CN117805356A - Method for screening cell coke death activator and inhibitor - Google Patents
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Abstract
The invention discloses a method for screening cell apoptosis activators and inhibitors, and belongs to the field of medicine. The invention can rapidly judge whether the cell is subjected to pyrodeath or not by detecting the relative ratio of P-ERK/ERK. Based on the above, if a certain compound to be screened makes the relative ratio of P-ERK/ERK be more than or equal to 4, the compound has the effect of activating cell apoptosis; if a compound reduces the relative ratio of P-ERK/ERK from 4 to below 2.6, it is shown that the compound has an effect of inhibiting cell apoptosis. The method can rapidly screen cell scorch activator and inhibitor. The method for screening the cell apoptosis activator and inhibitor is more convenient, quick and efficient, and can greatly shorten the screening time.
Description
Technical Field
The invention relates to the field of medicine, in particular to a method for screening cell scorch activators and inhibitors.
Background
Cell apoptosis is a unique pattern of apoptosis closely related to the formation of inflammatory small bodies, also known as inflammatory death. It is the main nonspecific defense mechanism in the organism, and plays an irreplaceable role in the processes of antagonizing invasion of external pathogens, sensing endogenous dangerous signals and the like. Research at home and abroad shows that cell apoptosis plays an important role in the occurrence and development processes of infectious diseases, nervous system diseases, cardiovascular diseases, tumors, digestive tract diseases and the like.
In classical pyro-death signaling pathways, the initiation step of cell pyro-death is the assembly of inflammatory bodies, i.e., nod-like receptor protein 3 (NLRP 3). The inflammatory body is mainly composed of Pattern Recognition Receptors (PRRs), apoptosis-related spotting proteins (ASCs), and a cysteine protease-1 precursor (pro-caspase-1). Inflammatory corpuscles are a multiprotein complex in cells, after relevant stimulation is identified, the inflammatory corpuscles are assembled and activate cysteine proteinase-1 (Caspase-1), the activated Caspase-1 promotes maturation and secretion of interleukin 1 beta (IL-1 beta) and interleukin 18 (IL-18), on the other hand, gasderm D (GSDMD) is taken as an executive protein of cell apoptosis, the activated Caspase-1 specifically cuts to generate N end and C end, the N end of the GSDMD is combined with cell membrane phospholipids, so that cell membrane forms a plurality of pores, cell integrity is damaged, water flows in, cells lose the ability of regulating and controlling substance in and out, intracellular and extracellular ion balance is damaged, cell swelling and rupture are caused, a large amount of contents are released, immune response of the organism is stimulated, inflammatory cells are recruited, and inflammatory response is enlarged. From the above, NLRP3, apoptosis-related spot-like proteins ASC, caspase-1, GSDMD are characteristic signal molecules for the occurrence of scorch in cells.
At present, the classical method for judging the cell apoptosis is to detect the levels of IL-1 beta and IL-18 in the culture supernatant of MODE-K cells by ELISA method, detect the expression quantity change of proteins such as NLRP3, ASC, caspase-1, GSDMD and the like in the cells by Western Blot method, and observe the morphological structure of the cell surface by combining a scanning electron microscope, namely, observing whether the cell membrane is swelled or ruptured and whether the cell structure is destroyed so as to confirm whether the cell apoptosis occurs. However, these methods are complicated and cumbersome in process, and are not conducive to rapid determination of whether or not apoptosis occurs, and also are not conducive to rapid screening of inhibitors and activators of apoptosis.
Disclosure of Invention
The invention aims to provide a method for screening a cell apoptosis activator and inhibitor, which solves the problems in the prior art, and judges the cell apoptosis condition by stimulating cells with a compound to be screened to detect the relative ratio of P-ERK/ERK, thereby judging whether the compound to be screened is the cell apoptosis activator or inhibitor.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for screening cell apoptosis activators, which comprises the following steps:
treating cells with a compound to be screened, detecting the expression levels of P-ERK and ERK proteins in the cells, determining the ratio of P-ERK to ERK, and recording as P-ERK Treatment of /ERK Treatment of ；
While using cells not treated with the compound to be screened as a control, detecting P-ERK in the control Control And ERK Control The ratio is recorded as P-ERK Control /ERK Control ；
According to the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Judging whether the cell is scorched or not, and further judging whether the compound to be screened can be used as a cell scorching activator or not.
