CN115919847B - Application of indole-3-methyl acetate in relieving radiation-induced pulmonary fibrosis - Google Patents
Application of indole-3-methyl acetate in relieving radiation-induced pulmonary fibrosis Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an application of methyl indole-3-acetate in relieving radiation-induced pulmonary fibrosis, wherein the radiation is completed by adopting ionizing radiation IR; the radiation source adopted by the IR is a Co-60 source; the dose of radiation is no greater than 10Gy; the dose of the radiation is 6Gy; the indole-3-acetic acid methyl ester has an effective concentration of 0.5-10mM in alleviating radiation-induced pulmonary fibrosis. The invention adopts I3MA to inhibit EMT to relieve the occurrence of radiation-induced pulmonary fibrosis.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an application of indole-3-methyl acetate in relieving radiation-induced pulmonary fibrosis.
Background
Radiation-induced pulmonary fibrosis is a chronic, progressive and fatal interstitial lung injury that occurs after radiation therapy, characterized by irreversible destruction of lung tissue, deterioration of lung function, impaired quality of life, respiratory failure, and even death. At present, the etiology of radiation-induced pulmonary fibrosis is not completely understood. It is known from the pathogenesis of radiation-induced pulmonary fibrosis that it is associated with activation of inflammatory transcription factors, differentiation of myofibroblasts, progression of collagen extracellular matrix and Epithelial Mesenchymal Transition (EMT).
EMT is a progressive process that confers the ability to metastasize and invade cells, including stem cell characteristics, reducing apoptosis and aging, and promoting immunosuppression; without intervention, lung injury is increased, followed by pulmonary fibrosis, and an irreversible loss of lung function. EMT results in reduced adhesion between cells and other epithelial cells, as well as loss of molecular characteristics of epithelial adhesion markers (e.g., E-cadherin) and acquisition of mesenchymal markers (e.g., N-cadherin, vildagliptin, and fibronectin).
Indole-3-acetic acid methyl ester (abbreviated as I3 MA) is a tryptophan catabolite converted by intestinal microorganisms. Tryptophan catabolites are taken up by intestinal epithelium and then enter the blood and are then excreted by urine. I3MA can be activated and upregulated by including herbal interventions to ameliorate ischemic stroke. In addition, the probiotic bacteria performed a dry prognosis of increased I3MA in high fat diet mice, suggesting that I3MA has the potential to prevent obesity. Furthermore, quercetin has been reported to have anti-inflammatory effects on colorectal cancer by upregulating I3MA levels. These properties make I3MA potentially useful for improving radiation-induced pulmonary fibrosis and reversing EMT. However, the role of I3MA in radiation-induced pulmonary fibrosis is currently unknown.
Disclosure of Invention
The invention aims to provide an application of indole-3-methyl acetate in relieving radiation-induced pulmonary fibrosis, and the specific technical scheme is as follows:
use of indole-3-acetic acid methyl ester for alleviating radiation-induced pulmonary fibrosis.
Optionally, the irradiation is accomplished with ionizing radiation IR; the radiation source used in the IR is a Co-60 source.
Optionally, the radiation dose is no greater than 10Gy.
Alternatively, the radiation dose is 6Gy.
Optionally, the indole-3-acetic acid methyl ester is present at an effective concentration of 0.5 to 10mM in alleviating radiation-induced pulmonary fibrosis.
The application of the technical scheme of the invention has at least the following beneficial effects:
The indole-3-methyl acetate is applied to relieving radiation-induced pulmonary fibrosis, the survival rate of cells is basically unchanged after the cells are pretreated by adopting I3MA, no obvious cytotoxicity is caused to the cells, and the cell morphology is kept elliptical; after the I3MA is adopted for pretreatment of the cells, the expression of the marker protein E-cadherin in the EMT process is increased, the expression of the N-cadherin is reduced, the invasion capacity and migration capacity of the cells are effectively reduced, the apoptosis of the cells after the radiation can be promoted, and then the invasive characteristics of the EMT induced by the radiation in the corresponding cells can be recovered; meanwhile, after the cells are pretreated by adopting I3MA, the endogenous factors, secretion factors and transcription factors in the cells are reduced, and the EMT of the corresponding cells can be inhibited. From these results, it can be seen that the use of I3MA for cells can suppress EMT to alleviate the occurrence of radiation-induced pulmonary fibrosis.
