SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparation of anti-new coronavirus drugs
阅读说明：本技术 含酯基芳香丙酰胺的SARMs类化合物及其代谢物在制备抗新冠病毒药物中的应用 (SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparation of anti-new coronavirus drugs ) 是由 朱新法 曹长清 朱然 于 2021-01-08 设计创作，主要内容包括：本发明属于医药技术领域,具体公开一种含酯基芳香丙酰胺的SARMs类化合物EG-017及其代谢物EG-2在制备抗新冠病毒药物以及抑制新冠病毒造成的肺部、心脏等多器官损伤和治疗肺纤维化中的应用。应用IFA评价化合物体外抗新型冠状病毒(SARS-CoV-2)活性和细胞毒性,很好的验证了EG-017对新型冠状病毒 βCoV/KOR/KCDC03/2020株在Vero细胞内的复制有明显抑制活性,表明其具有在制备抗新冠病毒药物中的应用前景；实验同时表明,EG-017可以很好的抑制高浓度雷帕霉素对细胞产生细胞毒性；通过MTT法验证EG-017能够显著提高大鼠心肌细胞的存活率。通过EG-017对TGF-β1诱导A549肺癌细胞纤维化过程的影响实验验证了EG-017及其代谢物对治疗新冠病毒引起的肺部病变,以及其它原因引起的肺纤维化等肺部疾病治疗的潜在应用价值。(The invention belongs to the technical field of medicines, and particularly discloses an application of SARMs compound EG-017 containing ester-based aromatic propionamide and a metabolite EG-2 thereof in preparation of anti-new coronavirus medicines, inhibition of multi-organ injuries of lung, heart and the like caused by new coronavirus and treatment of pulmonary fibrosis. IFA is applied to evaluate the activity and cytotoxicity of the compound against the novel coronavirus (SARS-CoV-2) in vitro, and the obvious inhibition activity of EG-017 on the replication of the novel coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells is well verified, which shows that the compound has application prospect in preparing anti-novel coronavirus medicines; experiments also show that EG-017 can well inhibit the cytotoxicity of high-concentration rapamycin to cells; the MTT method proves that EG-017 can obviously improve the survival rate of rat myocardial cells. The potential application value of EG-017 and metabolites thereof in treating lung diseases such as lung fibrosis caused by new coronavirus and lung diseases such as lung fibrosis caused by other reasons is verified by an experiment on the influence of EG-017 on a TGF-beta 1 induced A549 lung cancer cell fibrosis process.)
1. SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparing anti-new coronavirus medicines.
2. The use of claim 1, wherein: the SARMs compound containing the ester-based aromatic propionamide is EG-017 and has a chemical formula of C25H17F3N4O4。
3. The use of claim 1, wherein: the metabolite of the aromatic propionamide compound containing ester group is EG-2.
4. The use of claim 1, wherein: the application of the ester group-containing aromatic propionamide compound and the metabolite thereof in inhibiting the cytotoxicity of rapamycin on cells.
The application of EG-017 and metabolites thereof in preparing a medicine for treating lung lesions caused by new coronavirus.
Potential application of EG-017 and metabolites thereof in preparing drugs for treating pulmonary fibrosis lung diseases.
The use of EG-017 and its metabolites in the manufacture of a medicament for the treatment of disease caused by doxorubicin-damaged cardiomyocytes.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an SARMs compound containing ester-based aromatic propionamide and application of a metabolite thereof in preparation of a new coronavirus resistant medicine.
Background
At the initial stage, COVID-19 mainly shows fever, dry cough, hypodynamia and the like, and a few patients are accompanied with upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like, so that severe cases are developed; in the early stage, lung diseases are mainly caused and further spread to heart diseases and other organs of the whole body; the clinical symptom is dyspnea, and severe patients rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis and coagulation dysfunction and multiple organ failure. The main transmission route of the novel coronavirus is respiratory droplet transmission and contact transmission, and epidemiological investigation shows that the close contact with diagnosed cases can be tracked in many cases. Currently, no effective drug is available for control.
Disclosure of Invention
The invention aims to provide an application of SARMs (selective antigen receptors modulators) compound containing ester-based aromatic propionamides and a metabolite thereof in preparing anti-new coronavirus medicines.