Preferably, if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Is less than 2.6, indicating that the cell is not scorching, i.e., the compound to be screened is not a cell scorching activator;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control The ratio of (2) or more.6 and less than 4, indicating that the cell is suspected of undergoing apoptosis, at which time the compound to be screened may or may not be an activator of apoptosis;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control And the ratio of the compound to be screened is greater than or equal to 4, which indicates that the cells undergo pyrodeath, namely the compound to be screened is a cell pyrodeath activator.
Preferably, the concentration of the cells is the maximum concentration at which the cell viability is not less than 95%.
Preferably, the conditions of the treatment are: treatment is carried out at 37 ℃ for 5-10 h.
Preferably, the cells comprise mammalian cells.
The invention also provides a method for screening the cell apoptosis inhibitor, which comprises the following steps:
after treating cells with a compound to be screened, treating the cells with lipopolysaccharide and amide triphosphate, detecting the expression level of P-ERK and ERK proteins in the cells, determining the ratio of P-ERK to ERK, and recording as P-ERK Treatment of /ERK Treatment of ；
Simultaneously with cells treated with the compound to be screened alone as a control, P-ERK controls and ERK in the control are detected Control The ratio is recorded as P-ERK Control /ERK Control ；
According to the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Judging whether the cells undergo scorching or not, and further judging whether the compound to be screened can be used as a cell scorching inhibitor or not.
Preferably, if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Indicating that the cell does not undergo apoptosis, i.e., the compound to be screened is an inhibitor of apoptosis;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control A ratio of 2.6 to 4, indicating that the cell is suspected of undergoing apoptosis, wherein the compound to be screened may or may not be an inhibitor of apoptosis;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control And (2) a ratio of 4 or more, indicating that the cell is scorched, i.e., the compound to be screened is not a scorch inhibitor.
Preferably, the concentration of the cells is the maximum concentration at which the cell viability is not less than 95%.
Preferably, the method of the treatment comprises the following steps: after the cells were treated with the compound to be screened at 37℃for 20-25 and h, 2-5-h was treated with 1-3. Mu.g/mL LPS and 1-3-h was treated with ATP at a concentration of 1-6-mM.
Preferably, the cells comprise mammalian cells.
The invention discloses the following technical effects:
(1) The invention discloses a key index (relative ratio of P-ERK/ERK protein expression quantity) which can reflect the state of cell apoptosis. If the ratio of the expression quantity of the P-ERK/ERK protein in the target tissue, the target organ or the cell after the stimulation of the compound A to the expression quantity of the P-ERK/ERK protein in the normal target tissue, the target organ or the cell is more than or equal to 4, the compound A can activate the target tissue, the target organ or the cell to generate the cell coke death, and the compound A can be used as an activator of the cell coke death. On the premise that the cell apoptosis occurs, the compound B is added, if the ratio of the expression quantity of the P-ERK/ERK protein in the target tissue, the target organ or the cell to the expression quantity of the P-ERK/ERK protein in the target tissue, the target organ or the cell in which the cell apoptosis occurs is obviously reduced, or the relative ratio of the expression quantity of the P-ERK/ERK protein is less than 2.6, the compound B can inhibit the scorching process of the target tissue, the target organ or the cell, and the compound B can be used as an inhibitor of the cell apoptosis.
(2) The invention can rapidly judge whether the cell is subjected to pyrosis or not by detecting the ratio of P-ERK/ERK. Based on the above, if a certain compound makes the relative ratio of P-ERK/ERK be more than or equal to 4, the compound has the effect of activating the cell apoptosis; if a compound reduces the relative ratio of P-ERK/ERK from 4 to below 2.6, it is shown that the compound has an effect of inhibiting cell apoptosis. The method can rapidly screen cell scorch activator and inhibitor.