In addition to the objects, features and advantages described above, the present invention has other objects, features and advantages. The present invention will be described in further detail with reference to the drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a bar graph of HBE cell viability using the CCK8 kit after treatment of HBE cells with control DMSO and I3MA at the indicated concentrations for 48 hours in test A in example 1 of the present invention;
FIG. 2 is a graph showing the morphology of the cells of example 1 of the present invention after treatment of each group of HBE cells with different concentrations of I3MA and subsequent irradiation of each group of HBE cells with IR6Gy, before irradiation of each group with IR6 Gy;
FIG. 3 is a graph showing the effect of assay C in example 1 of the present invention on EMT-related biomarker expression in radiation induction after pretreatment of A549 cells with different concentrations of I3MA using Westernblotting;
FIG. 4 is a graph showing the effect of assay C in example 1 of the present invention on EMT-related biomarker expression in radiation induction after pretreatment of HCT cells with different concentrations of I3MA using Westernblotting;
FIG. 5 is a graph showing the effect of assay C in example 1 of the present invention on EMT-related biomarker expression in radiation induction after pretreatment of MDA-MB-231 cells with different concentrations of I3MA using Westernblotting;
FIG. 6 is a graph showing the results of a 10-day cross migration test of A549 cells using I3MA at the indicated concentrations for test D in example 1 of the present invention;
FIG. 7 is a graph showing the results of a 10-day crossover invasion assay of A549 cells using I3MA at the indicated concentrations for test E in example 1 of the present invention;
FIG. 8 is an apoptosis of A549 cells treated with or without I3MA (1 mM) prior to IR6Gy irradiation in example 1 of the present invention; wherein Q4-1 represents necrotic cells, Q4-2 represents late apoptotic cells, Q4-3 represents normal cells, and Q4-4 represents early apoptotic cells; perCP-A and FITC-A are two fluorescent channels, and the fluorescence intensity of PI and annexin V is detected respectively;
FIG. 9 is a graph showing the mRNA levels of pro-invasive cell endogenous factors detected by qRT-PCR in A549 cells treated with different concentrations of I3MA in test G of example 1 of the present invention;
FIG. 10 is a graph showing the mRNA levels of pro-invasive cytokine secretion in A549 cells treated with different concentrations of I3MA by qRT-PCR for test G in example 1 of the present invention;
FIG. 11 is a graph showing the mRNA levels of pro-invasive cell transcription factors detected by qRT-PCR in A549 cells treated with different concentrations of I3MA in example 1 of the present invention;
FIG. 12 is a graph showing the mRNA levels of pro-invasive cell endogenous factors detected by qRT-PCR in HCT cells treated with different concentrations of I3MA in test G of example 1 of the present invention;
FIG. 13 is a graph showing the mRNA levels of pro-invasive cell secretion factors detected by qRT-PCR in HCT cells treated with different concentrations of I3MA in example 1 of the present invention;
FIG. 14 is a graph showing the mRNA levels of pro-invasive cell transcription factors detected by qRT-PCR in HCT cells treated with different concentrations of I3MA in example 1 of the present invention;
FIG. 15 is a graph showing the mRNA levels of pro-invasive cell endogenous factors detected by qRT-PCR in different concentrations of I3MA treated MDA-MB-231 cells according to test G of example 1 of the present invention;
FIG. 16 is a graph showing the mRNA levels of pro-invasive cell secretion factors detected by qRT-PCR in MDA-MB-231 cells treated with different concentrations of I3MA in test G of example 1 of the present invention;
FIG. 17 is a graph showing the mRNA levels of pro-invasive cell transcription factors detected by qRT-PCR in different concentrations of I3MA treated MDA-MB-231 cells according to test G of example 1 of the present invention;
FIG. 18 is a protein expression pattern of immunoblots of test H of example 1 of the present invention on A549 cells after 10 days of treatment with or without I3MA (1 mM) prior to IR6Gy radiation;
FIG. 19 is a protein expression pattern of immunoblots of test H of example 1 of the present invention after 10 days of treatment with or without I3MA (1 mM) prior to IR6Gy irradiation of HCT cells;
FIG. 20 is a protein expression profile of test H in example 1 of the present invention following immunoblotting of MDA-MB-231 cells with or without I3MA (1 mM) for 10 days prior to IR6Gy irradiation.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
Example 1:
the use of methyl indole-3-acetate for alleviating radiation-induced pulmonary fibrosis is specifically demonstrated by the following test A-G development.
