CN114377024A

CN114377024A - Medicine for treating and/or preventing hyperlipemia

Info

Publication number: CN114377024A
Application number: CN202111610067.3A
Authority: CN
Inventors: 郭志刚; 曹世平; 屠燕; 潘杭雨; 郭凯; 张亚南; 杨婵娟; 刘丹
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-04-22
Anticipated expiration: 2041-12-27
Also published as: CN114377024B

Abstract

The invention discloses a medicine for treating and/or preventing hyperlipidemia diseases, which comprises one or the combination of more than two of mithramycin, mithramycin prodrug and pharmaceutically acceptable mithramycin salt. The medicine of the invention can inhibit the formation of foam cells, regulate the metabolism of cholesterol and lipid, and further effectively treat and prevent hyperlipidemia diseases.

Description

Medicine for treating and/or preventing hyperlipemia

Technical Field

The invention relates to the field of pharmacy, in particular to a medicament for treating and/or preventing hyperlipidemia diseases.

Background

Hyperlipidemia refers to a series of diseases seriously harmful to human health, such as atherosclerosis, coronary heart disease, fatty liver, etc. caused by high blood lipid level. Such as atherosclerosis, it has been found through research that (PSRC 1 overexpression regulates cholesterol transport and inflammation inhibition mouse atherosclerosis, a university of medical southern, doctor's academic thesis on level 2015, the author: Guokay, the filing date of the thesis: 2018, 5/26), macrophage formation, lipid metabolism disorder and cholesterol metabolism on the arterial wall are closely related to atherosclerotic diseases. Specifically, an increase in cholesterol uptake or a decrease in cholesterol efflux can result in an increase in lipid content in macrophages, forming foam cells; the research shows that the PSRC1 gene of the macrophage can down-regulate the SR-A1 and LDLR of the macrophage to reduce the intake of cholesterol by the macrophage, and can up-regulate the expression of ABCA1, ABCG1 and SR-B1 to promote the transfer of cholesterol, thereby reducing the content of cholesterol ester in the macrophage and inhibiting the formation of foam cells. In addition, the PSRC1 gene can increase cholesterol transfer rate and PON-1 activity, reduce MPO activity, MPO/PON-1 ratio and HII, and regulate cholesterol metabolism, including LDL-C and HDL-C levels, even HDL function; in addition, the overexpression of the liver cell PSRC1 can increase the expression of PPAR-gamma and LXR-alpha and play a role in lipid metabolism.

From the above, it is known that the prevention and treatment of hyperlipidemia diseases such as atherosclerosis can be achieved by regulating the expression of PSRC 1. Therefore, the search for a drug capable of improving the expression of the PSRC1 gene becomes the key for treating and preventing hyperlipidemia diseases.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a medicament for preventing and/or treating hyperlipidemia diseases (namely application/use of mithramycin in preparing medicaments for treating hyperlipidemia diseases).

In order to achieve the purpose, the invention adopts the following technical scheme:

a medicament for treating and/or preventing hyperlipidemic diseases, which comprises one or the combination of more than two of mithramycin, mithramycin prodrug and pharmaceutically acceptable mithramycin salt.

In the invention, a further preferable scheme is that the hyperlipemia disease is atherosclerosis, coronary heart disease or fatty liver.

In the present invention, it is further preferable that the drug is an oral solid preparation, an oral liquid preparation, or an injection.

In the present invention, it is further preferable that the oral solid preparation is a tablet, a capsule, a granule, a pill, or a powder.

In the present invention, it is further preferable that the drug is a sustained release preparation.

Compared with the prior art, the invention has the beneficial effects that through research, SP1 can be combined with the open region of PSRC1 gene and inhibit the expression of PSRC1 gene, and mithramycin serving as an inhibitor of SP1 can reduce the level of SP1 and further can up-regulate the expression of PSRC1 gene, so that the expression of ABCA1, ABCG1, SR-B1, PPAR-gamma and LXR-alpha is up-regulated, macrophages SR-A1 and LDLR are down-regulated, the cholesterol transfer rate and PON-1 activity can be increased, the MPO activity, the MPO/PON-1 ratio and HII are reduced, the formation of foam cells is inhibited, the metabolism of cholesterol and lipid is regulated, and then hyperlipidemia diseases (such as atherosclerosis) are treated and/or prevented.