In order to achieve the purpose, the invention adopts the technical scheme that:
SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparing anti-new coronavirus medicines.
Preferably, the SARMs compound containing ester-based aromatic propionamide is EG-017 and has the chemical formula C25H17F3N4O4。
Preferably, the metabolite of the ester group-containing aromatic propanamide compound is EG-2.
Preferably, the ester group-containing aromatic propionamide compound and the metabolite thereof are used for inhibiting the cytotoxicity of rapamycin on cells.
Preferably, EG-017 and its metabolites are used in the preparation of a medicament for treating lung lesions caused by new coronavirus.
Preferably, EG-017 and metabolites thereof are potentially applied to the preparation of the medicine for treating pulmonary fibrosis lung diseases.
Preferably, EG-017 and its metabolites are used in the preparation of a medicament for treating diseases caused by adriamycin damage to cardiomyocytes.
The invention has the advantages that: the activity and cytotoxicity of the compound against novel coronavirus (SARS-CoV-2) in vitro were evaluated using immunofluorescence assay (IFA). The EG-017 has obvious inhibitory activity on the replication of a novel coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells, and shows that the EG-017 has application prospect in preparing anti-novel coronavirus medicines.
Drawings
FIG. 1: histogram of cytotoxicity of different concentrations of EG-017.
FIG. 2: different concentrations of EG-017 induced cell damage histograms.
FIG. 3: effects of different concentrations of EG-017 on Vim protein expression in A549 cells.
FIG. 4: effect of different concentrations of EG-017 on TGF-. beta.1 induced proliferation of hepatic stellate cells.
Detailed Description
EG017 as referred to herein is a novel non-steroidal SARM of the formula C25H17F3N4O4, chinese chemical name (S) -1- ((4-cyano-3- (trifluoromethyl) phenyl) amino) -3- (4-cyanophenoxy) -2-methyl-1-oxoprop-2-ylnicotinate. The preparation method and the structural formula of EG017 are described in patent document CN 201410033958.0.
Mechanism of action of EG-017
Preliminary studies indicate that the new coronavirus causes damage to human body through ACE2 target; ACE2 is present in the epithelium of the nose, mouth and lungs as well as in most of the major tissue organs of the human body, and is the entry of new coronavirus infections into the body; the new coronavirus initially causes lung infection, causes serious damage to lung epithelium and endothelial tissues, and causes heart and other important tube lesions through blood diffusion; ACE2 contains androgen receptor, SARMs has effect target as androgen receptor, and exogenous androgen receptor regulator SARMs is supplemented in large amount, so that normal biological function of lung organ containing ACE2 can be rapidly activated, lung immune system can be regulated, and damage to lung caused by immune storm due to new coronavirus can be avoided; meanwhile, the exogenous sarms can be combined with the ACE2 competitive with the virus and relevant cells (so as to reduce the invasion probability of the virus to the cells and the invasion of the virus to the organism), thus indirectly enhancing the tolerance of the organism to the virus and promoting the organism to quickly recover health in the fight between the organism and the virus.
1) The activity of macrophage (containing androgen receptor) of lung is regulated by a large amount of exogenous non-steroidal androgen receptor regulator, and the virus removal capacity of lung tissues is enhanced; 2) the exogenous non-steroidal androgen regulator is combined with in-vivo ACE2 (containing androgen receptor), so that the activity and the spatial structure of ACE2 are changed, the binding point of virus and ACE2 is blocked, and the harm of the virus to a human body through an ACE2 channel is reduced; 3) the exogenous androgen receptor regulator is used to regulate the activity of cell nucleus, raise the antiviral self-repairing capacity of body and strengthen the self-repairing capacity of body.
In particular, studies have shown that the human body has a specific protein that enables the virus to infect human cells. This protein is known as angiotensin converting enzyme 2, or ACE2 is the "receptor" that provides an entry point for coronaviruses to enter and infect various human cells. ACE2 is present in many cell types and tissues, including lung, heart, blood vessels, kidney, liver, and gastrointestinal tract. It is present in epithelial cells that are in contact with certain tissue lines and form a protective barrier, and oxygen and carbon dioxide exchange between the lung and blood vessels occurs on the epithelial lining of the lung. ACE2 is present in the epithelium of the nose, mouth and lungs. In the lung, ACE2 is very abundant on type 2 pneumocytes, an important cell type, present in the chambers of the lung lungs where oxygen is absorbed and waste carbon dioxide is released.
