CN114686424B - Method for regulating and controlling C/EBP alpha gene expression and C/EBP alpha expression inhibitor - Google Patents
Method for regulating and controlling C/EBP alpha gene expression and C/EBP alpha expression inhibitor Download PDFInfo
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Abstract
The application discloses a method for regulating and controlling C/EBP alpha gene expression and a C/EBP alpha expression inhibitor, wherein cells are treated by 1, 7-diphenyl-4-hepten-3-one and/or derivatives thereof to regulate and control the C/EBP alpha gene expression of the cells. The derivative is 5-hydroxy-1, 7-diphenyl-4-hepten-3-one. The method has the advantages of simple operation, low administration concentration, obvious effect and easy popularization and application. The method provides a foundation for the research of the C/EBP alpha gene function and the treatment of related diseases.
Description
Technical Field
The application relates to the technical field of gene regulation, in particular to a method for regulating and controlling C/EBP alpha gene expression and a C/EBP alpha expression inhibitor.
Background
The C/EBP alpha gene is CCAAT/enhancer binding protein alpha gene, and the coded CCAAT/enhancer binding protein is an important transcription regulating factor and is an important member of transcription factor C/EBP family. The transcription factor C/EBP alpha exists in liver, lung and mammary gland and plays an important role in the processes of cell differentiation, cell metabolism, inflammatory reaction, tumorigenesis, apoptosis and the like. For example, YAMAMOTO et al analyzed the expression of C/ebpa in certain chronic inflammatory diseases and found that C/ebpa was expressed at higher levels in synovial tissue of rheumatoid arthritis and liver tissue of chronic viral hepatitis, whereas only a small number of C/ebpa positive cells were detected in mucosal cells of ulcerative colitis. Milan et al detected the expression of C/EBP alpha in colorectal cancer, found that C/EBP alpha was highly expressed in colorectal cancer tissue, down-regulating C/EBP alpha inhibited colon cancer cell proliferation, promoted apoptosis, and retarded cell cycle.
Currently, there is little research on how to regulate the expression of the C/EBP alpha gene. Zhang Tian and the like analyze possible action mechanisms of the left pill and the right pill for treating postmenopausal osteoporosis, and the results show that the left pill and the right pill can improve the bone marrow microenvironment of an ovariectomized rat, and the mechanisms of the left pill and the right pill can be related to the inhibition of femoral fat differentiation and the expression of bone marrow PPARgamma, C/EBP beta and C/EBP alpha proteins. Zhangyu et al examined the effect of inhaled formaldehyde exposure on mRNA and protein expression levels of the transcription factors GATA-2 and CCAAT/enhancer binding protein (C/EBPα) in mouse bone marrow tissue. The results show that high concentration formaldehyde exposure can inhibit normal expression of GATA-2 and C/EBP alpha mRNA and protein in mouse bone marrow tissue. There is no report on the treatment of cells with 1, 7-diphenyl-4-hepten-3-one and/or its derivatives to regulate the expression of the C/ebpα gene in the cells.
Disclosure of Invention
Based on the prior art, the application provides a method for regulating and controlling the expression of C/EBP alpha genes and a C/EBP alpha expression inhibitor, wherein 1, 7-diphenyl-4-hepten-3-one and/or derivatives thereof are used for treating cells to regulate and control the expression of the C/EBP alpha genes of the cells.
The scheme of the application comprises the following contents:
a method of modulating C/ebpα gene expression comprising: the cells are treated with 1, 7-diphenyl-4-hepten-3-one and/or derivatives thereof to regulate cellular C/ebpα gene expression.
Preferably, the derivative is 5-hydroxy-1, 7-diphenyl-4-hepten-3-one.
Preferably, the modulation is down-modulation.
Preferably, the cell is a 3T3-L1 cell.
Preferably, the treated cells are: cells were incubated for 16 hours for dosing.
Preferably, the administration concentration is 1 to 40. Mu.M.
More preferably, the concentration of administration is 30. Mu.M.
In another aspect, the present application also provides an inhibitor of C/ebpα expression, which contains 1, 7-diphenyl-4-hepten-3-one or a derivative thereof.
The 1, 7-diphenyl-4-hepten-3-one of the application may be prepared according to the method described in applicant's earlier patent application CN 112552155A. Of course, the skilled person can also carry out the artificial synthesis of the compound according to the general synthetic principles.
The preparation method of the 5-hydroxy-1, 7-diphenyl-4-hepten-3-one comprises the following steps:
preparing an aqueous extract of galangal: air drying and pulverizing rhizoma Alpiniae Officinarum, extracting 500g with 95% ethanol for 3 times (1 hr each time), air drying the residue, extracting with 2L water for 3 times (1 hr each time), mixing the water extracts, concentrating to 250ml, adding 937.5ml of 95% ethanol to obtain a solution, standing in 4deg.C refrigerator for 30 hr to obtain supernatant, and concentrating to obtain rhizoma Alpiniae Officinarum water extract.
