Pharmaceutical composition for resisting cell proliferation, fibrosis and epithelial-mesenchymal transition and application thereof

Abstract

The invention provides a pharmaceutical composition for resisting cell proliferation, fibrosis and epithelial-mesenchymal transition and application of the pharmaceutical composition, and belongs to the technical field of biological medicines. According to the invention, mitomycin-C (MMC) and doxorubicin (DOX) are combined for use according to a specific molar ratio, so that proliferation and migration of various visceral organ lesion cells can be effectively inhibited, and meanwhile, the intracellular oxidative stress level is sharply enhanced, so that obvious damage to a mitochondrial structure and breakage of an intracellular DNA chain are caused. The pharmaceutical composition can reduce the use concentration of a single drug, has the characteristics of resisting cell proliferation and fibrosis, and inhibits the occurrence and development of epithelial-mesenchymal transition, thereby having important application potential in the curative effect of clinically treating various eye diseases, cancers and organ fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition for resisting cell proliferation, fibrosis and epithelial-mesenchymal transition and its application. Background Art

[0002] Epithelial-mesenchymal transition (EMT) epitomizes cellular plasticity in embryonic development, cancer, and postoperative tissue repair and fibrosis. During EMT, cell-cell and cell-extracellular matrix interactions are remodeled, and epithelial cells undergo dramatic phenotypic changes, leading to separation of epithelial cells from each other and from the basement membrane, while simultaneously activating novel transcriptional programs to promote EMT. The most prominent features of EMT include changes in cell morphology and molecular markers: epithelial cells transform from a polygonal or cobblestone shape to an elongated spindle shape, cytoskeletal rearrangements, and a reduction in epithelial markers (e.g., E-cadherin, α-catenin, β-catenin, γ-catenin, CK, ZO-1) and a significant expression of mesenchymal markers (e.g., N-cadherin, Vimentin, α-SMA, fibronectin). These changes enhance cell migration and motility. In addition, cells are in a chronic inflammatory microenvironment for a long time, which aggravates the recruitment of fibroblasts. The activated fibroblasts secrete a large amount of growth factors, inflammatory factors and metalloproteinases, aggravating the deposition of extracellular matrix proteins (such as laminin, collagen IV, collagen I), causing epithelial cells to undergo EMT and enhancing cell proliferation and migration.

[0003] Taking posterior capsule opacification (PCO) as an example, a complication of cataract surgery, residual lens epithelial cells attached to the anterior capsule trigger a wound healing response after surgical trauma, initiating a fibrotic process characterized by excessive proliferation, matrix contraction and deposition, and epithelial-mesenchymal transition (EMT) to a disordered myofibroblast phenotype, ultimately leading to fibrotic PCOP. Alternatively, LECs may trigger lens fibrillation, forming pearly Elschnig bodies and Soemmering rings, also known as regenerative PCOP. EMT also plays a crucial role in tumor development and progression. It not only enables cancer cells to overproliferate, potentially leading to tumorigenesis and metastasis, but also conferring multidrug resistance to anticancer drugs.

[0004] The clinical use of small molecule chemotherapeutics to combat epithelial cell proliferation and fibrosis is an effective treatment for ocular and tumor diseases. For example, mitomycin-C (MMC), an antitumor antibiotic isolated from the culture medium of the actinomycete Streptomyces espitosus, is a cell cycle-nonspecific antitumor agent. MMC is also widely used in the treatment of ophthalmic diseases to prevent tissue adhesion and scarring at surgical incisions. MMC activates its benzoquinone ring through bioreduction, cross-linking DNA to form DNA adducts. Doxorubicin (DOX) is a first-line antitumor chemotherapy drug. Doxorubicin intercalates into DNA chains, inhibiting the action of topoisomerase II and leading to DNA strand breaks. Furthermore, doxorubicin can convert from a quinone structure to a semiquinone through redox cycling, generating superoxide radicals. However, the clinical benefits of monotherapy with MMC or doxorubicin are severely limited due to their high resistance at the cellular and tumor levels, complex extracellular matrix environments, and dose-limiting side effects. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a synergistic pharmaceutical composition that has anti-epithelial cell proliferation and fibrosis efficacy and can effectively inhibit the occurrence and development of EMT.

[0006] The present invention provides a pharmaceutical composition, characterized in that it comprises mitomycin-C and doxorubicin;

[0007] The molar ratio of mitomycin-C to doxorubicin is (1-3): (1-3).

[0008] Preferably, the molar ratio of mitomycin-C to doxorubicin is (1-3): (1-2).

[0009] Preferably, the molar ratio of mitomycin-C to doxorubicin is 3:1 or 1:2.

[0010] The present invention provides use of the pharmaceutical composition, mitomycin-C or doxorubicin in preparing drugs for resisting cell proliferation, resisting cell migration and / or resisting cell fibrosis.

[0011] Preferably, the anti-fibrosis activity includes inhibiting the differentiation of cells into fibroblasts.

[0012] Preferably, the disease caused by cell proliferation, cell migration and / or cell fibrosis includes at least one of the following: ocular fibrosis, organ fibrosis, in situ tumor or metastatic tumor.

[0013] The present invention provides use of the pharmaceutical composition, mitomycin-C or doxorubicin in preparing drugs for resisting epithelial-mesenchymal transition.

[0014] Preferably, the indications caused by epithelial-mesenchymal transition include at least one of the following: posterior capsule opacity, tumor development and metastasis, organ fibrosis, glaucoma, diabetic retinopathy, and macular degeneration.

[0015] Preferably, the epithelial-mesenchymal transition includes upregulation of the expression level of epithelial-mesenchymal transition markers.

[0016] Preferably, the epithelial-mesenchymal transition markers include vimentin and / or α-smooth muscle actin.

[0017] The present invention provides a medicine, the active ingredients of which include the pharmaceutical composition.

[0018] Preferably, the dosage form of the drug includes at least one of the following: injection solution, injection powder and nanoparticles.

[0019] Preferably, the nanoparticles are the shell material and the active ingredient encapsulated in the shell material;

[0020] The shell material includes polylactic acid-glycolic acid copolymer;

[0021] The constituent monomers of the polylactic acid-glycolic acid copolymer include lactic acid and glycolic acid;

[0022] The percentage of lactic acid to glycolic acid in the constituent monomers is 10-17:3-10.

[0023] The present invention provides the use of doxorubicin in preparing a medicine for synergistically improving mitomycin-C's ability to resist cell proliferation, migration and fibrosis.

[0024] Preferably, the synergistic enhancement of mitomycin-C's anti-cell proliferation and fibrosis effect includes at least one of the following: a sharp increase in oxidative stress levels, an imbalance in intracellular calcium ion homeostasis, and DNA strand breaks.