(3) The invention determines the index for evaluating the apoptosis of the cell coke as 1, namely the ratio of the P-ERK/ERK protein expression quantity, and compared with the method for detecting by using more than 6 indexes in the prior art, the detection index provided by the invention screens the activator or inhibitor of the apoptosis of the cell coke, is more convenient and rapid, can shorten a large amount of time and has high efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of inflammatory factor expression in LPS-induced cellular inflammation; a: TNF-alpha; b: IL-6;
FIG. 2 shows the results of expression of NLRP3 and GSDMD in LPS-induced cellular inflammation; a: NLRP3; b: GSDMD;
FIG. 3 shows the results of ERK phosphorylation in LPS-induced cellular inflammation; a: western blotting results; b: ERK proteins; c: P-ERK proteins; d: P-ERK/ERK ratio;
FIG. 4 is an electron microscope scan of the LPS/ATP-induced MODE-K cell apoptosis; a: scanning an electron microscope of a control group; b: electron microscope scanning images of the treatment group;
FIG. 5 shows the detection of protein expression of NLRP3, GSDMD-N and the like in the scorch of MODE-K cells induced by LPS/ATP; a: immunoblotting results of NLRP3, caspase-1, ASC and GSDMD-N in MODE-K cells; b: statistics of expression level of NLRP3 protein; c: calculating the expression level statistics of caspase-1 protein; d: calculating the expression level statistics of ASC protein; e: statistics of expression level of GSDMD-N protein;
FIG. 6 shows the variation of ERK phosphorylation in LPS/ATP-induced MODE-K apoptosis; a: immunoblotting results of ERK and P-ERK in MODE-K cells; b: counting the expression level of the P-ERK/ERK;
fig. 7 is a graph demonstrating the role of SCH772984 in cell apoptosis; a: immunoblotting results of NLRP3 and P-ERK in MODE-K cells; b: statistics of expression level of NLRP3 protein; c: counting the expression level of the P-ERK protein;
FIG. 8 is a graph showing the effect of detecting baicalin-butyl ester in apoptosis; a: immunoblotting results of NLRP3, caspase-1, ASC and GSDMD-N in MODE-K cells; b: statistics of expression level of NLRP3 protein; c: calculating the expression level statistics of caspase-1 protein; d: calculating the expression level statistics of ASC protein; e: statistics of expression level of GSDMD-N protein;
FIG. 9 is a graph showing the effect of baicalin-butyl ester on ERK phosphorylation in MODE-K apoptosis; a: immunoblotting results of ERK and P-ERK in MODE-K cells; b: counting the expression level of the P-ERK/ERK;
FIG. 10 is a graph showing the effect of NAC on NLRP3 protein expression in MODE-K apoptosis; a: immunoblotting results of NLRP3 in MODE-K cells; b: statistics of expression level of NLRP3 protein;
FIG. 11 is a graph showing the effect of NAC on ERK phosphorylation in MODE-K apoptosis; a: immunoblotting results of P-ERK and ERK in MODE-K cells; b: statistics of P-ERK/ERK expression levels.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1 phosphorylated extracellular regulatory protein kinase/extracellular regulatory protein kinase (P-ERK/ERK) use in MODE-K cell inflammation models
1. Expression of inflammatory factors in LPS-induced cellular inflammation
MODE-K cells were divided into Control (CON) and treatment (LPS).
Control group (CON): normally grown MODE-K cells were seeded at 37℃in 6-well plates at 80% concentration and cell supernatants were collected in sterile centrifuge tubes and centrifuged at 3000rpm for 20 min.
Treatment group (LPS): MODE-K cells were seeded at 37deg.C in 6 well plates at 80% concentration, washed twice with Phosphate Buffered Saline (PBS), treated with LPS at 40 μg/mL for 1h, and the cell supernatants were collected in sterile centrifuge tubes and centrifuged at 3000rps for 20 min.
The expression levels of TNF-alpha and IL-6 were determined according to the ELISA kit instructions. The results are shown in FIG. 1, where LPS stimulation significantly increased the expression of TNF- α (A in FIG. 1) and IL-6 (B in FIG. 1) in cells (P < 0.01) compared to the control group, indicating that LPS stimulated cells had an inflammatory response.
2. Expression of NLRP3 and GSDMD in LPS-induced cellular inflammation
MODE-K cells were divided into Control (CON) and treatment (LPS).
Control group (CON): normally growing MODE-K cells were seeded at 80% in 6-well plates at 37℃and washed twice with PBS and discarded for use.
Treatment group (LPS): MODE-K cells were seeded at 37deg.C in 6-well plates at 80% concentration, washed twice with PBS, treated with LPS at 40 μg/mL for 1h, and the DMEM medium was discarded, rinsed gently with PBS, and discarded for use.
After the cells of the control and treatment groups were treated according to the above method, total RNA was isolated from the cells using TRIzol reagent, and the purity of the RNA was measured at 260 nm wavelength using a spectrophotometer (by measuring A 260 /A 280 Is evaluated by ratio of (a) and selecting RNA with purity of 1.8-2.0, reversely transcribing the RNA into complementary DNA by a cDNA synthesis kit, detecting the expression of NLRP3 and GSDMD genes according to the specification of a fluorescence quantitative qPCR kit (the sequence of the primer is shown in the table and is synthesized by Shanghai Biotechnology Co., ltd.) and calibrating the result by Cq value of an internal gene beta-actin, namely DeltaCq=Cq value of the internal gene-Cq value of a target gene according to 2 -ΔΔCt The relative expression level of the target gene was calculated by the method.
