Application of medicine composition based on diammonium glycyrrhizinate in preparation of medicine for preventing and / or treating pulmonary fibrosis

Abstract

The invention discloses application of a medicine composition based on diammonium glycyrrhizinate to preparation of a medicine for preventing and / or treating pulmonary fibrosis. Under the administration condition that the dosage is halved, compared with single medication, the inhibition effect of combined medication on pulmonary fibrosis is remarkably enhanced, specifically, alveolar structure collapse and interstitial collagen deposition are relieved, and the content of hydroxyproline in lung tissue is remarkably reduced; the expression of an epithelial marker E-Cadherin is up-regulated, and the expression of N-Cadherin and alpha-SMA is inhibited at the same time, so that a fibrosis core driving mechanism is effectively blocked; the activity of collagen I / III synthesis key enzyme is inhibited, and the lung tissue collagen deposition area is effectively reduced. The pharmaceutical composition based on diammonium glycyrrhizinate is adopted, through bidirectional intervention of upstream inflammation inhibition and downstream fibrosis blocking, the target limitation of single-drug treatment is broken through, meanwhile, dosage optimization and toxic and side effect reduction are achieved, and a brand new solution is provided for pulmonary fibrosis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a drug combination based on diammonium glycyrrhizinate in the preparation of a drug for preventing and / or treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is a fatal disease characterized by progressive decline in lung function and irreversible remodeling of tissue structure. Its pathological mechanism involves a cascade reaction of multiple links and multiple pathways, which can be summarized as a three-stage process of "initial injury - abnormal repair - fibrotic remodeling". Under the induction of viral infection, oxidative stress or genetic factors, alveolar epithelial cells (AECs) are continuously damaged and release pro-inflammatory factors (such as TNF-α, IL-6), activating the innate immune system and forming a chronic inflammatory microenvironment. In this stage, transforming growth factor-β (TGF-β), as the core driving factor of fibrosis, induces epithelial-mesenchymal transition (EMT) of alveolar epithelial cells through Smad-dependent and independent pathways, manifested as down-regulation of the epithelial marker E-cadherin and significant up-regulation of the mesenchymal markers N-cadherin and α-smooth muscle actin (α-SMA), causing epithelial cells to lose polarity and transform into migratory fibroblasts. At the same time, activated fibroblasts abnormally proliferate and secrete a large amount of extracellular matrix (ECM) components (such as collagen I / III, fibronectin), resulting in an increase in hydroxyproline content and lung tissue stiffness, and ultimately forming irreversible fibrous scars.

[0003] Currently, the mainstream clinical treatment drugs include glucocorticoids, immunosuppressants, and the targeted drugs pirfenidone and nintedanib. However, their mechanisms of action have significant limitations. For example, although pirfenidone (PFD) and nintedanib can partially inhibit fibroblast activation or collagen deposition, they cannot effectively block the EMT process and the overactivation of the TGF-β / Smad pathway, and there are defects in the single-target regulation of the inflammation-fibrosis network. Data show that although pirfenidone and nintedanib have been approved by the FDA, multiple randomized controlled trials (such as the trials of TOMORROW and INPULSIS) have shown that they have not significantly improved the survival rate of patients (no statistical difference from the placebo group), and are accompanied by serious adverse reactions such as a diarrhea incidence rate as high as 60%, hepatotoxicity, and photosensitivity reactions, resulting in low treatment compliance. Glucocorticoids can inhibit the inflammatory response in the short term, but long-term use is likely to induce osteoporosis, metabolic disorders, and withdrawal rebound phenomena, making it difficult to meet the management needs of chronic diseases. Moreover, existing combination regimens are mostly limited to the dose superposition of the same type of drugs (such as the combination of glucocorticoids and immunosuppressants), which not only cannot cover multiple targets of "inflammation-EMT-ECM deposition", but also lead to low treatment compliance due to cumulative toxicity. The root causes of the above defects lie in three major bottlenecks in the existing drug system: (1) Limited efficacy: Single-target intervention cannot reverse the fibrosis cascade network; (2) Insufficient safety: High-dose single drugs or extensive combination use exacerbate the toxic side effects; (3) Single mechanism: Lack of the ability to synergistically regulate the EMT and TGF-β / Smad pathways.