Drawings

FIG. 1 is an electrophoretogram of 3.2 parts of western blot in Experimental example 1 for detecting the expression of a target protein;

FIG. 2 is a graph of ChIP-qPCR enrichment analysis data for the mithramycin-treated group and the control group in Experimental example 1;

FIG. 3 is a graph of ChIP-qPCR enrichment analysis data for SP 1-specific antibody and IgG non-specific antibody in Experimental example 1;

FIG. 4 is a diagram showing the binding sites of SP1 and PSRC1 promoter predicted in Experimental example 2;

FIG. 5 is a map of an SP1 protein expression vector plasmid (pcDNA3.1+ plasmid) in Experimental example 2;

FIG. 6 is a map of a luciferase expression vector plasmid (PGL-3 plasmid) in Experimental example 2;

FIG. 7 is a graph showing data on relative expression amounts of luciferase after cells were transfected with three different plasmids in Experimental example 2;

FIG. 8 is a graph showing electrophoretic data showing the change in the expression level of PSRC1 after THP-1 cells were stimulated with different concentrations of mithramycin detected by western blot in Experimental example 2;

FIG. 9 is a histogram of the statistical data of the gray scale values of the western blot in Experimental example 2;

in fig. 3, the columns in the odd-numbered columns are IgG-related test data, and the columns in the even-numbered columns are SP 1-related test data.

Detailed Description

The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment. Except as specifically noted, the materials and equipment used in this example are commercially available. The specific embodiments are merely illustrative and are not to be construed as limiting the scope of the invention.

Mithramycin, also known as mithramycin, with CAS number 18378-89-7, has a certain toxicity, and is used in the present clinical practice to treat tumors, such as testicular cancer, glioma, brain metastases, malignant lymphoma, chorioepithelial carcinoma, breast cancer, and the like.

According to the invention, through research, SP1 can be combined with the open region of a PSRC1 gene and inhibit the expression of a PSRC1 gene, and mithramycin serving as an inhibitor of SP1 can reduce the SP1 level and further can up-regulate the expression of a PSRC1 gene, so that the expressions of ABCA1, ABCG1, SR-B1, PPAR-gamma and LXR-alpha are up-regulated, macrophages SR-A1 and LDLR are down-regulated, the cholesterol transfer rate and PON-1 activity can be increased, the MPO activity, the MPO/PON-1 ratio and HII are reduced, the formation of foam cells is inhibited, the metabolism of cholesterol and lipid is regulated, and hyperlipidemia diseases (such as atherosclerosis) are further treated and/or prevented.

The hyperlipemia disease of the present invention can be called hyperlipemia, such as atherosclerosis, coronary heart disease or fatty liver disease, etc.

In order to facilitate the application of the medicine, the medicine can be prepared into oral solid preparations, oral liquid preparations or injections and other dosage forms according to requirements; correspondingly, the oral solid preparation is tablets, capsules, granules, pills or powder; the invention can also add corresponding auxiliary materials into the medicine to carry out prescription according to the requirement, and prepare the slow-release preparation.

EXPERIMENTAL EXAMPLE 1 ChIP-qPCR experiment

1. Laboratory apparatus and reagent

1.1 Primary reagents

1.2 Main instruments and Equipment

2. Proteins and predicted gene binding sites

2.1 protein information

The target protein: SP 1;

the species are as follows: a human;

antibody: SP1 (brand CST, cat # 9389S);

positive control protein: H3K27 ac;

the species are as follows: (human, mouse, rat);

antibody: Anti-Histone H3(acetyl K27) antibody (brand: abcam, cat # ab 4729).