ACE enzyme converts angiotensin I to angiotensin II. The primary role of ACE2 is to break down angiotensin II into molecules to counteract the deleterious effects of angiotensin II; that is, ACE2 helps regulate many activities of a protein called angiotensin ii (ang ii), which increases blood pressure and inflammation, increases damage to the vascular lining and various types of tissue damage. ACE2 converts ANG II into other molecules to counteract the effects of ANG II.
Also, studies have shown that most closely related to COVID-19, ANG II increases inflammation and alviginolide cell death, which is critical for the introduction of oxygen into the body, and taken together, these detrimental effects of ANG II are reduced by ACE 2.
In vitro cell experiment results show that the highest Dose Efficacy (Efficacy Max Dose) of EG02 and EG017 on HEK293 which is transferred into an androgen receptor by external source is more than 70%; EG02 and EG017 showed good tissue selectivity on human prostate cancer cells LNCaP endogenously expressing androgen receptor, with the highest Dose Efficacy (Efficacy Max Dose) EG02 being 66.7%; and 47.2% for EG 017. The results show that both EG017 and EG02 have tissue selectivity, reflect good selective androgen receptor regulation effect, and particularly have more remarkable EG017 effect.
Human metabolism studies have shown that EG-017 is metabolized to EG-2 in vivo, with concentrations reaching maximum in vivo for 1.5-3hrs and 6-7hrs, respectively.
In order to verify the effect of EG-017 on treating the pathological changes of organs such as the lung caused by the new coronavirus, the following specific examples are combined to demonstrate the beneficial effects of EG-017.
Test and control compounds
(1) Test compounds
Name: EG-017
The source is as follows: pharmaceutical source medicinal chemistry (Shanghai) Co., Ltd
Batch number: r191095
The preparation method comprises the following steps: 120mM stock solution prepared in 100% DMSO
(2) Test Compound 2
Name: rapamycin
The source is as follows: medchemiexpress
Batch number: 61925
The preparation method comprises the following steps: 40mM stock solution prepared in 100% DMSO
(3) Control Compound 1
Name: remdesivir, Reidesvir
The source is as follows: medchemiexpress
Batch number: CS-0028115
The preparation method comprises the following steps: made up in 10mM stock solution in 100% DMSO
(4) Control Compound 2
Name: lopinavir, Lopinavir
The source is as follows: selleckchem
Batch number: s138003
The preparation method comprises the following steps: made up in 10mM stock solution in 100% DMSO
(5) Control Compound 3
Name: chloroquinone diphosphate salt, Chloroquinone, Chloroquine diphosphate, Chloroquine phosphate
The source is as follows: Sigma-Aldrich
Batch number: 109M4003V
The preparation method comprises the following steps: 30mM stock solution was prepared in 100% DMSO
Viral strains
The novel coronavirus strain β CoV/KOR/KCDC03/2020 was provided by Korea Centers for Disease Control and preservation (KCDC), having the sequence number NCCP 43326.
Cells
African green monkey kidney (Vero) cells were from the American Type Culture Collection (ATCC), cat # CCL-81. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, WelGene) supplemented with 10% fetal bovine serum (Gibco) and 1% double antibody (Gibco). DMEM medium supplemented with 2% fetal bovine serum (Gibco) and 1% double antibody (Gibco) was used as the experimental medium.
Main instrument and reagent
(1) The main apparatus is as follows: the main instruments used in this project were a high content imaging analyzer (PerkinElmer, model Operetta), an automatic dispenser (ThermoFisher, model Multidrop Combi), a plate washer (BioTek, model ELx406) and a liquid workstation (CyBio, model CyBi-HummingWell).