Taking an aqueous extract of galangal, loading the aqueous extract of galangal into a sample by adopting a silica gel column chromatography dry method, and after loading the sample into the column, respectively eluting with chloroform according to the volume ratio: methanol 100: 1. 100: 2. 100: 3. 100: 6. 100: 10. 100: 15. 100: 20. 100: 25. 100:30 were flushed at 9 gradients. At 100:10, obtaining a first flushing material, carrying out rotary evaporation, taking 0.4g of the first flushing material after rotary evaporation, loading on a gel column, eluting with methanol to obtain 1-82-3 parts by weight of petroleum ether as a mixture: ethyl acetate 5:1.5 spread out dot plates are shown below in fig. 1. Spin-steaming 1-82-3 parts, filtering with methanol-dissolving microporous filter membrane, and preparing liquid phase instrument with mobile phase A of methanol and mobile phase B of water; when A: b=70: 30, grafting peak at 23-25.5min, developing agent petroleum ether: ethyl acetate 5:1.5 expansion Point plates were identified as monomers (see FIG. 2) identified as 5-hydroxy-1, 7-diphenyl-4-hepten-3-one by nuclear magnetic resonance.
The above preparation method of 5-hydroxy-1, 7-diphenyl-4-hepten-3-one is only an example provided by the present application, and the skilled person can make appropriate adjustments to obtain the compound more efficiently and rapidly, or can obtain the compound by artificial synthesis according to general synthesis principles.
Compared with the prior art, the application has the beneficial effects that:
the application discloses a method for regulating and controlling C/EBP alpha gene expression, which can effectively regulate and control the C/EBP alpha gene expression through 1, 7-diphenyl-4-hepten-3-one or derivatives thereof.
The method has the advantages of simple operation, low administration concentration, obvious effect and easy popularization and application. The method provides a foundation for the research of the C/EBP alpha gene function and the treatment of related diseases.
Drawings
Fig. 1:1-82-3 flow point plate diagram
Fig. 2: dot plate diagram after liquid phase preparation
Fig. 3: effect of 5-hydroxy-1, 7-diphenyl-4-hepten-3-one on C/ebpα gene expression (note: #p is blank vs model, #p <0.05, #p <0.01, #p < 0.001;: < p <0.05, < p <0.01, < p < 0.001: < p: < model vs dosing)
Fig. 4: effect of 1, 7-diphenyl-4-hepten-3-one on C/ebpα gene expression (note: #p is blank vs. model, #p <0.05, #p <0.01, #p < 0.001;: -p is model vs. dosing,: -p <0.05,: -p <0.01,: -p < 0.001)
Detailed Description
For a clear and complete description of the technical solutions of the present application, the inventors have described with reference to the examples and the accompanying drawings, but the following examples describe only some, but not all, of the examples of the present application.
Examples
1 materials, reagents and instruments
1.1 Experimental materials
3T3-L1 cells (available from Shanghai Biotechnology Co., ltd.), high-sugar DMEM medium (Gibco), low-sugar DMEM medium (Biosharap), 10% calf serum (BI), 10% fetal calf serum (BI) and 1% penicillin/streptomycin (Biosharp), 0.25% pancreatin (Biosharp), EDTA-free 0.25% pancreatin (Biosharp), serum-free cell cryopreservation (New Saimago Biotechnology Co., ltd.), insulin (Sigma), 3-isobutyl-1-methylxanthine (Sigma), dexamethasone (Sigma), PBS buffer (Biosharp), CCK8 reagent (APExBIO), 2-NBDG (APExBIO), oil red O kit (Solaibio), rosiglitazone (ma), 5-hydroxy-1, 7-diphenyl-4-hepten-3-one (laboratory preparation with a purity of 99%), 1, 7-diphenyl-4-hepten-3-one (laboratory preparation with a purity of 99%).
1.2. Instrument for measuring and controlling the intensity of light
Range-adjustable pipette, 0.22 mu m microporous filter membrane, freezing tube and constant temperature CO 2 Cell incubator, superclean bench, constant temperature water bath, centrifuge, precise electronic balance, molecular Devices full wavelength enzyme label instrument, molecular Devices fluorescence enzyme label instrument, -80 refrigerator, 24 pore plate, 96 pore plate.
