Application of beauveria bassiana in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention discloses application of beauveria bassiana in preparation of a medicine for treating pulmonary fibrosis. The invention proposes and verifies that the beauveria bassiana can inhibit abnormal epithelial-mesenchymal transition, regulate and control activation and proliferation of pulmonary epithelial cells and prevent deposition of fibrosis markers for the first time, so that the progress of pulmonary fibrosis is effectively relieved, and a new direction is provided for treatment of pulmonary fibrosis.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and more particularly to the application of a beauveria bassiana extract in the preparation of a therapeutic drug for pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a destructive chronic respiratory disease characterized by progressive scarring of the lung tissue, leading to irreversible loss of lung function and increased mortality. Idiopathic pulmonary fibrosis is the most common type, but its etiology and pathogenesis are not fully understood, and clinical treatment options are limited. Although nintedanib and pirfenidone can moderately slow the progression of the disease, they cannot cure it, and the 5-year mortality rate is as high as 70%.

[0003] The progression of this disease is closely related to chronic inflammation and epithelial-mesenchymal transition (EMT). Chronic inflammation, a hallmark of pulmonary fibrosis, continuously activates the immune response, promoting fibroblast activation and excessive extracellular matrix deposition, thus participating in irreversible tissue remodeling. Simultaneously, EMT plays a pivotal role in the process of pulmonary fibrosis: alveolar epithelial cells gradually lose their epithelial phenotype, transforming into mesenchymal cells with migratory and invasive capabilities, further differentiating into myofibroblasts, secreting large amounts of extracellular matrix, ultimately leading to lung structural destruction and functional failure. This process is regulated by multiple key signaling pathways and is intertwined with the inflammatory microenvironment, forming a complex pathological network.

[0004] Beauveria bassiana extract is a cyclic ester peptide natural product derived from Beauveria bassiana, known to possess various biological activities such as antibacterial, antiviral, and antitumor activity. Its application in the treatment of pulmonary fibrosis and related mechanisms have not been explored. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide the application of beauveria bassiana in the preparation of a drug that simultaneously inhibits three factors—abnormal epithelial-mesenchymal transition, abnormal activation and proliferation of lung epithelial cells, and deposition of fibrosis markers—thereby achieving the treatment of pulmonary fibrosis.

[0006] Technical solution: The application of the beauveria bassiana extract described in this invention in the preparation of drugs for the treatment of pulmonary fibrosis.

[0007] Preferably, the CAS number of the beauveria bassiana is 26048-05-5.

[0008] Preferably, the application is in the preparation of drugs that inhibit epithelial-mesenchymal transition abnormalities.

[0009] Preferably, the application is in the preparation of drugs that inhibit abnormal activation and proliferation of lung epithelial cells.

[0010] Preferably, the application is in the preparation of a drug that inhibits the deposition of fibrosis markers.

[0011] Preferably, the drug contains beauveria bassiana or its derivatives, or pharmaceutically acceptable salts, solvates, or hydrates of both as active ingredients, and pharmaceutically acceptable excipients.

[0012] Preferably, the beauveria bassiana derivative is a pharmaceutically acceptable peptide modified with side chains and / or intermediate residues; more preferably, the modification of the side chains and / or intermediate residues is any one or more of acylation, amidation, alkylation, esterification, glycosylation, and phosphorylation.

[0013] Preferably, the pharmaceutically acceptable excipients include any one or more of the following: excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0014] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that beauveria bassiana can inhibit abnormal epithelial-mesenchymal transition, regulate the activation and proliferation of lung epithelial cells, and prevent the deposition of fibrosis markers, thereby effectively alleviating the progression of pulmonary fibrosis and providing a new direction for the treatment of pulmonary fibrosis. Attached Figure Description

