Application of Miltefosine in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention relates to the technical field of biological medicines, and provides application of Miltefosine in preparation of a medicine for treating pulmonary fibrosis. According to the invention, the treatment effect of Miltefosine is systematically evaluated by constructing a plurality of mouse pulmonary fibrosis models. Research results show that Miltefosine can significantly reduce the degree of pulmonary fibrosis, and has no obvious toxicity to liver and kidney functions and important organs. Besides, in-vitro experiments prove that Miltefosine can play an anti-fibrosis role by inhibiting activation, proliferation and migration of fibroblasts induced by TGF-beta1 and promoting apoptosis of the fibroblasts. The researches provide important theoretical and experimental basis for Miltefosine'new use of old medicine 'in treatment of pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of Miltefosine in the preparation of a medicament for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a chronic, irreversible, and progressive fibrotic lung disease, which is the end-stage pathological change of various interstitial lung diseases (ILDs). It is characterized by abnormal remodeling of lung tissue, accompanied by continuous activation of fibroblasts and massive deposition of extracellular matrix (ECM), resulting in thickening of the lung tissue septum and ventilation dysfunction, and ultimately leading to respiratory failure.

[0003] Multiple ILDs can develop into pulmonary fibrosis. Among them, idiopathic pulmonary fibrosis (IPF) is the most common fibrotic disease. Other ILDs include exposure-related ILDs (such as silicosis), drug-induced pulmonary fibrosis, autoimmune-related ILDs (such as rheumatoid arthritis, systemic sclerosis), and idiopathic nonspecific interstitial pneumonia, etc. Patients with pulmonary fibrosis mostly present with progressive dyspnea, dry cough, and shortness of breath after activity, ultimately leading to respiratory failure and even death. With the progress of diagnostic techniques and the aggravation of population aging, the incidence and mortality of pulmonary fibrosis continue to rise. Currently, the incidence of IPF is approximately 10 cases per 100,000 people, the incidence of ILD is approximately 19.4 cases per 100,000 people, the median survival period of patients is about 3 - 5 years after diagnosis, and the 5-year mortality rate is as high as 55% - 75%.

[0004] The pathogenesis of pulmonary fibrosis is complex and involves the interaction of multiple cells (such as epithelial cells, endothelial cells, macrophages, and fibroblasts). Among them, the activation of fibroblasts and their differentiation into myofibroblasts are the core links in the progression of pulmonary fibrosis. The occurrence of pulmonary fibrosis begins with repeatedly damaged epithelial cells, leading to the release of pro-inflammatory factors and profibrotic mediators, which in turn activate fibroblasts and promote ECM deposition. The excessive deposition of ECM further increases tissue stiffness, forming a vicious cycle and resulting in the continuous progression of pulmonary fibrosis. Although there have been many studies on the regulatory mechanisms of fibroblast activation and ECM deposition, due to the complex interaction of multiple signaling pathways and multiple cell types involved in pulmonary fibrosis, developing drugs that can precisely intervene in key links with few side effects still faces huge challenges.

[0005] Currently, there are only two drugs approved by the FDA for the treatment of pulmonary fibrosis: pirfenidone (PFD) and nintedanib. These two drugs mainly delay disease progression by inhibiting fibroblast proliferation and ECM deposition, but their efficacy is limited, and they have significant side effects and high treatment costs. Specifically, pirfenidone reduces fibrosis by inhibiting the TGF-β signaling pathway, but its efficacy is weak, and common side effects include gastrointestinal reactions, photosensitivity, and abnormal liver function. Nintedanib, as a multi-target tyrosine kinase inhibitor, can inhibit fibroblast proliferation and angiogenesis, but its side effects include diarrhea, nausea, and hepatotoxicity, and it is expensive. In addition, existing treatment methods can only delay disease progression and cannot reverse pulmonary fibrosis or significantly improve the quality of life of patients. Therefore, the development of a new treatment plan with significant efficacy, few side effects, and economic feasibility is an urgent need in the current field of pulmonary fibrosis treatment. Summary of the Invention

[0006] In view of this, the present invention proposes the application of miltefosine in the preparation of drugs for the treatment of pulmonary fibrosis. Miltefosine can significantly reduce the degree of pulmonary fibrosis and has no obvious toxicity to liver and kidney functions and important organs, making it a potential drug in the current field of pulmonary fibrosis treatment.

[0007] The technical solution of the present invention is realized as follows: In the first aspect, the present invention provides the application of miltefosine in the preparation of drugs for the treatment of pulmonary fibrosis.

[0008] On the basis of the above technical solution, preferably, miltefosine inhibits bleomycin (BLM), silica (SiO2), and fluorescein isothiocyanate (FITC)-induced pulmonary fibrosis.

[0009] On the basis of the above technical solution, preferably, miltefosine inhibits fibroblast activation, proliferation, and migration, and then promotes fibroblast apoptosis.

[0010] In the second aspect, the present invention provides a drug for the treatment of pulmonary fibrosis, including miltefosine.

[0011] On the basis of the above technical solution, preferably, the dosage form of the drug is a capsule, powder, tablet, or solution preparation.

[0012] On the basis of the above technical solution, preferably, the drug is administered by injection, respiratory tract, skin, mucosa, or cavity.

