A polyketide compound and its application in preparing medicine for treating pulmonary fibrosis

Abstract

The present invention discloses a polyketide compound and its application in the preparation of a drug for treating pulmonary fibrosis. The polyketide compound or its pharmaceutically acceptable salt, ester, or solvate provided by the present invention can effectively inhibit fibrosis of tissues and organs in the respiratory system; the marine natural product has good application prospects in drugs for treating fibrosis of respiratory organs, especially drugs for treating pulmonary fibrosis, providing new means and new ideas for treating fibrotic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polyketide compound and application thereof in preparing a drug for resisting pulmonary fibrosis. Background Art

[0002] Fibrosis can occur in many organs. The main pathological changes are increased fibrous connective tissue and decreased parenchymal cells in organ tissues. Continuous progression can lead to organ structural destruction and functional impairment, and even failure, which seriously threatens human health and life. Among them, pulmonary fibrosis is a chronic progressive lung disease characterized by pulmonary interstitial fibrosis. Its pathogenesis has not yet been fully clarified, but many factors are believed to be related to its onset, including long-term inhalation of harmful particulate matter such as dust, chemicals, organic matter, etc., as well as certain drugs, radiation exposure, infection, genetic factors, etc., which can cause pulmonary fibrosis. Different causes may lead to different pathophysiological mechanisms, such as inflammatory response, oxidative stress, abnormal cell proliferation and apoptosis, which ultimately lead to persistent damage to lung structure and function. Pulmonary fibrosis is characterized by pulmonary interstitial fibrosis and structural destruction, accompanied by changes such as inflammatory cell infiltration, alveolar wall thickening, and collagen deposition. Typical pathological manifestations include honeycomb lung, fibrous bands, interstitial fibrosis, etc., which lead to progressive decline in lung function and limited gas exchange. There is currently no drug to cure pulmonary fibrosis.

[0003] The current treatment is mainly to relieve symptoms, delay disease progression and improve the quality of life. Commonly used drugs include immunosuppressants, anti-fibrotic drugs, anti-inflammatory drugs, etc. However, the effects of existing drug treatments are not satisfactory. Rehabilitation care is also an important treatment method, including respiratory rehabilitation, nutritional support, psychological intervention, exercise training, etc., which can improve patients' symptoms and functions. For patients with severe conditions, lung transplantation may be the last treatment option, but due to donor issues and surgical risks, its scope of application is limited. Therefore, the development of therapeutic drugs has been highly valued. Studies have found that wedelolactone inhibits the SMAD and non-SMAD signaling pathways induced by TGF-β1, thereby inhibiting the epithelial cell to mesenchymal cell transition (EMT) process and fibroblast transdifferentiation process induced by rhTGF-β1 recombinant protein. In addition, studies have found the application of leonurine in the preparation of drugs for the prevention or treatment of respiratory diseases. It has been verified that leonurine can enhance the lung function of animal models, inhibit the secretion of CD48 and IgE in serum, and can act on bronchial epithelial cells, reduce the content of lipoxygenase, inhibit PDE enzyme activity, etc., thereby preventing high airway responses, allergic reactions, tracheal rigidity, etc. caused by various mediator molecules, and effectively preventing or treating various respiratory diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis.

[0004] In summary, the development of new anti-fibrosis drugs provides new means and new ideas for the treatment of fibrotic diseases, which is of great significance in the field of fibrotic disease treatment. Summary of the invention

[0005] The object of the present invention is to overcome the disadvantages and shortcomings of the prior art and provide a polyketone compound.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned polyketone compound.

[0007] Another object of the present invention is to provide applications of the polyketone compound.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A polyketone compound having a structural formula of at least one of the following:

[0010]

[0011] Use of the above polyketide compound or its pharmaceutically acceptable salt, ester or solvate in the preparation of drugs for treating fibrotic diseases.