2. Experimental method
2.1 cell culture and differentiation
3T3-L1 cells were cultured in high-sugar DMEM complete medium containing 10% Calf Serum (CS) and 1% penicillin-streptomycin at 37deg.C, 5% CO 2 Is cultured in a cell culture vessel. After the number of the cells reaches about 80%, discarding the culture solution, adding 2mL of PBS solution for washing for 1 time, adding 0.25% of trypsin digestion solution for digestion, stopping digestion with a complete culture medium after 1-2 minutes, carrying out cell passage, and taking the cells in the logarithmic phase for subsequent experiments.
Cells were seeded in 96-well plates and cultured in high-sugar DMEM medium containing 10% cs and 1% penicillin-streptomycin until cells were inhibited by contact and fused for 2 days before induction of differentiation was initiated. Cells were fused for two days, and cultured for 4 days after adding 10% FBS high sugar DMEM medium containing 1. Mu.M dexamethasone (Dex), 10. Mu.g/mL insulin (Ins) and 0.5mM isobutyl methylxanthine (IBMX), the medium was changed once for two days, and the 10% FBS high sugar DMEM medium containing 10. Mu.g/mL insulin was added for 4 days, and then the medium was changed to DMEM medium containing 10% fetal bovine serum for culture, and the medium was changed once every two days until more than 90% of cells were differentiated and matured.
2.2 cell viability experiments
Taking cells in logarithmic growth phase, preparing single cell suspension, regulating cell concentration, inoculating 3T3-L1 cells into 96-well plate, and filling the edge with sterile PBS. 37 ℃ and 5% CO 2 Incubating under the condition, adding normal culture solution into blank group after cell monolayer is full of hole bottom by about 80%, adding freshly prepared culture solution of 5-hydroxy-1, 7-diphenyl-4-hepten-3-one (1, 5, 10, 20, 40, 80, 100. Mu.M) or culture solution of 1, 7-diphenyl-4-hepten-3-one (1, 5, 10, 20, 30, 40, 60. Mu.M) with different concentrations into drug group. 200 μl per well, 6 complex wells per group. After 16 hours of incubation for dosing, CCK-8 reagent (1. Mu.L/well) was added to the plate and the cells incubated for 2 hours at 37 ℃. Absorbance was measured at 450nm using a microplate reader.
2.3 cell grouping
After washing the cells induced to differentiate and mature with PBS, the cells were subjected to model culture for 96 hours in a conventional medium containing dexamethasone at a final concentration of 1. Mu.M, and a fat cell lipid metabolism disorder model was established. Taking the 3T3-L1 cells which induce differentiation and maturation, and dividing the 3T3-L1 cells into a blank group, a model group, a positive medicine group and a medicine group. Wherein the blank group is not modeled, and is cultured by a DMEM high-sugar culture medium containing 10% fetal bovine serum; the model group was the model set with no drug intervention, the positive drug group was the model set with 20. Mu.M rogridone intervention (16 hours post-dosing) and the drug group was the model set with 30. Mu.M 5-hydroxy-1, 7-diphenyl-4-hepten-3-one intervention (16 hours post-dosing).
2.4 detection of C/EBP alpha Gene mRNA levels
The gene expression level of C/EBP alpha was detected by quantitative polymerase chain reaction (qPCR). Using EastepSuper Total RNA Extraction Kit (Classification LS1040, shanghai Prolomei Biotechnology Co., ltd.) from mature 3T3-L1 pre-cellsTotal RNA was taken and the quality of the extracted RNA was confirmed using a nanophotometer-N50 ultra-microscopic spectrophotometer (Implen, germany). Using Hifair III 1 st Strand cDNA Synthesis SuperMix for qPCR (product No. 11141ES60, division of Saint Biotechnology (Shanghai)) RNA reverse transcription kit was used to reverse transcribe RNA samples into cDNA, and the PCR amplification kit was Hieff qPCR SYBR Green Master Mix (product No. 11202ES08, division of Saint Biotechnology (Shanghai)), and the C/EBP alpha primer was synthesized by the design of Seikovia Biotechnology, division of Korea, and the primer sequences are shown in Table 1. The PCR amplification was performed under the following conditions: the hot start DNA polymerase was initially activated at 95℃for 5 minutes, followed by 40 cycles of the second step (i.e., 95℃for 10 seconds, 58℃for 20 seconds, 72℃for 20 seconds). Then the melting curve stage is carried out, and finally the amplification primer is obtained. The relative expression of the C/EBP alpha gene was analyzed by the-DeltaCt method and GAPDH was used as an internal reference. The qPCR primer sequences are shown in Table 1.