Test materials:
1) Chemicals, antibodies and reagents
The antibodies used in example 1 were anti-ECADHERIN (3195 s; westernblotting diluted 1:1000; cell SignalingTechnology), anti-N-Cadherin (13116 s; westernblotting diluted 1:1000; CELLSIGNALING TECHNOLOGY) and anti-TGF-beta 1 (sc-130348; esternblotting diluted 1:1000; santaCruz Biotechnology).
2) Cell lines
Human HBE normal epithelial cell line (HBE cell for short), purchased from AmericanTypeCultureCollection (ATCC, manassas, va., USA), and cultured in Eagle medium supplemented with 10% fetal bovine serum, 100. Mu.g/mL streptomycin, and 100U/mL penicillin;
A549 human lung cancer cell line (abbreviated as a549 cells) obtained from ATCC and cultured in RPMI1640 medium (corning) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and penicillin-streptomycin (Gibco);
HCT116 (abbreviated as HCT cells) from ATCC in the same manner as a549 human lung cancer cell line;
MDA-MB-231 human breast cancer (triple negative breast cancer) cell line obtained from ATCC in the same manner as A549 human lung cancer cell line;
According to the manufacturer's instructions for each cell line, all cell lines were maintained at 37℃and 5% carbon dioxide, medium was changed every 24h, subculture was performed every 48h, and the whole cell culture was in a sterile state.
In a first aspect, to investigate whether I3MA was toxic to cells and whether cells were induced to develop EMT invasive characteristics in IR6Gy radiation after pretreatment of cells with I3MA, test a-test F was developed in example 1.
Test a (CCK-8 assay to detect cell viability):
CCK-8 (enhanced cell viability detection kit) is a rapid, high-sensitivity and non-radioactive colorimetric detection kit based on WST-8, and is widely applied to detection of cell proliferation or cytotoxicity. In the presence of an electron carrier, WST-8 is reduced by intramitochondrial dehydrogenase to generate water-soluble orange formazan, the amount of formazan generated is proportional to the number of living cells, and the amount of living cells can be calculated indirectly by detecting the absorbance at 450 nm.
Test A step: preparing a cell suspension from HBE cells with good growth state, and inoculating 100 mu L of the cell suspension (containing about 5000 cells) in each well into a 96-well plate;
Culturing the cell suspension inoculated in each hole by adopting a complete culture medium, wherein the complete culture medium is an Eagle culture medium of 10% fetal bovine serum, 100 mug/mL streptomycin and 100U/mL penicillin; after adding 100 mu L of complete culture medium into each well, placing the mixture in a constant temperature incubator with 5% CO 2 and 37 ℃ for culture;
Sequentially setting a test group A-0, a test group A-1, a test group A-2, a test group A-3, a test group A-4, a test group A-5, a blank control group (no cell suspension is added, but the same volume of complete culture medium is added) and a DMSO control group (the cell suspension is treated with the same volume of DMSO as a DMSO control group because I3MA is diluted with DMSO) for each cell suspension inoculated in each well; preparing I3MA into mother solutions with different concentrations in the test groups A-0 to A-5 by adopting DMSO, and diluting the mother solution to the required concentration by using the complete culture medium, wherein the concentrations of the I3MA corresponding to the test groups A-0 to A-5 are 0mM, 0.5mM, 1mM, 2.5mM, 5mM and 10mM in sequence;
Treating the cell suspensions inoculated in the test groups A-0 to A-5, the blank control group and the DMSO control group for 48 hours, respectively adding 10 mu LCCK-8 solution, and incubating for 1 hour under the same incubation conditions;
the absorbance at 450nm was measured with a microplate reader, and the HBE cell viability was calculated.
Conclusion of test a:
Referring to FIG. 1, CCk8 results show that I3MA is not significantly cytotoxic to HBE cells, and that I3MA is used at concentrations of 0.5-10mM, all without significant cytotoxicity to HBE cells.
Wherein, a blank control group was set for removing the effect of the complete medium on the CCk-8 result (CCk-8 result is that the 96-well plate was measured for absorbance at 450nm with a microplate reader, but the complete medium also had absorbance at 450nm, so CCk-8 calculated cell viability= (test group absorbance-blank control group absorbance)/(A-0 group absorbance-blank control group absorbance)).
