CN113533737A - Application of NFAM1 in screening drugs for preventing and treating coronary heart disease or preparing kit - Google Patents

Application of NFAM1 in screening drugs for preventing and treating coronary heart disease or preparing kit Download PDF

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CN113533737A
CN113533737A CN202010292380.6A CN202010292380A CN113533737A CN 113533737 A CN113533737 A CN 113533737A CN 202010292380 A CN202010292380 A CN 202010292380A CN 113533737 A CN113533737 A CN 113533737A
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heart disease
nfam1
coronary heart
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monocytes
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朱海波
龙捷
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Institute of Materia Medica of CAMS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
    • G—PHYSICS
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Abstract

The invention belongs to the technical field of medicines, and relates to application of NFAM1 in screening medicines for preventing and treating coronary heart disease or preparing a kit. In particular discloses an application of NFAT activating protein (NFAT activating protein with ITAM motif 1, NFAM1) with ITAM motif 1 in searching or screening drugs or biological agents for preventing and treating coronary heart disease and coronary heart disease related diseases and in preparing early warning, early diagnosis or prognosis evaluation kits. The invention proves that the expression of NFAM1 in peripheral blood mononuclear cells of patients with coronary heart disease is obviously increased for the first time. The invention also discovers that the inhibition of the NFAM1 level can effectively reduce the expression of chemokine receptors and the activation of p38 MAPK pathways, thereby inhibiting the chemotaxis of monocytes. Based on the discovery, the invention discloses that NFAM1 can be used as a new target for establishing a simple, convenient, safe and noninvasive early warning and drug treatment system for coronary heart disease.

Description

Application of NFAM1 in screening drugs for preventing and treating coronary heart disease or preparing kit
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of NFAT activated protein NFAM1 with ITAM motif 1 in searching or screening medicines or biological preparations for preventing and treating coronary heart disease and coronary heart disease related diseases, and application of NFAT activated protein NFAM1 with ITAM motif 1 in preparing a kit for early warning, early diagnosis or prognosis evaluation of coronary heart disease and coronary heart disease related diseases.
Background
Cardiovascular and cerebrovascular diseases are always major factors endangering human life. Among them, heart disease (coronary heart disease) caused by myocardial ischemia and hypoxia due to atherosclerosis and thrombosis thereof is an important factor causing myocardial infarction and stroke, and is associated with various risk factors and pathophysiological complexity. Due to the complex clinical manifestations, early warning and timely intervention of coronary heart disease always face huge challenges.
Various studies have shown that monocytes are not only the initiating factor for plaque formation, but also one of the triggers for atherosclerosis progression and instability. The recruitment and migration of monocytes in the blood to the damaged endothelium by chemokine attraction to the arterial wall has been considered an important process in the development of atherosclerotic plaques. The increased lipid burden and inflammatory stimuli caused by monocyte-derived foam cells accelerate the transition of lesions from stable to unstable lesions. Therefore, circulating monocytes can serve as an important discovery source for non-invasive early prediction and intervention methods for coronary heart disease.
The NFAT activating protein (NFAT activating protein with ITAM motif 1, NFAM1) having ITAM motif 1 is highly expressed in monocytes, and is a single transmembrane protein containing an Immunoreceptor Tyrosine Activated Motif (ITAM). The nucleotide sequence of the Gene is 150372 in Gene ID. The invention discovers for the first time that the expression level of NFAM1 in monocytes is related to the occurrence and development of coronary heart disease, and can be used as an important target of a targeted chemokine receptor, thereby providing support for finding a new target for early warning and other applications of coronary heart disease.
Disclosure of Invention
The technical problems solved by the invention are as follows:
the first aspect provides the application of NFAM1 in searching or screening drugs or biological preparations for preventing and treating coronary heart disease and diseases related to coronary heart disease.
The second aspect provides an application of NFAM1 in the preparation of a kit for early warning, early diagnosis or prognosis evaluation of coronary heart disease and coronary heart disease-related diseases.
In a third aspect, the application of shRNA shown in SEQ ID No.1 for inhibiting NFAM1 level in preparing a reagent for regulating and controlling chemokine receptors CCR2 and/or CCR5 is provided.