[0025] The present invention provides a pharmaceutical composition comprising mitomycin-C and doxorubicin; the molar ratio of mitomycin-C to doxorubicin is (1 to 3): (1 to 3). The present invention combines mitomycin-C with the reactive oxygen species generating small molecule chemotherapy drug doxorubicin to synergistically inhibit the activity of pathogenic characteristics such as cloning, overproliferation, EMT and fibrosis of various epithelial cells, endothelial cells and fibroblasts. The present invention combines mitomycin-C and doxorubicin in a specific synergistic ratio to effectively inhibit the proliferation and migration of epithelial cells. The synergistic effect of mitomycin-C and doxorubicin can significantly increase the level of intracellular oxidative stress. In the microenvironment where the semiquinone after reduction with doxorubicin competes for intracellular oxygen and reactive oxygen species accumulation, mitomycin-C switches from the double electron transfer reduction pathway to the single electron sequential transfer reduction pathway, continuously generating semiquinone free radical intermediates and hydroquinone intermediates, producing DNA adducts, leading to apoptosis of epithelial cells; in addition, mitomycin-C and doxorubicin synergistically reduce the effect of glutathione levels, enhance mitochondrial lipid peroxidation damage, and are accompanied by reduced ATP synthesis in epithelial cells, reduced mitochondrial membrane potential and damaged mitochondrial structure, thereby effectively inhibiting the occurrence of epithelial cell EMT. The pharmaceutical composition combines MMC and DOX to reduce the concentration of single drug use, has the potential to reduce the toxic and side effects of the combination, and has anti-epithelial cell proliferation and fibrosis properties, inhibiting the occurrence and development of epithelial-mesenchymal transition (EMT), thereby having important application potential in the clinical treatment of eye diseases and tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Results of screening the ratio of mitomycin-C combined with doxorubicin for the treatment of human lens epithelial cells by MTT assay; A. Combination index (CI) curves of three MMC-DOX combination ratios (1:1, 2:1, and 3:1) screened by MTT assay; B. Median effect plots of the interaction between single-agent MMC, single-agent DOX, and three MMC-DOX combination ratios (1:1, 2:1, and 3:1); C. Dose reduction index (DRI) curves of the three MMC-DOX combination ratios; red boxes (Log DRI < 0) indicate adverse reactions, and light purple boxes (Log DRI>0) indicates therapeutic efficacy; D. Cell viability of human lens epithelial cells treated with MMC alone, DOX alone, and three MMC-DOX combination ratios (1:1, 2:1, and 3:1); E. Drug concentrations that inhibited cell growth by 50% (IC) for MMC alone, DOX alone, and three MMC-DOX combination ratios (1:1, 2:1, and 3:1) 50 and IC 95 )Compare the results;

[0027] Figure 2The results of the screening of the ratio of mitomycin-C combined with doxorubicin in the treatment of human corneal epithelial cells are shown in Figure 3, where A is the combination index (CI) curve; B is the median effect equation; C is the dose reduction index (DRI) graph; D is the cell viability results of human lens epithelial cells treated with single-agent MMC, single-agent DOX, and four MMC-DOX combination ratios (1:1, 1:2, 2:1, and 3:1); E is the drug concentration that inhibits 50% cell growth (IC) for single-agent MMC, single-agent DOX, and four MMC-DOX combination ratios (1:1, 1:2, 2:1, and 3:1). 50 and IC 95 )Compare the results;

[0028] Figure 3 Figure 3 is a screening result of the ratio of mitomycin-C combined with doxorubicin for the treatment of retinal pigment epithelial cells, where A is the combination index (CI) curve; B is the median effect equation; C is the dose reduction index (DRI) graph; D is the cell viability results of human lens epithelial cells treated with single-agent MMC, single-agent DOX, and four MMC-DOX combination ratios (1:1, 1:2, 2:1, and 3:1); E is the drug concentration that inhibits 50% cell growth (IC) of single-agent MMC, single-agent DOX, and four MMC-DOX combination ratios (1:1, 1:2, 2:1, and 3:1). 50 and IC 95 )Compare the results;

[0029] Figure 4 The results of the screening of the ratio of mitomycin-C combined with doxorubicin for the treatment of colorectal cancer epithelial cells are shown, where A is the combination index (CI) curve; B is the median effect equation; C is the dose reduction index (DRI) graph; D is the cell viability results of human lens epithelial cells treated with single-agent MMC, single-agent DOX, and four MMC-DOX combination ratios (1:1, 1:2, 2:1, and 3:1); E is the drug concentration that inhibits 50% cell growth (IC) of single-agent MMC, single-agent DOX, and four MMC-DOX combination ratios (1:1, 1:2, 2:1, and 3:1). 50 and IC 95 )Compare the results;

[0030] Figure 5 The results are as follows: screening results of the ratio of mitomycin-C combined with doxorubicin for the treatment of human umbilical vein endothelial cells; A is the combination index (CI) curve; B is the median effect equation; C is the dose reduction index (DRI) graph; D is the cell viability results of human lens epithelial cells after treatment with single-drug MMC, single-drug DOX and four MMC-DOX combination ratios (1:1, 1:2, 2:1 and 3:1); E is the drug concentration (IC) that inhibits 50% cell growth of single-drug MMC, single-drug DOX and four MMC-DOX combination ratios (1:1, 1:2, 2:1 and 3:1).50 and IC 95 )Compare the results;

[0031] Figure 6 The results are as follows: screening results of the ratio of mitomycin-C combined with doxorubicin for the treatment of human colonic fibroblasts; A is the combination index (CI) curve; B is the median effect equation; C is the dose reduction index (DRI) graph; D is the cell viability results of human lens epithelial cells after treatment with single-drug MMC, single-drug DOX and four MMC-DOX combination ratios (1:1, 1:2, 2:1 and 3:1); E is the drug concentration (IC) that inhibits 50% cell growth of single-drug MMC, single-drug DOX and four MMC-DOX combination ratios (1:1, 1:2, 2:1 and 3:1). 50 and IC 95 )Compare the results;

[0032] Figure 7 Results of the validation experiment of the anti-proliferation and anti-migratory effect of the optimal synergistic ratio MMC-DOX (3:1) on HLE B-3 cells; A. / B. Colony formation results of 500 and 2000 HLE B-3 cells incubated with MMC-DOX (3:1) for 3h (A) and 24h (B) in the clone formation experiment; C. Survival score curves of low-density (500) and high-density (2000) HLE B-3 cells incubated with MMC-DOX (3:1) combination for 3h and 24h; D. IC values ​​of MMC-DOX (3:1) in low-density (500) and high-density (2000) HLE B-3 cells incubated for 3h and 24h 50 Compare the results;

