GB2627084A - Target for screening drug for inhibiting intestinal fatty acid intake and preventing fatty liver, and use thereof - Google Patents

Target for screening drug for inhibiting intestinal fatty acid intake and preventing fatty liver, and use thereof Download PDF

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GB2627084A
GB2627084A GB2404401.8A GB202404401A GB2627084A GB 2627084 A GB2627084 A GB 2627084A GB 202404401 A GB202404401 A GB 202404401A GB 2627084 A GB2627084 A GB 2627084A
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soat2
drug
screening
intestinal
target
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Gu Aihua
Jiang Zhaoyan
Shao Wentao
Liang Jingia
Liu Qian
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Nanjing University
Nanjing Medical University
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Nanjing University
Nanjing Medical University
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Abstract

Disclosed are a target for screening a drug for inhibiting the intestinal fatty acid intake and preventing fatty liver, and the use thereof. According to a knockout mouse experiment, provided in the present invention are a target Soat2 (Sterol O-acyl transferase) of drug action, and a drug screening model thereof. The inhibitory effect of a drug compound on Soat2 is determined by means of combined screening of high-throughput NBD-cholesterol screening, PCR and BODIPYTM FL C16 fluorescence. The drug compound with strong lipid-lowering effects and few toxic side effects can be screened out from the level of intestinal fatty acid intake by means of the drug screening model using Soat2 as the target.

Description

Target for screening drug for inhibiting intestinal fatty acid intake and preventing fatty liver, and use thereof
TECHNICAL FIELD
The present invention belongs to the field of biomedicine, and discloses a target for screening drugs that inhibit intestinal fatty acid uptake and preventing non-alcoholic fatty liver disease.
BACKGROUND
Non-alcoholic fatty liver disease is currently a common clinical disease. Abnormal liver metabolism leads to the accumulation of excessive triglycerides and the degeneration of liver cells, which then result in inflammatory responses. The progression of fatty liver may lead to liver fibrosis and ultimately lead to the malignant progression of liver cancer. The formation of fatty liver is associated with and affects diseases such as obesity, diabetes, and cardiovascular diseases. The mechanism of fatty liver is related to an increase in intestinal uptake of exogenous fatty acids and/or an increase in synthesis of in vivo fatty acids. Treatment measures for fatty liver are currently limited, mainly including intervention treatment for risk factors and drug treatment for regulating lipid metabolism. For example, in obese individuals, the incidence of non-alcoholic fatty liver disease (NAFLD) is as high as 75%. At present, besides weight loss and metabolic surgeries, the treatment for obesity and fatty liver mainly relies on lipid-lowering drugs. Most of the lipid-lowering drugs used in clinical practice target the liver and achieve lipid-lowering goals by promoting lipid clearance and metabolism. For example, the most widely used class of lipid-lowering drugs in clinical practice is statins. The statins reduce intracellular free cholesterol and accelerate the clearance of circulating lipoproteins by inhibiting a rate-limiting enzyme -3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the early synthesis stage of intracellular cholesterol. The effect of statins on reducing circulating lipoprotein cholesterol is not linearly correlated with dosage, although there is a certain correlation. The standard dosage of statins is often insufficient to achieve a therapeutic goal. The reason may be that the statins increase compensatory absorption of intestinal lipids while inhibiting cholesterol, and their lipid regulating effects are counteracted LH. In addition, fibrate drugs such as fenofibrate and benzafibrate activate peroxisome proliferator activated receptors (PPARa), induce lipoprotein esterase expression, promote the hydrolysis of triglycerides in triglyceride rich lipoprotein particles, and lead to a decrease in plasma very low-density lipoprotein (VLDL). And the fibrate drugs promote liver uptake of fatty acids, inhibit liver synthesis of triglycerides, and also inhibit hormone sensitive esterases in adipose tissues to reduce the generation of fatty acids, so as to further inhibit liver synthesis of triglycerides. Although the lipid-lowering drugs promote the clearance of lipoproteins to some extent, their large intestinal absorption still exists, which cannot fundamentally inhibit the formation of obesity. Moreover, long-term use of drugs has potential side effects that affect liver function. Other preparations such as choline, methionine, vitamins, and amino acids only play a role in protecting liver cells from further damage, delay the progression of fatty liver, but do not improve abnormal liver metabolism.