The fourth aspect provides application of shRNA shown as SEQ ID No.1 for inhibiting the level of NFAM1 in preparing a reagent for inhibiting chemotaxis or migration of monocytes.
The fifth aspect provides application of shRNA shown in SEQ ID No.1 for inhibiting NFAM1 level in preparation of MAPK inhibitor.
In order to solve the technical problem, the invention adopts the following technical scheme.
The first aspect of the technical scheme of the invention provides the application of NFAM1 in searching or screening drugs or biological preparations for preventing and treating coronary heart disease and diseases related to coronary heart disease.
Preferably, the application of searching or screening the medicine or biological agent for preventing and treating coronary heart disease and coronary heart disease related diseases is to screen the medicine or biological agent for inhibiting the expression of NFAM1 by determining the expression condition of NFAM1 in monocytes and comparing the expression level of NFAM1 with that of a control group.
The second aspect of the technical scheme of the invention provides application of NFAM1 in preparation of a kit for early warning, early diagnosis or prognosis evaluation of coronary heart disease and coronary heart disease-related diseases.
Preferably, the early warning, early diagnosis or prognosis for coronary heart disease is determined by determining whether the expression level of NFAM1 is increased in a blood sample of the subject compared to a control group by measuring the expression level of NFAM1 in monocytes.
Preferably, the reagent for determining the expression of NFAM1 in monocytes in a blood sample from the individual is a primer and/or probe and/or antibody of NFAM 1.
Preferably, the control group is healthy people who have no coronary heart disease and angina symptom and have coronary CT/contrast stenosis lesion of less than 50% stenosis in individuals.
The third aspect of the technical scheme of the invention provides application of shRNA shown as SEQ ID No.1 for inhibiting the NFAM1 level in preparing a reagent for regulating and controlling chemokine receptors CCR2 and/or CCR 5; the application in the preparation of the reagent for inhibiting the chemotaxis or migration of the monocyte; the application in preparing MAPK pathway inhibitor.
Preferably, the shRNA shown as SEQ ID No.1 for inhibiting the NFAM1 level can inhibit the expression of CCR2 and/or CCR 5.
Preferably, the shRNA shown in SEQ ID No.1 for inhibiting the level of NFAM1 can inhibit chemotaxis and/or migration of monocytes.
Preferably, the shRNA shown in SEQ ID No.1 that inhibits the level of NFAM1 inhibits chemokine-mediated activation of the p38 MAPK pathway.
The beneficial technical effects are as follows:
the invention establishes the correlation between NFAT activated protein NFAM1 with ITAM motif 1 and the occurrence and development of coronary heart disease for the first time, and finds that compared with a normal control group, the expression of NFAM1 in peripheral blood mononuclear cells of a coronary heart disease patient is obviously improved, and the high expression of NFAM1 can early warn the coronary heart disease, on one hand, the NFAT activated protein NFAM1 can be used as a new target spot of the coronary heart disease to screen medicines or preparations, and on the other hand, the NFAM activated protein NFAM1 can be used as a novel biomarker for early warning, early diagnosis or prognosis evaluation of the coronary heart disease. Molecular mechanism research further determines that the inhibition of the expression of NFAM1 can significantly reduce the expression levels of chemokine receptors CCR2 and CCR5 closely related to coronary heart disease, and inhibit chemokine-induced activation of the p38 MAPK pathway, thereby inhibiting the chemotactic migration effect of monocytes.
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FIG. 1 is a graph showing the correlation between the NFAM1 level of human peripheral blood mononuclear cells and the occurrence and development of coronary heart disease in example 1 of the present invention. (A) The quantitative results of flow analysis of the expression level of NFAM1 in peripheral blood mononuclear cells of patients with coronary heart disease and normal control groups are shown, wherein 79 cases of the control group, 70 cases of stable coronary heart disease, 183 cases of acute coronary syndrome and 253 cases of patients with total coronary heart disease (namely the sum of stable coronary heart disease and acute coronary syndrome). (B) Whether the NFAM1 can be used for early prediction of coronary heart disease is analyzed by using a receiver operating characteristic curve (ROC curve).
FIG. 2 shows the expression verification of the monocyte model for stably knocking down NFAM1 in example 2 of the present invention. The expression of NFAM1 was determined by stably knocking down NFAM1 cells using wild type U-937 cells, negative control shRNA cells, and NFAM1 shRNA, which were not transfected. (A) A real-time fluorescence quantitative PCR result comparison graph, (B) a flow analysis result comparison graph, and (C) a Western Blot result graph comparison graph.
