 5 minutes, add 31 μL of DEPC water to dissolve the precipitate, measure the total RNA concentration using nanodrop, and record the purity A260 / A230 and A260 / A280. For reverse transcription synthesis of cDNA, add 1 μg of total RNA to eight consecutive EP tubes, 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 10 seconds, and 4℃ to +∞. The reverse transcribed cDNA was amplified using a fluorescent quantitative PCR instrument. The relative expression level 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 internal reference gene in test group) - (average CT of target gene in blank group - average CT of internal reference gene in blank group). qPCR reaction system and reaction conditions are shown in Tables 1-3.

[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] To comprehensively evaluate the inhibitory effects of the synergistic components on MH-S cell polarization, we used a multi-index evaluation, assigning equal weight to each indicator. The combined efficacy (Output P) was derived by evaluating each combination based on its effects on macrophage-associated M1 markers IL-6, IL-1β, and 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 giving each combination drug.

[0060] 5. Animal Models and Treatment

[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 week 3 to week 6, 10 mice in the normal group and model group were gavaged with 0.5 mL of normal saline every day; 10 mice in the positive drug group were gavaged with tetrandrine (15 mg kg -1 ·d -1 The same number of mice in the drug combination group were orally administered 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 tissue was collected for pathological examination and immunohistochemistry. The dosage was adjusted every 3 days based on the body weight of each mouse.

[0062] 6. Histopathological examination

[0063] Lung tissue was fixed in 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 a light microscope. Quantitative assessment 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 scored using the Ashcroft score to assess the severity of pulmonary fibrosis.

[0064] 7. Immunohistochemistry

[0065] Lung tissue sections (4 μm thick) were blocked with 5% bovine serum albumin for 30 minutes 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 hours at 4°C. Integrated optical density was measured using IPP 6.0.

[0066] 8 Statistical methods

[0067] Experimental data were analyzed using IBM SPSS 22.0 statistical software. One-way ANOVA was used to compare intergroup data. For homogeneous variances, pairwise comparisons were performed using the least significant difference (LSD) method; for heterogeneous variances, the LSD method was used after data transformation. The significance level was set at α = 0.05. Data are presented as mean (χ) ± 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 isoliquiritin monomer on MH-S cells are as follows Figure 1 As shown in Figure A, isoliquiritin 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 Figure B, glycyrrhizic acid had no effect on the viability of MH-S cells at concentrations of 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 middle C, gallic acid has an effect on the viability of MH-S cells above 7.5 μg / mL, while 0-3.75 μg / ml has no significant effect.

[0073] 2. Monomer efficacy test results

[0074] The results of the inhibitory effect of isoliquiritin monomer on MH-S cell polarization are shown in Figure 2. Figure 2 As shown in the results, isoliquiritin 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 as follows Figure 3 As shown in the results, 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 data, 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. The comprehensive efficacy value of each combination ratio was calculated. It was found that combination 8, that is, the ratio of isoliquiritin, 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. Synergistic effect evaluation

[0082] We evaluated whether there is synergistic effect in a combination of isoliquiritin, glycyrrhizic acid, and gallic acid at 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 combined efficacy value of glycyrrhizic acid and gallic acid is significantly higher than the simple sum of the combined efficacy values ​​of glycyrrhizic acid and gallic acid when administered alone, indicating that glycyrrhizic acid and gallic acid have a synergistic effect when administered together. From another perspective, the combined use of gallic acid, which has 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 administered together.

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

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

[0086] 4. Pharmacodynamic evaluation of drug combination in 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 isoliquiritin, glycyrrhizic acid, and gallic acid at a mass ratio of 180:180:3.75). To evaluate its therapeutic effect on silicosis mice, the drug combination (e.g., Figure 9 Middle A). Figure 9 The HE and Masson staining results shown in B and C show that compared with the control group, the lungs of 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 showed an obvious dose effect. The positive drug group (hanfangjisu) 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. Figure 10 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 successful model establishment. 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 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 this specific embodiment.

Claims

1. A pharmaceutical composition for treating silicosis, characterized in that: The pharmaceutical composition consists of isoliquiritin, 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 medicament for treating silicosis.

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