[0004] In recent years, the multi-target intervention strategy of traditional Chinese medicine has provided new ideas for the treatment of fibrosis. As the third-generation licorice extract, diammonium glycyrrhizinate (DG) has been clinically verified for its anti-inflammatory and anti-fibrotic potential (such as improving liver fibrosis and COVID-19 lung injury). Its mechanism involves inhibiting the NF-κB pathway and regulating the Th1 / Th2 balance, and its safety is superior to that of traditional immunosuppressants. However, the efficacy of single use of diammonium glycyrrhizinate in pulmonary fibrosis is still limited by its insufficient regulation of the TGF-β / Smad signaling pathway and is difficult to comprehensively block the fibrosis cascade reaction. At the same time, the active metabolite of vitamin D3, calcitriol, has been proven to play an anti-fibrotic role by antagonizing TGF-β-induced fibroblast activation, inhibiting the EMT process, and regulating the expression of matrix metalloproteinases (MMPs). The relationship between the mechanism related to EMT and pulmonary fibrosis is one of the research hotspots. For example, the mechanisms related to STAT3 and HSP90AA1. It has been found that STAT3 / HIF-1α and the phosphorylation of STAT3 and Smad3 have a promoting effect on EMT. In addition, the HSP90AA1 / PI3K / AKT signaling pathway also has an obvious promoting effect on it. Epidemiological studies further suggest that vitamin D3 deficiency is significantly negatively correlated with the progression of multi-organ fibrosis.

[0005] Although both of them show certain anti-fibrotic potential when applied alone, there has been no research in the prior art exploring the feasibility of the synergistic combination of diammonium glycyrrhizinate and vitamin D3. Traditional combination therapies are mostly limited to the dose superposition of drugs of the same type (such as the combination of glucocorticoids and immunosuppressants), often ignoring the complementarity of drug mechanisms, resulting in limited improvement in efficacy and additive toxicity. Therefore, developing a treatment plan with multi-target synergistic effects of anti-inflammatory - anti-EMT - anti-ECM deposition is the key to breaking through the existing treatment bottleneck. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of a drug combination based on diammonium glycyrrhizinate in the preparation of a drug for preventing and / or treating pulmonary fibrosis. For the first time, a cross-mechanism synergistic scheme of diammonium glycyrrhizinate and vitamin D3 is proposed. Through the two-way intervention of "inhibiting inflammation upstream - blocking fibrosis downstream", the target limitation of single-drug treatment is broken through, and at the same time, dose optimization and reduction of toxic and side effects are achieved, providing a new solution for the treatment of pulmonary fibrosis.

[0007] The present invention is achieved by the following technical solutions:

[0008] The use of a drug combination based on diammonium glycyrrhizinate in the preparation of a drug for preventing and / or treating pulmonary fibrosis, wherein the drug combination based on diammonium glycyrrhizinate includes diammonium glycyrrhizinate and other anti-fibrotic drugs.

[0009] Preferably, the doses of both diammonium glycyrrhizinate and other anti-fibrotic drugs are half of the original doses.

[0010] Preferably, the other anti-fibrotic drug is one of pirfenidone, nintedanib, glucocorticoids, vitamins, ginseng, and bupleurum.

[0011] Preferably, the vitamins include vitamin A and vitamin D3.

[0012] Preferably, the solvent of the drug is one of normal saline, PBS buffer solution, and propylene glycol solution.

[0013] Preferably, the drug further includes a pharmaceutically acceptable drug delivery carrier, and the drug delivery carrier is one of phospholipid complexes, liposomes, lipid nanoparticles, and micelles.

[0014] Preferably, the drug delivery carrier is one of lecithin, sphingomyelin, cephalin, inositol phospholipid, phosphatidic acid, polyethylene glycol, polylactic acid, poly(D,L-lactide), polyaspartic acid, and polyglutamic acid.

[0015] Preferably, the administration route of the drug is oral, intraperitoneal injection, intravenous injection, pulmonary inhalation, or intramuscular injection.

[0016] A pharmaceutical composition for preventing and / or treating pulmonary fibrosis, comprising diammonium glycyrrhizinate and vitamin D3.

[0017] The beneficial effects of the present invention are as follows:

[0018] (1) Under the administration condition of halving the dose, the combined use of drugs in the present invention significantly enhances the inhibitory effect on pulmonary fibrosis compared with single drug use. Specifically, it is manifested as: reducing alveolar structure collapse and interstitial collagen deposition, and significantly decreasing the content of hydroxyproline (HYP) in lung tissue; up-regulating the expression of epithelial marker E-Cadherin, while inhibiting the expression of N-Cadherin and α-SMA, effectively blocking the core driving mechanism of fibrosis; inhibiting the activity of the key enzyme for collagen I synthesis, effectively reducing the collagen deposition area in lung tissue.