[0016] Figure 1 Figure 1 shows the results of cell viability assay in the BEAS-2B pulmonary fibrosis model after beautifier treatment. Figure 2 Figure 1 shows the results of cell viability assay in A549 pulmonary fibrosis model after beautifier treatment; Figure 3 Figure 1 shows the results of lactate dehydrogenase assay in BEAS-2B cells, a pulmonary fibrosis model after beautifier treatment. Figure 4 Figure 1 shows the results of lactate dehydrogenase assay in A549 cells, a pulmonary fibrosis model, after treatment with beauveria bassiana. Figure 5 Representative images of BEAS-2B cells in a pulmonary fibrosis model after beauveria bassiana treatment; Figure 6 Representative images of A549 cells in a pulmonary fibrosis model after beautifier treatment; Figure 7 Figure 1 shows the results of type I collagen assay in BEAS-2B cells, a lung fibrosis model after beautifier treatment. Figure 8 Figure 1 shows the results of type I collagen assay in A549 cells, a pulmonary fibrosis model, after treatment with beauveria bassiana. Figure 9 Figure 1 shows the results of α-SMA protein level measurement in BEAS-2B cells, a pulmonary fibrosis model, after treatment with beauveria bassiana. Figure 10 Figure 1 shows the results of α-SMA protein level measurement in A549 cells, a pulmonary fibrosis model, after beauveria bassiana treatment. Figure 11 Figure 1 shows the results of measuring the mRNA expression levels of genes related to the EMT pathway in BEAS-2B cells, a pulmonary fibrosis model after beautifier treatment. Figure 12 Figure 1 shows the results of mRNA expression levels of genes related to the EMT pathway in A549 cells, a pulmonary fibrosis model, after treatment with beauveria bassiana. Figure 13 Figure 1 shows the results of the migration ability assay of BEAS-2B cells in the pulmonary fibrosis model after treatment with beauveria bassiana. Figure 14 Figure 1 shows the results of the migration ability assay of A549 cells in the pulmonary fibrosis model after beauveria bassiana treatment. Figure 15 Figure 1 shows the results of the proliferation capacity assay of BEAS-2B cells, a lung fibrosis model, after treatment with beauveria bassiana. Figure 16 Figure 1 shows the results of the proliferation capacity assay of A549 cells, a pulmonary fibrosis model, after treatment with beauveria bassiana. Figure 17 Flowchart of the experiment on beauvericin treatment in a mouse model of pulmonary fibrosis. Figure 18 A graph showing the change in body weight in mice with pulmonary fibrosis after treatment with beauveria bassiana. Figure 19 A graph showing the statistical results of lung tissue coefficients in mice with pulmonary fibrosis after treatment with beauveria bassiana. Figure 20 Representative H&E staining images of lung tissue from mice with pulmonary fibrosis after treatment with beauveria bassiana; Figure 21 Representative Masson staining images of lung tissue from mice with pulmonary fibrosis after treatment with beauveria bassiana; Figure 22 Figure 1 shows the results of cytokine release levels in a mouse model of pulmonary fibrosis after treatment with beauveria bassiana. Figure 23 Image of lung tissue in a mouse model of pulmonary fibrosis after treatment with beauveria bassiana extract, showing the results of hydroxyproline determination. Figure 24 Figure 1 shows the results of measuring the mRNA expression levels of EMT pathway-related genes in lung tissue of a mouse model of pulmonary fibrosis after treatment with beauveria bassiana. Figure 25Figure 1 shows the results of measuring the expression levels of EMT pathway-related proteins in lung tissue of a mouse model of pulmonary fibrosis after treatment with beauveria bassiana. Detailed Implementation

[0017] The technical solution of the present invention will be further described below.

[0018] Example 1: In vitro efficacy verification of beauveria bassiana 1. Construction of a cell model of pulmonary fibrosis Human lung epithelial cells BEAS-2B and human lung adenocarcinoma cells A549 were cultured in a dedicated incubator at 37°C and 5% carbon dioxide using RPMI 1640 medium supplemented with 10% fetal bovine serum.

[0019] When the cell density reached 70%, TGFβ1 (purchased from PeproTech, Inc., catalog number PHG9202) at a final concentration of 10 ng / mL was added to the culture medium and treated for 24 h to construct a pulmonary fibrosis-related cell model.

[0020] 2. Cell viability assay after BEA treatment Beauvericin (BEA) was purchased from MedChemExpress LLC., catalog number HY-N6739; Pirfenidone (PFD) was purchased from MedChemExpress LLC., catalog number HY-B0673.

[0021] TGFβ1-induced BEAS-2B or A549 cells were seeded into 96-well plates. When the cell density reached 70%, the cells were treated with 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 µM BEA for 24 h. After treatment, each well was replaced with 100 μL of RPMI 1640 basal medium containing 10% CCK8 reagent and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the IC50 value was calculated.