[0013] The application of miltefosine of the present invention in the preparation of drugs for the treatment of pulmonary fibrosis has the following beneficial effects compared with the prior art:

[0014] The present invention systematically evaluated the therapeutic effect of Miltefosine by constructing multiple mouse models of pulmonary fibrosis (such as bleomycin-induced, silica-induced, and FITC-induced models). The research results showed that Miltefosine could significantly reduce the degree of pulmonary fibrosis and had no obvious toxicity to liver and kidney functions and important organs. In addition, in vitro experiments confirmed that Miltefosine could play an anti-fibrotic role by inhibiting the activation, proliferation, and migration of fibroblasts induced by TGF-β1 and promoting their apoptosis. These findings provide important theoretical and experimental bases for the "new use of old drugs" of Miltefosine in the treatment of pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0016] Figure 1 It is a result diagram showing that Miltefosine significantly reduces bleomycin-induced pulmonary fibrosis in mice. Figure A is a schematic diagram representing the protein expression levels of fibronectin and collagen 1 in the lung tissue of mice detected by Western Blot, and the right side is quantitative analysis and statistical analysis using Image J software; Figure B is the detection of the expression of fibronectin (Fn1) and collagen (Col1a1) by RT-qPCR; Figure C is the quantitative analysis of Ashcroft score after pathological staining; Figure D is the pathological staining of lung tissue sections: the upper part is H&E staining, the middle part is Masson staining, and the lower part is Sirius red staining, with a magnification of 200 times; Figure E is immunofluorescence staining indicating the expression of fibronectin, collagen1, and α-SMA in the lung tissue of mice, with a magnification of 400 times.

[0017] Figure 2 It is a result diagram showing that Miltefosine reduces silica-induced pulmonary fibrosis in mice. Figures A - E are the same as Figure 1 .

[0018] Figure 3 It is a result diagram showing that Miltefosine reduces FITC-induced pulmonary fibrosis in mice. Figures A - E are the same as Figure 1 .

[0019] Figure 4It is a safety assessment diagram of miltefosine for the treatment of pulmonary fibrosis. In Figure A, the average levels of AST (left) and ALT (right) in mouse serum were detected; in Figure B, the average levels of UREA (left) and CR (right) in mouse serum were detected; in Figure C, pathological H&E staining of mouse internal organs was performed.

[0020] Figure 5 It is a diagram of miltefosine inhibiting fibroblast activation. In Figure A, the CCK8 experiment of miltefosine interfering with fibroblasts was carried out; in Figure B, a schematic diagram representing the protein level expressions of fibrosis indicators fibronectin, collagen 1 and activation indicator (α-SMA) in fibroblasts was detected by Western blot method; in Figures C-D, progressive quantitative analysis of the expressions of fibronectin, collagen 1 and α-SMA was performed using Image J software; in Figure E, the RNA expression level of α-SMA was detected by real-time fluorescence quantitative PCR technology; in Figure F, the fibrosis expression and activation level of fibroblasts were detected by immunofluorescence staining of cell slides.

[0021] Figure 6 It is a diagram of miltefosine inhibiting fibroblast activation. In the left side of Figure A, a schematic diagram representing the protein level expressions of fibrosis indicators fibronectin, collagen 1 and activation indicator (α-SMA) in fibroblasts was detected by Western blot method, and in the right side, progressive quantitative analysis of the expressions of fibronectin, collagen 1 and α-SMA was performed using Image J software; in Figure B, the RNA expression level of α-SMA was detected by real-time fluorescence quantitative PCR technology.

[0022] Figure 7 It is a diagram of miltefosine inhibiting fibroblast proliferation. In Figure A, the proliferation of fibroblasts was detected by EdU staining, with red being the proliferating cells and blue being the cell nuclei; in Figure B, the EdU positive rate was analyzed and statistically calculated using Image J software.

[0023] Figure 8 It is a diagram of miltefosine inhibiting fibroblast migration. In Figure A, a cell scratch experiment was carried out; in Figure B, the fibroblast migration rate was analyzed and statistically calculated using Image J software.

[0024] Figure 9It is a figure showing that miltefosine promotes apoptosis of fibroblasts. Figure A is the AnnexinV-FITC / PI double staining of fibroblasts with PBS; Figure B is the Annexin V-FITC / PI double staining of fibroblasts with L-milte; Figure C is the Annexin V-FITC / PI double staining of fibroblasts with H-milte; Figure D is the analysis and statistics of the apoptosis rate of fibroblasts using Image J software. Detailed implementation mode

[0025] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention.

[0026] Miltefosine is a phosphatidylcholine synthesis inhibitor, initially developed as an anti-parasitic drug and currently approved by the FDA for the treatment of leishmaniasis and American trypanosomiasis. In recent years, studies have found that Miltefosine has a wide range of biological activities, including:

[0027] Anti-inflammatory effect: It shows significant anti-inflammatory effects in endothelial cells by inhibiting the PI3K / Akt signaling pathway and inducing IL-12-dependent Th1 responses.

[0028] Immunomodulatory effect: It exerts anti-tumor effects in cancer cells by regulating the immune response.