FIG. 3 is a graph showing the effect of inhibiting the expression of NFAM1 on the expression of monocyte chemokine receptor in example 3 of this invention. Expression of chemokine receptors CCR2 and CCR5 were determined by stable knock-down of NFAM1 cells using non-transfected wild type U-937 cells, negative control shRNA cells, NFAM1 shRNA cells, respectively. (A) Real-time fluorescence quantitative PCR result comparison graph, and (B) flow analysis result comparison graph.
FIG. 4 is a graph comparing the effect of the inhibition of NFAM1 expression on the ability of monocytes to chemotactic migration under the action of the chemokines MCP-1 or CCL5 in example 4 of the present invention.
FIG. 5 shows that inhibition of NFAM1 expression inhibits the activation of the p38 MAPK pathway in example 5 of the present invention. (A) A comparison of the effect of inhibiting NFAM1 expression on MCP-1-induced activation of the p38 MAPK pathway. (B) A comparison of the effect of inhibiting NFAM1 expression on CCL5 induced activation of the p38 MAPK pathway.
Fig. 6 is a schematic diagram of the chemical structure of AMPK agonists M1, MP, 119 used in example 6 of the present invention.
FIG. 7 is a comparative graph of Western blot results of an experimental example of screening compounds capable of regulating the expression of NFAM1 by using NFAM1 as a target in example 6 of the present invention.
Detailed Description
The following specific examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods and experimental materials used in the following examples are conventional means well known to those skilled in the art, and the reagents used therein are commercially available.
The overall experimental scheme of the invention is as follows:
the NFAT activating protein NFAM1 with ITAM motif 1 is obtained by performing transcriptome sequencing and subsequent bioinformatics analysis on monocytes selected from peripheral blood of coronary heart disease patient groups and control groups. Analysis of transcriptome results shows that the expression of NFAM1 is remarkably up-regulated in peripheral blood mononuclear cells of patients with coronary heart disease. In order to research the correlation between the NFAM1 and the occurrence and development of coronary heart disease, the inventor further collects the peripheral blood samples of 332 patients (including 79 control groups, 70 stable coronary heart diseases and 183 acute coronary syndromes), and performs flow cytometry analysis to confirm that the NFAM1 protein has significantly increased expression of peripheral blood mononuclear cells of the patients with coronary heart disease. The combination with clinical index analysis shows that NFAM1 can be used as a biomarker or a therapeutic target for early warning or prevention and treatment of coronary heart disease. Furthermore, the inventor constructs a monocyte strain for knocking down NFAM1, researches the influence of NFAM1 on the biological functions of monocytes, and finds that the inhibition of the expression level of NFAM1 can effectively reduce the expression of chemokine receptors CCR2 and CCR5 and inhibit the activation of a p38 MAPK pathway, thereby inhibiting the chemotactic migration of monocytes.
The statistical method related by the invention comprises the following steps:
the quantitative variables of normal distribution are represented by mean value plus standard deviation, and the quantitative variables of non-normal distribution are represented by median + interquartile range (IQRS); differences between groups for the quantitative variables were calculated using Student's t test, One-way or Two-way ANOVA; the Spearman test is used to assess the level of NFAM1 expression and clinical indicators or risk factors; the ROC curve is used for evaluating the prediction degree of the NFAM1 expression on the occurrence of the coronary heart disease. The expression of NFAM1 in monocytes in flow analysis was expressed as percent (%) positive, and other indices were expressed as Median Fluorescence Intensity (MFI). Data analysis using Graphpad Prism 7 and SPSS Statistics 26, p <0.05 indicated statistical differences.
Example 1 correlation of the level of human peripheral blood mononuclear cells NFAM1 with the development of coronary heart disease
The invention is applied to patients suspected of coronary atherosclerotic disease and subjected to coronary artery CT/radiography examination in the group extramons hospital between 4 months in 2018 and 11 months in 2018. The study was approved by the ethical committee of the hospital on the outside of the house, and all enrolled patients signed informed consent.