(2) The main reagents are as follows: the main reagents used in the project comprise novel coronavirus N protein anti-Human SARS-CoV-2N protein antibody (Beijing Yiqian Shenzhou science and technology Co., Ltd., product No. 40143-T62), ｃA sheep anti-rabbit IgG secondary antibody (Molecular Probes, product No. MOP-A-11034) marked by AlexｃA Fluor 488, and ｃA cell nucleus staining solution Hoechst 33342(Molecular Probes, product No. MOP-H-3570).
Test method
IFA experiments are used in the research to evaluate the in vitro antiviral activity of a tested compound on the new coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells. In addition to testing the antiviral activity of the test compounds EG-017 and rapamycin when treated individually, the combined antiviral activity of EG-017 in the presence of 100. mu.M rapamycin was also tested. Rituxivir, lopinavir and chloroquine phosphate were used as positive control compounds. Test compounds and control compounds were tested at 10 concentration points. The concentrations tested are shown in table 1.
TABLE 1 test concentrations of test and control Compounds
The average antiviral inhibition and average cell viability rates of the compounds at the above different concentrations are shown in the following table:
cell plating
Vero cells were trypsinized and diluted to 480,000 cells per ml with the experimental medium. The diluted cells were added to 384-well cell assay plates using an automated dispenser, 25. mu.l per well, 12,000 cells. Cells in 5% CO2And cultured overnight in an incubator at 37 ℃.
Compound treatment and viral infection
Compounds were diluted with DMSO and then the diluted compounds were added to the wells of test cells using a liquid station. Then 25. mu.l of the experimental culture medium was added to each well to dilute the SARS-CoV-2 virus, MOI 0.0125. Cell controls (cells, no compound treatment or viral infection) and no compound treatment controls (cells infected with virus, no compound treatment, 0.5% DMSO added) were set. The final volume of cell culture broth per well was 50. mu.l. Cells in 5% CO2Then, the culture was continued in an incubator at 37 ℃ for 24 hours.
Immunofluorescence staining
(1) 24 hours after viral infection, 17. mu.l of 16% paraformaldehyde was added to each well. Then standing for 30 minutes at room temperature;
(2) the supernatant was aspirated off and the plate washed twice with DPBS;
(3) adding 25 mul of 0.25 percent TritonX-100 into each hole, and standing for 20 minutes at room temperature;
(4) 0.25% TritonX-100 was aspirated off, and the plate was washed twice with DPBS;
(5) mu.l of diluted primary antibody (1:3000 times diluted) was added to each well and incubated at 37 ℃ for 1 hour;
(6) absorbing the primary antibody, and washing the plate twice by using DPBS;
(7) mu.l of diluted secondary antibody Alexa Fluor 488-labeled goat anti-rabbit IgG (1: 2000-fold dilution) and 2.5. mu.g/ml (1: 4000-fold dilution) of Hoechst 33342 were added to each well and incubated at 37 ℃ for 1 hour;
(8) absorbing the secondary antibody and Hoechst, and washing the plate twice by using DPBS;
(9) using a high content imaging analyzer Operetta reading plate, instrument settings: 488/405emission,20 times objective, 5 fields per well.
Data analysis
The total number of cells (Hoechst stained cells) and the number of cells infected with the novel coronavirus (Alexa Fluor 488-labeled cells) in the pictures obtained by plate reading with the high content imaging analyzer were quantitatively analyzed using Columbus software. The proportion of infected cells and total cell number data were used for compound antiviral activity and cytotoxicity assays.
The calculation formula is as follows:
inhibition (%) of 100 ═ 100 — (test well infected cell ratio-cell control well average infected cell ratio)/(no-compound-treated control well average infected cell ratio-cell control well average infected cell ratio) × 100
Cell viability (%). Total cell number in test wells/average Total cell number in control wells without Compound treatment x 100
Inhibition and cell viability of the compounds were analyzed by nonlinear fit using XLFit 4 software and IC's of the compounds were calculated50And CC50The fitting method is "Sigmoidal dose-response". IC (integrated circuit)50And CC50The calculation formula of (2) is as follows: y ═ Bottom + (Top Bottom)/(1+ (IC)50/X) Hillslope). Wherein, IC50Indicating a 50% inhibitory concentration.
Results and conclusions
The results of the single drug test of test and control compounds for inhibiting the replication activity of the novel coronavirus in vitro are shown in table 2. The experiment was performed 1 time.