[0019] (2) In the combined drug use scheme of the present invention, the dose of diammonium glycyrrhizinate is reduced by 50%, avoiding the potential liver toxicity risk caused by high dose of single drug; the dosage of vitamin D3 is reduced to the physiological supplement level (200 - 400 IU / day), avoiding adverse reactions such as hypercalcemia and vascular calcification, and significantly improving the patient tolerance.

[0020] (3) The combined drug use scheme of the present invention has complementary mechanisms. The synergistic effect of DG and VD3 against EMT depends on the crosstalk between STAT3 and HSP90AA1, thereby inhibiting the downstream HIF-1α pathway. The two form a two-way interactive regulatory network, breaking through the limitation of insufficient targeting of single drugs. Description of the Drawings

[0021] Figure 1 For Example 1: A is a schematic diagram of the modeling experiment and grouping; B is a schematic diagram of the influence on the body weight change of mice; C is the lung coefficient of mice in each treatment group; D is a schematic diagram of the detection result of the hydroxyproline content in mouse lung tissue;

[0022] Figure 2 For Example 1: A is a schematic diagram of the observation of the morphology, Masson staining and H&E staining of mouse lung tissue; B is the quantitative result of Masson staining; C is the quantitative result of H&E staining;

[0023] Figure 3For Example 1: A is the immunohistochemical analysis of the protein expressions of E-Cadherin, N-Cadherin, α-SMA, and Collagen I; B is the expression level of E-Cadherin; C is the expression level of N-Cadherin; D is the expression level of α-SMA; E is the expression level of Collagen I; F is the immunoblot experimental analysis of the protein expressions of E-Cadherin, N-Cadherin, and α-SMA; G is the protein expression level of E-Cadherin; H is the protein expression level of N-Cadherin; I is the protein expression level of α-SMA;

[0024] Figure 4 For Example 2: A is the cytotoxicity of A549 cells after being treated with different doses of BLM for 48 h; B is the quantitative analysis of the cell migration rate after being treated with different doses of BLM; C is the quantitative analysis of cell invasion after being treated with different doses of BLM; D is the change in cell morphology of A549 cells after being treated with different doses of BLM for 48 h; E is the effect of different doses of BLM on the migration ability of A549 cells at 0 h and 48 h, and the invasion ability through the transwell chamber at 24 h;

[0025] Figure 5 For Example 3: A is the toxicity of the drug to A549 cells after administering different doses of DG for 48 h; B is the toxicity of the drug to A549 cells after administering different doses of VD3 for 48 h; C is the toxicity of the drug to A549 cells after administering different doses of PFD for 48 h;

[0026] Figure 6 For Example 3, the changes in the morphology of A549 cells after the action of low (L), medium (M), high (H) doses of DG, VD3, PFD, and DG+VD3 for 48 h;

[0027] Figure 7 For Example 3: A is the inhibitory effect of different concentration treatment groups on the migration ability of BLM-induced A549 cells; B is the quantification of the inhibitory effect on the migration ability of the low-dose group; C is the quantification of the inhibitory effect on the migration ability of the medium-dose group; D is the quantification of the inhibitory effect on the migration ability of the high-dose group;

[0028] Figure 8 For Example 3: A is the inhibitory effect of different concentration treatment drugs on the invasion ability of BLM-induced A549 cells; B is the quantification of the inhibitory effect on the invasion ability of the low-dose group; C is the quantification of the inhibitory effect on the invasion ability of the medium-dose group; D is the quantification of the inhibitory effect on the invasion ability of the high-dose group;

[0029] Figure 9For Example 3: A is the representative western blot image expression of E-Cadherin, N-Cadherin, and α-SMA proteins in A549 cells treated with different therapies; B is the quantification of E-Cadherin protein expression; C is the quantification of N-Cadherin protein expression; D is the quantification of α-SMA protein expression;

[0030] Figure 10 For Example 4: A is the representative western blot image expression of STAT3, HSP90AA1, p-STAT3, and HIF-1α proteins in A549 cells treated with different therapies; B is the quantification of STAT3 protein expression; C is the quantification of HSP90AA1 protein expression; D is the quantification of p-STAT3 protein expression; E is the quantification of HIF-1α protein expression;