TABLE 1 primer sequences
The results are shown in figure 2, where LPS treatment caused a significant increase in NLRP3 expression (P < 0.05) (a in figure 2) but no significant change in GSDMD expression (B in figure 2) compared to the control group, indicating that LPS stimulation did not cause scorch to the cells.
3. Expression of ERK phosphorylation in LPS-induced cellular inflammation
MODE-K cells were divided into Control (CON) and treatment (LPS).
Control group (CON): normally growing MODE-K cells were seeded at 80% in 6-well plates at 37℃and washed twice with PBS and discarded for use.
Treatment group (LPS): MODE-K cells were seeded at 37deg.C in 6-well plates at 80% concentration, washed twice with PBS, treated with LPS at 40 μg/mL for 1h, and the DMEM medium was discarded, rinsed gently with PBS, and discarded for use.
After the cells of the control group and the treated group were treated according to the above method, whole cell extracts of the control group and the treated group were obtained by lysis with a protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and the samples were transfected onto nitrocellulose membrane, and blocked, and incubated at 4℃for 2 h with primary antibodies (anti-ERK 1/2 antibody: cat#4695, cell Signaling Technology, USA; anti-P-ERK 1/2 antibody: cat#4370,Cell Signaling Technology,USA; anti-beta-actin antibody: cat#HC201, tranGen Biotech, china; dilution ratio 1:1000) at 4℃for 12 h (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio 1:1000). After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The results are shown in fig. 3, with LPS treatment significantly increased ERK protein expression compared to the control group (P<0.05 Extremely significantly increases the expression of P-ERK protein (P)<0.01 (a-C in fig. 3). P-ERK Treatment of /ERK Treatment of And P-ERK Control /ERK Control The ratio of (2) was 1.1 (D in FIG. 3), indicating that LPS was not able to act as an activator of apoptosis.
Example 2 LPS/ATP acts as an activator of MODE-K apoptosis
1. LPS/ATP-induced MODE-K cell apoptosis
MODE-K cells were divided into Control (CON) and treatment (LA).
Control group (CON): normally grown MODE-K cells were seeded at 37deg.C in 6-well plates containing cell slide at 80% concentration, washed twice with PBS, and discarded.
Treatment group (LA): MODE-K cells were seeded at 37deg.C in 6-well plates containing cell slide plates at 80% concentration, washed twice with PBS, treated with LPS at 2 μg/mL 4h, treated with ATP at 5mM at 1h, and the DMEM medium was discarded, rinsed gently with PBS, and the PBS was discarded for use.
And adding electron microscope fixing solution into 6 pore plates of the control group and the treatment group, fixing at 25 ℃ in a dark place for 2 h, transferring to 4 ℃ for preservation, and finally observing cell morphology under a scanning electron microscope.
The results are shown in FIG. 4, and the treated group cell membrane swelling, perforation and cell surface rupture are obvious, which indicates that LPS/ATP induction can cause cell apoptosis.
2. Expression of proteins NLRP3, GSDMD-N and the like in LPS/ATP-induced MODE-K cell pyrosis
After MODE-K cells of the control and treatment groups were treated according to the method of "1, LPS/ATP-induced MODE-K cell apoptosis", whole cell extracts of the control and treatment groups were obtained by lysis with a protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and transfected onto nitrocellulose membrane, blocking was performed, and primary antibody (anti-NLRP 3 antibody: cat#ab263899, abcam. Uk; anti-ASC antibody: cat# 67824,Cell Signaling Technology,USA; anti-caspase-1 antibody: cat#24232,Cell Signaling Technology,Inc. USA; anti-GSDMD-N antibody: cat#df 13758, affinity. USA; anti- β -actin antibody: cat#hc201, tranGen biotech. China; dilution ratio of 1:1000) was incubated for 12 h, and secondary antibody (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio of 1:1000) at 4 ℃ was incubated for 2 h. After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The results are shown in FIG. 5A-E, where protein expression of NLRP3 (P < 0.001), ASC (P < 0.01), caspase-1 (P < 0.05), GSDMD-N (P < 0.001) was significantly elevated in the treatment group, indicating that LPS/ATP induction caused apoptosis.