[0029] Based on its anti-inflammatory and immunomodulatory effects, Miltefosine may play a therapeutic role by intervening in the dual mechanisms of inflammation and fibrosis in pulmonary fibrosis. However, its specific efficacy and mechanism in pulmonary fibrosis have not been fully studied. In particular, whether it can inhibit fibroblast activation, reduce ECM deposition, and reverse the process of pulmonary fibrosis still needs to be further verified.

[0030] In view of this, the present invention proposes the application of Miltefosine in the preparation of drugs for the treatment of pulmonary fibrosis. Miltefosine can significantly reduce the degree of pulmonary fibrosis and has no obvious toxicity to liver and kidney functions and important organs, and it is a potential drug in the current field of pulmonary fibrosis treatment.

[0031] Next, in combination with experiments, the technical solutions of the present invention will be further described.

[0032] The Miltefosine and BLM (bleomycin) used in the present invention were purchased from Shanghai MCE Company. FITC was purchased from Sigma Company, USA.

[0033] Example 1 Miltefosine Alleviates BLM (Bleomycin)-induced Pulmonary Fibrosis in Mice

[0034] Mouse pulmonary fibrosis model: Male wild-type C57BL / 6 mice aged 6 - 8 weeks were selected as experimental subjects. For mice induced by BLM (5 μg / mL), the required dosage was calculated at a dose of 1.5 U / kg per mouse, and the required amount of BLM was aspirated with a micro syringe for standby.

[0035] The injection method was as follows: The mouse was fixed on a wooden table with an inclination of 45°, so that the front teeth of the mouse hung above the table, the body was straightened and parallel to the inclined plane of the table. The operator carefully and gently stretched out the mouse's tongue with forceps to fully open the glottis of the mouse, and another hand held the airway syringe and inserted it into the airway through the glottis, and quickly injected BLM into the airway. Then the mouse was gently removed and placed in a cage with its upper body in a high position and waited to wake up.

[0036] On the 14th day after BLM induction, Miltefosine 30 mg / kg (prepared with PBS) or PFD (Pirfenidone) 100 mg / kg (prepared with PBS) was administered by gavage for 7 days, and then the mice were euthanized. The protein of the upper lobe of the right lung tissue of the mice was extracted, and Western blot technology was used to detect the expression levels of fibrosis-related indicators (fibronectin, collagen type I) protein levels. The results are shown in Figure 1 。

[0037] The specific steps of Western Blot technology are as follows:[[]]END]]

[0038] During this process, different protein gel concentrations and required transfer membrane times are used for different molecular weights. Therefore, different concentrations of gels and transfer membrane conditions are selected according to the molecular weight of the target protein required.

[0039] 1. Preparation of SDS-PAGE gel

[0040] (1) Clean the glass plates with dishwashing liquid, rinse them with double-distilled water, and dry them for standby.

[0041] (2) Place the dried glass plates on the glass plate rack and seal them with the prepared low-concentration agarose gel to prevent liquid leakage.

[0042] (3) Prepare the lower-layer separating gel with the required concentration. Taking the required 8% separating gel as an example, add the following reagents to a 50 mL centrifuge tube in turn and mix them by inverting up and down (Table 1-4).

[0043] Table 1 Preparation of 8% separating gel

[0044] Reagent Amount required for 6 gels <![CDATA[ddH2O]]> 20.7 mL 30% Acrylamide 12 mL 1.5 mM Tris-HCL 11.4 mL 10% SDS 450 μL AP 450 μL TEMED 22.5 μL

[0045] (4) Use a Pasteur pipette to inject the prepared separating gel into the glass plate, then slowly inject an appropriate amount of absolute ethanol for liquid surface equilibration, and let it stand for 20 minutes.

[0046] (5) After 20 minutes, slowly discard the absolute ethanol and blot the residual absolute ethanol dry with filter paper. Prepare the stacking gel, and the specific reagents required for preparation are shown in Table 1-5.

[0047] Table 2 Preparation of 8% stacking gel

[0048]

[0049]

[0050] (6) Slowly inject the stacking gel into the glass plate, carefully insert a 10-well or 15-well gel comb, and it can be removed from the gel plate for use after 25 minutes.

[0051] 2. Protein electrophoresis

[0052] (1) Gently fix the prepared gel plate on the electrophoresis rack, and slowly pour the prepared 1× electrophoresis buffer into the electrophoresis tank.

[0053] (2) Gently and vertically pull out the gel comb, and slowly add the target protein and protein marker.

[0054] (3) First, perform electrophoresis at a constant voltage of 80V for about 30 minutes. After the marker is separated, adjust the voltage to 110V and continue until the target protein is completely separated, then stop electrophoresis.

[0055] 3. Blotting

[0056] (1) Place the PVDF membrane cut to the appropriate size into methanol for activation.

[0057] (2) Lay the blotting cassette in a tray filled with pre-cooled blotting buffer. Carefully take out the protein gel with a template, cut off the stacking gel part, pick up the PVDF membrane with forceps, mark it with a ballpoint pen, cover it on the surface of the protein gel, pay attention to no bubbles, and the gel corresponds to the black clamping plate, and the PVDF membrane corresponds to the transparent side. Then use a blotting roller to gently apply pressure, and finally clamp the buckle tightly.

[0058] (3) Put the blotting cassette into the blotting tank, with the black clamping plate corresponding to the black surface of the blotting tank and the transparent side corresponding to the red surface of the blotting tank.