As a key enzyme in cholesterol esterification, Soat2 (Sterol O-acyl transferase) is known to play an important role in cholesterol metabolism. However, in fact, the mechanisms of absorption of cholesterol and fatty acids by the intestine are different. Throughout the digestive tract, the absorption of cholesterol mainly occurs in the proximal intestinal segments. This is related to the high expression of a key cholesterol absorption protein Niemann-Pick Cl Likel (NPC1L1) in the small intestine. The NPC1L1 can specifically bind to cholesterol, expose endocytic signals, and absorb free cholesterol in the intestinal cavity through vesicular endocytosis. The intestinal absorption of fatty acids mainly relies on protein dependent transport. Leukocyte differentiation antigen 36 (CD36) and fatty acid binding protein (FATP4) are considered fatty acid transport related proteins involved in intestinal fatty acid uptake.
The CD36 is the optimal transporter of fatty acids in enterocytes. In addition, studies found that inhibiting the absorption of cholesterol in the intestine has little effect on the intestinal fatty acid uptake [2.31. Saturated fatty acids (SFA) in the diet promote the expression of cholesterol transport related proteins in the intestine, promote the absorption of cholesterol in the intestine, and increase plasma low density lipoprotein cholesterol (LDL-C)14,51. Therefore, if only the absorption of cholesterol in the intestine is limited, the desired lipid-lowering effect is often not achieved. Most of the lipid-lowering drugs for inhibiting intestinal absorption, put forward in recent years, achieve lipid-lowering goals by inhibiting the absorption of cholesterol in diet and bile by the intestine, and such drugs are rarely marketed. Ezetimibe is currently the only drug found to have a brush border attached to the small intestinal villous epithelium, inhibit the sterol carrier NPC1L1 in a targeting manner, and inhibit the absorption of cholesterol, thereby reducing the uptake of cholesterol in the intestine and promoting the clearance of blood cholesterol. However, clinical studies found that the combination of ezetimibe and statins can reduce the LDL-C level of serum to some extent, but cannot block the progression of subsequent obesity related diseases H. If targets can be found to alleviate obesity and fatty liver by inhibiting intestinal fatty acid uptake, and lipid-lowering drug screening models and methods targeting corresponding proteins can be developed, they will be strong impetuses for the treatment of lipid metabolism related diseases such as fatty liver and obesity.
SUMMARY
To solve the above technical problems, a first objective of the present invention is to provide a drug target screening method for inhibiting intestinal fatty acid absorption and a lipid-lowering drug screening model targeting the protein.
A second objective is to provide a method for screening a lipid-lowering drug targeting intestinal Soat2 protein using the drug screening model.
The objectives of the present invention can be achieved by the following technical solutions: A use of Soat2 as a target in screening a drug for inhibiting intestinal fatty acid uptake. A use of Soat2 as a target in screening a lipid-lowering drug.
A use of Soat2 as a target in screening a drug for treating fatty liver.
As a key enzyme for cholesterol esterification, Soat2 is only expressed in intestinal absorptive cells and liver cells. The present invention found that, in addition to previous studies on inhibiting the absorption of free cholesterol in the intestine, Soat2 also plays a key role in inhibiting the absorption of intestinal fatty acids. Small intestinal Soat2 deficient mice have milder liver steatosis (intestinal and full knockout mice). However, the deficiency of Soat2 only in the liver (liver knockout mice) reduces cholesterol esters, but still leads to or even exacerbates the accumulation of triglycerides (TG) (FIGS. 3, 4, and 5). This indicates that even if liver Soat2 is normally present, the deficiency of small intestinal Soat2 is sufficient to resist diet induced fatty liver. The key mechanism may be that the deficiency of intestinal Soat2 promotes the ubiquitination degradation of fatty acid transport protein CD36, thereby reducing the intake of exogenous fatty acids. Therefore, the present invention provides Soat2 protein as a drug target.
A use of Soat2 as a target in screening a drug for treating fatty liver.
A lipid-lowering drug screening model is a colon cancer cell Caco2 that stably expresses a Soat2 protein.
A method for screening a lipid-lowering drug includes first selecting intestinal epithelial cells CaCo2 that stably express a Soat2 protein, inoculating the cells to a dish for 24 hours, adding a compound, culturing for a period of time, and then determining a quantity of the Soat2 protein by high throughput NBD-cholesterol screening, PCR, and BODLPY" FL C16 fluorescence. The NBD-cholesterol intensity indicates changes in Soat2 activity, the PCR result indicates a relative expression quantity of Soat2, and the BODIPY" FL C16 intestisty indicates uptake rate of free fatty acids. If the above results show a decrease, it indicates that the compound can inhibit the expression and activity of Soat2 in the intestine.