3. LPS/ATP-induced changes in ERK phosphorylation in MODE-K cell apoptosis
After MODE-K cells of the control and treatment groups were treated according to the method of "1, LPS/ATP-induced MODE-K cell apoptosis", whole cell extracts of the control and treatment groups were obtained by lysis with a protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and the samples were transfected onto nitrocellulose membrane, and blocked, and incubated at 4℃for 2 h with primary antibodies (anti-ERK 1/2 antibody: cat#4695,Cell Signaling Technology,USA; anti-P-ERK 1/2 antibody: cat#4370, cell Signaling Technology, USA; anti-beta-actin antibody: cat#HC201, tranGen Biotech. China; dilution ratio 1:1000) at 4℃for 12 h (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio 1:1000). After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The results are shown in FIG. 6, where protein expression of P-ERK was significantly increased (A in FIG. 6), the ratio of P-ERK/ERK was significantly increased (P<0.01 (B in FIG. 6), P-ERK Treatment of /ERK Treatment of And P-ERK Control /ERK Control The ratio was 4. The above shows that LPS/ATP can act as an activator of apoptosis.
Example 3 SCH772984 plays an inhibitory role in apoptosis of cell coke
SCH772984 is an ERK inhibitor and can inhibit ERK phosphorylation.
MODE-K cells were divided into the control group (CON), the apoptosis group (LA), and the SCH772984 drug group (SCH).
Control group (CON): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, the medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
Cell focal death group (LA): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, then treated with LPS (2. Mu.g/ml, 4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
SCH772984 pharmaceutical group (SCH): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, pretreated with SCH772984 (ERK inhibitor) for 1h, then treated with LPS (2 μg/mL,4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
After MODE-K cells were treated according to the above method, the adherent cells were rinsed with PBS and lysed with protein lysate (RIPA: protease inhibitor at a volume ratio of 100:1) to obtain whole cell extracts. After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and the samples were transfected onto nitrocellulose membrane, and blocked, and incubated at 4℃for 2 h with primary antibodies (anti-NLRP 3 antibody: cat#ab263899, abcam. UK; anti-P-ERK 1/2 antibody: cat#4370,Cell Signaling Technology,USA; anti-beta-actin antibody: cat#HC201, tranGen Biotech. China; dilution ratio 1:1000) at 4℃for 12 h (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio 1:1000). After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The results are shown in FIG. 7, where protein expression of NLRP3 was significantly increased (P < 0.01) after LPS/ATP treatment, while protein expression of NLRP3 was significantly decreased (P < 0.01) after addition of SCH772984 (A-B in FIG. 7). Protein expression of P-ERK was significantly increased after LPS and ATP treatment, phosphorylation of ERK was significantly inhibited after SCH772984 pretreatment, and protein expression of P-ERK was reduced (P < 0.01) (C in fig. 7). SCH772984 was shown to inhibit ERK phosphorylation, reduce NLRP3 protein expression, and inhibit cell apoptosis.
Example 4 Baicalin N-butyl ester (BNE) plays an inhibitory role in apoptosis
1. Effect of BNE on expression of proteins such as NLRP3 and GSDMD-N in MODE-K cell apoptosis
MODE-K cells were divided into control group (CON), apoptosis group (LA), baicalin-n-butyl ester (low dose, medium dose, high dose) drug group.
Control group (CON): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, the medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
Cell focal death group (LA): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, then treated with LPS (2. Mu.g/mL, 4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
Baicalin n-butyl ester pharmaceutical group (BNE): MODE-K cells were inoculated at 37deg.C and 80% in 6-well plates containing DMEM medium, washed twice with PBS, treated with n-butyl baicalin (L, M, H) at different concentrations (3, 6, 12 μm) for 24 h, treated with LPS at a concentration of 2 μg/mL for 4h, treated with ATP at a concentration of 5mM for 1h, and the DMEM medium was discarded, the adherent cells were washed with PBS, and the PBS was discarded for use.
After MODE-K cells were treated according to the above method, whole cell extracts were obtained by lysis with protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and transfected onto nitrocellulose membrane, blocking was performed, and primary antibody (anti-NLRP 3 antibody: cat#ab263899, abcam. Uk; anti-ASC antibody: cat# 67824,Cell Signaling Technology,USA; anti-caspase-1 antibody: cat#24232,Cell Signaling Technology,Inc. USA; anti-GSDMD-N antibody: cat#df 13758, affinity. USA; anti- β -actin antibody: cat#hc201, tranGen biotech. China; dilution ratio of 1:1000) was incubated for 12 h, and secondary antibody (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio of 1:1000) at 4 ℃ was incubated for 2 h. After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
As shown in fig. 8 a-E, protein expression of NLRP3 (P < 0.001), ASC (P < 0.01), caspase-1 (P < 0.05), GSDMD-N (P < 0.001) was significantly elevated in the apoptotic group, whereas both medium (6 μm) and high (12 μm) doses of N-butyl baicalin reduced ASC (P < 0.05). Both medium (6 μm) and high (12 μm) doses of N-butyl baicalin reduced protein expression of NLRP3 (P < 0.01), caspase-1 (P < 0.05), GSDMD-N (P < 0.01) and significantly reduced in a dose-dependent manner. The result shows that the LPS/ATP induction can cause cell apoptosis, and the baicalin-butyl ester (6-12 mu M) has remarkable inhibition effect on the cell apoptosis induced by the LPS/ATP.