[0059] (4) Perform blotting in an ice-water bath, and the blotting conditions are 200 mA for 2 hours (adjust according to the required molecular weight).

[0060] 4. Closure

[0061] (1) Prepare 5% skimmed milk powder.

[0062] (2) Take out the PVDF membrane and place it in 5% skimmed milk powder. The two membranes can be placed "back to back" and then placed on a shaker for 1 hour.

[0063] 5. Primary Antibody Incubation

[0064] (1) Discard the 5% skimmed milk powder and wash the membrane twice with 1× TBST washing solution.

[0065] (2) After cutting the film gloves, place the PVDF membrane on top of the film gloves, use a ballpoint pen to mark the molecular weight and the name of the target protein molecule, then cover the film gloves, use a blade to cut open, and place the strip into the corresponding antibody tube. Incubate overnight on a shaker in a 4°C cold room.

[0066] (3) After incubation, the primary antibody was recovered and stored in a 4°C cold room.

[0067] 6. Anti-Washing Membrane

[0068] (1) After removing the strip from the antibody tube, place it in a membrane washing incubation box, add 1×TBST membrane washing solution and place it on a shaker. (2) At room temperature, replace the 1×TBST membrane washing solution every 7 minutes, and repeat 6 times.

[0069] 7. Secondary Antibody Incubation

[0070] (1) Prepare the secondary antibody incubation solution according to the different secondary antibody species at a ratio of 1:1000.

[0071] (2) Incubate on a shaking table at room temperature for 1 hour.

[0072] 8. Secondary antibody washing membrane

[0073] The membrane washing method was carried out according to the primary antibody washing method.

[0074] 9. Development

[0075] (1) Using ECL developer, mix solution A and solution B in a ratio of 1:1.

[0076] (2) Use filter paper to absorb the moisture of the strip, place it on the exposure plate, add ECL developer, and place it in the exposure instrument dark box for exposure.

[0077] (3) Use automatic exposure mode for development and adjust the exposure time according to the fluorescence intensity of the strip.

[0078] Figure 1Results showed that compared with the PBS control group, after stimulation with BLM, the expressions of fibronectin and collagen1 were significantly increased. After treatment with Miltefosine, the expressions of fibronectin and collagen1 were significantly lower than those in the BLM group, and the difference was statistically significant ( Figure 1 A). Subsequently, we also extracted mRNA to prove at the RNA level that after treating BLM-induced mice with Miltefosine, the fibrosis indexes Fn1 and Col1a1 were significantly lower than those in the simple BLM stimulation group, and the difference was statistically significant. There was no significant difference compared with the effect of PFD in treating pulmonary fibrosis ( Figure 1 B).

[0079] To further directly observe the therapeutic effect of Miltefosine in treating BLM-induced pulmonary fibrosis in mice, in this study, the left lungs of the mice were fixed and embedded, and lung tissue sections were made for H&E, Masson, and Sirius red staining. The specific steps of tissue pathological staining are as follows:

[0080] 1. Tissue dehydration and embedding

[0081] The lung tissue fixed with 4% paraformaldehyde for more than 24 hours was sent to the dehydration machine in the Experimental Medicine Research Center for dehydration treatment. After the dehydration treatment was completed, embedding was carried out in the Experimental Medicine. The lung lobe was separated from the middle cross-section with scissors, placed in the embedding frame, liquid wax was injected on the embedding machine, and it was placed on the cooling table for wax coagulation until the wax block could be easily taken out of the mold.

[0082] 2. Paraffin sectioning and drying

[0083] (1) Turn on the tissue spreading machine and preheat the temperature to 42 °C. Carefully install the blade of the paraffin slicer and label the glass slides in groups.

[0084] (2) Place the paraffin in a -80 °C refrigerator for freezing. After freezing, cut the wax block with a blade, leaving 1-2 mm around the lungs.

[0085] (3) Place the trimmed wax block on the paraffin slicer. First, trim it to a thickness of 20 μm until the lung contour is complete, then adjust it to a thickness of 5 μm for continuous sectioning. Use forceps and a brush to gently place the slices carefully into the spreading machine, flatten the spread sections completely, and carefully pick them up onto the glass slides.

[0086] (4) After sectioning, place it in an oven at 65 °C for baking for 1 hour and 30 minutes, and then proceed with staining or store for later use.

[0087] 3. Dewaxing and making water

[0088] Immerse the dried tissue sections in xylene Ⅰ, Ⅱ, and Ⅲ in the fume hood for 15 minutes each, then sequentially place them in absolute ethanol Ⅰ and Ⅱ for 10 minutes each, and finally in 95% absolute ethanol Ⅰ for 10 minutes and Ⅱ for 10 minutes. Subsequently, place them in 80% absolute ethanol for 10 minutes, and finally rinse slowly with running water for 5 minutes.

[0089] 4. H&E staining

[0090] (1) After dehydration of the tissue sections with paraffin, place the sections in hematoxylin for staining for 5 minutes. The specific time can be adjusted according to the staining effect. Then, rinse with running water for 30 seconds, immerse the sections in 1% hydrochloric acid alcohol for 2 seconds for differentiation, and rinse again with running water for 5 minutes for blueing.

[0091] (2) Immerse the sections from step 1 in eosin staining solution for 1 minute, then rinse with running water to remove the excess eosin.