2.2 qPCR primer design

See table 1 below for specific PCR amplification primer sequences:

TABLE 1 primer sequences for predicted sites

3. Experimental methods

3.1 cell Cross-linking

1) After counting the cells, culturing overnight until the cells are attached to the wall, wherein the number of the cells is not less than 1x10⁷And (4) respectively.

2) Add 16% formaldehyde (methanol free) to the cell culture medium to a final concentration of 1%, gently shake to homogenize the liquid, incubate in a fume hood at room temperature for 10min, and see the cross-linking under microscope.

3) Glycine (10X) was added to a final concentration of 1X and incubated in a fume hood at room temperature for 5min to terminate the crosslinking reaction.

4) The medium was aspirated in a fume hood and the cells were washed twice with an equal volume of precooled PBS.

5) Add 10. mu.l/ml of Hot Cocktail in pre-cooled PBS, scrape cells, aspirate into 1.5ml of pre-cooled centrifuge tube, centrifuge off supernatant.

3.2 Western blot detection of expression of target proteins

1) Extracting total protein from small amount of cells, adding 5x SDS loading buffer, and decocting at 100 deg.C for 10 min.

2) Preparing 12% separation gel, adding isopropanol, standing for 20min, removing isopropanol when the separation gel is solidified, washing residual isopropanol with water, and removing residual liquid with absorbent paper. Adding 5% of prepared concentrated gel, inserting into a comb, standing for 20min, and pulling out the comb after the concentrated gel is solidified.

3) The electrophoresis device is installed and the sample is loaded, firstly 70V and 15min, and then 120V and 90 min.

4) And (5) rotating the membrane, and wet-rotating the membrane to 120V and stabilizing the pressure for 100 min.

5) And (3) sealing, namely putting the PVDF membrane into 5% skimmed milk, and sealing for 30 min.

6) The membrane was washed 3 times for 5min each time with TBST.

7) Primary antibody was incubated (TBST dilution) and incubated overnight at 4 ℃.

8) The membrane was washed 3 times with TBST for 10min each time.

9) Incubation secondary antibody (1: 5000, TBST dilution), incubation at room temperature for 2 h.

10) The membrane was washed 3 times with TBST for 10min each time.

11) According to the following steps: 1, adding two luminescent agents in the kit, and incubating for 1 min.

12) Development, fixing, and scanning results are shown in fig. 1.

3.3 cell lysis and nuclease digestion

1) Mu.l of lysine Buffer 1 containing protease inhibitor was added to the cells, vortexed for 15s, ice-cooled for 10min, centrifuged at 9000g, and the supernatant removed.

2) The pellet was resuspended in 100. mu.l of MNase gels Buffer working solution.

3) Adding 0.25 μ l Micrococcus nucleic, mixing, water bathing at 37 deg.C for 15min, and turning over every 5 min.

5) The reaction was stopped by adding 10. mu.l MNase Stop Solution and ice-cooled for 5 min. 9000g was centrifuged for 5min and the supernatant was removed.

6) The pellet was resuspended in 50. mu.l lysine Buffer 2 containing protease/phosphatase inhibitor and vortexed for 15s every 5min in an ice bath for 15 min.

7)9000g was centrifuged for 5min and the supernatant was transferred to a fresh 1.5ml pre-cooled centrifuge tube.

3.4 chromatin immunoprecipitation

1) 5 μ l to 1.5ml of the supernatant obtained in the above step was collected and stored at-20 ℃ as Input.

2) Mu.l of the supernatant obtained in the above step was taken into a 1.5ml centrifuge tube containing 450. mu.l of 1XP Dilution Buffer.

3) For each IP, 500. mu.l of Diluted lysate was added to a plug spin column, primary antibody was added, and the mixture was tumbled overnight at 4 ℃.