2. Effect of BNE on ERK phosphorylation in MODE-K apoptosis
According to the method of 'influence of 1 and BNE on protein expression of NLRP3, GSDMD-N and the like in MODE-K cell apoptosis', MODE-K cells are treated, and then are lysed by protein lysate (RIPA: protease inhibitor with the volume ratio of 100:1) to obtain whole cell extract. After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and the samples were transfected onto nitrocellulose membrane, and blocked, and incubated for 12 h at 4℃with primary antibody (anti-ERK 1/2 antibody: cat#4695,Cell Signaling Technology,USA; anti-P-ERK 1/2 antibody: cat#4370,Cell Signaling Technology,USA; anti-beta-actin antibody: cat#HC201, tranGen Biotech. China; dilution ratio 1:1000) and for 2 h with secondary antibody (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio 1:1000). After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The results are shown in FIG. 9, where protein expression of P-ERK is significantly increased in the apoptotic group (A in FIG. 9), the relative ratio of P-ERK/ERK is significantly increased (P<0.01 (B in fig. 9). After pretreatment of baicalin-butyl ester, the relative ratio of P-ERK/ERK is obviously reduced by dose dependence (P<0.05 (a-B in fig. 9). Medium dose of n-butyl baicalin P-ERK Baicalin-butyl ester /ERK Baicalin-butyl ester And P-ERK Control /ERK Control The ratio is 2.6, and the high dose of baicalin n-butyl ester P-ERK Baicalin-butyl ester /ERK Baicalin-butyl ester And P-ERK Control /ERK Control The ratio was 2.1. The above shows that baicalin-butyl ester (6-12 mu M) can be used as an inhibitor of cell apoptosis.
Example 5 NAC (ROS scavenger) acts as an inhibitor in apoptosis of the cell
1. Effect of NAC on expression of NLRP3 protein in MODE-K apoptosis
MODE-K cells were divided into control group (CON), apoptosis group (LA), NAC drug group (NAC).
Control group (CON): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, the medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
Cell focal death group (LA): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, then treated with LPS (2. Mu.g/mL, 4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
NAC drug group (NAC): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, pre-treated with NAC for 1h, then treated with LPS (2 μg/mL,4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
After MODE-K cells were treated according to the above method, whole cell extracts were obtained by lysis with protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and transfected onto nitrocellulose membrane, blocking was performed, primary antibodies (anti-NLRP 3 antibodies: cat#ab263899, abcam. Uk; anti-beta-actin antibodies: cat#hc201, tranGen biotech. China; dilution ratio was 1:1000) were incubated for 12 h, secondary antibodies (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio was 1:1000) at 4 ℃ were incubated for 2 h. After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
As shown in fig. 10 a-B, protein expression of NLRP3 (P < 0.01) was significantly increased after LPS and ATP treatment, and protein expression of NLRP3 (P < 0.05) was significantly decreased after NAC pretreatment, indicating that NAC can inhibit protein expression of NLRP3 and reduce cell apoptosis.
2. Effect of NAC on ERK phosphorylation in MODE-K apoptosis
MODE-K cells were divided into control group (CON), apoptosis group (LA), NAC drug group (NAC).
Control group (CON): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, the medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
Cell focal death group (LA): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, then treated with LPS (2. Mu.g/ml, 4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
NAC drug group (NAC): MODE-K cells were seeded at 37deg.C in 6-well plates containing DMEM medium at 80% concentration, washed twice with PBS, pre-treated with NAC for 1h, then treated with LPS (2 μg/mL,4 h) and ATP (5 mM,1 h), medium was discarded, adherent cells were washed with PBS, and PBS was discarded for use.