[0092] (3) Sequentially place the sections in 70%, 80%, 95%, and 100% ethanol for dehydration, with 1 minute of dehydration for each concentration. Then, sequentially immerse them in xylene Ⅰ and Ⅱ for clearing, about 2 minutes each time. Finally, mount the sections with neutral balsam and take pictures after drying.

[0093] 5. Sirius red staining

[0094] (1) After making the tissue sections water-compatible with paraffin, immerse them in Sirius red staining solution for 2 - 3 hours, and then quickly pass them through water to remove the excess dye.

[0095] (2) Place the sections in hematoxylin staining solution for nuclear staining for 30 seconds. The specific time is adjusted according to the staining effect. Immerse the sections in 1% hydrochloric acid alcohol for 1 second for differentiation, and rinse again with running water for 5 minutes for blueing.

[0096] (3) After air-drying, mount the sections with neutral balsam and take pictures.

[0097] 6. Masson staining

[0098] In this experiment, Sevier Masson trichrome staining solution kit was used for staining. After staining with Masson trichrome staining solution, collagen fibers appear sky blue or bright dark blue.

[0099] (1) First, dewax and make the paraffin sections water-compatible, then place them in 2.5% potassium dichromate mordant solution and soak at room temperature overnight (about 15 hours).

[0100] (2) Place the sections from step 1 in an oven at 65°C (note that the sections need to be immersed in the staining solution), incubate for 30 minutes. After taking them out of the oven, rinse slowly with tap water for 30 seconds until the yellow color on the lung tissue fades.

[0101] (3) To ensure the smooth progress of the experiment, the ponceau acid fuchsin reagent and 2.5% aniline blue solution were placed in an oven at 65 °C for preheating at the same time as step 2 for standby.

[0102] (4) Immerse the sections in step 2 in Weigert iron hematoxylin stain (note that this solution needs to be prepared and used immediately) for 1 minute, and then rinse slightly with running water.

[0103] (5) Differentiate the sections in step 4 with 1% hydrochloric acid alcohol for about 30 seconds (the time can be appropriately extended). Observe under the microscope that the cell nuclei are grayish black, and the tissue background is almost colorless or light gray to the naked eye.

[0104] (6) Gently rinse with running water, gently blot the residual water on the sections with absorbent paper, and then place the sections in the preheated ponceau acid fuchsin solution for impregnation for about 6 minutes. Then observe under the microscope. If the color of the lung tissue shows bright red, stop staining. If the red color is still light, the time can be extended according to the specific situation. Then gently rinse the sections with running water for about 20 seconds, and observe the color of the running water during the rinsing process. Stop when the rinsed water is colorless.

[0105] (7) Gently drain the water from the sections (note to avoid complete drying), immerse them in the preheated 1% phosphomolybdic acid solution for differentiation (for about 1 minute) until the collagen fibers turn light red. The soaking time of the 1% phosphomolybdic acid solution can be appropriately adjusted according to the required staining depth, and the time is usually 1 - 2 minutes.

[0106] (8) Take out the sections from the 1% phosphomolybdic acid solution, drain slightly, without washing with water, and directly transfer them to the 2.5% aniline blue solution for staining for 30 seconds, and adjust according to the actual staining effect.

[0107] (9) Rinse and differentiate the sections in step 8 with 1% aqueous acetic acid solution in three consecutive cylinders for about 7 seconds each to remove the excess aniline blue. When rinsing to the third cylinder of 1% aqueous acetic acid, observe the staining situation of the sections under the microscope to avoid over-differentiation of blue.

[0108] (10) Dehydrate the sections in step 9 in absolute ethanol, 3 seconds in absolute ethanol I, and 5 seconds each in absolute ethanol II and III. Soak in xylene for 5 minutes for section transparency treatment, and finally mount with neutral gum. Take pictures after drying. The results are shown in Figure 1C-E. The results showed that the lung tissues of the mice in the BLM group were severely damaged, and a large amount of collagen deposition formed dense fibrotic tissues, resulting in the loss of the normal lung structure. However, the degree of lung tissue damage in the mice treated with Miltefosine was lower than that in the BLM group alone. The amount of collagen deposition was significantly reduced compared with the BLM group. The degree of pulmonary fibrosis in the PFD treatment group was also significantly alleviated compared with the BLM group. Consistent with this, the Ashcroft score showed that the fibrosis levels in the Miltefosine treatment group and the PFD treatment group were significantly lower than those in the BLM group alone, and the treatment effects between the PFD group and the Miltefosine treatment group were comparable ( Figure 1 C-D). In addition, immunofluorescence staining was used again to prove that the expression levels of fibrosis indicators such as fibronectin, collagen1, and α-SMA in the lung tissues of the mice in the Miltefosine treatment group were significantly lower than those in the BLM group alone ( Figure 1 E). Therefore, Miltefosine is effective in treating BLM-induced pulmonary fibrosis in mice.

[0109] Example 2 Miltefosine alleviates SiO2-induced pulmonary fibrosis in mice

[0110] Pulmonary fibrosis is an end-stage pathological change caused by more than 200 reasons. Pulmonary fibrosis caused by dust and SiO2 is a typical representative of occupation-related pulmonary fibrosis. To explore whether Miltefosine can alleviate occupation-induced pulmonary fibrosis, a mouse model of SiO2-induced pulmonary fibrosis was constructed in this example. The required dosage was calculated according to the dose of 200 mg / kg of SiO2 per mouse, and the required dosage was aspirated with a microsyringe for standby. The specific method was the same as that for constructing the mouse model of BLM-induced pulmonary fibrosis.