The results showed that the control compounds chloroquine phosphate, lopinavir and ridciclovir all showed anti-novel coronavirus activity, IC50The values were 4.3. mu.M, 14.1. mu.M and 5.82. mu.M, respectively, in agreement with literature data (Sangeun J., et al. 2020). None of the three control compounds showed significant cytotoxicity, CC, at the concentrations tested50Values greater than the highest tested concentration are shown in table 2.
The tested compounds EG-017 and rapamycin have obvious inhibitory activity on the replication of the novel coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells, and the IC50 values are respectively 17.09 mu M and 9.34 mu M. The test compound EG-017 showed no significant cytotoxicity on Vero cells with CC50 values greater than the highest tested concentration, >300 μ M. rapamycin showed significant cytotoxicity with a CC50 value of 29.5 μ M.
In the combined antiviral activity test of 100 mu M Rapamycin and EG-017, the inhibitory activity of the combined drug on viruses cannot be distinguished because Rapamycin has obvious cytotoxicity at the concentration of 100 mu M (Table 7); however, the combined administration result shows that EG-017 can inhibit the toxicity of Rapamycin to cells at the concentration of 300 mu M-11.11 mu M; the efficacy of the combination of the two against viruses requires further evaluation by lowering the test concentration of Rapamycin.
TABLE 2 anti-New coronavirus Activity of test and control Compounds
Compound (I)
Unit of
IC50
CC50
EG-017
μM
17.09
>300
rapamycin
μM
9.34
29.5
Chloroquine
μM
4.30
>150
Lopinavir
μM
14.10
>50
Remdesivir
μM
5.82
>50
The MTT method is adopted to further verify that EG-017 and metabolites thereof repair the cytotoxicity of adriamycin on myocardial cells
Subject: H9C2 rat cardiomyocytes
The experimental method comprises the following steps: MTT method
The experimental principle is as follows:
the MTT method is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of formazan at the wavelength of 550nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for screening antitumor drugs and cytotoxicity tests.
Experimental procedure
1. Cytotoxicity of EG-017 at various concentrations
(1) Logarithmic phase H9C2 cells were collected and cell suspension concentration was adjusted to 5X 104cells/mL, 100ul per well, plated to density test cells to 5000 cells/well, and marginal wells filled with sterile PBS.
(2)5％CO2Incubate at 37 ℃ until the cell monolayer is well-bottom (96-well flat bottom plate), add the concentration gradient drug, plate the day before afternoon, and add the drug the next day in the morning. EG-017 sets up six gradients of 0, 1, 10, 50, 100 and 500uM, and 100ul of each hole sets up 4 ~ 6 compound holes. And setting a zero setting hole (culture medium, MTT and dimethyl sulfoxide) and a control hole (cells, a drug dissolving medium with the same concentration, a culture solution, MTT and dimethyl sulfoxide).
(3)5％CO2Incubated at 37 ℃ for 24 hours and observed under an inverted microscope.
(4) 20ul MTT solution (5mg/ml, i.e. 0.5% MTT), 5% CO was added per well2The incubator was incubated at 37 ℃ for 4 hours.
(5) The culture was terminated and the culture medium in the wells was carefully aspirated.
(6) 150ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at OD 490nm in an ELISA detector.
The experimental results are shown in fig. 1, wherein a: the MTT method is used for detecting the survival condition of the cells with the EG-017 action concentration of 1, 10, 50, 100 and 500M. P <0.05 compared to control group; p <0.01 compared to control group; p <0.001 compared to control group; p <0.0001 compared to control. The result shows that the A.EG-017 can obviously promote the proliferation of the myocardial cells when the concentration is 100 and 500M.
2. Different concentrations of EG-017 induce cell damage
(1) Logarithmic phase H9C2 cells were collected and cell suspension concentration was adjusted to 5X 104cells/mL, 100ul per well, plated to density test cells to 5000 cells/well, and marginal wells filled with sterile PBS.