[0031] Figure 11 For Example 4: A is the screening of si-STAT3 bands with different sequences; B is the screening of si-HSP90AA1 bands with different sequences;

[0032] Figure 12 For Example 4: A is the western blot analysis of the effects of si-STAT3 on the expression of STAT3, E-cadherin, N-cadherin, and α-SMA proteins in A549 cells; B is the western blot analysis of the effects of si-HSP90AA1 on the expression of HSP90AA1, E-cadherin, N-cadherin, and α-SMA proteins in A549 cells; C is the quantification of the effect of si-STAT3 on STAT3 expression; D is the quantification of the effect of si-STAT3 on E-cadherin expression; E is the quantification of the effect of si-STAT3 on N-cadherin expression; F is the quantification of the effect of si-STAT3 on α-SMA expression; G is the quantification of the effect of si-HSP90AA1 on HSP90AA1 expression; H is the quantification of the effect of si-HSP90AA1 on E-cadherin expression; I is the quantification of the effect of si-HSP90AA1 on N-cadherin expression; J is the quantification of the effect of si-HSP90AA1 on α-SMA expression;

[0033] Figure 13In Example 4: A shows the effects of si-STAT3 on the expressions of HSP90AA1, p-STAT3, and HIF-1α in A549 cells; B shows the effects of si-HSP90AA1 on the expressions of STAT3, p-STAT3, and HIF-1α in A549 cells; C shows the quantification of the effect of si-STAT3 on the expression of HSP90AA1; D shows the quantification of the effect of si-STAT3 on the expression of p-STAT3; E shows the quantification of the effect of si-STAT3 on the expression of HIF-1α; F shows the quantification of the effect of si-HSP90AA1 on the expression of STAT3; G shows the quantification of the effect of si-HSP90AA1 on the expression of p-STAT3; H shows the quantification of the effect of si-HSP90AA1 on the expression of HIF-1α. Detailed implementation mode

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art. The experimental methods without specific conditions indicated are all conventional methods in the art.

[0036] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0037] The experimental animals used in the following examples are C57BL / 6 mice, female, 7 - 8 weeks old, weighing 20 - 25 g, purchased from the Experimental Animal Center of Nantong University, and the experiments have been carried out in accordance with the ethical requirements of experimental animals (filing number No: P20250223 - 007).

[0038] Example 1

[0039] 1. Experimental steps

[0040] As Figure 1 shown in A below, for C57BL / 6 mice at 6 - 8 weeks old, the trachea was surgically separated, and bleomycin (5 mg / kg) was injected intratracheally using an insulin syringe. The day of modeling was recorded as the 1st day, and drug treatment started on the 8th day after surgery. The combined drug treatment was carried out by gavage at a dose of 37.5 mg / kg of diammonium glycyrrhizinate (half of the dose of the single - drug treatment group, i.e., 75 mg / kg), and at the same time, a vitamin D3 propylene glycol solution (half of the dose of the single - drug treatment group, i.e., 1 μg / kg) was injected intraperitoneally for synergistic treatment (DG + VD3 combined administration group). Diammonium glycyrrhizinate was administered once a day, and vitamin D3 was administered once every two days. The treatment continued for 14 days, and the mice were sacrificed on the 22nd day. The lung tissue was weighed, and half of the fresh lung tissue was used for the determination of hydroxyproline (HYP) content, and the other half of the lung tissue was fixed for pathological analysis and immunohistochemical detection.

[0041] At the same time, set the following experimental groups ( Figure 1 in A):

[0042] Blank control group (Control): Inject 50 μL of 0.9% normal saline into the tracheal cartilage space with an insulin syringe;

[0043] BLM model group: Inject bleomycin 5 mg / kg into the tracheal cartilage space with an insulin syringe;

[0044] DG group: Treat by gavage at a dose of 75 mg / kg of diammonium glycyrrhizinate, once a day;

[0045] VD3 group: Intraperitoneally inject 2 μg / kg of vitamin D3 propylene glycol solution, once every two days;

[0046] Positive control group (PFD): Treat by gavage at 300 mg / kg of pirfenidone, once a day.

[0047] The experimental data of this example and the following examples were statistically analyzed using GraphPad Prism software (Version 8.0.8), and all data were expressed as mean ± standard error of the mean. Among them, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicates a significant difference compared with the control group. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 indicates a significant difference compared with the BLM group. ^ P < 0.05, ^^ P < 0.01, ^^^ P < 0.001, ^^^^ P < 0.0001 indicates a statistically significant difference compared with the DG group. + P < 0.05, ++ P < 0.01, +++ P < 0.001, ++++ P < 0.0001 indicates a significant difference compared with the VD3 group. Data analysis was performed using Prism 8 software (GraphPad).