Test B:
And (3) a test B step:
First, HBE cells were equally plated in six well plates, and divided into test groups B-1, B-2, B-3 and B-4;
Next, each group of HBE cells was treated with I3MA at concentrations of 0mM, 0.5mM, 1mM and 5mM, respectively, prior to irradiation of each group with IR6 Gy;
Finally, after irradiation of each group of HBE cells with IR6Gy, each group of HBE cells was photographed at 24h and 72h, respectively.
Conclusion of test B:
Referring to FIG. 2, if HBE cells were not treated with I3MA prior to IR6Gy irradiation, the phenotype of the HBE cells changed from oval to spindle; if the HBE cells are treated with I3MA prior to IR6Gy irradiation, the I3MA can protect morphological changes and maintain the ellipticity of the HBE cells.
Test C (Westernblot test (western blot test)):
Test C:
first, the subcultured A549 cells, HCT cells and MDA-MB-231 cells were plated in equal amounts in different six well plates, respectively, and the A549 cells, HCT cells and MDA-MB-231 cells were treated with I3MA at concentrations of 0mM, 0.5mM, 1mM and 5mM, respectively, before the IR6Gy was irradiated to each group of cells.
Extracting A549 cells, HCT cells and MDA-MB-231 cell proteins respectively:
Extracting A549 cells, HCT cells and MDA-MB-231 cells from the culture media respectively, specifically, firstly, pouring out the culture media in each culture dish, and sucking the residual culture media by a pipette; subsequently, adding a proper amount of PBS solution pre-cooled in advance along the walls of each culture dish, and carefully cleaning each cell three times; finally, the PBS solution in each culture dish is sucked and discarded by a pipetting gun;
Tilting the culture dishes, carefully scraping the cells in each culture dish by using a cell scraper according to the sequence of the two sides and the center; then, adding a proper amount of PBS solution into each culture dish, and preparing scraped cells and PBS into a cell suspension;
Transferring the cell suspension in the culture dish into a new 1.5mlEP pipe by adopting a 1ml pipetting gun, putting the EP pipe into a centrifuge, and balancing; the centrifugation conditions were set as follows: the temperature is 4 ℃, the rotating speed is 3000rpm, and the time is 5min;
sucking out supernatant in the EP tube, observing the cell precipitation amount in the EP tube, adding a proper amount of protein lysate according to the cell precipitation amount, and placing on ice for cracking for 30min;
during the cracking period, the EP tube is taken out from ice every 5-10min, placed on a vortex oscillator for vortex oscillation for 10s, and repeated until the cracking is completed;
After the pyrolysis is finished, the EP pipe is put into a centrifuge at the temperature of 4 ℃ for balancing; the centrifugation conditions were set as follows: the temperature is 4 ℃, the rotating speed is 12000rpm, and the time is 15min;
carefully aspirate the supernatant using a pipette, transfer to another new EP tube, carefully avoid aspiration of cell pellet, and mark the EP tube cap;
The protein concentration of the sample is measured and recorded by using the Nanodrop, 1/4 of the sample volume of loading buffer (5 xLoadingBuffer) is added according to the sample volume, and the sample is boiled at 100 ℃ for 10min; if the next experimental operation is not carried out, the sample can be stored at the temperature of minus 20 ℃;
Firstly, cleaning the glass plate by double distilled water to ensure that the two sides of the glass plate have no residual impurities; then, placing the glass plate in an oven for airing; fixing the long and short glass plates in a clamping groove for glue making, horizontally shaking the table top for several times to enable the bottom surfaces of the two glass plates to be level, pulling a clamping groove clamp, and clamping the glass plates to avoid liquid leakage during glue pouring; a rubber pad is arranged on the glue making device, the assembled device is vertically arranged on a frame, the glass plate is pressed down to enable the bottom of the glass plate to be tightly attached to the rubber strip, and the glass plate is fixed by a clamp of the glue making device; after double distilled water is poured into a glass plate for leak detection, 10% SDS-PAGE separating gel is prepared:
Running gel to transfer film, firstly determining total protein loading quantity (40 ug), calculating the loading volume of each protein sample according to a formula according to the concentration of the protein sample measured before, and taking notes;
Taking out the protein sample frozen in a refrigerator at the temperature of minus 20 ℃, standing at room temperature, boiling for 1-2min after the protein sample is melted, centrifuging at a low speed for 1-2min, centrifuging the protein sample on the tube wall, and adding 4ul protein markers into comb holes after the comb is pulled out, and adding the protein sample with a calculated volume in advance;
The electrophoresis tank is covered according to the positive and negative electrodes, the voltage is set to 80V, and electrophoresis is carried out for 30-40min; after the protein Marker is separated, suspending electrophoresis, and resetting the voltage to 120V; continuing electrophoresis for 50-80min; after electrophoresis is finished, the power supply is turned off;
Taking out the glass plates in the electrophoresis tank, temporarily soaking the redundant glass plates in the electrophoresis tank, carefully prying up the glass plates between the two glass plates by using a seesaw, cutting the glue according to the molecular weight of the target protein and the Marker strip position, and discarding the redundant parts;