(2)5％CO2Incubate at 37 ℃ until the cell monolayer is well-bottom (96-well flat bottom plate), add the concentration gradient drug, plate the day before afternoon, and add the drug the next day in the morning. EG-017 is provided with 0, 1, 10,50. And (4) setting 4-6 multiple holes in 100 uM and 500uM gradients, wherein each hole is 100 ul. After 2 hours of pretreatment, the culture medium was changed and DOX (1uM) was added to continue the culture, while the zero-setting wells (medium, MTT, DMSO) and the control wells (cells, drug-dissolving medium of the same concentration, culture medium, MTT, DMSO) were set.
(3)5％CO2Incubated at 37 ℃ for 48 hours and observed under an inverted microscope.
(4) 20ul MTT solution (5mg/ml, i.e. 0.5% MTT), 5% CO was added per well2The incubator was incubated at 37 ℃ for 4 hours.
(5) The culture was terminated and the culture medium in the wells was carefully aspirated.
(6) 150ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at OD 490nm in an ELISA detector.
The results are shown in FIG. 2, where A: the MTT method is used for detecting the survival condition of the cells with the EG-017 action concentration of 1, 10, 50, 100 and 500M under the condition that the adriamycin is 1M. P <0.05 compared to control group C; p <0.01 compared to control group C; p <0.001 compared to control group C; p <0.0001 compared to control C. # P <0.05 compared to control group D; # P <0.01 compared to control group D; # P <0.001 compared to control D; # P <0.0001 compared to control group D. C in the figure is control; d, control + DOX (1M).
The results show that the A.EG-017 can obviously improve the survival rate of the myocardial cells under DOX treatment when the concentration is 100 and 500M.
In order to further verify the effectiveness of EG-017 in preparing anti-new coronavirus medicines, experiments are carried out to verify the influence of EG-017 on the process of TGF-beta 1 inducing A549 lung cancer cell fibrosis.
Subject: a549 lung cancer cell
The experimental method comprises the following steps: WB method
The experimental principle is as follows:
western blot, a Western blot technique, is a protein detection technique in which the total proteins of cells or tissues after electrophoretic separation are transferred from a gel onto a solid support NC membrane or PVDF membrane, and then a specific antigen is detected using a specific antibody, and the position of the staining and the depth of staining of the bands of the specific protein are obtained by analysis, thereby obtaining the expression of the specific protein in the cells or tissues.
Transforming growth factor-beta (TGF-beta 1) is the most critical cytokine for liver fibrosis. TGF-beta 1 is used for inducing A549 lung cancer cell fibrosis in vitro, vimentin (Vim) in the fibrotic lung cancer cell is obviously expressed, and the expression of cadherin E (E-Cad) is obviously reduced. And detecting protein expression levels of Vim and E-Cad by using a WB technology, and verifying the fibrosis level of A549 cells.
Experimental procedure
Western blot is used for detecting the influence of EG-017 with different concentrations on the expression of Vim and E-Cad proteins in cells,
1. protein sample preparation
(1) A549 cells were cultured to log phase and plated in six well plates after digestion. A blank control group and a dosing group were set. After 24h, when the cell monolayer is uniformly attached to about 80% of the bottom of the plate, adding 2ml of culture medium containing TGF-beta 1 and having the concentration of 20ng/ml, wherein the serum concentration of the culture medium is 2%, adding the culture medium containing EG-017 with different concentrations after 48h, adding the culture medium and then placing the culture medium in an incubator for 48 h.
(2) And (3) extracting total cell protein: after 48h, the six-well plate is taken out, the culture medium is sucked off, the plate is washed for 2 times by PBS, pancreatin is added to digest the cells, the cells are collected and centrifuged, the supernatant is discarded, the plate is washed by PBS for two times, then the prepared RIPA lysate is added, and the cell lysate is shaken back and forth or blown by a gun for several times to ensure that the lysate is fully contacted with the cells and is cracked on ice for 30min and shaken every 5 min. After the lysis is finished, the EP tube is placed in a low-temperature centrifuge for 5min at 12000rpm, and colorless transparent supernatant is the total protein of the cells. The supernatant after centrifugation was concentrated by BCA protein assay, and the diluted protein sample was mixed with 5 XSDS loading buffer as 4: adding into the mixture at a ratio of 1, boiling in boiling water for 5min to completely denature protein, and storing at-70 deg.C.