[0033] Figure 8 Results of the experimental validation of the anti-migration effect of the optimal synergistic ratio of MMC-DOX (3:1) on HLE B-3 cells; A. Transwell assay for anti-migration images of HLE B-3 cells after 3 hours of incubation with MMC alone, DOX alone, and MMC-DOX (3:1); B. Image J quantitative analysis of cell migration results;

[0034] Figure 9 The results of the experimental verification of the anti-EMT effect of the optimal synergistic ratio MMC-DOX (3:1) on HLE B-3 cells were shown in Figure 2. A. Western Blot was used to detect the effects of single-agent MMC, single-agent DOX, and MMC-DOX (3:1) on the D 50 The expression level of EMT marker proteins in HLE B-3 cells after 3h incubation with different doses; B. The expression level of vimentin in single-drug MMC, single-drug DOX and MMC-DOX (3:1) in D 50Quantitative analysis of the expression levels of HLE B-3 cells after incubation with different doses for 3 h; C. α-smooth muscle actin (α-SMA) in single-agent MMC, single-agent DOX and MMC-DOX (3:1) in D 50 Quantitative analysis results of expression levels after incubation of HLE B-3 cells for 3 h at different doses;

[0035] Figure 10 MMC-DOX (3:1) drug in D 50 and D 95 The results of the effects of different doses on the oxidative stress level of HLE B-3 cells; Figure A shows the effects of single-agent MMC, single-agent DOX and MMC-DOX (3:1) on the oxidative stress level of HLE B-3 cells. 50 (i~iii) and D 95 (iv~vi) Changes in total intracellular reactive oxygen species levels in HLE B-3 cells after treatment with different doses for 3 h; B. MMC alone, DOX alone, and MMC-DOX (3:1) in D 50 and D 95 Quantitative detection results of total intracellular ROS in 3hHLE B-3 cells after treatment with 400 mg / kg of MMC; C. Quantitative detection results of total intracellular ROS in D 50 Results of intracellular lipid peroxidation levels in HLE B-3 cells after treatment with different doses for 3 h; D. The results of intracellular lipid peroxidation levels in HLE B-3 cells after treatment with different doses of MMC, DOX and MMC-DOX (3:1) in D 50 Quantitative detection results of intracellular lipid peroxidation levels in HLE B-3 cells after treatment with different doses for 3 h;

[0036] Figure 11 To quantitatively analyze the antioxidant GSH levels in HLE B-3 cells treated with MMC-DOX (3:1) and MMC / DOX alone for 3 h in response to DTNB;

[0037] Figure 12 Fluorescence images of mitochondrial lipid peroxidation in HLE B-3 cells treated with single drugs and MMC-DOX (3:1) at a dose of D50 for 3 h (A) and mitochondrial localization analysis results (B);

[0038] Figure 13 TUNEL assay was used to determine the activity of single drug and MMC-DOX (3:1) in D 50 The results of DNA damage in HLE B-3 cells after treatment with different doses for 3h and 24h, where i-xiii in A show the single drug and MMC-DOX (3:1) in D 50 Changes in intracellular DNA damage in HLE B-3 cells after 3 h of treatment with different doses; i-xiii in B show the changes in intracellular DNA damage in D between single drug and MMC-DOX (3:1)50 The results of the changes of intracellular DNA damage in HLE B-3 cells treated with different doses for 24 hours; C. The changes of intracellular DNA damage in D 50 Fluorescence quantitative analysis results of DNA damage in HLE B-3 cells after treatment with different doses for 3h and 24h;

[0039] Figure 14 The results of mitochondrial structure and function damage detection are shown in Figure 2. A.MMC / DOX single drug and MMC-DOX (3:1) in D 50 Electron microscopic images of mitochondrial structure in HLE B-3 cells after treatment with different doses for 1 hour; Quantitative analysis results of mitochondrial area in the electron microscopic images of mitochondria in Figure BA; C. MMC / DOX single drug and MMC-DOX (3:1) drug in D 50 The results of ATP production and mitochondrial membrane potential measurement in HLEB-3 cells after treatment with different doses for 3h, 8h, 12h and 24h. DETAILED DESCRIPTION

[0040] The present invention provides a pharmaceutical composition comprising mitomycin-C and doxorubicin; the molar ratio of mitomycin-C to doxorubicin is (1-3): (1-3).

[0041] In the present invention, the molar ratio of mitomycin-C and doxorubicin is preferably (2-3):1, more preferably 3:1 or 1:2. In one embodiment of the present invention, the Chou-Talalay mathematical model was used to screen the optimal drug synergistic ratio of mitomycin-C and doxorubicin. The combination index (CI) curve results showed that the MMC-DOX drug was less than 1 at a molar ratio of 1:1, 2:1, 1:2 and 3:1, but MMC-DOX was most stable at a molar ratio of 3:1 or 1:2. The median effect diagram showed that the doses of MMC and DOX in the MMC-DOX combination ratio that inhibited 50% cell growth were all less than the doses of single-drug MMC and single-drug DOX. The DRI diagram showed that the three MMC-DOX combination ratios showed no adverse reactions in the process of inhibiting cell growth with higher inhibition rates, but the trend of MMC-DOX (3:1 or 1:2) was the most stable. It can be seen that the optimal synergistic ratio is when the molar ratio of mitomycin-C and doxorubicin is 3:1 or 1:2. It can be seen that the MMC-DOX combination can effectively inhibit the proliferation of corneal epithelial cells, retinal pigment epithelial cells, colorectal cancer epithelial cells, human colonic fibroblasts and human umbilical vein endothelial cells, and greatly reduce the dosage of the drug, greatly reducing the cytotoxicity of the drug. In addition, compared with single-drug MMC and DOX, the MMC-DOX combination can effectively inhibit cell migration, downregulate the expression of epithelial-mesenchymal transition markers (vimentin and α-smooth muscle actin), increase epithelial cell oxidative stress levels and reduce glutathione levels, and the differences are significant.

[0042] The present invention provides use of the pharmaceutical composition, mitomycin-C or doxorubicin in preparing drugs for resisting cell proliferation, migration, fibrosis or epithelial-mesenchymal transition.

[0043] In the present invention, the anti-fibrosis activity includes inhibiting the differentiation of cells into fibroblasts.

[0044] In the present invention, cell proliferation preferably refers to enhanced cell proliferation ability. Cell migration preferably refers to enhanced cell migration activity. Epithelial cell fibrosis includes enhanced differentiation of epithelial cells into myofibroblasts. In the present invention, the cells preferably include at least one of the following: lens epithelial cells, corneal epithelial cells, retinal pigment epithelial cells, colorectal cancer epithelial cells, human colonic fibroblasts, and human umbilical vein endothelial cells.