[0012] The above-mentioned polyketide compounds or their pharmaceutically acceptable salts, esters, and solvates can effectively inhibit the fibrosis of lung epithelial cells, indicating that marine natural products can provide new ideas for the treatment of diseases related to fibrosis of respiratory tissues and organs.

[0013] Use of the above-mentioned polyketide compound or its pharmaceutically acceptable salt, ester, solvate in the preparation of alpha-SMA protein (alpha smooth muscle actin, also known as ACTA2) and / or Fibronectin protein expression inhibitor for the purpose of non-disease diagnosis and / or treatment.

[0014] Use of the above polyketide compound or its pharmaceutically acceptable salt, ester or solvate in the preparation of a cell fibrosis inhibitor.

[0015] Preferably, the fibrotic disease comprises pulmonary fibrosis.

[0016] Preferably, the daily adult dosage of the polyketide compound or its pharmaceutically acceptable salt, ester, solvate is 20 to 200 μM.

[0017] Preferably, the polyketide compound or a pharmaceutically acceptable salt, ester or solvate thereof is used to reduce the expression level of α-SMA protein and / or Fibronectin protein in cells.

[0018] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0019] Preferably, the auxiliary materials include any one or a combination of at least two of a carrier, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0020] Preferably, the dosage form of the drug includes any one of tablets, dispersible tablets, lozenges, orodisintegrating tablets, sustained-release tablets, capsules, soft capsules, pills, granules, injections, powder injections or aerosols.

[0021] A method for preparing a polyketone compound comprises the following steps:

[0022] (1) constructing a non-reducing polyketide synthase gene, an α, β-hydrolase gene, and a post-modification oxidase gene onto a vector, transferring the genes into engineering bacteria, and screening positive clones to obtain recombinant transformants;

[0023] (2) inoculating the recombinant transformant into a culture medium for cultivation, and extracting the obtained culture fluid to obtain an extract concentrate;

[0024] (3) The extract is added to a silica gel column for separation, and the eluate is purified by HPLC to obtain a polyketone compound.

[0025] The sequence of the non-reducing polyketide synthase gene in step (1) is shown in SEQ ID NO.1.

[0026] The sequence of the α,β-hydrolase gene described in step (1) is shown in SEQ ID NO.2.

[0027] The sequence of the post-modification oxidase gene described in step (1) is shown in SEQ ID NO.3.

[0028] The vector described in step (1) is at least one of pTAex3, pUSA and pTRI.

[0029] The engineered bacteria described in step (1) is Aspergillus oryzae NSAR1.

[0030] The culture medium described in step (2) is DPY culture medium.

[0031] The culture conditions of step (2) are culturing at 25-30° C. for 5-12 days.

[0032] The extraction in step (2) is performed using ethyl acetate.

[0033] The eluent in step (3) is a mixed solution of ethyl acetate / petroleum ether; preferably a mixed solution of ethyl acetate / petroleum ether in a volume ratio of 40:60 or 60:40.

[0034] The polyketone compound described in step (3) is at least one of formulas I to II.

[0035]

[0036] When the polyketide compound is of formula I, the HPLC conditions are:

[0037] Mobile phase: acetonitrile-0.1% acetic acid aqueous solution (volume ratio 50:50); flow rate: 3 mL / min, chromatographic column: Welch Ultimate XB-C18, 10×250 mm, 5 μm; instrument Essentia LC-16.

[0038] When the polyketone compound is of formula II, the HPLC conditions are:

[0039] Mobile phase: acetonitrile-0.1% acetic acid aqueous solution (volume ratio 30:70); flow rate: 3 mL / min, chromatographic column: ZORBAXSB-C18, 10×250 mm, 5 μm; instrument: Essentia LC-16.