measuring the size of the cut separating glue by a ruler, and cutting the NC film according to the size of the separating glue; then, placing the glue and NC film into 1x film transferring liquid;
soaking a filter paper board in a film transferring liquid, soaking the film transferring liquid in a film transferring liquid with the film being sandwiched, and paving a sponge cushion, the filter paper board, glue, an NC film, the filter paper board and the sponge cushion in sequence; note that the black surface of the film transfer groove corresponds to the glue, and the white surface corresponds to the NC film;
putting the film transfer groove into the washbasin, and fully spreading ice around the film transfer groove; then, putting the film transfer clamp into a film transfer groove according to the positive and negative electrodes, and adding 1x film transfer liquid pre-cooled by 4 ℃ in advance into the film transfer groove until the liquid level is over the platinum wire in the film transfer groove; the membrane transfer tank cover is covered, a power supply is connected, the membrane transfer voltage is set to be 100V, and the membrane transfer time is 90min (the membrane transfer time is properly adjusted according to the molecular weight of the target protein).
After incubation and development of the primary antibody are finished, carefully recovering the primary antibody, taking care of not mixing, and putting the primary antibody back to a refrigerator at the temperature of minus 20 ℃ for preservation; taking out all the target strips by using tweezers, putting the target strips into 1xTBST, putting the target strips on a shaking table at room temperature, rapidly shaking the target strips so as to clean the target strips, taking the cleaning time for 10min each time, and repeating the cleaning step for 3 times; after washing, the target strip was taken out and placed in an antibody cassette, and the corresponding secondary antibody (GADPH, E-cadherin, N-cadherin) was added thereto, and incubated on a shaker at room temperature for 60min with shaking at low speed. It should be noted that the selection principle of the secondary antibody should be matched correspondingly according to the source of the primary antibody. After the secondary antibody incubation is finished, taking out the NC membrane, putting the NC membrane into 1xTBST, putting the NC membrane on a shaking table at room temperature, shaking slowly, and cleaning for 3 times, wherein each time is 10min. The secondary antibodies were carefully recovered in sequence and returned to the-20℃refrigerator for storage. And (3) preparing a proper amount of ECL developing working solution (A solution: B solution=1:1) in advance, sucking the strips by using filter paper before formal development, putting the strips on a full-automatic imaging instrument plate, and adding a proper amount of developing working solution on the strips for on-machine development.
Conclusion of test C:
Referring to FIGS. 3-5, by the Westernblot test, the A549 cells, the HCT cells and the MDA-MB-231 cells were pretreated with I3MA at different concentrations of 0.5-5mM, and then subjected to IR (6 Gy) radiation, and the expression changes of the marker proteins (E-cadherin and N-cadherin) in the EMT process of the corresponding cells were substantially consistent, i.e., the E-cadherin expression was increased and the N-cadherin expression was decreased; among them, the pretreatment with I3MA at a concentration of 1mM is most effective.
Test D (cell migration test):
And D, testing:
First, subcultured a549 cells were equally plated in six well plates, and a549 cells were treated with I3MA at concentrations of 0mM, 1mM, and 5mM, respectively;
Culturing the treated A549 cells in a constant temperature incubator at 37 ℃ for 24 hours, and pre-cooling the 1.5mLEP tube, the gun head and the cell chamber (which are put in the pore plate in advance) on ice;
Serum-free medium=1:7.5 diluted on ice to use liquid according to ECMGel;
Cutting off a small section (about 3 μm) of the gun head, sucking 40 mu L/hole ECMGel of using liquid on ice, lightly adding the liquid into the upper chamber of the small chamber, slowly moving the gun head during adding, and ensuring that the liquid is flatly paved at the bottom;
placing in a incubator at 37 ℃ for 15min to solidify the glue;
three groups of A549 cells (0 mM,1mM and 5 mM) were digested, centrifuged and the cells were counted, and the cells were diluted with serum-free medium at 2.5X10 4 cells/mL to prepare a cell suspension;
adding 200 mu L of cell suspension into the upper chamber, adding 500 mu L of 10% FBS+ culture medium into the lower chamber, and culturing in a incubator at 37 ℃;
taking out after a plurality of hours, sucking out redundant liquid in the upper chamber, washing twice by PBS, lightly rotating in the upper chamber by a cotton stick, sucking up water and wiping out cells on the inner side of the membrane;
adding crystal violet dye liquor into the upper chamber, dyeing for 5min, recovering the dye liquor, slowly buffering the dye liquor by flowing water, lightly rotating the cotton swab in the upper chamber again, and absorbing water;
placing a glass slide on the upright microscope, inversely placing a cell chamber on the upright microscope, and photographing;
the films were counted up, down, left, right, and middle at 100 times the field of view, and averaged.