[0022] The results are as follows Figure 1 , 2 As shown, BEA significantly inhibited the activity of BEAS-2B and A549 cells after TGFβ1 induction in a concentration-dependent manner, with an IC50 value of 2.59 µM for BEAS-2B cells and 3.72 µM for A549 cells.

[0023] 3. Measurement of lactate dehydrogenase (LDH) after BEA treatment TGFβ1-induced BEAS-2B or A549 cells were seeded in 6-well plates. When the cell density reached 70%, the cells were treated with 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 µM BEA for 24 h. Each concentration was used in triplicate. The cell culture medium from each well was collected, and the lactate dehydrogenase (LDH) release level was detected using the Beyotime Lactate Dehydrogenase Cytotoxicity Assay Kit (Catalog No. C0016) to assess the cytotoxicity of BEA to the cells.

[0024] The results are as follows Figure 3 , 4 As shown, with the increase of BEA concentration, the relative level of LDH release also gradually increases. The amount of LDH release can reflect the degree of cell damage, thereby assessing the effect of BEA on the activity of pulmonary fibrosis cells. This experimental result once again shows that BEA can inhibit the activity of fibrosis cells, thereby alleviating the symptoms of pulmonary fibrosis.

[0025] Normal cells and TGFβ1-induced BEAS-2B or A549 cells were seeded in 6-well plates. When the cell density reached 70%, the induced cells were divided into the following groups: model group (TGFβ1), model group + 1.25 µM BEA treatment group (TGFβ1 BEA1.25), model group + 2.5 µM BEA treatment group (TGFβ1 BEA 2.5), and model group + 15 µM PFD treatment group (TGFβ1 PFD). After adding the corresponding drugs, the cells were cultured for another 24 h. This was used for cell morphology evaluation, determination of pulmonary fibrosis-related markers, and determination of mRNA expression levels related to the epithelial-mesenchymal transition (EMT) pathway.

[0026] 4. Evaluation of cell morphology after BEA treatment The morphology, arrangement, and protrusions of each group of cells were observed under a microscope, and images were collected and compared.

[0027] The results are as follows Figure 5 , 6 As shown, the normal group cells had regular morphology and good growth status, while the model group cells showed abnormal morphological changes. After BEA treatment, the cell morphology gradually recovered to the normal state, which was similar to the effect of the positive drug PFD. This preliminarily indicates that BEA has an ameliorative effect on the abnormal state of pulmonary fibrosis cells.

[0028] 5. Determination of pulmonary fibrosis-related markers after BEA treatment 5.1 Measurement of Type I Collagen Levels Cell culture medium was collected, and type I collagen levels were detected using the Human Pro-Collagen I α1 ELISA Kit (catalog number EK1C01) from Linko Biotechnology.

[0029] The results are as follows Figure 7 , 8 As shown, the level of type I collagen in the model group cells was significantly increased, while the level of BEA decreased in a concentration-dependent manner in each concentration group. The results suggest that BEA can inhibit collagen deposition in pulmonary fibrosis cells and reduce the degree of fibrosis.

[0030] 5.2 Determination of α-SMA protein expression level after BEA treatment Cells were collected and lysed using RIPA lysis buffer (WB3100) containing 0.1 volume of cyprotease inhibitor mixture (P001) and 0.1 volume of cyprotease inhibitor mixture (P003), and incubated at 4°C for 30 min. After centrifugation at 12000 rpm for 30 min at 4°C, the supernatant was collected and quantified using a cyprotein BCA protein quantification kit (WB6501). Subsequently, 5× Loading buffer (WB2001) was added proportionally, and the mixture was boiled in a metal bath for 10 minutes. After a period of time, SDS-PAGE electrophoresis was performed. After transfer and blocking, α-SMA primary antibody (Proteintech, catalog number 14395-1-AP) diluted 1:5000 or GAPDH primary antibody (Proteintech, catalog number 10494-1-AP) diluted 1:10000 was added and incubated overnight at 4°C. After rinsing, secondary antibody (Proteintech, catalog number SA00001-2) diluted 1:10000 was added and incubated at room temperature for 2 h. Finally, the images were developed and acquired using the New Semiconductor Ultrasensitive ECL Chemiluminescence Kit (catalog number P10100).

[0031] The results are as follows Figure 9 , 10 As shown, the expression of α-SMA protein in the model group was significantly higher than that in the normal group. After BEA treatment, the protein expression level decreased, and the high concentration group showed better inhibitory effect than the low concentration group, confirming the inhibitory effect of BEA on α-SMA expression in A549 cells.