Positive control IP:5ug H3K27ac Antibody。

Negative control IP:5ug Normal Rabbit Ig G。

Target-specific IP：5ug antibody。

4) Add 20. mu.l ChIP Grade Protein A/G plus Agarose to each IP and incubate at 4 ℃ for 1h on a shaker. Plug columns were placed into a 2ml collection tube.

5) Centrifuge at 3000g for 30s and discard the pool.

6) Add 500. mu.l of IP Wash Buffer 1, centrifuge at 3000g for 30s and discard the pool.

7) Add 500. mu.l of IP Wash Buffer 2, centrifuge at 3000g for 30s and discard the pool.

8) And 7, repeating the step.

9) Add 500. mu.l of IP Wash Buffer 3, centrifuge at 3000g for 30s and discard the pool.

10) Centrifuge at 3000g for 1 min.

3.5 elution of chromatin immunoprecipitate

1) The plug column was replaced in the 1.5ml centrifuge tube, 150. mu.l of 1 × IP Elution Buffer wash resin was added, and incubated at 65 ℃ for 40min, flicked every 10min to resuspend the Agarose magnetic beads.

2) Centrifuge at 6000g for 1min and discard the column.

3) Mu.l of 5M NaCl and 2. mu.l of 20mg/mL proteinase K were added to the tube and mixed.

4) Input was thawed and 150. mu.l of IP Elution Buffer, 6. mu.l of 5M NaCl, and 2. mu.l of 20mg/mL proteinase K were added.

5) The metal bath is carried out for 1.5h at 65 ℃.

3.6 DNA purification recovery

1) 750. mu.l of DNA Binding Buffer was added.

2) Pipette 500. mu.l into a DNA Clean-Up column, which is placed on a 2ml collection tube. Centrifuge at 10,000g for 1min and discard the collected liquid.

3) The remaining sample was added to the same DNA Clean-Up column, repeat 2.

4) The Column was placed in a collection tube, followed by addition of 750. mu.l DNA Column Wash Buffer, 10,000g centrifugation for 1min, and the liquid in the collection tube was discarded.

5) Centrifuge at 10,000g for 2 min.

6) The column was placed in a new 1.5ml collection tube, 20. mu.l of DNA Elution Solution was added, and 10,000g was centrifuged for 1min to obtain a collection tube, which was purified DNA, and qPCR was performed.

3.7 qPCR

1) The total reaction system for qPCR was 20 μ l, as shown in table 2:

TABLE 2

2) qPCR used a two-step amplification system, the reaction conditions are shown in Table 3, and the number of cycles is 49 cycles.

TABLE 3

95 deg.C (pre-denatured)	10min
		95 deg.C (modified)	15sec
60 deg.C (extension)	1min

3) q-pcr data processing

ΔCt[normalized IP]＝(Ct[IP]-(Ct[Input]-Log₂(Input Dilution Factor)))

％Input＝2^(-ΔCt[normalized IP])*100％

Our input is usually1/10 in total amount, i.e. 10-fold dilution, Log₂(Input Dilution Factor)≈3.2

Example of calculation:

input SNP70

U1 14.05 17.55

ΔCt＝17.55-(14.05-3.2)＝6.7 ％input＝2^(-6.7)*100％＝0.961831573％

the experimental method is adopted to test the gene enrichment condition of the THP-1 cells in the mithramycin treatment group and the control group, wherein the mithramycin treatment group comprises the following specific steps: after the THP-1 cells are starved for 6-8h by using a serum-free culture medium, the THP-1 cells are added with the mithramycin at the concentration of 100nM for treatment for 24h, the cells of a control group are not treated with the mithramycin, and the specific data are shown in a table 4 and figures 2-3 through tests;

table 4: promoter gene enrichment data sheet

Referring to fig. 2-3, the data in table 4 above show that cells SP1 in the site 1 control group and the treated group can be specifically enriched, the enrichment rate is not high, the enrichment in the site 2 and 4 treated groups is reduced, and the enrichment in the site 3 treated group is most obviously reduced; SP1 was able to bind to the PSRC1 promoter region.