[0111] Miltefosine 30 mg / kg (prepared with PBS) or PFD 100 mg / kg (prepared with PBS) was continuously administered for 14 days starting 28 days after injecting SiO2 (at a dose of 200 mg / kg of SiO2 per mouse) into the airways of the mice. The mice were euthanized on the 42nd day, and their tissue specimens were taken for detection. By extracting the proteins from the mouse lung tissues, the expression differences of fibronectin and collagen1 at the protein level in the lung tissues of the mice in different groups were detected (the detection method was the same as before), and the results are shown in Figure 2 A-B.

[0112] Results showed that the protein and RNA levels of fibronectin and collagen in the lung tissues of mice induced by SiO2 were significantly higher than those of the control wild-type mice. After treatment with Miltefosine, the protein and RNA levels of fibrosis indicators in the lung tissues of mice decreased significantly, and the difference was statistically significant. The lung fibrosis-related indicators in the group of mice treated with PFD also decreased significantly, but the effects of the Miltefosine treatment group and the PFD treatment group were comparable( Figure 2 A-B).

[0113] To further prove the therapeutic effect of Miltefosine, pathological staining was used to visually observe the lesion changes to evaluate the therapeutic efficacy. Through H&E staining, Masson staining, and Sirius red staining (the detection methods were the same as before), it was found that in the lung tissues of mice induced by SiO2, the area, collagen content, and number of silicotic nodules were significantly higher than those of the littermate wild-type mice. The results showed that after treatment with Miltefosine and PFD, the formation of SiO2 nodules was significantly reduced, the nodule area was also significantly reduced, and the collagen content decreased.( Figure 2 C-D). Subsequently, immunofluorescence staining of paraffin sections was performed in this study to evaluate the expression of fibronectin, collagen1, and α-SMA in the lung tissues of each group. The fluorescence intensity of the Miltefosine group was significantly weaker than that of the SiO2 group( Figure 2 E).

[0114] Example 3 Miltefosine alleviates FITC-induced pulmonary fibrosis in mice

[0115] To explore whether Miltefosine can treat pulmonary fibrosis in mice caused by various reasons, we induced a pulmonary fibrosis model in mice by injecting fluorescein isothiocyanate (FITC) into the airways. The required dosage was calculated according to the dose of 10 mg / kg of FITC per mouse, and the required dosage was aspirated with a microinjector for standby. The specific method was the same as that of the BLM-induced pulmonary fibrosis model in mice.

[0116] On the 14th day after intratracheal injection in mice, mice were treated with miltefosine 30 mg / kg (prepared with PBS) or PFD 100 mg / kg (prepared with PBS) continuously for 7 days, and the mice were euthanized on the 21st day. First, similar to the previous two models, we extracted proteins and mRNAs from mouse lung tissues and detected the expression differences of fibronectin and collagen1 among the wild-type mouse group, FITC group, miltefosine-alone group, FITC+miltefosine treatment group, and FITC+PFD treatment group by western blot and RT-qPCR techniques. The results showed that in mice induced by FITC, the expression of fibronectin and collagen in lung tissues increased significantly, and the difference was statistically significant compared with the control group. After treatment with miltefosine and PFD, the expression of fibrin and collagen decreased significantly compared with the FITC group, and the difference was statistically significant. The effects of the miltefosine treatment group and the PFD group were comparable( Figure 3 A-B).

[0117] Subsequently, H&E, Masson, and Sirius red pathological staining methods were used to observe the pathological structure changes of lung tissues among different groups. The results showed that in the lung tissues of mice induced by FITC, a large amount of extracellular matrix was deposited, the expression of collagen fibers increased significantly, the normal lung tissues were replaced by dense fibrotic tissues, and the lung tissue structure was severely damaged. After treatment with miltefosine, the expression of collagen deposition in lung tissues decreased significantly compared with the FITC group, and the damaged lung tissues were reduced. After treatment with PFD, the lung tissue damage also improved significantly. The therapeutic effect of miltefosine was basically similar to that of PFD in treating pulmonary fibrosis. The Ashcroft score, which was consistent with the pathological staining, also showed that the score of the FITC+miltefosine treatment group was significantly lower than that of the FITC group, and the difference was statistically significant( Figure 3 C-D).

[0118] For further confirmation of the pathological changes, immunofluorescence staining was used to evaluate the expression of fibrosis indexes Fibronectin, Collagen1, and α-SMA in lung tissues of each group. The results showed that the expression of Fibronectin, Collagen1, and α-SMA in lung tissues of the FITC group was significantly higher than that of the PBS group. After treatment with the miltefosine group and the PFD group, the expression of fibrosis indexes decreased significantly compared with the FITC group( Figure 3 E)

[0119] Example 4 Safety evaluation of miltefosine in the treatment of pulmonary fibrosis

[0120] In the mouse model of pulmonary fibrosis induced by the above-mentioned multiple factors, the present study demonstrated the potential feasibility of miltefosine in the treatment of pulmonary fibrosis. However, whether miltefosine is safe for the treatment of pulmonary fibrosis remains unknown.