[0048] 2. Experimental results

[0049] (1) Body weight change, lung coefficient and HYP content

[0050] After modeling and treatment, the general conditions of the mice were observed. There were no significant differences in the body weights of the mice in each group. As the experiment progressed, the mice after modeling showed obvious symptoms such as coughing. The mice were euthanized on the 22nd day. The changes in the body weights of the mice during the entire treatment process were observed.

[0051] As Figure 1 shown in B below, there were no significant differences in the body weights of the mice in each group. As Figure 1 shown in C and D below, compared with the BLM group, the lung coefficients of the mice in each treatment group were decreased, but the inhibitory effects of the DG group and the VD3 group on the HYP content were not obvious. The treatment effect of the DG+VD3 group was significant, inhibiting the significant decrease in the lung coefficient (P<0.0001) and HYP content (P<0.001) caused by BLM.

[0052] (2) Appearance of lung tissue, Masson staining and H&E staining

[0053] After dissection, the appearance of the lung tissue of each group was observed. As Figure 2 shown in A below, the lung tissue of the mice in the normal group was tender in texture, smooth on the surface and had good elasticity. After BLM modeling, the texture of both lungs became hard, and the whitening of the lung tissue indicated fibrosis. After treatment in the DG group, the color of the lung tissue was significantly improved, and the texture was slightly soft, but obvious red inflammation could still be observed. The lung tissue of the VD3 group remained white, and the improvement effect was not obvious. The lung tissue of the DG+VD3 combined treatment group was significantly improved, with rosy, smooth tissue and good elasticity.

[0054] Masson staining was used to observe the severity of lung tissue fibrosis. As Figure 2 shown in A and B below, the collagen deposition in the lung tissue of the mice after BLM stimulation was significantly increased compared with the normal group. The collagen deposition in the lung tissue of the mice in the DG+VD3 group was significantly lower than that in the BLM model group, and lower than that in the DG group and the VD3 group.

[0055] H&E staining was used to observe the lung inflammation. As Figure 2 shown in A and C below, similar to the results of the appearance of lung tissue and Masson staining, the original normal lung reticular structure could hardly be observed in the lung tissue of the BLM group, and a large number of inflammatory cells aggregated in the lungs. After drug treatment, the degree of inflammation in the DG group was significantly reduced, and the degree of inflammation in the VD3 group was also reduced. The treatment effects of the PFD group and the DG+VD3 group were significant.

[0056] (3) Expression of EMT-related factors

[0057] To explore the effects of DG and VD3 on the expression of EMT-related factors induced by BLM, IHC and WB were used to detect the expression of EMT marker proteins.

[0058] As Figure 3As shown in Figure A, IHC results showed that compared with the normal group, BLM induction significantly promoted the downregulation of E-Cadherin expression in lung tissue ( Figure 3 in Figure B, P < 0.0001), and promoted the upregulation of the protein expressions of N-Cadherin, α-SMA and Collagen I ( Figure 3 in Figures C-E, P < 0.0001). After treatment intervention, each drug administration group could reverse this trend to varying degrees, manifested as the upregulation of E-Cadherin expression and the downregulation of N-Cadherin, α-SMA and Collagen I expressions. Among them, the regulatory effect of the DG + VD3 combination treatment group was the most significant ( Figure 3 in Figures B-E). It indicates that DG and VD3 may affect EMT in lung tissue and anti-lung fibrosis by affecting EMT in lung tissue.

[0059] To verify this result, we further detected the expression levels of EMT-related proteins by WB. As Figure 3 shown in Figures F-H, the changing trends of the expressions of E-Cadherin, N-Cadherin and α-SMA were consistent with the IHC results, further verifying the effect of DG + VD3 in synergistically anti-EMT.

[0060] Example 2

[0061] In this example, the CCK-8 method was used to detect the effects of different concentrations (0, 0.1, 0.2, 0.3, 0.5 and 1 μg / mL) of BLM on the viability of A549 cells (lung cancer cells) after treatment for 48 h.

[0062] As Figure 4 shown in Figure A, with the increase of the BLM concentration, the viability of A549 cells showed a dose-dependent decreasing trend. In the concentration range of 0-0.5 μg / mL, BLM had no toxic effect on the cells.