[0045] In the present invention, diseases caused by cell proliferation, cell migration, and / or cell fibrosis preferably include at least one of the following: ocular fibrosis, organ fibrosis, in situ tumors, or metastatic tumors. The ocular fibrosis preferably includes lens fibrosis, corneal fibrosis, and retinal fibrosis. The organ fibrosis preferably includes colon fibrosis, liver fibrosis, pulmonary fibrosis, cardiac fibrosis, and the like. The in situ tumor or metastatic tumor includes colorectal cancer.

[0046] In the present invention, the epithelial-mesenchymal transition preferably includes upregulation of the expression level of epithelial-mesenchymal transition markers. The indications caused by the epithelial-mesenchymal transition preferably include at least one of the following: posterior capsule opacification, tumor development and invasion, organ fibrosis, glaucoma, diabetic retinopathy, and macular degeneration.

[0047] In the present invention, in order to further explore the drug molecular mechanism of mitomycin-C and / or doxorubicin, the mitochondrial localization of mitochondrial lipid peroxidation is detected, and the co-localization fluorescence image analysis by Pearson coefficient shows that MMC-DOX drug combination obviously causes lipid peroxidation damage on mitochondrial membrane, and the lipid peroxidation caused by both the single drug group and the drug combination group is located on mitochondria. At the same time, in the embodiment of the present invention, the TUNEL method is used to measure the DNA double-strand break situation in the cell after MMC-DOX drug combination or single drug treatment of HLE B-3 cells. The results show that MMC-DOX drug combination and single drug can cause DNA double-strand break, leading to cell apoptosis, and MMC-DOX drug combination is more significant than single drug in terms of DNA double-strand break. TUNEL method is to look at the breakage apoptosis of DNA by the 3'-OH sticky end produced after dUTP labeling DNA break. Experimental results showed that when MMC and DOX were combined to treat cells, a single hydroquinone intermediate was generated through a two-electron reduction pathway and embedded in DNA. Mitomycin-C likely switched from a two-electron reduction pathway to a one-electron reduction pathway, generating two semiquinone radical intermediates and a hydroquinone intermediate that were embedded in DNA, exacerbating DNA damage. Accumulated reactive oxygen species caused lipid peroxidation within the mitochondria, further enhancing the anti-proliferative and EMT effects. Due to the different spatial distributions of MMC and DOX and the different treatment durations, the residual 3'-OH sticky ends were more labeled with TUNEL after combination treatment compared to single-drug treatment, indicating DNA chain breaks.

[0048] The present invention provides a medicine, the active ingredients of which include the pharmaceutical composition.

[0049] In the present invention, the dosage form of the drug preferably includes an injection solution, an injection powder, and nanoparticles. The drug also preferably includes an excipient. When preparing an injection solution, the excipient preferably includes water for injection, such as a 0.9% by weight sodium chloride solution. When preparing an injection powder, the excipient preferably includes a lyoprotectant. The present invention does not specifically limit the type of lyoprotectant; any lyoprotectant known in the art can be used.

[0050] In the present invention, the nanoparticles are a shell material and an active ingredient encapsulated in the shell material. The shell material preferably includes polylactic acid-co-glycolic acid (PLGA). The constituent monomers of the PLGA include lactic acid and glycolic acid. The percentage of lactic acid and glycolic acid in the constituent monomers is preferably 10-17:3-10, and can be 5:1. By adjusting the ratio of PLGA constituent monomers, different release rates of the active pharmaceutical ingredient are achieved. In an embodiment of the present invention, microfluidics and experimental design (DoE) strategies are used to encapsulate the MMC-DOX combination drug in polylactic acid-co-glycolic acid (PLGA)-based nanoparticles to screen out the optimal synergistic ratio (3:1) to obtain intravenously injectable PLGA nanoparticles containing MMC-DOX (D / M PLGANPs). Within the effective concentration range, the released drug concentration ratio is closest to the synergistic ratio at which the drug combination works best, thereby exerting an effective therapeutic effect against cell proliferation and migration.

[0051] In the present invention, the effective concentration of mitomycin-C in the drug is not less than 0.3 μM, and can be 0.372 μM to 5.53 μM. The effective concentration of doxorubicin is not less than 0.1 μM, and can be 0.124 μM to 3.08 μM. The present invention has no particular limitations on the preparation method of the drug, and any drug preparation method known in the art can be used.

[0052] The present invention provides the use of doxorubicin in preparing a medicine for synergistically improving the anti-cell proliferation and fibrosis effects of mitomycin-C.

[0053] In the present invention, the synergistic enhancement of the anti-cell proliferation and fibrosis effect of mitomycin-C includes at least one of the following: a sharp increase in oxidative stress levels, an imbalance in intracellular calcium ion homeostasis, and DNA chain breaks.

[0054] In the embodiment of the present invention, compared with single-drug mitomycin-C, the combined use of doxorubicin and mitomycin-C, which are small molecule drugs for generating reactive oxygen species, can significantly reduce the IC of each single drug. 50 Dosages of 10 mg / dL significantly inhibited epithelial cell proliferation and EMT migration. Doxorubicin and mitomycin-C synergistically enhanced oxidative stress in epithelial cells, leading to mitochondrial lipid peroxidation and loss. This suggests that the combination of doxorubicin and mitomycin-C at a specific ratio can exert a synergistic effect.

[0055] The following is a detailed description of a pharmaceutical composition for inhibiting epithelial cell proliferation, fibrosis and epithelial-mesenchymal transition and its application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Screening test for the optimal drug synergistic ratio of mitomycin-C combined with doxorubicin

[0058] Human lens epithelial cells (HLE B-3), corneal epithelial cells (HCE-T), retinal pigment epithelial cells (ARPE-19), colorectal cancer epithelial cells (Caco-2), human umbilical vein endothelial cells (HUVEC), and human colonic fibroblasts (CCD-18co) were cultured at 1×10 4 Cells were seeded at a density of 100 μg / mL onto 96-well plates and incubated at 37°C for 24 h. The cells were washed with fresh serum-free MEM and treated with MMC and DOX at molar ratios of 1:1, 1:2, 2:1, and 3:1, with single-drug MMC concentrations ranging from 0 μM to 32 μM and single-drug DOX concentrations ranging from 0 μM to 32 μM, respectively. After 24 h, 10 μL of MTT (5 mg / mL) was added to each well and incubated at 37°C for 3 h. After removing the supernatant, 150 μL of DMSO was added to the plate. The sample solution was mixed for 10 min to completely dissolve the precipitate at room temperature. The effect of the drug on cell viability was measured by absorbance at 490 nm using a microplate reader.