[0121] In this example, the safety was evaluated by obtaining the serum of mice for the detection of liver function indexes and renal function indexes.

[0122] I. Detection of liver function indexes

[0123] Kit preparation: Take out the liver function detection kit from the refrigerator and restore it to room temperature. Carefully read the kit instruction manual to understand the detection principle, operation steps, reagent composition, precautions, etc.

[0124] Standard curve drawing: Prepare a series of standard products with different concentrations according to the requirements of the kit instruction manual. Generally, the kit will provide the dilution method and concentration range of the standard products. Add the standard products into the corresponding wells of the enzyme-linked immunosorbent assay (ELISA) plate, and set duplicate wells for each concentration. Then, according to the steps in the kit instruction manual, add the corresponding reagents for reaction, and finally measure the absorbance value on the microplate reader. Use the concentration of the standard product as the abscissa and the absorbance value as the ordinate to draw the standard curve.

[0125] Sample detection: Dilute the serum samples of the mice to be detected according to the requirements of the kit instruction manual. Then add the diluted serum samples into the wells of the ELISA plate, and set duplicate wells for each sample. Follow the same steps as in the standard curve drawing, add the reagents for reaction, and measure the absorbance value on the microplate reader. Calculate the content of the liver function indexes in the samples according to the standard curve.

[0126] II. Detection of renal function indexes

[0127] Kit preparation: Follow the same steps as in the preparation of the liver function detection kit. Take out the renal function detection kit, restore it to room temperature, and carefully read the instruction manual.

[0128] Standard curve drawing: Prepare the standard products and draw the standard curve according to the requirements of the renal function detection kit. Generally, the renal function detection kit will provide standard products for indexes such as creatinine (Cr) and blood urea nitrogen (BUN).

[0129] Sample detection: Mouse serum samples were processed and diluted according to the requirements of the kit instructions. Then the diluted samples were added to the wells of the microplate reader for detection. The content of renal function indexes in the samples was calculated according to the standard curve. Experiments showed that miltefosine did not damage liver function during the treatment of pulmonary fibrosis. Specifically, there was no significant statistical difference in the liver function-related indexes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) between the treatment group and the control group ( Figure 4 A).

[0130] In addition, renal function-related indexes such as urea (UREA) and creatinine (CR) were also detected to evaluate the normal concentration and filtration functions of renal function. The results showed that, similar to liver function, there was no significant statistical difference among the control group, the simple drug administration group, and each treatment group ( Figure 4 B).

[0131] To further evaluate the safety of miltefosine in the treatment of pulmonary fibrosis, important internal organs such as the heart, liver, kidney, spleen, and small intestine of mice were obtained for pathological H&E staining (the steps were the same as before). H&E staining showed that after treatment with miltefosine (starting on the 14th day after injection of BLM, PBS or miltefosine at 30 mg / kg was administered by gavage to the corresponding mouse groups), there were no obvious pathological changes in the structures of the important organs of the mice ( Figure 4 C), proving the safety of miltefosine in the treatment of pulmonary fibrosis in mice and providing a feasible basis for the use of miltefosine in the treatment of pulmonary fibrosis.

[0132] Example 5 In vitro experiment

[0133] To explore whether miltefosine can affect fibroblast activation. With the approval of the hospital ethics committee, we collected lung tissues from normal patients, extracted their fibroblasts for subculture in medium, and induced fibroblast activation with TGF-β1.

[0134] In this study, the toxicity of miltefosine intervention on fibroblasts was evaluated by CCK8 experiment to select an appropriate intervention concentration. Figure 5A). The fibroblasts in 12-well plates were pre-stimulated with PBS or miltefosine, and after 2 hours, 10 ng / mL TGF-β1 was added to the corresponding groups to induce fibroblast differentiation. The cells were divided into a PBS group, a TGF-β1 group, an L-milte group (20 μM), and an H-milte (40 μM) group. After 24 hours, cell proteins and RNAs were extracted for detection, and immunofluorescence detection of cell smears was performed.

[0135] First, we extracted the proteins of fibroblasts after 24 hours of TGF-β1 stimulation and miltefosine intervention, and used Western blot to detect the protein level expressions of fibrosis indexes such as fibronectin, collagen 1, and the activation marker α-SMA in each tissue. The results showed that compared with the unstimulated control group, TGF-β1 stimulation significantly increased the protein expression levels of fibronectin, collagen 1, and α-SMA. However, when the cells were treated with miltefosine for 24 hours on the basis of TGF-β1 stimulation, the above-mentioned fibrosis indexes and the degree of fibroblast activation were significantly decreased compared with the group stimulated by TGF-β1 alone, and the difference was statistically significant ( Figure 5 B-D).

[0136] In addition, we also extracted the RNAs of the cells, prepared cDNA through the reverse transcription process, and used real-time fluorescence quantitative PCR technology to detect the expression of α-SMA. Similar to the protein level, after TGF-β1 stimulation, the level of α-SMA was significantly higher than that of the control group, while after 24 hours of miltefosine intervention, the α-SMA at the RNA level was significantly decreased compared with the TGF-β1 stimulation group, and there was a statistical difference ( Figure 5 E).