Acute coronary syndromes grouped according to the invention, including ST elevation myocardial infarction (STEMI), non-ST elevation myocardial infarction (NSTEMI) and unstable angina (Brauwald typing was determined), 183 cases; coronary artery CT/radiography shows that at least one main coronary artery (the diameter is more than or equal to 2.5mm) has more than or equal to 50 percent of stenosis, and 70 cases are diagnosed as stable coronary heart disease; the control group is 79 normal people without coronary heart disease and angina pectoris symptoms and with coronary artery CT/radiography of less than 50% stenosis.
The invention extracts peripheral blood sample and uses BD containing sodium citrate
Figure BDA0002450911980000041
CPTTMPeripheral blood mononuclear cells were isolated using vacuum blood collection tubes (BD, usa, cat # 362761) and were specifically manipulated according to the supplier's instructions. 100 μ L of NFAM1 antibody (Abcam Ebol, cat # ab203206) diluted 1:100 in PBS was added and incubated at 4 ℃ for 1 hour. PBS was washed 2 times, and a fluorescent secondary goat anti-rabbit FITC antibody FITC (BD Co., USA, cat # 554020) diluted 1:2500 in PBS was added thereto and incubated at 4 ℃ for 40 minutes. The cells were washed twice with PBS 2 times, resuspended in 100. mu.L PBS, filtered through a 40 μm filter in a flow tube, and the expression level of NFAM1 was examined using a FACSELODY flow cytometer (BD Co., USA).
The results show (fig. 1A) that the expression of NFAM1 was significantly upregulated in peripheral blood mononuclear cells from patients with coronary heart disease (p <0.0001) compared to normal controls. Further classified by disease severity, NFAM1 expression was significantly elevated in peripheral blood mononuclear cells (p <0.0001) compared to normal controls (65.31% (49.87% -78.97%)) both in stable coronary heart disease (85.44% (75.22% -91.43%)) and acute coronary syndrome (86.14% (77.71% -91.58%)). The ROC curve analysis result (fig. 1B) shows that the area under the curve (AUC) of the prediction of the coronary heart disease by the NFAM1 is 0.816 (specificity is 84.8%, and sensitivity is 65.6%), which proves that the early warning of the occurrence of the coronary heart disease can be performed by detecting the expression level of the peripheral blood mononuclear cell NFAM 1.
TABLE 1 correlation of NFAM1 expression levels in human peripheral blood mononuclear cells with clinical features of coronary heart disease
Figure BDA0002450911980000051
In table 1, the statistical correlation coefficient is Spearman correlation coefficient. When p is less than 0.05, the larger the absolute value of the correlation coefficient is, the better the correlation is, positive numbers are positive correlations, and complex numbers are negative correlations. Denotes p <0.01, denotes p < 0.05. As shown in Table 1, the expression of NFAM1 has no or weak correlation tendency with each clinical index and risk factor, and the absolute value of all correlation coefficients except total white blood cell count (WBC) is below 0.3, which indicates that the expression of NFAM1 is not influenced by other indexes and can be used as an independent early warning biomarker of coronary heart disease.
Example 2 establishment and validation of stably knockdown NFAM1 monocyte model
1. Culture of monocytic cell lines
Human histiocytic lymphoma cells (U-937) were cultured in RPMI-1640 medium (Corning, USA) containing 10% FBS, and passaged by changing the medium every 3-4 days. Centrifuge for 4 minutes at 800 rpm, and replace fresh medium. Cells grown in log phase were taken for subsequent experimental manipulations.
2. Construction of U-937 cell line stably knocking down NFAM1
U-937 cells in logarithmic growth phase are taken to be respectively infected with recombinant lentivirus (the sequence is shown as SEQ ID No. 1) containing shRNA for inhibiting NFAM1 expression and corresponding negative control (the sequence is shown as SEQ ID No. 2), the infection Multiplicity (MOI) is 10, and the infected lentivirus vector and virus particles are constructed by Beijing synbiotic gene technology Co. The medium was replaced with fresh medium 16 hours after infection, and 250ng/mL puromycin was administered 96 hours after infection for selection. After 1 week of resistance selection, the cells were plated into 96-well cell culture plates using medium containing 250ng/mL puromycin at a concentration of 0.8-1 cells/well. After culturing for 4-5 days, the wells of the single cells were observed under an inverted microscope and labeled once. After another week of culture, cell monoclonals for generating single cell populations were selected and placed in 24-well culture plates for expansion culture. The knockdown efficiency is verified by three methods, namely real-time fluorescence quantitative PCR, flow analysis and Western blot. The cell population obtained after verification is the U-937 cell strain for stably knocking down NFAM1, and the cell strain for stably transfecting the negative control lentivirus particles is used as a negative control.