[0063] As Figure 4 shown in Figure D, it was found by microscopic observation that when the BLM concentration reached 0.2 μg / mL, the cell morphology began to change significantly, gradually changing from the cobblestone-like epithelial morphology to the long spindle-like mesenchymal-like morphology. This morphological change was concentration-dependent. With the increase of the BLM concentration, the proportion of long spindle-shaped cells increased significantly, suggesting that BLM may induce the EMT process in A549 cells.

[0064] To further evaluate the effects of BLM on the migration and invasion abilities of A549 cells, we detected the scratch healing and Transwell invasion assays of the cells under the above concentration gradients. As Figure 4 shown in Figures B, C, E, with the increase of the BLM concentration, the cell migration area in the scratch assay decreased significantly ( Figure 4In B), at the same time, the number of cells that penetrated the Transwell chamber increased significantly ( Figure 4 In C), indicating that BLM may promote the migration and invasion ability of A549 cells.

[0065] Based on the above results, we selected 0.2 μg / mL of BLM for subsequent experiments.

[0066] Example 3

[0067] 1. Experimental grouping

[0068] After determining the BLM dose, this example used the CCK-8 experiment to screen for therapeutic drugs. As observed from the concentration-survival rate fitting curves of each therapeutic drug, as Figure 5 shown in A-C, the half-maximal effective concentration of the DG drug was 1.52 μmol / mL, the half-maximal effective concentration of the VD3 drug was 0.019 nmol / mL, and the half-maximal effective concentration of the PFD drug was 4.23 μmol / mL. Three doses of low, medium, and high were selected, and a combined treatment group was added at the same time. The drugs included DG and VD3 (specifically, the doses of the DG group and the VD3 group in each group were halved).

[0069] The low-dose group (L) included six subgroups, which were, in sequence: Control group, BLM group, L-DG group (0.3125 μmol / mL), L-VD3 group (0.01 nmol / mL), L-PFD group (0.25 μmol / mL), L-DG+VD3 group (0.1563 μmol / mL + 0.005 nmol / mL).

[0070] Similarly, the medium-dose group (M) was also divided into six subgroups: Control group, BLM group, M-DG group (0.625 μmol / mL), M-VD3 group (0.012 nmol / mL), M-PFD group (0.5 μmol / mL), M-DG+VD3 group (0.3125 μmol / mL + 0.006 nmol / mL).

[0071] Finally, the high-concentration group (H) was divided into Control group, BLM group, H-DG group (1.25 μmol / mL), H-VD3 group (0.014 nmol / mL), H-PFD group (1 μmol / mL), H-DG+VD3 group (0.625 μmol / mL + 0.007 nmol / mL).

[0072] 2. Experiment and result analysis

[0073] (1) A549 cell morphology

[0074] Observe the effects of each group on the morphology of A549 cells, asFigure 6 As shown, the cells in the Control group were cobblestone-shaped with clear cell structures. Compared with the Control group, the cell morphology in the BLM group changed significantly, with the cells being long spindle-shaped and the cell edges being blurred.

[0075] As Figure 6 shown, in the low-dose group, the cells in the L-DG group were still long spindle-shaped, even more obvious than those in the BLM group. Approximately 30% of the cells in both the L-VD3 group and the L-PFD group restored to the cobblestone shape. Approximately 50% of the cells in the L-DG+VD3 group restored to the cobblestone shape.

[0076] As Figure 6 shown, in the medium-dose group, the M-DG group, the M-VD3 group, the M-PFD group, and the M-DG+VD3 group significantly inhibited the cell morphological changes induced by BLM. Approximately 70%, 70%, 60%, and 90% of their cells respectively restored to the cobblestone shape.

[0077] As Figure 6 shown, in the high-dose group, the H-DG group, the H-VD3 group, the H-PFD group, and the H-DG+VD3 group significantly inhibited the cell morphological changes induced by BLM. Approximately 70%, 80%, 90%, and 90% of their cells respectively restored to the cobblestone shape. Cell fragmentation and a decrease in cell number were found in the H-VD3 group.

[0078] (2) Scratch assay

[0079] The scratch assay was used to detect the changes in cell migration ability after treatment with different therapeutic drugs combined with BLM (0.2 μg / mL) for 48 h.