EXAMPLE 2 Dual luciferase assay

1. Experimental Material

1.1 Primary reagents

1.2 Main instruments and Equipment

1.3 Gene information

Protein information: SP 1;

the species are as follows: a human;

length of inserted plasmid fragment: 2214 bp;

a target gene promoter: PSRC 1;

the species are as follows: a human;

fragment length: 724 bp;

reference is made to FIG. 4 for predicting the binding site of SP1 and the PSRC1 promoter (specifically: site A: 1899-1904; site B: 2111-2120; site C: 2186-2196; site D: 2413-2418);

1.4 primer sequences

PCR gene amplification primer sequence in the preparation process of SP1 protein expression plasmid (pcDNA3.1 +):

forward primer sequence (5 '-3'): atgagcgaccaagatcactcc, respectively;

reverse primer sequence (5 '-3'): tcagaagccattgccactg, respectively;

PCR gene amplification primer sequence in the preparation of luciferase expression plasmid (PGL-3 plasmid):

forward primer sequence (5 '-3'): cccacaactgaggaacctc, respectively;

reverse primer sequence (5 '-3'): acctccgggaacgatacg, respectively;

each primer sequence is synthesized intelligently by Jinwei Suzhou.

1.5 vector information (plasmid map)

SP1 protein expression plasmid: pcDNA3.1+, the plasmid map is shown in FIG. 5;

luciferase expression plasmid: the plasmid map of the PGL-3 plasmid is shown in FIG. 6.

2. Experimental methods

2.1 PCR amplification of fragments of the Gene

In 0.2ml EP tube, wild type and MUT gene 3' UTR region fragments were PCR-amplified using genomic DNA as a template, and the amplification reaction system is shown in Table 5. The PCR reaction conditions are 94 ℃ pre-denaturation for 5min, the cycle parameters are 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 2min respectively, 35 cycles are totally carried out, the temperature is 72 ℃ and the amplification condition is fully extended for 10min, and the 1.5% agarose gel electrophoresis is used for detecting the amplification condition.

TABLE 5 PCR reaction System

2.2 recovery of Gene fragments

1) Column balancing: to the adsorption column CB2 (adsorption column put in the collection tube), 500. mu.l of the equilibrium solution BL was added, centrifuged at 12,000rpm for 1min, the waste liquid was discarded, and the adsorption column was replaced in the collection tube.

2) A single band of the DNA of interest was cut from the agarose gel, placed in a clean EP tube and weighed.

3) Adding equal volume of solution PC into the gel block, and standing in 50 deg.C water bath for about 10min to dissolve the gel block sufficiently.

4) Adding the solution obtained in the previous step into an adsorption column CB2, centrifuging at 12,000rpm for 1min, pouring off waste liquid, and placing adsorption column CB2 into a collection tube.

5) Add 600. mu.l of the rinsing solution PW to the adsorption column, centrifuge at 12,000rpm for 1min, pour off the waste liquid, place the adsorption column CB2 in the collection tube.

6) Operation 5 is repeated.

7) The adsorption column CB2 was put into a collection tube, centrifuged at 12,000rpm for 2min, and the adsorption column was left at room temperature for 3 min.

8) The adsorption column CB2 was put into a new centrifuge tube, 30. mu.l of elution buffer EB was dropped into the middle of the adsorption film, and the resulting solution was left at room temperature for 2min, centrifuged at 12,000rpm for 1min, and the DNA solution was collected.

2.3 double enzyme gel cutting recovery fragment and vector

The fragments were recovered according to the enzyme system, double digestion vector and gel shown in Table 6. After gentle mixing, incubation was carried out overnight at 37 ℃ and detection was carried out by 1.5% agarose gel electrophoresis.

TABLE 6 double enzyme digestion System

2.4 gel recovery of cleaved vector and target fragment

The specific steps are the same as 2.2, and the content of the gene segments is recovered;

2.5 destination fragment to vector

Ligation systems as shown in Table 7 were prepared, mixed gently and ligated overnight at 4 ℃.