The present invention provides a method for screening a lipid-lowering drug targeting an intestinal Soat2 protein using the above drug screening model: A. preliminarily screening all target drugs using high throughput screening analysis test on fluorescence intensity of NBD-cholesterol: inoculating cells to a 96-well plate, adding each preliminarily screened drug to a medium, using a blank reagent as a control group, using a Soat2 specific inhibitor PPPA to completely inhibit the activity of Soat2 in the cells as a background value, incubating for a period of time, then adding NBD-cholesterol for incubation, discarding the medium, washing twice with cold PBS, and detecting the fluorescence intensity using an ELISA (excitation wavelength 485 nm, emission wavelength 535 nm), and subtracting the background value from ELISA readings incubated with various natural compounds as the basis for Soat2 activity; B. using a qRT-PCR method to expand the sample size for verification, and further screening the preliminarily screened drugs for the expression of Soat2; and C. testing free fatty acid uptake using BODIPYIM FL Clotluorescence: inoculating intestinal epithelial cells CaCo2 that stably express a Soat2 protein to a confocal dish, then adding each preliminarily screened drug to a medium, using a blank reagent as a control group, incubating for a period of time, then adding BODIPYTM FL C16 for incubation, and using a fluorescence microscope for capture and comparing the fluorescence levels of various groups.
A use of a material for inhibiting intestinal Soat2 in preparation of a drug for inhibiting intestinal fatty acid uptake. c
The material for inhibiting intestinal Soat2 may be selected from small molecule compounds for inhibiting intestinal Soat2, or from biological drugs such as siRNA, shRNA, lentivirus, and adenovirus for inhibiting intestinal Soat2 expression.
A use of a material for inhibiting intestinal Soat2 in preparation of a lipid-lowering drug.
The material for inhibiting intestinal Soat2 may be selected from small molecule compounds for inhibiting intestinal Soat2, or from biological drugs such as siRNA, shRNA, lentivirus, and adenovirus for inhibiting intestinal Soat2 expression.
A use of a material for inhibiting intestinal Soat2 in preparation of a drug for treating fatty liver.
The material for inhibiting intestinal Soat2 may be selected from small molecule compounds for inhibiting intestinal Soat2, or from biological drugs such as siRNA, shRNA, lentivirus, adenovirus, plasmids that edit Soat2 genes, and gRNA for inhibiting intestinal Soat2 expression.
Beneficial effects of the present invention are as follows: 1) The drug target Soat2 and drug screening model provided by the present invention can screen drug compounds having strong lipid-lowering effects and low toxic and side effects from the level of intestinal fatty acid uptake.
2) In the 96-well plate, the drug screening model for compound screening has the advantages of high throughput, strong specificity, convenient operation, etc. 3) The experiments of the present invention prove that the intestinal Soat2 can serve as a target for screening drugs that inhibit intestinal fatty acid uptake, lipid-lowering drugs, fatty liver treatment drugs, and obesity treatment drugs, and plays a role in drug screening.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates construction and verification of Soat2 knockout mice in the present invention.
A. represents CRISPR/Cas9 genome editing technology for constructing full knockout mice, B. represents genotype test of full knockout mice, C. represents embryonic stem cell targeting technology for constructing conditional knockout mice, D, E, and F. represent genotype test of conditional knockout mice, G, 11, and I. represent efficiency test of Soat2 knockout mice.
FIG. 2 illustrates a target plasmid map in the construction of Soat2 knockout mice in the present invention.
FIG. 3 illustrates liver steatosis of high-fat diet induced Soat2 full knockout mice in the present invention.
A. represents body size changes of Soat2 full knockout mice after 12 weeks of high-fat feeding, B. represents liver steatosis of the mice, C. represents staining of liver sections with hematoxylin and eosin (H&E) and oil red, D. represents non-alcoholic fatty liver disease activity score (NAS) of liver tissue, and E. represents triglycerides (TG) content of the mouse liver.
FIG. 4 illustrates liver steatosis of high-fat diet induced Soat2 liver knockout mice in the present invention.
A. represents body size changes of Soat2 liver knockout mice after 12 weeks of high-fat feeding, B. represents liver steatosis of the mice, C. represents staining of liver sections with H&E and oil red, D. represents NAFLD activity score (NAS) of liver tissue, and E. represents triglycerids (TG) content of the mouse liver.
FIG. 5 illustrates liver steatosis of high-fat diet induced Soat2 intestinal knockout mice in the present invention.
A. represents body size changes of Soat2 intestinal knockout mice after 12 weeks of high-fat feeding, B. represents liver steatosis of the mice, C. represents staining of liver sections with H&E and oil red, D. represents NAFLD activity score of liver tissue, and E. represents TG content of the mouse liver.