3. Expression validation following NFAM1 knockdown
In the experiment, the knockdown efficiency is verified by adopting real-time fluorescence quantitative PCR, flow cytometry analysis and a Western blot method, and a cell strain of the negative control lentivirus particles is stably transfected to serve as a control.
3.1 detection of changes in expression of NFAM1 mRNA by real-time fluorescent quantitative PCR
Taking stably knocked down NFAM1 cells, negative control cells and U-937 cells which are not subjected to transfection treatment in logarithmic growth phase, counting and collecting 1 × 10 cells6Individual cell, 800 turning centrifugeAfter 4 minutes, the supernatant was discarded. For the experiment, total RNA of cells was extracted by TRIzol method (American Satemishuhell technology, cat # 15596-
Figure BDA0002450911980000063
One-step cDNA Synthesis kit, Beijing Quanji gold organism, cat # AT311), use
Figure BDA0002450911980000064
The qPCR kit (Beijing Quanji gold biology, Cat. AQ142) is placed in the fluorescent quantitative PCR instrument of ABI7900HT USA (applied biosystems, USA) to detect the gene expression level. Relative expression of the gene was calculated by the Δ Δ Ct method using GAPDH as an internal control. Specific amplification NFAM1 primer was synthesized by Jinzhi Biotechnology, Inc., Suzhou, with the following sequence:
Figure BDA0002450911980000061
the qPCR reaction system was as follows:
Figure BDA0002450911980000062
Figure BDA0002450911980000071
the qPCR reaction conditions were as follows:
Figure BDA0002450911980000072
3.2 flow cytometry analysis method is adopted to detect the expression change of NFAM1 protein
Taking stably knocked down NFAM1 cells, negative control cells and U-937 cells which are not subjected to transfection treatment in logarithmic growth phase, counting and collecting 1 × 10 cells6The cells were centrifuged at 800 rpm for 4 minutes and the supernatant was discarded. The cell pellet was washed 2 times with 1 × PBS buffer and centrifuged for 4 minutes at 800 rpm. Adding 100 muL1: 500 Primary anti-NFAM 1 antibody (Abcam Ebol, cat # ab203206) diluted in PBS was incubated at 4 ℃ for 1 hour. PBS washing 2 times, adding 1:100 u L1: 2500 diluted in PBS Alexa
Figure BDA0002450911980000073
647 goat anti-rabbit secondary antibody (Eboanti, cat # ab150079), incubated at 4 ℃ for 40 minutes. The cells were washed twice with PBS 2 times, resuspended in 100. mu.L PBS, filtered through a 40 μm filter in a flow tube, and the expression level of NFAM1 was measured using a BD FACSIMODY flow cytometer.
3.3 detection of NFAM1 protein expression Change by Western blot method
Taking stably knocked down NFAM1 cells, negative control cells and U-937 cells which are not subjected to transfection treatment in logarithmic growth phase, counting and collecting 1 × 10 cells6The cells were centrifuged at 800 rpm for 4 minutes and the supernatant was discarded. The cell pellet was washed 2 times with pre-chilled 1 × PBS buffer and centrifuged for 4 min at 800 rpm each time. mu.L of a pre-cooled RIPA cell lysate (Beijing prilley Gene technology, Inc., cat # C1053+) containing a protease inhibitor (Roche, cat # 04693159001) and a phosphatase inhibitor (Roche, cat # 4906845001) was added, total protein in the cells was extracted, and the protein concentration was measured using a BCA method kit (Beijing prilley gene technology, Inc., cat # P1511).