TABLE 7 connection system

2.6 transformation of ligation products

1) Taking 200 ul TOP10 competent bacteria, and adding the ligated product;

2) standing on ice for 30 min;

3) heat shock at 42 ℃ for 90 s;

4) rapidly moving to ice and standing for 2 min;

5) in a clean bench, 1ml of LB medium (Amp)^－) Shaking at 37 deg.C and 160rpm for 1-2 h;

6) centrifuging the bacterial liquid at 3000rpm for 10min, discarding most of supernatant, resuspending the precipitate, adding all the bacterial liquid into LB plate (Amp)⁺) Uniformly coating, and performing inverted culture at 37 ℃ (overnight for 12-15 h);

7) the plates were removed and single colonies picked in a sterile worktop into LB medium (Amp) containing 1ml⁺) In a 15ml centrifuge tube, the bacteria are shaken in a shaker at 37 ℃ and 200rpm for 3 to 4 hours.

2.7 PCR identification of Positive clones with bacterial liquid

1) Taking 2 mul of bacterial liquid as a template, and carrying out PCR reaction according to the PCR system in the table 5 and the PCR reaction conditions in the step 2.1. Amplification was detected by 1.5% agarose gel electrophoresis.

2) And selecting positive clones to extract plasmids, and sequencing and checking.

2.8 plasmid petiole

1) Column balancing: to the adsorption column CP3 (the adsorption column was put into the collection tube), 500. mu.l of the equilibrium solution BL was added, and the mixture was centrifuged at 12,000rpm for 1min to remove the supernatant as much as possible.

2) 1.5ml of overnight-cultured bacterial suspension was taken, added to a centrifuge tube, centrifuged at 12,000rpm for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated off.

3) To the centrifuge tube where the pellet of the cells remained, 250. mu.l of the solution P1 was added and the pellet of the cells was thoroughly precipitated using a pipette.

4) 250 μ l of the solution P2 was added to the tube and gently turned upside down 6-8 times to lyse the cells thoroughly.

5) Add 350. mu.l of solution P3 to the tube, gently turn it up and down 6-8 times immediately, mix well until a white flocculent precipitate appears, centrifuge at 12,000rpm for 10min, at which time a precipitate forms at the bottom of the tube.

6) Transferring the supernatant collected in the previous step to adsorption column CP3 with a pipette, centrifuging at 12,000rpm for 30-60s, pouring off waste liquid, and placing adsorption column CP3 into a collection tube.

7) Adding 500 μ l deproteinized solution PD into adsorption column CP3, centrifuging at 12,000rpm, centrifuging for 30-60s, discarding waste liquid, and replacing adsorption column CP3 in the collection tube.

8) Adding 600 μ l deproteinized solution PW 3 into adsorption column CP3, centrifuging at 12,000rpm, centrifuging for 30-60s, discarding waste liquid, and replacing adsorption column CP3 in the collection tube. And repeating the steps once.

9) The adsorption column CP3 was replaced in the collection tube and centrifuged at 12000rpm for 2 min.

10) The adsorption column CP3 was placed in a clean centrifuge tube, 50. mu.l of elution buffer EB was added dropwise to the middle of the adsorption membrane, the membrane was left at room temperature for 2min, centrifuged at 12,000rpm for 2min, the plasmid solution was collected in the centrifuge tube, placed in an ice box, and the concentration was measured. Storing at-20 deg.C for use.

3. Luciferase assay

3.1 purpose of experiment: in mouse hepatocytes, binding of the SP1 protein to the PSRC1 promoter was verified by luciferase assay.

3.2 materials of the experiment

3.2.1 Primary reagents

3.2.2 Main instruments and Equipment

Instrument name instrument source cat.No

Cell super clean bench Jiabao purified JB-CJ-2FC

Cell culture box Thermo 8000

3.3 Experimental methods

3.3.1 cell plating

1) Cells were cultured to a confluency of 70%.