[0040] Compared with the prior art, the present invention has the following advantages and effects:

[0041] The present invention discovers for the first time that a class of marine natural products or their pharmaceutically acceptable salts, esters, and solvates can effectively inhibit fibrosis of respiratory system tissues and organs; the marine natural products have good application prospects in anti-fibrosis drugs for respiratory system organs, especially therapeutic drugs for pulmonary fibrosis, providing new means and new ideas for treating fibrotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the H NMR spectrum of compound R25 (CDCl 3 );

[0043] Figure 2 is the C NMR spectrum (CDCl 3 );

[0044] Figure 3 The H NMR spectrum (CD 3 OD);

[0045] Figure 4 The C NMR spectrum (CD 3 OD);

[0046] Figure 5 This is the result of rhTGF-β recombinant protein induction model detection;

[0047] Figure 6It is a quantitative diagram of the test results of the TGF-β recombinant protein-induced model;

[0048] Figure 7 This is the result of WB test of natural product anti-fibrosis;

[0049] Figure 8 This is a quantitative graph of the anti-fibrosis WB test results of natural products;

[0050] Fig. 9 This is the result of natural product anti-fibrosis immunofluorescence test;

[0051] Fig.10 This is a quantitative graph of the results of natural product anti-fibrosis immunofluorescence detection;

[0052] Fig.11 This is a graph showing the results of natural product anti-fibrotic cytotoxicity testing;

[0053] Fig.12 This is a graph showing the test results of the effective concentration of the natural product R25 in anti-fibrosis;

[0054] Fig.13 This is a graph showing the test results of the effective concentration of the natural product R28 in anti-fibrosis; DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0056] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0057] Example 1 Extraction of marine natural products

[0058] (1) After whole genome sequencing of the sea squirt fungus Diaporthe sp.SYSU-MS4722, the sequencing results were analyzed to identify a biosynthetic gene cluster whose core gene polyketide synthase has 72% protein sequence similarity with the core gene of the reported compound Clavatol, and the gene cluster is not expressed under conventional culture conditions and is a silent gene cluster. To this end, the quadruple-defective Aspergillus oryzae NSAR1 was used as a heterologous expression host to activate the silent gene cluster.

[0059] (2) After extracting the DNA of Diaporthe sp.SYSU-MS4722, the following primers F-claG and R-claG were used for PCR in vitro amplification to obtain the core gene non-reducing polyketide synthase gene claG (sequence as shown in SEQ ID NO.1), and constructed on the KpnI / SmaI double-enzyme linearized vector pTAex3 to form the recombinant plasmid pTAex3-claG.

[0060] F-claG:AATTCGAGCTCGGTACcgcgtggtattacttttcctact

[0061] R-claG: CTACTACAGATCCCCGGgcaggagatactcctcgtctg

[0062] The α,β-hydrolase gene claF (sequence shown in SEQ ID NO.2) was constructed on the KpnI / SmaI double-digested linearized vector pUSA to form the recombinant plasmid pUSA-claF. The upstream and downstream primers were:

[0063] F-claF:AATTCGAGCTCGGTACCagtgccctagtttactttgatc

[0064] R-claF: TACTACAGATCCCCGGGactgatctatccgtacatcgc

[0065] At the same time, the post-modification gene α-ketoglutarate-dependent (α-KG) non-heme iron oxygenase (PRB-D) (sequence shown in SEQ ID NO.3) from the reported clavatol biosynthesis gene cluster in the fungus Penicillium crustosum PRB-2 was constructed into the HindIII / SmaI double-enzyme linearized vector pTRI to form the recombinant plasmid pTRI-PRB-D. The upstream and downstream primers are:

[0066] F-PRB-D:CTATGACCATGATTACGCCAAGCTTcgactccaatcttcaagagc

[0067] R-PRB-D: AGTGAATTCGAGCTCGGTACCCGGGTgtaagatacatgagcttcgg

[0068] (3) Then, a 1% Yatalase, 0.6 M (NH 4 )2 SO 4 Protoplasts of Aspergillus oryzae NSAR1 were prepared by incubating in a medium containing 50 mM maleic acid and pH 5.5 at 30°C for 4 hours. The plasmids pTAex3, pUSA and pTRI carrying the gene were transfected into the protoplasts of quadruple-deficient Aspergillus oryzae NSAR1 using a PEG-4000-mediated method. Finally, the protoplasts were grown in M ​​selection medium (0.2% NH 4 Cl, 0.1% (NH 4 ) 2 SO 4 ,0.05%KCl,0.05%NaCl,0.1%KH 2 PO 4 ,0.05%MgSO 4 .7H 2 O, 0.002% FeSO 4 .7H 2 O, 2% glucose, 1.2M sorbitol, 0.01% adenine, 0.1μg / mL Pyrithiamine hydrobromide, 1.2% Agar) were cultured at 28°C for 6 days, and the obtained transformants were subcultured 3 times on M medium without sorbitol, and genomic DNA was extracted for verification, and transfected strains containing clavatol biosynthesis genes and exogenous post-modification oxidase genes were screened. The Penicilliumcrustosum PRB-2 has a deposit number of GDMCC NO: 809030, and the genebank sequence number of the α-ketoglutarate-dependent (α-KG) non-heme iron oxygenase gene is MK360918.

[0069] (4) The recombinant transformants obtained by screening were inoculated into 5 × 10 mL of DPY medium (2% dextrin, 1% polyvalent peptone, 0.5% yeast extract, 0.5% KH 2 PO 4 , 0.05% MgSO 4 .7H 2 O) and cultured at 28°C and 180 rpm for 2 days, then the culture solution was transferred to 5×200 mL DPY medium and cultured at 28°C and 180 rpm for another day as seed solution. The seed solution was transferred to 15 L CD-starch medium (0.3% NaNO 3 , 0.2% KCl, 0.05% MgSO 4 .7H 2 O, 0.1% KH2 PO 4 , 0.002%FeSO 4 .7H 2 O, 1% polypeptide, 2% starch, pH 5.5), inoculate each bottle with 20 mL of seed solution, inoculate 50 bottles in total, and induce expression for 6 days at 28°C and 200 rpm. Extract the culture medium with ethyl acetate 5 times and concentrate under reduced pressure to obtain the extract.

[0070] (5) The extract obtained in step (4) was mixed with 100-200 mesh silica gel in a mass ratio of 1:1, wet loaded onto a column, and gradient eluted with ethyl acetate / petroleum ether (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to obtain elution portions containing components of different polarity, and the 40:60 component was purified by high performance liquid chromatography to obtain compound R25, and the 60:40 component was purified by high performance liquid chromatography to obtain compound R28. Then, high performance liquid chromatography was used for purification;

[0071] Wherein, the conditions of high performance liquid chromatography of compound R25 are:

[0072] Mobile phase: acetonitrile-0.1% acetic acid aqueous solution (volume ratio 50:50); flow rate: 3mL / min, chromatographic column: Welch Ultimate XB-C18, 10×250mm, 5μm; instrument Essentia LC-16. Compound R25 was separated at 39.2min as a light yellow oil. The obtained light yellow oil was detected by nuclear magnetic resonance and mass spectrometry. The spectrum is shown in Figures 1-2 From the results of mass spectrometry and nuclear magnetic resonance, it can be determined that the molecular formula of compound R25 is C 18 H 22 O 5 , the structural formula is shown in Formula I. Its mass spectrum and NMR data are as follows: HR-ESI-MS (m / z) 317.1395 [MH] - (C 18 H 21 O 5 ,calcd for 317.1394). 1 HNMR (600MHz, CDCl 3)δ7.34(s,1H),4.08(m,1H),3.01(dd,J=16.8,6.3,1H),2.76(dd,J=14.5,1.9,1H),2.54(s,3H),2.50(m,1H ),2.47(m,1H),2.46(m,1H),2.30(m,1H),2.28(m,1H),2.07(s,3H),1.22(d,J=6.2,3H),0.95(d,J=7.1,3H); 13 C NMR (150 MHz, CDCl 3 )δ205.05,203.10,160.99,155.26,129.73,117.06,113.52,109.05,1 02.82,67.28,48.56,47.33,33.07,26.43,23.63,21.88,15.36,14.89.