[0059] Chou-Tatalay median effect analysis

[0060] The Chou-Talalay mathematical model is a widely recognized method for quantitative analysis of drug synergy. CompuSyn, a drug combination dose-effect analysis software, generates a plot of the fraction affected (fa) / combination index (CI). The Chou-Talalay method, derived from the law of mass action, derives the median effect equation for mitomycin C-doxorubicin at different combination ratios and calculates the CI and DRI values ​​using the following formula:

[0061]

[0062] where f a (affect fraction) is the rate of effect of the drug on cell viability. u (unaffected fraction) is the rate of cell viability not affected by the drug. D is the dose of a single drug. m Same as D 50, is the dose of a single drug that affects 50% of cell viability. (Dx)1 is the dose of single drug 1 (e.g., mitomycin-C) that inhibits x% of cell viability, (Dx)2 is the dose of single drug 2 (e.g., doxorubicin) that inhibits x% of cell viability, and (D)1 and (D)2 are the doses of single drug 1 (e.g., mitomycin-C) combined with single drug 2 (e.g., doxorubicin) that inhibit x% of cell viability, respectively.

[0063] The CI value and DRI value are indicators proposed by Chou-Talalay based on the law of mass action to evaluate the effects of drug interactions: a CI value of 1 when the drugs are combined indicates an antagonistic effect between the drugs; the DRI value of each drug is obtained by algorithm fitting after rearranging the CI equation. When the DRI value is >1, it indicates that the dosage of the drug in combined use can be reduced, thereby reducing drug-related toxic side effects in therapeutic applications.

[0064] See the results Figures 1 to 6 .Depend on Figure 1 As shown in Figure A, the combination index (CI) values ​​of MMC-DOX combination drugs at ratios of 1:1, 2:1, and 3:1 are all less than 1, but the trend of MMC-DOX (3:1) is the most stable. Figure 1 As shown in Figure B, the doses of MMC and DOX that inhibited 50% of HLE B-3 cell growth in the three MMC-DOX combination ratios of 1:1, 2:1, and 3:1 were all lower than the doses of single-agent MMC and single-agent DOX. Figure 1 As shown in Figure C, the three MMC-DOX combination ratios of 1:1, 2:1 and 3:1 showed no adverse reactions in the process of inhibiting the growth of HLE B-3 cells, but MMC-DOX (3:1) showed the most stable trend. Figure 1 IC of ZhongE 50 and IC 95 The results showed that at the MMC-DOX (3:1) ratio, the MMC dose in the combination was 1.67 times lower than that of MMC alone, and the DOX dose in the combination was 3.5 times lower than that of DOX alone. In summary, MMC-DOX (3:1) was screened as the optimal synergistic ratio for the treatment of human lens epithelial cells.

[0065] Depend on Figure 2 As shown in Figure A, the combination index (CI) values ​​of MMC-DOX combination drugs at ratios of 1:1, 1:2, 2:1, and 3:1 are all less than 1, but the trend of MMC-DOX (1:2) is the most stable. Figure 2 As shown in Figure B, the doses of MMC and DOX that inhibited 50% of HLE B-3 cell growth in the four MMC-DOX combination ratios of 1:1, 1:2, 2:1, and 3:1 were all lower than the doses of single-agent MMC and single-agent DOX. Figure 2As shown in Figure C, the four MMC-DOX combination ratios of 1:1, 1:2, 2:1 and 3:1 showed no adverse reactions in the process of inhibiting the growth of HLE B-3 cells, but MMC-DOX (1:2) showed the most stable trend. Figure 2 Zhong E and Figure 8 IC 50 and IC 95 The results showed that at the MMC-DOX (1:2) ratio, the MMC dose in the combination was 1.66 times lower than that of MMC alone, and the DOX dose in the combination was 1.74 times lower than that of DOX alone. In summary, MMC-DOX (1:2) was screened as the optimal synergistic ratio for inhibiting human corneal epithelial cell viability.

[0066] according to Figure 1 and Figure 2 The analysis method of Figures 3 to 6 Analysis was performed and MMC-DOX (3:1) was screened out as the optimal synergistic ratio for inhibiting the activity of retinal pigment epithelial cells, colorectal cancer epithelial cells, human umbilical vein endothelial cells and human colon fibroblasts.

[0067] Table 1 shows the IC values ​​of MMC alone, DOX alone, and MMC-DOX combination therapy in human lens epithelial cells (HLE B-3), corneal epithelial cells (HCE-T), retinal pigment epithelial cells (ARPE-19), colorectal cancer epithelial cells (Caco-2), human umbilical vein endothelial cells (HUVEC), and human colonic fibroblasts (CCD-18co) 50 and IC 95 The data showed that the MMC and DOX doses in the MMC-DOX combination were superior to those in the monotherapy group.

[0068] Table 1 IC values ​​of MMC-DOX combination drug or single drug on different cell types 50 and IC 95 Dosage Schedule

[0069]

[0070]

[0071] Example 2

[0072] MMC-DOX (3:1) anti-proliferation and anti-migration experiments on HLE B-3 cells

[0073] 1. Anti-proliferation experiment of MMC-DOX (3:1) on low-density and high-density HLE B-3 cells

[0074] HLE B-3 cells were cultured at 1 × 10 6 The cells were seeded into 6-well plates at a density of 100 cells per well and adhered overnight. Subsequently, the cells were incubated for 3 h and 24 h with the screened combined drug MMC-DOX (3:1) synergistic concentration ratio. After treatment, a single cell suspension was obtained and re-seeded in 6-well plates with 500 cells / well and 2000 cells / well. The treated cells were cultured in an incubator with 20% FBS-MEM for 14 days to form colonies and stained with 1% crystal violet. The stained cells from each well were dissolved with 1 mL of 10% acetic acid, and the optical density of the solution was measured at a wavelength of 590 nm on a microplate reader.

[0075] Antiproliferation test results Figure 7 As shown. Under the same incubation time, the anti-proliferative effect of MMC-DOX (3:1) combination on low-density cells (500 cells / well) is better than that on high-density cells (2000 cells / well). When the incubation time is 3h, the IC 50 Compared with the IC of high-density cells (2000) 50 When the incubation time is 24h, the IC value of the cells at low density (500 / well) is 0.13 times smaller. 50 Compared with the IC of high-density cells (2000 cells / well) 50 At the same cell density, the anti-proliferative effect of MMC-DOX (3:1) combined with HLE B-3 cells incubated for 24 hours was better than that incubated for 3 hours. At low cell density (500 cells / well), the IC 50 Compared with IC after 3 h incubation 50 23 times smaller; at high cell density (2000 cells / well), the IC 50 Compared with IC after 3 h incubation 50 The reduction of drug dose was cell density-dependent and incubation time-dependent, but the incubation time had a greater impact on the antiproliferative effect of the combination drug.