After MODE-K cells were treated according to the above method, whole cell extracts were obtained by lysis with protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and the samples were transfected onto nitrocellulose membrane, and blocked, and incubated at 4℃for 2 h with primary antibodies (anti-ERK 1/2 antibody: cat#4695,Cell Signaling Technology, USA; anti-P-ERK 1/2 antibody: cat#4370, cell Signaling Technology, USA; anti-beta-actin antibody: cat#HC201, tranGen Biotech. China; dilution ratio 1:1000) at 4℃for 12 h (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio 1:1000). After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The results are shown in FIG. 11A-B, where the ratio of P-ERK/ERK is significantly increased after LPS and ATP treatment (P<0.01），P-ERK LA /ERK LA And P-ERK Control /ERK Control The ratio was 4.2, demonstrating that the cells underwent pyrodeath under this condition. NAC, P-ERK was added after LPS and ATP treatment NAC /ERK NAC And P-ERK Control /ERK Control The ratio is 2.4 (P<0.05). As indicated above, NAC can act as an inhibitor of apoptosis.
Example 6P application of ERK/ERK in screening for Coke death activators
MODE-K cells were used as subjects and were divided into control and treatment groups. The normal growing cell group was the control group (CON), and the group treated with compound a was the treatment group. First, the concentration x of compound A is determined by detecting the cell activity rate, and at this time, x is the maximum value when the cell activity rate is not less than 95%.
The treatment mode of the treatment group is as follows: cells were seeded at 80% in 6-well plates at 37 ℃. Cells were seeded at 80% in 6-well plates at 37℃and washed twice with PBS and then treated with compound A at x concentration (cell viability. Gtoreq.95%) 6 h.
The whole cell extract was obtained by lysis with a protein lysate (RIPA: protease inhibitor in a volume ratio of 100:1). After protein concentration was detected using BCA protein detection kit, SDS-PAGE electrophoresis was performed, and the samples were transfected onto nitrocellulose membranes, blocked, and incubated at 4℃for 2 h with primary antibodies (anti-ERK 1/2 antibodies: cat#4695, cell Signaling Technology, USA; anti-P-ERK 1/2 antibodies: cat#4370, cell Signaling Technology, USA; anti-beta-actin antibodies: cat#HC201, tranGen Biotech. China; dilution ratio 1:1000) at 4℃for 12 h (HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG, dilution ratio 1:1000). After washing the membrane, the relative ratio of P-ERK/ERK was calculated for the control and A treated cells using a high sensitivity chemiluminescent substrate kit exposure.
The cell apoptosis was judged according to the criteria in the following table. If after treatment A, according to P-ERK Treatment of /ERK Treatment of
And P-ERK Control /ERK Control The ratio of (2) can judge whether the cell is scorched or not, and further judge whether A can be used as an activator of the scorching of the cell or not.
Table 2 evaluation criteria
Note that: -, represents that no scorching occurred; ++ represents suspected scorching; ++, which indicates that scorching occurred.
2. Application of P-ERK/ERK in screening of pyrosis inhibitor
Cells were seeded at 80% in 6-well plates at 37 ℃. Cells were seeded at 80% in 6-well plates at 37 ℃. Compounds of different concentrations were added to 6-well plates and after treatment of cells 24 and h, the maximum value at which the cell viability was greater than or equal to 95% was determined to be y.
Cells were seeded at 37℃in 6-well plates at 80% concentration, washed twice with PBS, treated with compound B at y concentration for 24 h, treated with LPS at 2. Mu.g/mL for 4h, treated with ATP at 5mM for 1h, and lysed with protein lysate (RIPA: protease inhibitor at a volume ratio of 100:1) to obtain whole cell extracts. After protein concentration is detected by using a BCA protein detection kit, SDS-PAGE electrophoresis is carried out, and then transfection is carried out on a nitrocellulose membrane, and the membrane is closed and incubated for the primary antibody and the secondary antibody at 4 ℃. After washing the membrane, the high sensitivity chemiluminescent substrate kit is used for exposure.
The control group (CON) was treated with neither an unknown compound nor LPS nor ATP, the cell coke death group (LA) was treated with LPS and ATP, and the treatment group was treated with compound B at the concentrations of LPS, ATP and y. And judging the apoptosis of the cell coke according to the relative ratio of P-ERK/ERK.
If P-ERK Treatment of /ERK Treatment of And P-ERK Control /ERK Control If the ratio of (2) is significantly reduced or less than 2.6, then it is considered that the cell does not undergo pyrosis, and B is an inhibitor of pyrosis.
Table 3 evaluation criteria
Note that: -, represents that no scorching occurred; ++ represents suspected scorching; ++, which indicates that scorching occurred.
From the above, it can be seen from the experimental results of the examples that the relative ratio of the expression levels of the P-ERK/ERK proteins can reflect the state of apoptosis. If the relative ratio of the expression quantity of the P-ERK/ERK protein in the cell is more than or equal to 4 after the compound A stimulates the cell, the compound A can activate the cell to generate the cell coke death, and the compound A can be used as an activator of the cell coke death.