10% separation gel and 5% concentrated gel were used in this experiment, with a protein loading of 15 μ g. After membrane transfer, the PVDF membrane was incubated with NFAM1 antibody (Sigma Aldrich, cat # HPA031812,1:1250 dilution) overnight at 4 ℃. The cells were washed three times with 1 XTSS-T, and incubated for one hour at room temperature with horseradish-conjugated goat anti-rabbit secondary antibody (Ebos, cat # ab6721,1:5000 dilution). After washing three times at 1 × TBS-T, the NFAM1 expression level was detected by exposure to light using Shanghai office ChemiScope series chemiluminescence imaging System (3300mini) and ECL detection kit (GE general-purpose company, cat # RPN2235) with β -actin as an internal reference.
4. Results
The invention adopts a slow virus infection mode, and inoculates the strain in a 96-well plate to screen cell monoclonals at the concentration of 0.8-1 cell/well after resistance screening is added for one week. By matching with a flow analysis method or a Western blot and a real-time fluorescence quantitative PCR method, the method can obtain the cell monoclonal of the stably knocked-down target protein within 3-4 weeks. Compared with the traditional infinite dilution method, the method saves more reagents and consumables, about 30 monoclonal cell strains can be obtained from each 96-well plate, and the protein level expression is stable. In the experiment, a U-937 monoclonal cell strain which stably knockdown NFAM1 is selected and identified for the function and application research of the protein through real-time fluorescence quantitative PCR (figure 2A), flow cytometry (figure 2B) and Western blot (figure 2C).
Example 3 inhibition of the Effect of NFAM1 expression on monocyte chemokine receptor expression
1. Chemokine receptor transcript level detection
The invention uses real-time fluorescence quantitative PCR to detect the transcription levels of the chemokine receptors CCR2 and CCR5 related to coronary heart disease. For the experiment, total RNA of cells was extracted by TRIzol method (American Satemishuhell technology, cat # 15596-
Figure BDA0002450911980000081
One-step cDNA Synthesis kit, Beijing Quanji gold organism, cat # AT311), use
Figure BDA0002450911980000082
The qPCR kit (Beijing Quanji gold biology, Cat. AQ142) is placed in the fluorescent quantitative PCR instrument of ABI7900HT USA (applied biosystems, USA) to detect the gene expression level. Relative expression of the gene was calculated by the Δ Δ Ct method using GAPDH as an internal control. Primers for specific amplification CCR2 and CC5 were synthesized by Soviet Jinzhi Biotechnology, Inc., and the sequences were as follows, and the qPCR reaction system and reaction conditions were the same as those in example 2.
Figure BDA0002450911980000083
2. Chemokine receptor protein level detection
Taking stably knocked down NFAM1 cells, negative control cells and U-937 cells which are not subjected to transfection treatment in logarithmic growth phase, counting and collecting 1 × 10 cells6Centrifuging for 4 minutes at 800 rpm, discarding the supernatant. The cell pellet was washed 2 times with 1 × PBS buffer and centrifuged for 4 min at 800 rpm. 100 μ L of either a coupled APC anti-human CD192(CCR2) antibody or a coupled PE/Cy7 anti-human CD195(CCR5) antibody (BioLegend) diluted 1:200 in PBS was added and incubated at 4 ℃ for 40 minutes. The cells were washed twice with PBS 2 times, resuspended in 100 μ L PBS, filtered through a 40 μm filter in a flow tube, and the expression levels of chemokine receptor proteins CCR2 and CCR5 were detected using a BD facmolody flow cytometer.
3. Results
The invention proves that chemokine receptors CCR2 and CCR5 related to coronary heart disease endogenously expressed by monocytes are remarkably reduced in both transcription level (figure 3A) and protein level expression (figure 3B) by inhibiting the expression of NFAM1 in the monocyte cell line, and p is less than 0.0001. Compared with the negative control group, the CCR2 mRNA level was reduced by 77.8%, the protein level was reduced by 58.2%, the CCR5 mRNA level was reduced by 64.4%, and the protein level was reduced by 51.6%.