[0076] 2. MMC-DOX (3:1) anti-migration experiment on HLE B-3 cells

[0077] HLE B-3 cells were treated with MMC-DOX combination (D 50 ), single-agent MMC (the same concentration as the MMC in the combination drug), single-agent DOX (the same concentration as the DOX in the combination drug) were treated for 3 h, and the cells were digested with 0.25% trypsin-EDTA solution and resuspended in serum-free MEM. The cell suspension was plated at 2.5×10 4Cells were evenly seeded into the upper chamber of a 24-well transwell insert with a fibronectin-coated polycarbonate membrane. Culture medium (600 μL) containing 10% FBS and 5 ng / mL TGF-β2 was added to the bottom of the lower chamber. After 48 hours of incubation, the top of the upper chamber of the transwell insert was removed and cells that had not migrated from the top of the membrane were gently removed using a cotton swab. The lower surface of the top of the transwell insert was placed in 4% paraformaldehyde solution (Novonature, NL0617) for half an hour at room temperature to fix the migrated cells. The top chamber was immersed in PBS solution and fixed three times for 2 minutes each. 1 mL of 0.1% crystal violet was added to the 24-well plate and the top chamber was immersed in the stain. After incubation at room temperature for 30 minutes, the stain solution was removed. The upper surface of the top of the insert was immersed in PBS for 2 minutes and washed three times to remove excess crystal violet. Liquid from the lower surface of the top of the insert was gently removed using a cotton swab and the bottom of the insert was allowed to dry at room temperature. The images were observed under an inverted microscope at 10× magnification and analyzed using Image J.

[0078] See the results Figure 8 MMC-DOX (3:1) can effectively inhibit the proliferation and migration activity of HLE B-3 cells during their growth.

[0079] Example 3

[0080] Western blot detection of epithelial-mesenchymal transition (EMT) marker proteins

[0081] Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells transform into mesenchymal cells. Epithelial cells lose their apical-basal cell polarity, lose their adhesion and acquire the phenotype of mesenchymal cells, acquiring the ability of mesenchymal cells to migrate to promote metastasis and drug resistance. During EMT, the expression levels of α-SMA and vimentin proteins are upregulated. During wound healing and tumor angiogenesis, α-SMA protein is expressed in large quantities in fibroblasts and myofibroblasts, promoting the development of EMT. Vimentin protein promotes the occurrence of EMT by changing cell shape and movement. This example verifies the effect of MMC-DOX (3:1) drug on the process of epithelial-mesenchymal transition in HLE B-3 cells.

[0082] HLE B-3 cells were treated with MMC-DOX (3:1) in D 50Cells were pretreated for 3 hours with either MMC (at the same concentration as in the combination) or DOX (at the same concentration as in the combination) at the same dose. Subsequently, cells were pretreated with MEM medium containing 5 ng / ml TGF-β2 for 48 hours. Proteins were lysed in a mixture of protease and phosphatase inhibitors and RIPA lysis buffer, centrifuged at 13,400 × g for 30 minutes at 4°C, and heated to 100°C for 8 minutes. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 2 hours, the target proteins on the PVDF membranes were incubated with primary antibodies against α-SMA (1:1000), vimentin (1:600), and GAPDH (1:1000), respectively, overnight at 4°C. The PVDF membrane was incubated with HRP-conjugated goat anti-mouse IgG (H+L) (1:5000) secondary antibody at room temperature, and finally photographed with a chemiluminescence imaging system, and protein expression was quantitatively analyzed using Image J software.

[0083] See the results Figure 9 Single-drug MMC, single-drug DOX and MMC-DOX (3:1) in D 50 After incubating HLE B-3 cells with the doses of MMC-DOX (3:1) for 3 h, the expression levels of α-SMA and vimentin proteins decreased, indicating that MMC-DOX (3:1) has a significant anti-EMT effect on HLE B-3 cells.

[0084] Example 4

[0085] Effects of MMC-DOX (3:1) on oxidative stress in HLE B-3 cells

[0086] 1. Reactive oxygen species detection

[0087] HLE B-3 cells were cultured at a rate of 1 × 10 4 The cells were seeded at a density of 100 cells / well into a 96-well black plate with a clear bottom overnight. The cells were washed twice with Hank's balanced salt solution (HBSS) and then incubated with MMC-DOX (D 50 ) / (D 95 ), and their corresponding single-agent MMC and single-agent DOX at different doses for 3 hours to induce ROS generation. The supernatant was aspirated and washed twice with HBSS. 100 μL / well of high-sensitivity DCFh-DA dye working solution was added to the wells and incubated for 30 minutes. The supernatant was aspirated and washed twice with HBSS. The cells were quantified using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Fluorescence images of ROS generation were observed using a fluorescence microscope.

[0088] 2. Quantitative Analysis of Lipid Peroxidation

[0089] HLE B-3 cells were cultured at a rate of 1×10 6 The cells were seeded at a density of 100 cells / well in 6-well plates and cultured overnight. 50 ), MMC (same concentration as in the combination), and DOX (same concentration as in the combination) were treated for 3 hours. Supernatants were removed. The lipid peroxidation product, malondialdehyde (MDA), was measured using the TBARS assay kit and expressed as nanomoles per milligram of protein.

[0090] See the results Figure 10 As shown in Figures A and B, the single drug MMC / DOX and MMC-DOX (3:1) 50 and D 95 The treatment of HLE B-3 cells with different doses significantly increased the intracellular oxidative stress level, but MMC-DOX (3:1) could induce oxidative stress more significantly than single drug: MMC-DOX (3:1) in D 95 At the dose of 40 mg / dL, the combined drug caused the oxidative stress level in HLE B-3 cells to be approximately D 50 The dose caused 1.67 times the level of oxidative stress in HLE B-3 cells.

[0091] exist Figure 10 In C and D, red fluorescence indicates the reduced state of cells after treatment, and green fluorescence indicates the oxidized state after treatment. That is, the stronger the green fluorescence, the stronger the lipid peroxidation. The results showed that the environment of the untreated cells was in a normal state, and the cells showed low-intensity lipid peroxidation. After MMC / DOX monotherapy, the red fluorescence intensity decreased and the green fluorescence increased, indicating a significant increase in lipid peroxidation. In comparison, the oxidized green fluorescence intensity was highest in the MMC-DOX (3:1) treatment group, indicating that the combination drug caused more significant intracellular lipid peroxidation than either drug alone, with the combination drug being approximately three times that of either drug alone.