Conclusion of test D:
Referring to FIG. 6, pretreatment of A549 cells (A549 cells are cancer cells and have strong cell migration ability) with I3MA at concentrations of 1mM and 5mM effectively reduced the migration ability of A549 cells, and the higher the concentration of I3MA was, the more strongly the migration ability of A549 cells was reduced.
Test E (cell invasion test):
Test E step:
Subcultured a549 cells were plated in 6-well plates and treated with 0mM,1mM and 5mM of I3MA, respectively;
Culturing the treated A549 cells in a constant temperature incubator at 37 ℃ for 23 hours, thawing ECMGel stock solution in a refrigerator at 4 ℃ for 1 hour, and transferring to an ice box before the experiment;
placing 1.5mLEP tube, gun head and cell chamber (which are put into pore plate in advance), and pre-cooling on ice;
Serum-free medium=1:7.5 diluted on ice to use liquid according to ECMGel;
cutting off a small section (about 3 μm) of the gun head, sucking 40 mu L/hole ECMGel of using liquid on ice, lightly adding the liquid into the upper chamber of the small chamber, slowly moving the gun head during adding, and ensuring that the liquid is flatly paved at the bottom;
placing in a incubator at 37 ℃ for 15min to solidify the glue;
three groups of A549 cells (0 mM,1mM and 5 mM) were digested, centrifuged and the cells were counted, and the cells were diluted with serum-free medium at 2.5X10 4 cells/mL to prepare a cell suspension;
adding 200 mu L of cell suspension into the upper chamber, adding 500 mu L of 10% FBS+ culture medium into the lower chamber, and culturing in a incubator at 37 ℃;
taking out after 20 hours, sucking out redundant liquid in the upper chamber, washing twice by PBS, lightly rotating a cotton stick in the upper chamber, sucking up water and wiping out cells on the inner side of the membrane;
adding crystal violet dye liquor into the upper chamber, dyeing for 5min, recovering the dye liquor, slowly buffering the dye liquor by flowing water, lightly rotating the cotton swab in the upper chamber again, and absorbing water;
placing a glass slide on the upright microscope, inversely placing a cell chamber on the upright microscope, and photographing; the films were counted up, down, left, right, and middle at 100 times the field of view, and averaged.
Test E conclusion:
referring to FIG. 7, pretreatment of A549 cells (A549 cells are cancer cells and have strong cell invasion ability) with I3MA at concentrations of 1mM and 5mM effectively reduced the invasion ability of A549 cells, and the higher the concentration of I3MA was, the stronger the invasion ability of A549 cells was reduced.
Assay F (apoptosis detection):
test F:
first, subcultured A549 cells were equally plated in different six well plates, and the A549 cells were treated with I3MA at concentrations of 0mM and 1mM, respectively, and divided into three groups of control group (0 mMI MA), IR (6 Gy) group and IR (6 Gy) +1mMI MA group.
Briefly, a549 cell culture supernatants were collected after 0 and 48 hours of irradiation of a549 cells using a Fluorescein Isothiocyanate (FITC) -annexin V apoptosis detection kit according to manufacturer's instructions (BDPharmingen, sanDiego, CA, USA);
after digestion of a549 cells with trypsin (EDTA-free), the a549 cells were centrifuged;
the supernatant was collected and washed 3 times with PBS;
A549 cells were resuspended in binding buffer at a concentration of about 5 x 10 5 cells/mL and FITC-conjugated annexin V and Propidium Iodide (PI) solution were added according to manufacturer's instructions;
incubating the a549 cells at room temperature in the dark for 15 minutes;
Finally, flow cytometry analysis of cells (AgilentNovoCyte, USA)
Conclusion of test F:
Referring to FIG. 8, pretreatment of A549 cells with 1mM I3MA prior to IR6Gy radiation promoted apoptosis of post-radiation A549 cells compared to A549 cells without I3MA pretreatment. Thus, it was shown that pretreatment of A549 cells with I3MA prior to IR6Gy radiation slowed the IR6Gy radiation-induced EMT process.