2. SDS-PAGE electrophoretic separation of proteins
Preparing separation gel and concentrated gel according to the instruction of a gel preparation kit and the molecular weight of target protein, adding each extracted protein into a sample adding hole by using a liquid transfer gun, adding 10 mu L of each sample, adding 3 mu L of protein marker, assembling an electrophoresis apparatus, adjusting the electrophoresis apparatus to 80V, adjusting the electrophoresis apparatus to 120V to continue gel running when a sample strip runs to the vicinity of the boundary of the compressed gel and the separation gel, and stopping gel running until the bottom reaches 1 cm. After electrophoresis is finished, cutting out a needed strip according to the marker indication.
3. Rotary film
(1) Sufficient transfer buffer (100mL of 10 × transfer solution +200mL of methanol +700mL of ultrapure water) was prepared for use.
(2) After electrophoresis, the glass plate was taken out and gently pried open with tweezers. And (3) soaking the target strip by using the membrane transferring solution, cutting the target strip according to the position of a protein marker, making a mark, and putting the mark into the membrane transferring solution. The gel was transferred to transfer buffer. And cutting out a PVDF membrane with the size consistent with that of the cut gel, soaking in methanol for 5min for activation, and then putting in a transfer buffer solution for balancing for 2min for later use.
(3) Making a sandwich structure: in the electrotransfer liquid, opening the clamp, horizontally placing the clamp, padding a layer of sponge on the black clamp, and rolling the black clamp by using a glass rod to drive bubbles; then putting thick filter paper, fixing, removing bubbles by using a glass rod, then putting gel, covering the gel with a PVDF film, upwards sequentially arranging three layers of filter paper and sponge, and finally closing and clamping the white clip.
(4) The clamp is placed in a transfer tank, glue is applied to the negative electrode, and the membrane is applied to the positive electrode.
(5) Film transferring conditions: constant current of 200mA for 100min, heat generation during electric conversion, and film transfer in ice bath.
4. Immune response
(1) Sealing milk: after the membrane transfer was completed, the PVDF membrane was taken out and placed in an incubation box containing 5% skim milk prepared with 5ml of LTBST on a shaker and sealed at room temperature for 4 hours.
(2) Primary antibody incubation: with 1 × TBST at 1: GAPDH, Vim and E-Cad antibodies were diluted at 1000, the blocking solution was decanted after blocking was complete, the prepared primary antibody dilution was added to allow the primary antibody dilution to sink through the PVDF membrane, overnight at 4 deg.C, and the membrane was removed the next day and rinsed 3 times with 1 × TBST for 15min each.
(3) And (3) secondary antibody incubation: with 1 × TBST at 1: 10000 dilution of secondary antibody, recovery of primary antibody dilution after night, addition of secondary antibody dilution, placing on a shaking table, incubation for 2h at room temperature, and rinsing with TBST for 3 times, 15min each time.
5. ECL luminescence detection
(1) Mixing the two reagents of the solution A and the solution B in the luminescence kit in equal volume in a centrifuge tube.
(2) The PVDF membrane is placed on a sample tray, and then the mixed luminescent liquid is uniformly dropped on the PVDF membrane.
(3) Placed in the instrument and the instrument is manipulated to expose it.
The results of the effect of EG-017 at different concentrations on Vim protein expression in A549 cells are shown in FIG. 3, and the WB method is used for detecting the Vim protein expression in cells after EG-017 action concentrations are 0.4, 2 and 10M for treating the cells for 48h, and the results show that EG-017 inhibits Vim protein expression after treating the cells for 48 h. The results of the effect of different concentrations of EG-017 on TGF-beta 1-induced proliferation of hepatic stellate cells are shown in FIG. 4, and the WB method is used for detecting the expression condition of the E-cad protein in the cells after the cells are treated for 48h by using EG-017 action concentrations of 50, 100 and 200. The results show that EG-017 promotes the expression of the E-cad protein.
Finally, the experiments show that EG-017 can inhibit A549 cell fibrosis induced by TGF-beta 1 and shows certain dose dependence.
The invention has various embodiments, and all technical solutions formed by adopting equivalent transformation or equivalent transformation are within the protection scope of the invention.