[0092] Example 5

[0093] Effect of MMC-DOX (3:1) combination on antioxidant GSH levels in HLE B-3 cells

[0094] Glutathione content detection

[0095] HLE B-3 cells were cultured at a rate of 1 × 10 6 The density of the cells was inoculated into culture dishes and grown overnight. 50), single-drug MMC (the concentration is the same as the MMC concentration in the combination drug), single-drug DOX (the concentration is the same as the DOX concentration in the combination drug) for 3 hours. Gently scrape the cells on ice with a scraper and collect them in a centrifuge tube. Centrifuge twice at 800×g at 4°C for 5 minutes each time. After aspirating the supernatant, add 300μL 0.1M PBS to resuspend the cells. After ultrasonic treatment of the cell suspension (30s / cycle, 5 cycles), mix it with reagent I and centrifuge it at 1000×g for 10 minutes at 4°C. Mix the obtained supernatant with the reagent prepared in the kit and let it stand at room temperature for 5 minutes for complete color development. Measure the absorbance (A) at a wavelength of 405nm using an enzyme reader. Determine the glutathione content based on the protein concentration in the supernatant.

[0096] See the results Figure 11 Single drug MMC / DOX and MMC-DOX (3:1) in D 50 The intracellular antioxidant glutathione level was significantly reduced at all doses, but there was no significant difference in the effect of single drug and combination drug in reducing glutathione level. 95 At the dose of MMC / DOX (3:1), MMC-DOX (3:1) has a stronger effect on reducing glutathione levels than single drug. 50 Dose ratio D 95 The dose significantly reduced glutathione levels.

[0097] Example 6

[0098] Localization of mitochondrial lipid peroxidation in living cells

[0099] HLE B-3 cells were cultured at 3 × 10 4 The cells were seeded at a density of 10 cells / plate, 48 wells in a 20 mm confocal culture dish and incubated for more than 48 h. 50), MMC alone, and DOX alone for 3 h. After treatment, Bodipy 581 / 591C11 (Thermo) probes were added to the culture dishes at a final concentration of 5 μM. Cells were washed twice with prewarmed PBS and incubated with 1 μg / ml Hoechst 33342 for 10 min. Additionally, 250 nM MitoTracker Deep Red dye was added and incubated for 30 min to analyze mitochondrial localization of lipid peroxidation. Fluorescence images were observed under a confocal laser scanning microscope using the following reference wavelengths: 1) Bodipy 581 / 591C11 sensor (oxidized state): excitation wavelength 488 nm, emission wavelength 510 nm; 2) Bodipy 581 / 591C11 sensor (reduced state): excitation wavelength 581 nm, emission wavelength 591 nm; 3) nuclear localization: excitation wavelength 405 nm, emission wavelength 488 nm; 4) mitochondrial localization: excitation wavelength 644 nm, emission wavelength 665 nm. The colocalization of lipid peroxidation in mitochondria was assessed by the Pearson correlation coefficient (PCC), with values ​​closer to 1 indicating a higher correlation in the localization of lipid peroxidation in mitochondria. Fluorescence images were analyzed using the colocationization-coloc2 plugin in Image J software (win64, National Institutes of Health).

[0100] See the results Figure 12 The fourth column of images in Panel A shows mitochondrial localization. Quantitative analysis using the Pearson coefficient reveals that after 3 hours of treatment with MMC / DOX alone and the MMC-DOX (3:1) combination in HLE B-3 cells, intracellular lipid peroxidation was localized to the mitochondria, confirming that the combination significantly induces lipid peroxidation damage on the mitochondrial membrane. In Panel B, the closer the coefficient is to 1, the higher the degree of overlap with the mitochondria. The results in Panel B indicate that lipid peroxidation damage occurs in the mitochondria. In summary, lipid peroxidation in both the single-agent and combination groups is localized to the mitochondria.

[0101] Example 7

[0102] DNA damage detection

[0103] 2×10 4 HLE B-3 cells were seeded into 20 mm confocal culture dishes and incubated for 48 h. 50HLE B-3 cells were treated with MMC or DOX alone for 3 hours and then washed twice with PBS. 4% paraformaldehyde was added to the culture dish and incubated at room temperature for 30 minutes. The cells were washed three times with PBS and then incubated with 0.3% TritonX-100 at room temperature for 30 minutes. The cells were washed twice with BSA working solution and the reaction mixture prepared in advance according to the TUNEL kit (200 μL / well) was added to the culture dish and incubated at 37°C for 2 hours. The cells were washed twice with PBS and then DAPI working solution was added to the culture dish and incubated for 10 minutes. The dye solution was aspirated and the cells were washed twice with PBS. Fluorescence images of DNA damage were observed using a confocal laser scanning microscope.

[0104] See the results Figure 13 . In the detection results of cell DNA damage, DAPI blue represents the cell nucleus, and green fluorescence represents DNA breakage damage. The results showed that the MMC / DOX single-drug group and the MMC-DOX (3:1) combination group treated HLE B-3 cells for 3h and 24h showed more obvious DNA damage than the untreated group, and indicated that the drug's damage response to DNA occurred almost entirely in the cell nucleus. After a treatment time of 3h, the MMC-DOX (3:1) combination group significantly enhanced DNA damage compared to the single-drug group. After a treatment time of 24h, both the combination group and the single-drug group caused DNA damage in the cell nucleus, and the fluorescence intensity was lower than that after 3h of treatment, but the fluorescence intensity of DNA damage caused by the combination group was still stronger than that of the single-drug group. (MMC and DOX both exert their damaging effects by binding to guanine and / or cytosine bases. When we use the drugs, very obvious oxidative stress damage will occur in the cells. When a large amount of reactive oxygen species is produced, the 3'-OH sticky ends will be damaged or even decomposed. Our long-term treatment will further cause more damage and decomposition of the 3'-OH sticky ends than three hours, so the fluorescence of 24 hours will be weaker than that of 3 hours. Tunel uses dUTP to label the 3'-OH sticky ends produced after DNA breakage to observe DNA breakage and apoptosis. Based on this, when MMC and DOX are combined to treat cells, MMC and DOX compete for oxygen consumption. After activation, MMC will switch from the one-electron reduction pathway to the two-electron reduction pathway under the conditions of rapid production of reactive oxygen species and large-scale oxygen consumption. MMC is embedded in DNA, resulting in DNA fragments with sticky ends unable to bind to other fragments. DOX more obviously causes lipid peroxidation in mitochondria, and some of them will produce 3'-OH sticky ends through topoisomerase II. Due to the different spatial distribution of MMC and DOX and the different treatment durations, the residual 3'-OH sticky ends after combined drug treatment of cells will be more than those of single drugs and will be more labeled by Tunel.