[0032] 6. Measurement of EMT pathway-related mRNA expression levels after BEA treatment Cells were collected, and total RNA was extracted using the Novizan RNA isolater Total RNA Extraction Reagent kit (catalog number R401-01). The RNA was then reverse transcribed into cDNA using the Novizan HiScript III All-in-one RT SuperMix Perfect for qPCR kit (catalog number R333-01), using the following primers: N-Cadherin upstream primer: 5'-gagacattggggacttcatt-3'; N-Cadherin downstream primer: 5'-caaacactaacagggagtca-3'; E-Cadherin upstream primer: 5'-ggagaagaggaccaggactt-3'; E-Cadherin downstream primer: 5'-agtatcagccgctttcagat-3'; 18S upstream primer: 5'-aaacggctaccacatccaag-3'. The 18S downstream primer, 5'-cctccaatggatcctcgtta-3', was used for RT-PCR detection using the Novizan ChamQ Universal SYBR qPCR Master Mix kit (catalog number Q711-02). The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing extension for 30 s, for 35 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. Using 18S as an internal control, Ct values ​​were calculated, and the relative mRNA expression levels of N-Cadherin and E-Cadherin were calculated using the ΔΔCt method.

[0033] The results are as follows Figure 11 , 12 As shown, compared with the normal group, the mRNA level of E-Cadherin in the model group was decreased and the mRNA level of N-Cadherin was increased. However, after BE treatment, this trend was reversed, that is, E-Cadherin expression was upregulated and N-Cadherin expression was downregulated, and the effect was similar to that of the PFD group. That is, BEA can regulate the expression of EMT-related genes in BEAS-2B cells and A549 cells in a concentration-dependent manner and block the EMT process.

[0034] 7. Determination of cell migration ability after BEA treatment Normal cells and TGFβ1-induced BEAS-2B or A549 cells were seeded in 6-well plates. When the cell density reached 70%, the induced cells were divided into the following groups: model group (TGFβ1), model group + 1.25 µM BEA treatment group (TGFβ1 BEA1.25), model group + 2.5 µM BEA treatment group (TGFβ1 BEA 2.5), and model group + 15 µM PFD treatment group (TGFβ1 PFD). Cells were scratched in each well using a sterile 10 µL pipette tip. After washing with PBS to remove detached cells, the corresponding drugs were added and the cells were cultured for 24 h. Subsequently, the medium was replaced with RPMI 1640 medium containing 1% FBS and cultured for another 48 h. Cell migration ability was assessed by observation under a microscope and images were acquired.

[0035] The results are as follows Figure 13 , 14 As shown, the normal group heals slowly, while the model group heals quickly. The degree of wound healing in the BEA-treated group decreases with increasing concentration, and the healing rate in the high-concentration group is comparable to that in the PFD group, indicating that BEA can inhibit the migration ability of BEAS-2B cells and A549 cells.

[0036] 8. Determination of cell proliferation capacity after BEA treatment Normal cells and TGFβ1-induced BEAS-2B or A549 cells were seeded in 24-well plates. When the cell density reached 70%, the induced cells were divided into the following groups: model group (TGFβ1), model group + 1.25 µM BEA treatment group (TGFβ1 BEA1.25), model group + 2.5 µM BEA treatment group (TGFβ1 BEA 2.5), and model group + 15 µM PFD treatment group (TGFβ1 PFD). After adding the corresponding drugs, the cells were cultured for 24 h. After treatment, 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well, and the mixture was incubated at 37℃ for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 15 min, the mixture was permeated with Beyotime immunostaining permeabilization buffer (catalog number P0096) at room temperature for 20 min. Then, Click reaction solution (purchased from Novizan, catalog number A413-01) was added, and the mixture was incubated in the dark for 30 min. The slides were then mounted with Beyotime anti-fluorescence quenching mounting buffer (catalog number P0131), and the slides were observed and images were acquired using a fluorescence microscope.

[0037] The results are as follows Figure 15 , 16 As shown, the proportion of EdU-positive cells was low in the normal group and significantly increased in the model group; the proportion of positive cells in the BEA-treated group decreased with increasing concentration, and the proliferation inhibition effect of the high-concentration group was close to that of the PFD group.