[0080] As Figure 7 shown in A of Figure 7 , in the low-dose treatment group, except for the L-DG+VD3 group, there were no significant differences in the effects on cell migration ability among the other treatment groups compared with the BLM group. However, quantitative analysis showed that, compared with the BLM group, all treatment groups could significantly inhibit cell migration (

[0081] Note that, as Figure 7 shown in A and C of

[0082] In the medium-dose group, the inhibitory effect of the M-DG+VD3 group was significantly better than that of the single-drug treatment groups M-DG and M-VD3 (P < 0.05). Figure 7As shown in A and D, each treatment group showed a strong inhibition of cell migration (P < 0.001). Notably, the inhibitory effect of the H-DG+VD3 group was significantly better than that of the H-DG group and the H-VD3 group (P < 0.001), indicating that the combined use of DG and VD3 has a synergistic inhibitory effect on cell migration at high doses.

[0083] The above results suggest that DG and VD3 can dose-dependently inhibit the migration of BLM-induced A549 cells, and the effect of combined drug use is better than that of single drug treatment.

[0084] (3) Transwell invasion assay

[0085] As Figure 8 shown, except for the L-DG+VD3 group ( Figure 8 in A and B, P < 0.0001) having an obvious inhibitory effect compared with BLM, the low-concentration treatment groups of various drugs had no obvious inhibitory effect on the BLM-induced cell invasion. However, the medium- and high-dose treatment groups both had a significant reversal of the BLM-induced cell invasion effect ( Figure 8 in A, C, and D, P < 0.0001). As the concentration of the treatment drug increased, the inhibitory effect became more obvious, and the DG+VD3 treatment had the best effect.

[0086] (4) Western Blot analysis

[0087] As Figure 9 shown, compared with the normal control group, the BLM treatment group significantly down-regulated the expression of the epithelial marker E-Cadherin ( Figure 9 in A and B, P < 0.001), and at the same time significantly up-regulated the expression of the mesenchymal markers N-Cadherin ( Figure 9 in A and C, P < 0.0001) and α-SMA ( Figure 9 in A and D, P < 0.001). Notably, the treatment with DG combined with VD3 could effectively reverse the above-mentioned changes in the expression of EMT-related proteins induced by BLM, including significantly restoring the expression level of E-Cadherin and significantly inhibiting the up-regulation of the expression of N-Cadherin (P < 0.0001) and α-SMA (P < 0.0001).

[0088] The above in vitro experimental results prove the therapeutic effect of DG combined with VD3 in inhibiting the EMT process.

[0089] Example 4

[0090] 1. Research on the anti-fibrotic mechanism

[0091] To investigate the mechanism of the combined anti-fibrotic EMT effect of DG + VD3, we set up a total of five experimental groups, namely the Control group, the BLM group, the DG group, the VD3 group, and the DG+VD3 group, in the manner of Example 1, and performed Western Blot analysis on the protein expression levels of STAT3, HSP90AA1, p-STAT3, and HIF-1α.

[0092] The WB detection results are as Figure 10 shown in A - E below. Compared with the Control group, BLM treatment significantly upregulated the protein expression levels of STAT3, HSP90AA1, p-STAT3, and HIF-1α. After drug intervention, both the DG group and the VD3 group could reduce the upregulation of the above-mentioned protein expression induced by BLM, and the inhibitory effect of the DG group was more obvious. It is worth noting that the inhibitory effect of the DG+VD3 combination group on these key proteins was significantly stronger than that of the single-drug treatment group.

[0093] 2. Screening of siRNA genes

[0094] To clarify the specific mechanism by which STAT3 and HSP90AA1 exert anti-PF effects by regulating the HIF-1α pathway, we used siRNA to silence STAT3 and HSP90AA1. First, the interference efficiency of different siRNAs was detected by WB. Five groups were set up in the experiment: the Control group, the NC group, and the si-STAT3 group (three different siRNAs of STAT3).

[0095] As Figure 11 shown in A below, si-STAT3-2 had the most significant inhibitory effect on STAT3 protein expression. As Figure 11 shown in B below, all three siRNAs of HSP90AA1 could effectively inhibit the expression of the target protein.

[0096] Based on the above results, we selected si-STAT3-2 and si-HSP90AA1-1 with the best interference efficiency for subsequent mechanism studies.

[0097] 3. Interaction between key targets STAT3 and HSP90AA1

[0098] To clarify the regulatory roles of STAT3 and HSP90AA1 in the EMT process, we used the above si-STAT3-2 and si-HSP90AA1-1 to silence STAT3 and HSP90AA1, and performed Western Blot analysis on the protein expression levels of E-Cadherin, N-Cadherin, and α-SMA.