[0105] Example 8

[0106] Mitochondrial structure and function damage detection test

[0107] 1. Mitochondrial structure observation

[0108] HLE B-3 cells were collected at 3×10 6 The cells were inoculated at a density of 100 mm in a 100 mm culture dish and grown overnight in a 37°C incubator. 50 ), MMC alone, or DOX alone was used to treat HLE B-3 cells for 1 hour. The treated cells were washed three times with preheated PBS and chemically fixed with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) on polystyrene pretreated culture plates for 1 hour. The cells were then fixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 20 minutes. The fixed cells were dehydrated in graded ethanol and embedded in Epon618. The embedded HLE B-3 cells were cut into ultrathin sections 80-90 nm thick and stained with 5% uranyl acetate for 10 minutes, followed by 5% lead citrate for 10 minutes. The stained sections were mounted on transmission electron microscope grids and the morphological changes in mitochondrial structure after drug treatment were observed by transmission electron microscopy at an accelerating voltage of 120 kV. Quantitative analysis was performed using Image J.

[0109] 2. ATP detection

[0110] HLE B-3 cells were cultured at a rate of 1 × 10 4 The cells were seeded at a density of 100 μg / mL in a 96-well white plate with a clear bottom and incubated overnight at 37°C under 5% CO2. 50 ), single-agent MMC, and single-agent DOX treated cells for 3h, 8h, 12h, and 24h. A reaction mixture was prepared using an ADP / ATP ratio assay kit. After aspirating the drug solution, the ATP reagent was added to the wells and mixed with MEM. Incubated at room temperature for 1 min, the luminescent signal of ATP (RLUA) was read on a microplate reader. Incubated at room temperature for another 10 min, the residual ATP signal (RLUB) was read on a microplate reader, and the ADP reagent was immediately added to the wells and mixed. After incubation for 1 min, the luminescent signal of the ADP signal (RLUC) was read on a microplate reader.

[0111] 3. Mitochondrial Membrane Potential Measurement

[0112] HLE B-3 cells were cultured at a rate of 5 × 10 5 The cells were seeded in 6-well plates at a density of 100 μg / mL and incubated overnight. 50), single-agent MMC, single-agent DOX, and CCCP (positive control) were incubated for 3 h, 8 h, 12 h, and 24 h, and the culture medium was aspirated from the culture plates. Working solutions were prepared according to the mitochondrial membrane potential assay kit. After aspirating the supernatant from the drug culture medium, the JC-1 working solution was diluted and added to the cell culture wells. Incubate at 37°C for 30 min, and wash the cells twice with JC-1 staining buffer. Mitochondrial membrane potential was measured using a microplate reader at the following wavelengths: 1) JC-1 monomer: excitation wavelength 514 nm, emission wavelength 529 nm; 2) JC-1 aggregates: excitation wavelength 585 nm, emission wavelength 590 nm.

[0113] See the results Figure 14 . Figure A shows that after 1 hour of treatment of HLE B-3 cells with MMC / DOX monotherapy and MMC-DOX (3:1) combination therapy, the mitochondrial structure was destroyed, including loose cristae, matrix swelling, matrix depression, and the nuclear membrane was not intact. The degree of mitochondrial structural damage in the MMC-DOX (3:1) combination therapy was more severe than that of the two drugs alone, such as swelling of the interstitial space and interference of cristae; the nucleus of the cell treated with the combined drugs was completely destroyed; DOX monotherapy entered the cell nucleus but did not destroy the nucleolus, causing the contents of the nucleolus to be dispersed in the inner nuclear membrane; that of the MMC monotherapy group entered the cell nucleus and was divided into two parts. As shown in Figure B, the mitochondrial area of ​​damaged HLE B-3 cells treated with MMC / DOX monotherapy and MMC-DOX (3:1) combination therapy was quantitatively analyzed. As shown in Figure C, the ATP synthesis produced by mitochondria supports the proliferation of HLE B-3 cells. The MMC-DOX (3:1) combination therapy was significantly associated with the proliferation of HLE B-3 cells. 50 The cells were treated with the dose for 3h, 8h, 12h and 24h. The level of ATP synthesized in the cells decreased with the extension of treatment time, inhibiting cell proliferation. At the same time, the mitochondrial membrane potential also decreased with the extension of treatment time, affecting the function of mitochondria.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that These include mitomycin-C and doxorubicin; The molar ratio of mitomycin-C to doxorubicin is (1-3): (1-3).

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of mitomycin-C to doxorubicin is (1-3): (1-2).

3. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of mitomycin-C to doxorubicin is 3:1 or 1:

2.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3, mitomycin-C or doxorubicin in the preparation of drugs for anti-cell proliferation, anti-cell migration and / or anti-cell fibrosis.

5. The application according to claim 4, characterized in that: The anti-fibrotic activity includes inhibiting the differentiation of cells into fibroblasts.

6. The application according to claim 4, characterized in that: Diseases caused by cell proliferation, cell migration and / or cell fibrosis include at least one of the following: ocular fibrotic lesions, organ fibrotic lesions, in situ tumors or metastatic tumors.

7. Use of the pharmaceutical composition according to any one of claims 1 to 3, mitomycin-C or doxorubicin in the preparation of a drug for resisting epithelial-mesenchymal transition.

8. The application according to claim 7, characterized in that: The indications caused by the epithelial-mesenchymal transition include at least one of the following: posterior capsule opacification, tumor development and metastasis, organ fibrosis, glaucoma, diabetic retinopathy, and macular degeneration.

9. The application according to claim 7, characterized in that: The epithelial-mesenchymal transition includes upregulation of the expression level of epithelial-mesenchymal transition markers.

10. The application according to claim 9, characterized in that: The epithelial-mesenchymal transition markers include vimentin and / or α-smooth muscle actin.

11. A drug, characterized in that The active ingredient comprises the pharmaceutical composition according to any one of claims 1 to 3.

12. The drug according to claim 11, characterized in that The dosage form of the drug includes at least one of the following: injection solution, injection powder and nanoparticles.

13. The drug according to claim 12, characterized in that The nanoparticles are a shell material and an active ingredient encapsulated in the shell material; The shell material includes polylactic acid-glycolic acid copolymer; The constituent monomers of the polylactic acid-glycolic acid copolymer include lactic acid and glycolic acid; The percentage of lactic acid to glycolic acid in the constituent monomers is 10-17:3-10.

14. Use of doxorubicin in the preparation of drugs that synergistically enhance the anti-cell proliferation, migration and fibrosis effects of mitomycin-C.

15. The use according to claim 14, characterized in that: The synergistic enhancement of mitomycin-C's anti-cell proliferation and fibrosis effect includes at least one of the following: a sharp increase in oxidative stress levels, an imbalance in intracellular calcium ion homeostasis, and DNA chain breaks.

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