2) The medium was aspirated off, 10ml of 1XPBS was added, the dish was gently shaken, and PBS was aspirated off.

3) 1ml of 0.25% pancreatin was added, the culture dish was gently shaken to allow the pancreatin to spread evenly over the bottom of the dish, and the digestion of the cells was observed under a microscope.

4) After digestion was complete, digestion was stopped by adding 5ml of complete medium (DMEM + 10% FBS + 10% double antibody) and the cell suspension was gently pipetted with a pipette tip.

5) Cell density was counted on a hemocytometer and cells were diluted 1X10 with complete medium⁶One per ml.

6) The diluted cell suspension was added to six well plates, 2ml per well. The cells are evenly distributed by shaking back and forth or left and right for several times.

7)37℃，5％CO₂The cells were incubated at constant temperature for 16h, at which time the degree of cell fusion was approximately 80%.

3.3.2 cell transfection

1) And marking the six-hole plate cover.

2) The medium in the six-well plate was aspirated off, 2ml of opti-MEM was added to each well, and the mixture was placed at 37 ℃ under 5% CO₂And (5) continuing culturing in the constant-temperature incubator.

3) Mu.g of the plasmid was taken and mixed with 0.5ml of opti-MEM.

4) Mu.l lipofectmin2000 transfection reagent was mixed with 0.5ml opti-MEM.

5) The plasmid mixture and the transfection reagent mixture were mixed well and left to stand for 20 min.

6) The mixture was uniformly dropped into a six-well plate marked.

7)37℃，5％CO₂Culturing at constant temperature for 6h, removing opti-MEM containing mixed reagent by suction, adding 2ml complete medium, 37 deg.C, and 5% CO₂Culturing at constant temperature for 24 h.

3.3.3 luciferase assay

1) The medium in the six-well plate was aspirated, washed twice with PBS, digested with 0.25% pancreatin, and after complete digestion, the complete medium was added to stop digestion.

2) The cell counting plate counts the cells and dilutes the cells to 1x10⁵One per ml.

3) 100ul of fluorogenic substrate was added to each 96-well plate, followed by 100ul of cell suspension.

4) Every 10 minutes, the OD was measured with an ultraviolet spectrophotometer₄₉₀。

5) And selecting a numerical value with a higher fluorescence value for analysis.

The experimental method is adopted to detect the fluorescence intensity of cells after plasmid transfection, and the PSRC1 expression levels of cells of a mithramycin treatment group and a control group with different concentrations, wherein the mithramycin treatment group comprises: after the THP-1 cells are starved for 6-8h by using a serum-free culture medium, adding mithramycin at final concentrations of 100nM, 200nM and 300nM respectively for treatment for 24 h; cells of the control group were not treated with mithramycin; the test results are shown in fig. 7-9, and tests show that SP1 can be combined with the PSRC1 open region and inhibit the expression of the PSRC1 gene, and the expression of the PSRC1 gene can be up-regulated after the treatment of mithramycin, so that the hyperlipidemia disease can be effectively treated.

Finally, it should be noted that: the above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention should not be limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. A medicament for treating and/or preventing hyperlipidemia diseases, which comprises one or the combination of more than two of mithramycin, mithramycin prodrug and pharmaceutically acceptable mithramycin salt.

2. The medicament for treating and/or preventing hyperlipemia disease as described in claim 1, wherein said hyperlipemia disease is atherosclerosis, coronary heart disease or fatty liver.

3. The agent for the treatment and/or prevention of hyperlipemia disease as described in claim 1 or 2, wherein said agent is an oral solid preparation, an oral liquid preparation or an injection.

4. The medicament for treating and/or preventing hyperlipemia disease as described in claim 3, wherein said oral solid preparation is tablet, capsule, granule, pill or powder.

5. The agent for the treatment and/or prevention of hyperlipemia disease as described in claim 1 or 2, wherein said agent is a sustained release preparation.