[0073]

[0074] Wherein, the conditions of high performance liquid chromatography of compound R28 are:

[0075] Mobile phase: acetonitrile-0.1% acetic acid aqueous solution (volume ratio 30:70); flow rate: 3mL / min, chromatographic column: ZORBAXSB-C18, 10×250mm, 5μm; instrument Essentia LC-16. Compound R28 was separated at 26.9min as a white needle-shaped solid. The obtained white needle-shaped solid was detected by nuclear magnetic resonance and mass spectrometry. The spectrum is shown in Figures 3-4 From the results of mass spectrometry and nuclear magnetic resonance, it can be determined that the molecular formula of compound R28 is C 13 H 16 O 5 , the structural formula is shown in Formula II. Its mass spectrum and NMR data are as follows: HR-ESI-MS (m / z) 251.0926 [MH] - (C 13 H 15 O 5 ,calcd for 251.0925). 1 H NMR (600MHz, CD 3 OD)δ7.49(s,1H),2.91(m,1H),2.80(m,1H),2.80(m,1H),2.52(s,3H),2.17(s,3H),1.16(d,J=6.6,3H); 13 C NMR (150MHz, CD 3OD)δ204.56,181.73,162.74,162.23,132.14,117.74,114.43,113.92,40.22,27.40,26.26,17.70,16.65.

[0076]

[0077] Example 2 Cell culture (human lung fibroblasts, HFL-1) and establishment of fibrosis induction model FMT induction model: Human lung fibroblasts (HFL-1) were used in the experiment, with a cell number of 0.7×10 5 The density of the cells was evenly plated in a 12-well plate, and 1 mL of HFL-1 complete medium was added to each well. The plates were placed at 37°C and 5% CO. 2 The cells were cultured in an incubator for 48 h. When the density reached 80%, the complete medium was replaced with F-12K basal medium for 24 h. The original F-12K basal medium was then discarded and 1 mL of F-12K basal medium containing 10 ng / mL rhTGF-β1 was added to each well. The cells were stimulated for 0 h (CON group), 12 h, 24 h, 36 h and 48 h, respectively. Cell proteins were extracted and western blotting analysis was performed to examine the changes in cell α-SMA protein levels at different stimulation times.

[0078] The results are as follows Figures 5-6 As shown, under the treatment of TGF-β-induced model, the expression of fibrosis marker protein α-SMA protein increased with the increase of time, indicating that the induced model was successfully constructed and the degree of fibrosis gradually increased with the increase of time.

[0079] Example 3 Detection of the anti-pulmonary fibrosis effect of marine natural products

[0080] 1. Drug Screening

[0081] Human lung fibroblasts (HFL-1) were used in the experiment, with a cell number of 0.7×10 5 The density of the cells was evenly plated in a 12-well plate, and 1 mL of HFL-1 complete medium was added to each well. The plates were placed at 37°C and 5% CO. 2The cells were cultured in an incubator for 48 h. After the density reached 80%, the complete culture medium was replaced with F-12K basal culture medium for 24 h, and then the original F-12K basal culture medium was discarded. First, F-12K basal culture medium was added to select one group as the sham. group; then a group was added with 0.1% DMSO as the veh. group; a group was selected to add 10 μM SB431542 on the basis of the related substances added to the veh. group to become the SB group; two groups were selected to add 20 μM of the compound R25 or R28 separated in Example 1 on the basis of the related substances added to the veh. group (marked as R25 and R28 groups), and after culturing for 30 minutes, 10 ng / mL rhTGF-β1 was added to each group except the sham. group, and a group without solvent or drug was selected as the treat. group (including F-12K basal culture medium + rhTGF-β1). After culturing for 36 hours, cell proteins were extracted, and protein immunoblotting analysis was performed to detect cell α-SMA and Fibronectin protein expression.