Example 4 inhibition of the Effect of NFAM1 expression on monocyte chemotaxis
This example uses
Figure BDA0002450911980000084
The chamber (Corning Corp.) measures the chemotactic capacity of monocytes. One day before the experiment, stable knock-down NFAM1 cells and negative control U-937 cells in logarithmic growth phase are taken, and the cell concentration is adjusted to be 1 × 106mL, starved for 24 hours in RPMI-1640 medium with 0.5% fetal bovine serum. On the day of the experiment, 600. mu.L of a medium containing 100ng/mL of recombinant human MCP-1 (i.e., CCL2, cat # 300-04) or 100ng/mL of recombinant human RANTES (i.e., CCL5, cat # 300-06) (both available from PeproTech) in a chamber (diameter 6.5mm, pore size of a semi-permeable polycarbonate membrane 5.0 μm) containing 0.5% fetal bovine serum RPMI-1640 was added to the chamber, and a blank medium containing no chemokine was used as a negative control for migration background, and the medium was equilibrated at 37 ℃. Add 100. mu.L of 2X 10 to the upper chamber5Cells were cultured in a 37 ℃ incubator for 4 hours, and then the cells in the lower chamber were collected, centrifuged, resuspended in PBS, and the number of cells transferred to the lower chamber was measured using a BD FACCSmolody flow cytometer. The relative mobility of the cells was calculated.
Figure BDA0002450911980000091
The results show (fig. 4) that NFAM1 is closely related to monocyte chemotaxis, and the chemotactic ability of monocytes under the action of human recombinant chemokines MCP-1 and CCL5 was reduced by 81% (p <0.0001) and 42.4% (p ═ 0.0003), respectively, after knockdown of its expression.
Example 5 Effect of inhibiting NFAM1 expression on chemokine-induced activation of the p38 MAPK pathway
This example examined the effect of inhibiting NFAM1 expression on the p38 MAPK signaling pathway by using the Western blot method.
One day before the experiment, stable knock-down NFAM1 cells and negative control U-937 cells in logarithmic growth phase are taken, and the cell concentration is adjusted to be 1 × 106mL, starved for 24 hours in serum-free RPMI-1640 medium. On the day of the experiment, serum-free FBS RPMI-1640 medium containing 100ng/mL of recombinant human MCP-1 (i.e., CCL2, cat # 300-04) or 100ng/mL of recombinant human RANTES (i.e., CCL5, cat # 300-06) (both purchased from PeproTech) was added to the cells and equilibrated in a 37 ℃ incubator. Cells were harvested after 0, 10, 30, 60, 120, 240 minutes of chemokine action, respectively. The specific implementation steps of cell lysis, total protein extraction and determination and Western blot are the same as in example 3. Primary antibodies included phospho-p 38 MAPK rabbit monoclonal antibody (cat 4511) and p38 MAPK rabbit monoclonal antibody (cat 8690) (both available from CST). Beta-actin mouse monoclonal antibody (Beijing holotype Jinbiol, cat # HC201-01) is used as the internal reference. The corresponding secondary antibody is goat anti-secondary antibody (Aibo anti company, cat # ab6721) marked by horseradish enzyme or goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao biotechnology, Co., cat # ZB2305) marked by horseradish enzyme, and the dilution ratio is 1: 5000. The strips were exposed using the Shanghai flight ChemiScope series chemiluminescence imaging System (3300mini) and ECL detection kit (GE general Inc., cat # RPN2235), and subjected to grayscale analysis using Image J software.
The results show (FIG. 5) that the expression of phosphorylated p38 MAPK in monocytes increases gradually with time upon stimulation by the chemokines MCP-1 and CCL 5. 30 minutes after MCP-1 stimulation, the activation degree of the p38 MAPK pathway in NFAM1 knockdown cells shows a remarkable inhibition trend compared with a control group, the activation level is reduced by 58.2 percent (p is 0.047), the inhibition is 49.8 percent (p is 0.036) after 60 minutes, and the inhibition degree is highest at 120 minutes and reaches 69.1 percent (p is less than 0.0001). After inhibiting NFAM1 expression, activation of the p38 MAPK pathway by CCL5 stimulation also showed inhibition tendency, which was 52.8% (p ═ 0.0008) after 120 min and reached 56.6% (p ═ 0.0006) at 240 min.
Example 6 example of screening for Compounds that modulate the expression of NFAM1 with NFAM1 as target
In the embodiment, a Western blot method is used for detecting the regulation and control effect of a series of compounds on the expression of NFAM1, and the regulation and control effect is taken as an application example of using NFAM1 as a target in searching or screening medicines or biological preparations for preventing and treating coronary heart disease and coronary heart disease related diseases.