The results of experiments C-F show that the aggressive characteristics of the EMT induced by IR6Gy radiation in corresponding cells can be restored after pretreatment of A549 cells, HCT cells and MDA-MB-231 cells with I3 MA.
In a second aspect, to investigate how I3MA inhibits EMT of cells, test G and test H were developed in example 1.
Test G (real-time fluorescent quantitative PCR, abbreviated qRT-PCR):
test G step:
Firstly, respectively paving sub-cultured A549 cells, HCT cells and MDA-MB-231 cells in different six-hole plates in equal quantity, respectively adopting I3MA with the concentration of 0mM and 1mM to treat the A549 cells, the HCT cells and the MDA-MB-231 cells before radiating the cells of each group by IR6Gy, and dividing the cells into three groups of control groups (0 mMI MA), IR (6 Gy) groups and IR (6 Gy) +1mMI MA; harvesting the above three cells 1-5×10 7 respectively, adding 1mlTrizol (guanidine isothiocyanate/phenol) (taking out white blood cells beside refrigerator, adding Trizol), and mixing;
Repeatedly withdrawing with a 1ml syringe (about 30 times) to disrupt cells and shear DNA, and standing at room temperature for 5min (to completely separate nucleic acid protein complex);
Adding 0.2ml chloroform, shaking vigorously for 15-30s, standing for 2-3min; obtaining a sample by centrifugation at 4 ℃, wherein the centrifugation condition is 12000rpm multiplied by 15min;
The samples were divided into three layers: the bottom layer is a yellow organic phase, and the upper layer is a colorless aqueous phase and a middle layer;
RNA is predominantly in the aqueous phase, which is about 60% of the TRIzol reagent used, i.e., about 600ul;
the supernatant was carefully aspirated into a new DEPC-treated 1.5mlEP tube (if the organic phase was retained for DNA and protein isolation), 0.5ml of isopropanol was added, the tube was gently mixed, and the tube was allowed to stand at room temperature for 10min. Centrifuging at 4deg.C, 12000rpm×10min; no RNA precipitate was seen before centrifugation, and colloidal precipitate appeared on the tube side and bottom after centrifugation;
Removing the supernatant, adding 1ml of 75% ethanol (ice-cold) into the precipitate, shaking, and washing the precipitate thoroughly;
centrifuging at 4deg.C, 12000rpm×5min; discarding the supernatant, briefly centrifuging, carefully sucking the discarded supernatant;
adding a proper amount (20 μl) DEPC (RnaseFree) H20 to dissolve RNA (dissolving for 10-15 min at 65 ℃);
Reverse transcription of the RNA into cDNA using a cDNA synthesis kit;
The primer sequence of qRT-PCR was designed by PrimerPremierSoftware5.0 (PremierBioSoftInternational, USA); the polymerase chain reaction was performed in 96-well plates, 10 μl of reaction per well, containing 1 μl of diluted cDNA, 5 μl LFastStartUniversalSYBRGreenMaster (ROX), 0.3 μl of upstream and downstream primers (10 μl) and 3.4 μl of water;
Using Light The system (barcello, switzerland) evaluates the mRNA expression level of the target gene; the internal reference for mRNA expression level was beta-action, and the relative expression level was calculated by the method of 2 -△△CT.