[0082] 2. Extract protein.

[0083] The cells cultured in step 1 were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors for 30 min, all on ice. Then, a cell scraper was used to scrape the cells, and the lysate was centrifuged at 12,000 rpm for 10 min at 4°C to obtain protein lysate. The supernatant was collected and stored at -80°C until needed.

[0084] 3.WB experiment.

[0085] The protein concentration was determined using the BCA protein assay kit according to the instructions in the manual. 8 μg of protein lysate was denatured and separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Electrophoresis was performed at 75V first, and then adjusted to 150V after marker separation. After electrophoresis, the protein was transferred to a PVDF membrane and transferred to a mold at 100V for 90min. Then, a 5% skim milk TBST solution was used to block at 25°C for 1h. After that, TBST was used to wash 3 times, each for 10min. The membrane was incubated overnight at 4°C. The main antibodies used included α-SMA (1:1000 dilution, A2547, Sigma) and GAPDH (1:1000 dilution, 2118S, CST). On the second day, after antibody recovery, the cells were washed three times with TBST for 10 min each time, and then incubated at 25 °C for 1 h with horseradish peroxidase (HRP)-labeled secondary immunoglobulins (1:3000 dilution of HRP-labeled goat anti-rabbit antibody (7074S, CST); 1:5000 dilution of HRP-labeled goat anti-mouse antibody (7076S, CST). The cells were washed three times with TBST for 10 min each time. Enhanced chemiluminescence (ECL) was performed using the GenSuper ECL kit (JXE0011, Jingxin, China), and protein bands were visualized on an iBright FL1500 system (Thermofisher, CN, USA). The results are shown in Table 1. Figures 7-8 The results showed that compounds R25 and R28 were potential anti-fibrotic compounds.

[0086] 4. Cell immunofluorescence experiment.

[0087] (1) Cell culture: Human lung fibroblasts (HFL-1) were used in the experiment, with a cell number of 0.7×10 per well. 5 The density of the plate was evenly spread into a 12-well plate, and 1 mL of HFL-1 special complete medium (F12K + 10% fetal bovine serum) was added to each well. The plate was placed at 37°C and 5% CO 2 cultured in an incubator.

[0088] (2) Fixing cells: Treat cells with formaldehyde or other fixatives for 30 minutes to fix proteins and structures within the cells. Fixation preserves the morphology of cells and the location of molecules.

[0089] (3) Permeabilization treatment: Wash with PBS containing Triton X-100 to increase the permeability of the cell membrane, allowing the antibody to enter the cell.

[0090] (4) Blocking nonspecific binding: Treat cells with 5% BSA blocking solution for 1 hour to prevent the antibodies added subsequently from nonspecifically binding to cell components.

[0091] (5) Primary antibody incubation: Incubate the cells with the primary antibody against the target protein (Fibronectin) at 4°C overnight.

[0092] (6) Washing: Wash the cells with PBS or other buffer to remove unbound primary antibody.

[0093] (7) Secondary antibody incubation: Incubate the cells with a fluorescently labeled secondary antibody that specifically binds to the primary antibody for 1 hour.

[0094] (8) Wash again: Wash the cells to remove unbound secondary antibody.

[0095] (9) Nuclear staining: Use DAPI fluorescent dye to stain the cell nucleus for observation under a microscope.

[0096] (10) Microscope observation: Use a fluorescence microscope to observe and photograph cells and analyze the distribution and intensity of fluorescent markers. Figures 9-10 As shown, compounds with cell immunofluorescence detection results similar to those of positive drugs are considered to be meaningful alternative anti-fibrotic compounds. Combined with the WB results, it is confirmed that the positive compounds R25 and R28 are effective anti-pulmonary fibrosis compounds.

[0097] Example 4 This example conducts cytotoxicity detection of marine natural products.