On the premise of the occurrence of cell apoptosis, the compound B is added, for example, the relative ratio of the P-ERK/ERK protein expression quantity in cells is obviously reduced, or the relative ratio of the P-ERK/ERK protein expression quantity is less than 2.6, which indicates that the compound B can inhibit the cell apoptosis process and can be used as an inhibitor of cell apoptosis. That is, if a certain compound makes the relative ratio of P-ERK/ERK more than or equal to 4, the compound has the effect of activating the cell apoptosis; if a compound reduces the relative ratio of P-ERK/ERK from 4 to below 2.6, it is shown that the compound has the effect of inhibiting cell apoptosis. The method of the invention can rapidly screen cell scorch activators and inhibitors.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. A method of screening for an activator of apoptosis comprising the steps of:
treating cells with a compound to be screened, detecting the expression levels of P-ERK and ERK proteins in the cells, determining the ratio of P-ERK to ERK, and recording as P-ERK Treatment of /ERK Treatment of ；
While using cells not treated with the compound to be screened as a control, detecting P-ERK in the control Control And ERK Control The ratio is recorded as P-ERK Control /ERK Control ；
According to the P-ERK Treatment of /ERK Treatment of And the P-ERK Control And ERK Control Judging whether the cell is scorched or not, and further judging whether the compound to be screened can be used as a cell scorching activator or not.
2. The method of screening for an activator of apoptosis of claim 1,
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Is less than 2.6, indicating that the cell is not scorching, i.e., the compound to be screened is not a cell scorching activator;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control A ratio of 2.6 to 4, indicating that the cell is suspected of undergoing pyrosis;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control And the ratio of the compound to be screened is greater than or equal to 4, which indicates that the cells undergo pyrodeath, namely the compound to be screened is a cell pyrodeath activator.
3. The method of screening for a cell death activator according to claim 1, wherein said cell concentration is a maximum concentration at which said cell viability is not less than 95%.
4. The method of screening for an activator of apoptosis of claim 1, wherein the treatment is performed under conditions of: treatment is carried out at 37 ℃ for 5-10 h.
5. The method of screening for an activator of apoptosis of claim 1, wherein said cells comprise mammalian cells.
6. A method of screening for inhibitors of apoptosis comprising the steps of:
after treating cells with a compound to be screened, treating the cells with lipopolysaccharide and amide triphosphate, detecting the expression level of P-ERK and ERK proteins in the cells, determining the ratio of P-ERK to ERK, and recording as P-ERK Treatment of /ERK Treatment of ；
While taking cells treated with only the compound to be screened as a control, detecting P-ERK in the control Control And ERK Control The ratio is recorded as P-ERK Control /ERK Control ；
According to the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Judging whether the cells undergo scorching or not, and further judging whether the compound to be screened can be used as a cell scorching inhibitor or not.
7. The method of screening for inhibitors of apoptosis of claim 6, wherein if said P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control Indicating that the cell does not undergo apoptosis, i.e., the compound to be screened is an inhibitor of apoptosis;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control A ratio of 2.6 to 4, indicating that the cell is suspected of undergoing pyrosis;
if the P-ERK Treatment of /ERK Treatment of And the P-ERK Control /ERK Control And (2) a ratio of 4 or more, indicating that the cell is scorched, i.e., the compound to be screened is not a scorch inhibitor.
8. The method of screening for a cytostatic agent according to claim 6, wherein said concentration of cells is the maximum concentration at which said cell viability is greater than or equal to 95%.
9. The method of screening for inhibitors of apoptosis of claim 6, wherein said treating is by: after the cells were treated with the compound to be screened at 37℃for 20-25 and h, 2-5-h was treated with 1-3. Mu.g/mL LPS and 1-3-h was treated with ATP at a concentration of 1-6-mM.
10. The method of screening for inhibitors of apoptosis of claim 6, wherein said cells comprise mammalian cells.
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| CN112210017A (en) * | 2020-10-12 | 2021-01-12 | 中国药科大学 | Group of tar death executor fusion proteins and tar death activity detection method based on luciferase fragment complementation analysis technology |
| CN113583956A (en) * | 2021-07-27 | 2021-11-02 | 浙江大学 | Construction method and application of cell scorch model |
| CN114984224A (en) * | 2022-06-14 | 2022-09-02 | 中国中医科学院中药研究所 | Application of biomarker-targeted agent in preparation of medicine for relieving/treating neuropathic pain |
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