[0137] To further prove that the activation of fibroblasts was inhibited by miltefosine, we performed immunofluorescence staining of cell smears to evaluate the expressions of the fibrosis indexes fibronectin, collagen 1, and fibroblast activation (α-SMA). Consistent with the protein level and RNA level, the activation of fibroblasts induced by TGF-β1 was significantly inhibited after miltefosine treatment. Figure 5 F). The above results showed that miltefosine could significantly inhibit the activation of fibroblasts induced by TGF-β1 in normal patients.

[0138] To further prove that miltefosine can significantly inhibit the activation of fibroblasts, this study not only verified it at the cellular level of normal patients, but also collected lung tissues from IPF lung transplant patients for fibroblast extraction and culture. And according to the above method, TGF-β1 stimulation and miltefosine treatment were given. After 24 hours of TGF-β1 stimulation, the total protein of fibroblasts from IPF patients was collected. We found that compared with the group stimulated by TGF-β1 alone, the miltefosine treatment group could also significantly inhibit the protein expression of fibrosis indicators fibronectin and collagen 1, as well as the protein expression of activation indicator (α-SMA) ( Figure 6 A).

[0139] Similarly, after collecting the cellular RNA of IPF patients for reverse transcription, the real-time fluorescence quantitative PCR technique was used to detect the RNA expression level of α-SMA. The results showed that after 24 hours of TGF-β1 stimulation, the level of α-SMA was significantly higher than that of the non-stimulated group. After treatment with miltefosine, the expression of α-SMA was significantly decreased compared with the group stimulated by TGF-β1 alone, and there was a statistical difference between the two groups ( Figure 6 B). Therefore, miltefosine can inhibit the activation of fibroblasts in IPF patients.

[0140] The activation of fibroblasts plays a crucial role in pulmonary fibrosis. However, after tissue damage, the excessive proliferation of fibroblasts is also a key process promoting the occurrence of pulmonary fibrosis. In this study, the proliferation of primary fibroblasts was detected by the EdU proliferation detection kit of Guangzhou Ribobio Co., Ltd. First, we set up 3 groups of cells, including the PBS group, the low-dose drug group, and the high-dose drug group. After adding drug stimulation for 24 hours, the EdU proliferation detection was carried out. The study found that after 24 hours, whether it was the low-dose drug administration group or the high-dose drug administration group, the number of EdU-positive cells was significantly reduced compared with the control group (see Figure 7 A-B, Figure 7 the red in A are proliferating cells and the blue are cell nuclei). Therefore, miltefosine can significantly inhibit the proliferation of fibroblasts.

[0141] The migration of fibroblasts to the damaged area and their differentiation into myofibroblasts are a core process in wound healing and a key driver of excessive tissue remodeling and the progression of fibrotic diseases. During this process, numerous cytokines and growth factors are synthesized and released. For example, TGF-β1, as a key mediator promoting cell migration, can effectively induce the aggregation of fibroblasts at the damaged tissue site and activate their functions. In this study, the effect of miltefosine on the migration function of fibroblasts was evaluated through a cell scratch assay, and the migration was recorded and compared at 0 hours, 12 hours, and 24 hours. It was found that miltefosine could significantly inhibit the migration of fibroblasts. At 24 hours, the migration rate of the PBS group was 72.59%, the migration rate of the low-dose drug administration group was 53.95%, and the migration rate of the high-dose drug administration group was 46.38%, showing statistically significant differences ( Figure 8 A-B). Therefore, miltefosine can effectively inhibit the migration of fibroblasts.

[0142] During tissue development and the maintenance of homeostasis, apoptosis is an indispensable physiological mechanism, and the imbalance of its regulation will lead to cell dysfunction or malfunction. During pulmonary fibrosis, the resistance of fibroblasts to apoptosis leads to increased fibrosis. Therefore, the apoptosis of fibroblasts is also extremely important during pulmonary fibrosis. In this study, the AnnexinV-FITC / PI double staining method was used to detect the effect of miltefosine on the apoptosis of fibroblasts. It was found that after miltefosine intervention, the apoptosis of fibroblasts could be effectively promoted. The average apoptosis rate of the PBS group was 1.87%, the average apoptosis rate of the L-milte group was 8.47%, and the average apoptosis rate of the H-milte group was 36.56%, showing statistically significant differences ( Figure 9 A-D).

[0143] In summary, miltefosine can inhibit the activation, proliferation, and migration of TGF-β1-induced fibroblasts and promote their apoptosis.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of miltefosine in the preparation of a medicament for treating pulmonary fibrosis.

2. The application according to claim 1, wherein: Miltefosine inhibits bleomycin, silica, and FITC-induced pulmonary fibrosis.

3. The application according to claim 1, characterized in that: Miltefosine promotes fibroblast apoptosis by inhibiting TGF-β1-induced fibroblast activation, proliferation, and migration.

4. A drug for treating pulmonary fibrosis, characterized in that: It includes miltefosine.

5. The drug for treating pulmonary fibrosis according to claim 4, characterized in that: The dosage form of the medicament is a capsule, powder, tablet, or solution preparation.

6. The medicament for treating pulmonary fibrosis according to claim 4, characterized in that: The medicament is administered by injection, respiratory tract administration, skin administration, mucosal administration, or cavity administration.

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