[0038] Example 2: In vivo efficacy verification of beauveria bassiana 1. Construction of animal models of pulmonary fibrosis and drug treatment Ten-week-old female C57BL / 6 mice, weighing 20-22 g, were purchased from the Experimental Animal Center of Nantong University and randomly divided into a saline group (n=10) and a model group (n=40). Mice in the model group were induced by intratracheal infusion of 100 µL of 0.4 mg / mL bleomycin (BLM, purchased from MedChemExpress LLC., catalog number HY-108345) solution, once every 4 days for 4 consecutive weeks; mice in the normal control group were infused with an equal volume of saline.

[0039] After successful model construction, the model group mice were randomly divided into 4 groups (n=10 per group): BLM group (intraperitoneal injection of saline), BLM+PFD group (intraperitoneal injection of 100 mg / kg PFD), BLM-BEA group 5 (intraperitoneal injection of 5 mg / kg BEA), and BLM-BEA group 10 (intraperitoneal injection of 10 mg / kg BEA). Mice in the saline and BLM groups received an equal volume of saline intraperitoneally. All groups were administered the medication every 3 days for up to day 30, following the protocol as follows: Figure 17 As shown.

[0040] 2. Weight monitoring Starting from day 0 of the drug treatment, the body weight of mice in each group was measured every 3 days, and the trend of body weight change was recorded.

[0041] The results are as follows Figure 18 As shown in the figure, the mice were weighed every 3 days starting from day 6 until day 30. The figure shows that the weight of the mice in the normal group increased steadily. Compared with the saline group, the weight of the mice in the model group and the drug treatment group did not change significantly, which means that the concentration of the drug used in the experiment did not have significant biological toxicity.

[0042] 3. Organ coefficient detection After treatment, the mice were euthanized, and the intact lung tissue was removed. The surface moisture was absorbed with filter paper and the tissue was weighed. The lung tissue coefficient was calculated according to the following formula: Lung tissue coefficient (%) = wet weight of lung tissue ÷ mouse body weight × 100.

[0043] The results are as follows Figure 19 As shown, the lung tissue coefficient in the model group was abnormal, and the lung tissue coefficient tended to be normal after BEA treatment, indicating that BEA can reduce pathological damage to lung tissue.

[0044] 4. Lung tissue pathological analysis 4.1 H&E staining Mouse lung tissue was fixed in 4% paraformaldehyde solution for 48 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 5 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0045] The results are as follows Figure 20 As shown, the alveolar structure of normal mice was intact and there was no significant thickening of the lung interstitium. The alveolar structure of model mice was destroyed, the lung interstitium was thickened, and there were obvious pathological changes such as inflammatory cell infiltration. After BEA treatment, the alveolar structural damage was reduced, the thickening of the lung interstitium was alleviated, the inflammatory infiltration was reduced, and the pathological state was significantly improved.

[0046] 4.2 Masson staining The preliminary steps were the same as those for H&E staining. In the staining section, the collagen deposits in the liver tissue were stained using the Beyotime Masson Tricolor Staining Kit (catalog number C0189S). After mounting with neutral resin, the tissue was observed and images were acquired under a microscope.

[0047] The results are as follows Figure 21 As shown, in the normal group, only a small amount of blue collagen fibers were distributed around blood vessels in the lung tissue; in the model group, a large amount of blue collagen fibers were deposited in the lung tissue and diffusely distributed in the lung interstitium and around the alveoli; collagen deposition was reduced in the low-dose BEA group and significantly reduced in the high-dose group. Similar to the PFD group, there was only a small amount of collagen distributed around blood vessels, indicating that BEA can effectively inhibit collagen deposition in lung tissue.

[0048] 5. Determination of relevant indicators in mouse peripheral blood and lung tissue 5.1 Peripheral blood cytokine assay At the end of treatment, blood samples were collected from mice and centrifuged to obtain peripheral blood serum. Serum cytokine levels were measured using the Solarbio mouse tumor necrosis factor α assay kit (catalog number SEKM-0034), Solarbio mouse interferon γ assay kit (catalog number SEKM-0031), Solarbio mouse interleukin 6 assay kit (catalog number SEKM-0007), and Solarbio mouse interleukin 18 assay kit (catalog number SEKM-0019).

[0049] The results are as follows Figure 22 As shown, BEA treatment reduced the levels of pro-fibrotic and pro-inflammatory cytokines in peripheral blood, while increasing the levels of anti-inflammatory and anti-fibrotic cytokines, indicating that BEA can alleviate inflammatory responses and the degree of fibrosis by regulating the balance of cytokine secretion.