Test G conclusion:
Referring to FIGS. 9-17, mRNA transcription levels of endogenous factors (CDH 1, CDH2, and TGF beta 1), secreted factors (MMP 2, IL32, and CCL 2), and transcription factors (JUNB, TP63, snail, KLF9, HOXD, and FOSB) in A549 cells, HCT cells, and MDA-MB-231 cells were examined using qRT-PCR;
the endogenous factor, the secreted factor and the transcription factor are highly expressed in cells in which EMT occurs;
for endogenous factors in A549 cells, HCT cells, and MDA-MB-231 cells, CDH1, CDH2, and TGF-beta 1mRNA transcript levels of each cell increased upon IR irradiation; however, after pretreatment of each cell with I3MA prior to IR irradiation, CDH1, CDH2 and tgfβ1mRNA transcript levels of each cell were reduced by IR irradiation;
For secreted factors in A549 cells, HCT cells, and MDA-MB-231 cells, MMP2, IL32, and CCl2mRNA transcript levels in each cell increased upon IR irradiation; however, after pretreatment of each cell with I3MA prior to IR irradiation, MMP2, IL32 and CCl2 levels were reduced in each cell by IR irradiation;
For secreted factors in A549 cells, HCT cells, and MDA-MB-231 cells, transcript levels of JUNB, TP63, snail, KLF9, HOXD9, and FOSBmRNA were elevated in each cell following IR irradiation; however, after pretreatment of each cell with I3MA prior to IR irradiation, the transcript levels of JUNB, TP63, snail, KLF9, HOXD9 and FOSBmRNA were reduced for each cell by IR irradiation;
Taken together, tgfβ1 gene mRNA transcript levels in the endogenous factors are most down-regulated. Notably, tgfβ1 is a marker of pre-invasion and poor prognosis, contributing to the formation of EMT and radiation-induced pulmonary fibrosis, is abnormally high expressed in a variety of cancer-related fibrosis, and is a major regulator of EMT.
Test H (immunoblotting test for detection of TGF-beta 1 expression after 10 days of I3MA treatment on A549 cells, HCT cells and MDA-MB-231 cells):
Test H step:
First, subcultured A549 cells, HCT cells and MDA-MB-231 cells were spread in different six-well plates in equal amounts, respectively, and before each group of cells was irradiated with IR6Gy, the A549 cells, HCT cells and MDA-MB-231 cells were treated with I3MA at concentrations of 0mM and 1mM, respectively, and divided into three groups of control group (0 mMI MA), IR (6 Gy) group and IR (6 Gy) +1mMI MA. Then, proteins of each group of cells were extracted respectively by referring to the procedure of test C, and Westernblot test was performed.
Test H conclusion:
Referring to FIGS. 18-20, for A549 cells, HCT cells, and MDA-MB-231 cells, TGF-beta 1 protein expression was increased for each cell after IR irradiation; however, after pretreatment of each cell with I3MA prior to IR irradiation, TGF-beta 1 protein expression was decreased in each cell by IR irradiation.
The results of the test G-H show that the pretreatment of the A549 cells, the HCT cells and the MDA-MB-231 cells by adopting the I3MA can inhibit the EMT of the corresponding cells.
In example 1, the results of experiments A-H show that after the pretreatment of the cells by using I3MA, the cell survival rate is basically unchanged, no obvious cytotoxicity is caused to the cells, and the cell morphology is kept elliptical; after the I3MA is adopted for pretreatment of the cells, the expression of the marker protein E-cadherin in the EMT process is increased, the expression of the N-cadherin is reduced, the invasion capacity and migration capacity of the cells are effectively reduced, the apoptosis of the cells after the radiation can be promoted, and then the invasive characteristics of the EMT induced by the radiation in the corresponding cells can be recovered; meanwhile, after the cells are pretreated by adopting I3MA, the endogenous factors, secretion factors and transcription factors in the cells are reduced, and the EMT of the corresponding cells can be inhibited. From these results, it can be shown that the use of I3MA has radioprotection against both bronchial epithelial HBE cells and lung cancer A549 cells, and that the use of colorectal cancer HCT cells and breast cancer MDA-MB-231 cells is to demonstrate the general applicability of I3MA to radiation damage protection, where I3MA can protect cells from IR-induced morphological changes of cells and reduce IR-induced EMT processes. Whereas the EMT process is an important pathogenesis of tissue fibrosis, it is involved in the onset of fibrotic disease, and thus the present invention employs I3MA to inhibit EMT by cells to alleviate the onset of radiation-induced pulmonary fibrosis.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. The use of indole-3-acetic acid methyl ester for preparing a medicament for alleviating radiation-induced pulmonary fibrosis, wherein the concentration of indole-3-acetic acid methyl ester is 0.5-10mM.
2. The use according to claim 1, wherein the irradiation is accomplished with ionizing radiation IR; the radiation source used in the IR is a Co-60 source.
3. The use according to claim 2, wherein the radiation dose is not more than 10Gy.
4. The use according to claim 3, wherein the radiation dose is 6Gy.
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