[0099] The experimental groups were: negative control group (NC), BLM-induced model group, control group of BLM + NC siRNA, and treatment group of BLM + si-STAT3 / si-HSP90AA1.

[0100] As Figure 12 shown, both si-STAT3-2 and si-HSP90AA1-1 could effectively reverse the upregulation of STAT3 ( Figure 12 in A, C, P < 0.0001) and HSP90AA1 ( Figure 12 in B, G, P < 0.01) protein expression induced by BLM.

[0101] Importantly, STAT3 gene knockout significantly inhibited the expression of BLM-stimulated mesenchymal markers N-Cadherin ( Figure 12 in A, E, P < 0.05) and α-SMA ( Figure 12 in A, F, P < 0.01), and at the same time promoted the expression of epithelial marker E-Cadherin ( Figure 12 in A, D, P < 0.001). Similarly, HSP90AA1 gene silencing also showed a similar regulatory pattern ( Figure 12 in B, G-J).

[0102] The above experimental results not only confirmed that STAT3 and HSP90AA1 play key regulatory roles in the EMT process, but also suggested that the two may participate in the development of EMT in PF through a mechanism of interaction.

[0103] 4. Effects on the HIF-1α pathway

[0104] The experimental groups were: negative control group (NC), BLM-induced model group, control group of BLM + NC siRNA, and treatment group of BLM + si-STAT3 / si-HSP90AA1.

[0105] As Figure 13 shown, si-STAT3-2 effectively blocked the expression of the HIF-1α signaling pathway ( Figure 13 in A, E), and si-HSP90AA1-1 also had the effect of inhibiting the HIF-1α signaling pathway ( Figure 13 in B, H). At the same time, si-STAT3-2 had the effect of inhibiting HSP90AA1 expression ( Figure 13 in A, C), and si-HSP90AA1-1 also had the effect of inhibiting STAT3 expression ( Figure 13 in B, F). This indicates that there is an inhibitory interaction between STAT3 and HSP90AA1, which also seems to explain the reason for the synergistic inhibitory effect of DG and VD3 on PF.

[0106] As shown Figure 13 in A, B, D, and G, both si-STAT3-2 and si-HSP90AA1-1 have the effect of inhibiting the expression of p-STAT3 and reversing the expression of HIF-1α. This indicates that the inhibitory effect on the downstream HIF-1α pathway can be produced through the mutual inhibition between STAT3 and HSP90AA1.

[0107] The experimental results of this example show that both si-STAT3-2 and si-HSP90AA1-1 can intervene in the HIF-1α pathway related to EMT.

[0108] The embodiments described above are only a part of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents the selected embodiments of the present invention. The protection scope of the present invention shall be subject to the scope claimed in the claims. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

Claims

1. Use of a pharmaceutical combination based on diammonium glycyrrhizinate in the preparation of a medicament for preventing and / or treating pulmonary fibrosis, characterized in that, The drug combination based on diammonium glycyrrhizinate includes diammonium glycyrrhizinate and other anti-fibrotic drugs.

2. The application according to claim 1, wherein The dosages of both diammonium glycyrrhizinate and other anti-fibrotic drugs are half of the original dosages.

3. The application according to claim 1, characterized in that, The other anti-fibrotic drug is one of pirfenidone, nintedanib, glucocorticoid, vitamins, ginseng, and bupleurum.

4. The application according to claim 3, characterized in that, The vitamins include vitamin A and vitamin D3.

5. The application according to claim 1, wherein The solvent of the drug is one of normal saline, PBS buffer solution, and propylene glycol solution.

6. The application according to claim 1, characterized in that, The drug further includes a pharmaceutically acceptable drug delivery carrier, and the drug delivery carrier is one of phospholipid complex, liposome, lipid nanoparticle, and micelle.

7. The application according to claim 6, wherein The drug delivery carrier is one of lecithin, sphingomyelin, cephalin, phosphatidylinositol, phosphatidic acid, polyethylene glycol, polylactic acid, poly D,L-lactide, polyaspartic acid, and polyglutamic acid.

8. The application according to claim 1, wherein The administration route of the drug is oral administration, intraperitoneal injection, intravenous injection, pulmonary inhalation, or intramuscular injection.

9. A pharmaceutical composition for preventing and / or treating pulmonary fibrosis, characterized in that, It includes diammonium glycyrrhizinate and vitamin D3.

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