TABLE 1qPCR C/EBP alpha primer sequences
2.5 statistical analysis
Analytical data were collated with statistical analysis software graphpadprism5.0 and experimental data were expressed as mean ± standard deviation (x±s). The difference between groups is tested by t test, the variance is uneven by non-parameter test, the comparison between groups is analyzed by single factor variance, and P is less than 0.05, namely the difference is considered to have statistical significance.
2.6 results and analysis
Cell viability assay results:
the 5-hydroxy-1, 7-diphenyl-4-hepten-3-one significantly inhibited the cell activity at the administration concentration of 80-100. Mu.M, with cell viability of 95.01% (80. Mu.M) and 88.63% (100. Mu.M), respectively. While the dosing concentration of 1-40 μm had no significant effect on the cells. Thus, a concentration of 30. Mu.M was used as the dosing concentration for subsequent experiments with 5-hydroxy-1, 7-diphenyl-4-hepten-3-one.
1, 7-diphenyl-4-hepten-3-one significantly inhibited this cell activity at an administration concentration of 60. Mu.M, with cell viability of 87.32%, respectively. While the dosing concentration of 1-40 μm had no significant effect on the cells. Thus, 30. Mu.M was used as the dosing concentration for subsequent experiments with 1, 7-diphenyl-4-hepten-3-one.
C/EBP alpha gene mRNA level detection results:
the C/EBP alpha gene expression was significantly increased in the model group compared to the blank group, and rosiglitazone as a PPARgamma agonist could up-regulate C/EBP alpha gene expression by agonizing PPARgamma, consistent with our results. And after pretreatment of 5-hydroxy-1, 7-diphenyl-4-hepten-3-one and 1, 7-diphenyl-4-hepten-3-one for 16 hours, the C/EBP alpha gene expression of the administration group is obviously reduced, which indicates that the 5-hydroxy-1, 7-diphenyl-4-hepten-3-one can inhibit the C/EBP alpha gene expression. (FIG. 3, FIG. 4)
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.
Claims (2)
- Use of 5-hydroxy-1, 7-diphenyl-4-hepten-3-one for the preparation of a reagent for down-regulating C/EBP alpha gene expression in a cell in vitro, characterized in that the cell is 3T3-L1 cells and that the concentration of 5-hydroxy-1, 7-diphenyl-4-hepten-3-one in the reagent is 30 μm by treating the cell in vitro with the reagent to down-regulate C/EBP alpha gene expression in the cell.
- 2. The use according to claim 1, wherein the time to treat the cells is 16 hours.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6434942A (en) * | 1987-07-30 | 1989-02-06 | Yakult Honsha Kk | Cycloheptenone derivative and remedy for hepatopathy containing said derivative |
| JP2000217576A (en) * | 1999-02-02 | 2000-08-08 | Herikkusu Kenkyusho:Kk | Induction of differentiation into adipocyte, compound which controls differentiation into adipocyte, and its screening |
| CA2930973A1 (en) * | 2013-11-22 | 2015-05-28 | Pal SAERTROM | C/ebp alpha short activating rna compositions and methods of use |
| CN107841556A (en) * | 2017-12-19 | 2018-03-27 | 武汉大学 | A kind of kit and application based on C/EBP α and IGF1R genescreen medicine development toxicities |
| CN112552155A (en) * | 2020-12-23 | 2021-03-26 | 海南医学院 | 1, 7-diphenyl-4-heptylene-3-ketone separated from galangal and application thereof |
| CN112645808A (en) * | 2021-01-05 | 2021-04-13 | 海南医学院 | 5-hydroxy-1, 7-diphenyl-3-heptanone separated from galangal and application thereof |
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Patent Citations (6)
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|---|---|---|---|---|
| JPS6434942A (en) * | 1987-07-30 | 1989-02-06 | Yakult Honsha Kk | Cycloheptenone derivative and remedy for hepatopathy containing said derivative |
| JP2000217576A (en) * | 1999-02-02 | 2000-08-08 | Herikkusu Kenkyusho:Kk | Induction of differentiation into adipocyte, compound which controls differentiation into adipocyte, and its screening |
| CA2930973A1 (en) * | 2013-11-22 | 2015-05-28 | Pal SAERTROM | C/ebp alpha short activating rna compositions and methods of use |
| CN107841556A (en) * | 2017-12-19 | 2018-03-27 | 武汉大学 | A kind of kit and application based on C/EBP α and IGF1R genescreen medicine development toxicities |
| CN112552155A (en) * | 2020-12-23 | 2021-03-26 | 海南医学院 | 1, 7-diphenyl-4-heptylene-3-ketone separated from galangal and application thereof |
| CN112645808A (en) * | 2021-01-05 | 2021-04-13 | 海南医学院 | 5-hydroxy-1, 7-diphenyl-3-heptanone separated from galangal and application thereof |
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| Title |
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