[0050] 5.2 Determination of hydroxyproline in lung tissue 100 mg of mouse lung tissue was collected, ground with liquid nitrogen, and the hydroxyproline content in the lung tissue was detected using the Solarbio Hydroxyproline Content Assay Kit (catalog number BC0250).

[0051] The results are as follows Figure 23 As shown, the level of hydroxyproline in lung tissue decreased after BEA treatment, further confirming that BEA can reduce collagen synthesis and deposition in lung tissue and alleviate fibrosis.

[0052] 5.3 Determination of mRNA and protein expression levels related to pulmonary fibrosis 5.3.1 Determination of mRNA expression levels related to lung fibrosis in lung tissue 100 mg of mouse lung tissue was collected, ground in liquid nitrogen using a mortar and pestle, and total RNA was extracted using the Novizan RNA isolater Total RNA Extraction Reagent kit. The RNA was then reverse transcribed into cDNA using the Novizan HiScript III All-in-one RTSuperMix Perfect for qPCR kit, using the following mouse primers: N-Cadherin upstream primer: 5'-gagcctgatgccatcaagc-3'; N-Cadherin downstream primer: 5'-tcattcaggtagtcatagtcctgg-3'; E-Cadherin upstream primer: 5'-catggtttcgttcatacgcgaaattc-3'; E-Cadherin downstream primer: 5'-ggaagcaattccttagtgttgtcc-3'; The aforementioned primers were used... See 18S Primers were used for RT-PCR detection using the Novizan ChamQ Universal SYBR qPCR Master Mix kit, under the same reaction conditions as before. Fluorescence values ​​were collected during the extension phase of each cycle. 18S was used as an internal control, and Ct values ​​were calculated. The relative mRNA expression levels of N-Cadherin and E-Cadherin were calculated using the ΔΔCt method.

[0053] The results are as follows Figure 24 As shown, BEA treatment can regulate the mRNA levels of these two molecules, suggesting that BEA may exert its anti-pulmonary fibrosis effect by inhibiting the EMT pathway.

[0054] 5.3.2 Determination of expression levels of pulmonary fibrosis-related proteins 100 mg of mouse lung tissue was collected, ground in liquid nitrogen using a mortar and pestle, and then lysed in 300 µL of RIPA lysis buffer containing 0.1 volume of cyprotease inhibitor mixture and 0.1 volume of cyprotease inhibitor mixture. The mixture was incubated at 4°C for 30 min, centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was collected. Quantification was performed using a cyprotease BCA protein quantification kit. Subsequently, 5× loading buffer was added in proportion, and the mixture was boiled in a metal bath for 10 minutes. After a period of time, SDS-PAGE electrophoresis was performed. After transfer and blocking, N-Cadherin primary antibody (purchased from Proteintech, catalog number 22018-1-AP) diluted 1:5000, or E-Cadherin primary antibody (purchased from Proteintech, catalog number 22018-1-AP) diluted 1:20000, or GAPDH primary antibody diluted 1:10000 was added and incubated overnight at 4°C. After rinsing, secondary antibody diluted 1:10000 was used and incubated at room temperature for 2 h. Finally, the images were developed and acquired using the NewSemi high-sensitivity ECL chemiluminescence kit.

[0055] The results are as follows Figure 25 As shown, the results are consistent with those detected by qPCR, further clarifying that BEA participates in the treatment of pulmonary fibrosis by regulating the EMT pathway.

Claims

1. The application of a beauveria bassiana in the preparation of a drug for the treatment of pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The CAS number for the beauveria bassiana is 26048-05-5.

3. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit epithelial-mesenchymal transition abnormalities.

4. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit abnormal activation and proliferation of lung epithelial cells.

5. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit the deposition of fibrosis markers.

6. The application according to claim 1, characterized in that, The drug contains beauveria bassiana or its derivatives, or pharmaceutically acceptable salts, solvates, or hydrates of both as active ingredients, and pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The beauveria bassiana derivative is a peptide modified with pharmaceutically acceptable side chains and / or intermediate residues.

8. The application according to claim 7, characterized in that, The modification of the side chain and / or intermediate residues is any one or more of acylation, amidation, alkylation, esterification, glycosylation, and phosphorylation.

9. The application according to claim 6, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

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