The day before the experiment, taking U-937 cells in logarithmic growth phase, adjusting cell concentration to 1 × 106mL, starved overnight in serum-free RPMI-1640 medium. On the day of the experiment, the specific inhibitors of AMPK, compound C (20. mu.M/L), were added to the cells, respectively. After incubation in a cell culture chamber at 37 ℃ for 2h, DMSO (Sigma Aldrich, cat # D2650), AICAR (Shanghai ceramic Biotechnology Co., Ltd., cat # T1477,1mmol/L), A-769662 (Shanghai ceramic Biotechnology Co., Ltd., cat # T2468,100. mu. mol/L), M1 (100. mu. mol/L), 119 (100. mu. mol/L) and MP (1. mu. mol/L) were added to each well. Wherein M1 is a main metabolite of IMM-H007 in vivo, which is a novel AMPK agonist developed by the institute of medicine of Chinese medical sciences, MP is a metabolite and a main active molecule of M1, and 119 is a homolog of IMM-H007. M1, 119 and MP were synthesized by the pharmaceutical institute and the structures are shown in FIG. 6. After addition of the compound, the cells were collected after incubation in a cell incubator at 37 ℃ for 24 hours.
The specific implementation steps of cell lysis, total protein extraction and determination and Western blot are the same as in example 3. The first antibody is NFAM1 rabbit polyclonal antibody (Sigma Aldrich, cat # HPA031812,1:1250 dilution), and beta-actin mouse monoclonal antibody (Beijing holo-type gold organism, cat # HC201-01) as internal reference. Primary antibody was incubated overnight at 4 ℃. The corresponding secondary antibody is goat anti-secondary antibody (Aibo anti company, cat # ab6721) marked by horseradish enzyme or goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao biotechnology, Co., cat # ZB2305) marked by horseradish enzyme, and the dilution ratio is 1: 5000. The strips were exposed using the Shanghai flight ChemiScope series chemiluminescence imaging System (3300mini) and ECL detection kit (GE general Inc., cat # RPN2235), and subjected to grayscale analysis using Image J software.
The result is shown in fig. 7, the compound used in this example has no significant effect on the protein expression level of NFAM1 in U-937 monocytes, but the experimental method used in this example can be used as an example of the application of NFAM1 as a target in searching or screening drugs or biological agents for preventing and treating coronary heart disease and diseases related to coronary heart disease.
The foregoing is merely a general description and preferred embodiments of the invention, and is not intended to limit the invention thereto. Any modification, equivalent replacement or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
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Claims (10)

1. The application of NFAT activation protein NFAM1 with ITAM motif 1 in searching or screening drugs or biological preparations for preventing and treating coronary heart disease and diseases related to coronary heart disease.
2. The use according to claim 1, wherein said coronary heart disease comprises occult or asymptomatic coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease and sudden death; the coronary heart disease related diseases comprise atherosclerosis.
3. The use according to any one of claims 1 or 2, wherein said use for finding or screening a drug or biological agent for preventing or treating coronary heart disease or a disease associated with coronary heart disease is to screen a drug or biological agent for inhibiting the expression of NFAM1 by determining whether the expression level of NFAM1 in monocytes is changed or not as compared to a control group.
4. The NFAT activation protein NFAM1 with ITAM motif 1 is applied to the preparation of a kit for early warning, early diagnosis or prognosis evaluation of coronary heart disease and coronary heart disease-related diseases.
5. The use according to claim 4, wherein said coronary heart disease comprises occult or asymptomatic coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease and sudden death; the coronary heart disease related diseases comprise atherosclerosis.
6. The use according to any of claims 4 or 5, wherein said early warning, early diagnosis or prognosis of coronary heart disease, or a coronary heart disease-associated disease is determined by determining whether the expression level of NFAM1 in monocytes is increased in a blood sample of said subject compared to a control group.
7. The use according to claim 6, wherein said reagents for determining the expression of NFAM1 on monocytes in a blood sample from said individual are primers and/or probes and/or antibodies for the detection of NFAM 1.
8. Application of shRNA shown as SEQ ID No.1 in preparation of a reagent for regulating and controlling chemokine receptor CCR2 and/or CCR 5.
9. The shRNA shown as SEQ ID No.1 is applied to the preparation of a reagent for inhibiting the chemotaxis or migration of monocytes.
10. The shRNA shown as SEQ ID No.1 is applied to the preparation of MAPK inhibitor.
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