[0098] (1) Cell culture: Human lung fibroblasts (HFL-1) were used in the experiment, with a cell number of 0.7×10 per well. 5 The cells were evenly plated at a density of 1 mL into a 12-well plate, 1 mL of HFL-1 complete culture medium was added to each well, and the plates were cultured in an incubator at 37°C and 5% CO2.

[0099] (2) Drug treatment: Prepare drug solutions of compounds R25 and R28, add the drugs to the cell culture wells at a final concentration of 20 μM, and usually include a control group (untreated cells or cells containing only solvent). For specific group settings, please refer to the experimental grouping in step 1 of Example 3.

[0100] (3) Place the cell culture dish in the Incucyte real-time cell imaging system and incubate: Incubate the treated cells at 37°C and 5% CO2 for 0 to 40 hours. The Incucyte system will automatically take images of the cells in the dish every 10 hours. These images can be used to observe cell morphological changes, growth status, and degree of fusion.

[0101] (4) Data analysis: Incucyte software was used to analyze cell images, calculate cell coverage area, and evaluate drug cytotoxicity. Fig.11As shown, there was little difference between the compound group and the control group, and no obvious cytotoxicity was shown.

[0102] Example 5 Detection of IC50 of marine natural products.

[0103] Detection of effective concentration of marine natural products R25 / R28.

[0104] (1) Cell culture: Human lung fibroblasts (HFL-1) were used in the experiment, with a cell number of 3×10 per well. 3 The density of the cells was evenly plated in a 96-well plate, and 0.2 mL of HFL-1 complete medium was added to each well. The plate was placed at 37°C and 5% CO. 2 cultured in an incubator.

[0105] (2) Drug treatment: In order to further clarify that the anti-fibrotic effect of the compound is not caused by cytotoxicity, different concentrations of drug solutions (0, 0.02, 0.2, 2, 20, 200 uM) are prepared. Different concentrations of drugs are added to the cell culture wells, usually including a control group (untreated cells or cells containing only solvent).

[0106] (3) Incubation: The treated cells were incubated at 37°C and 5% CO2 for 40 hours.

[0107] (4) Add CCK-8 reagent: After the incubation is completed, add CCK-8 reagent to each well. Continue incubation for 2 hours to allow CCK-8 to react with the active enzyme in the cells to produce a visible color change.

[0108] (5) Determine absorbance: Use a microplate reader to measure the absorbance (OD value) of each well, usually at a wavelength of 450 nm.

[0109] (6) Calculate the relative cell survival rate: Cell survival rate (%) = (OD value of control group - OD value of blank group) / (OD value of treatment group - OD value of blank group) × 100

[0110] (7) Draw a dose-response curve: Draw a curve of drug concentration (horizontally) and relative cell survival rate (vertically) on a graph.

[0111] (8) Calculation of IC50 value: Use graphical analysis software or nonlinear regression analysis statistical methods to determine the IC50 value from the dose-response curve. Figures 12-13 As shown, the effective concentrations of the candidate compounds R25 and R28 are between 20 and 200 μM, and their IC50 values ​​are 115.6 μM and 151.7 μM, respectively, indicating that the anti-fibrotic effect at a concentration of 20 μM is not caused by cytotoxicity.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polyketone compound, characterized in that The structural formula is shown in Formula I: 。 2. Use of a polyketide compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The structural formula of the polyketone compound is at least one of the following: 。 3. Use of a polyketide compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for reducing the expression level of α-SMA protein and / or Fibronectin protein in cells, characterized in that: The structural formula of the polyketone compound is at least one of the following: 。 4. Use of a polyketide compound or a pharmaceutically acceptable salt thereof in the preparation of a cell fibrosis inhibitor, characterized in that: The structural formula of the polyketone compound is at least one of the following: ； The cells are human lung fibroblasts HFL-1.

5. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients; The auxiliary materials include any one or a combination of at least two of carriers, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

6. The use according to claim 2, characterized in that: The dosage form of the drug includes any one of tablets, capsules, pills, granules, injections, powder injections or aerosols.

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