FIG. 6 illustrates screening results of 41 drugs using a screening system in Example 1 of the present invention.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The following examples are used for the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means familiar to those skilled in the art, and the raw materials used are commercially available commodities.
Example 1: Based on various Soat2 knockout mouse model experiments, this study provided Soat2 protein as a target for screening a lipid-lowering drug that can be used for inhibiting intestinal fatty acid uptake.
1. Feeding and treatment of mice: Soat2 full knockout mouse (Soat2-'-) models were generated through CRISPR/Cas9 genome editing (FIG. 1A). Cas9 mRNA and gRNAs were micro-injected into fertilized eggs of C57BL/6J mice by in vitro transcription, and then the fertilized eggs were implanted into C57BL/6 pseudo pregnant female mice to obtain FO generation mice. Upstream and downstream sequence information of knockout sites and Giude RNA design information were shown in Table 1. Genotypes of the FO generation mice were identified to obtain FO generation mice with positive knockout of target genes. PCR products were confirmed to succeed in knockout by Sanger sequencing. The FO generation positive mice were mated with wild type mice to obtain Soat2' heterozygous mice for maintenance and breeding. Genetic typing was performed on the Soat2" mice using Fl: 5'-ACAGCCTTTCAAGAACCCTCAG-3' and R2: 5'-AAGACCTGCCTTGCCCACA-3', and 1028 by wild type alleles and 217 or 193 by knockout alleles can be obtained by amplification (FIG. 1B). The knockout effect of Soat2 in mouse liver was verified at the RNA level, with a knockout efficiency of over 99% (FIG. 1G). Flox modification was performed on Soat2 genes using the principle of homologous recombination in a manner of targeting embryonic stem cells to construct conditional knockout mouse models (FIG. 1C). An embryonic stem cell targeting vector was first constructed (as shown in the targeting plasmid map in FIG. 2), where the vector included a 5.1kb 5' homologous ann, a 0.8kb fox region, PGK-Neo-polyA, exon 6, a 3.6kb 3' homologous arm, and a MCI-TK-polyA negative screening marker (plasmids). The target vector was linearized and electroporated into JAIVI8A3 ES cells. After screening, a total of 144 G418 and Ganc resistant clones were obtained. Correct integration of the clones was analyzed through long fragment PCR. The correct targeted clones were injected into C57BL/6 blastocysts to obtain chimeric mice. The chimeric mice were mated with Flp mice to obtain positive de-Neo Soat2r1°x'n°x mice. The Soatr""x mice were hybridized with Vill-Cre mice and Alb-Cre mice respectively to obtain intestinal specific Soat2 knockout mice (Soat2'-''O) and liver specific Soat2 knockout mice (Soat2'). Genetic typing was performed on the mice carrying the Soat2l1" alleles using primers Fl: 5'-TCGTCCCAGCCCAGTCTTT-3' and R2: 5'-CTGCCTTGCCCACAGTTTCT-3', and 289 by wild type alleles and 344 by knockout alleles can be obtained by amplification (FIG. ID). Genetic typing was performed on the mice expressing Vill-Cre using primers Vill-Cre-Fl: 5'-TCGATGCAACGAGTGATGAG-3', Vill-Cre-R1: 5'-TCCATGAGTGAACGAACCTG-3', Control -Fl: 5'-CAAATGTTGCTTGTCTGGTG-3' and Control-R1: 5'-GTCAGTCGAGTGCACAGTTT-3' to obtain a target product of approximately 400 by and a control product of approximately 200 by (FIG. 1E). Genetic typing was performed on the mice expressing Alb-Cre using primers Alb-Cre-PI: 5'-TGGCAAACATACGCAAGGG-3', Alb-Cre-P2: 5'-CGGCAAACGGACAGAAGCA-3', and Alb-Cre-P3: 5'-GGCAATGGTTCCTCTCTGCT-3' to obtain a target product of approximately 450 by and a control product of approximately 800 by (FIG. IF). The knockout effects of Soat2 in the small intestines of intestinal knockout mice and in the livers of liver knockout mice were verified at the RNA level, with a knockout efficiency of over 99% (FIG. 1H, O. The construction of the Soat2 full knockout mouse (Soat2') models and the conditional knockout mouse models was entrusted to Shanghai Model Organisms Center, Inc. The models may alternatively be constructed according to conventional technical means in the art.
The constructed Soat2 full knockout, liver knockout, and intestinal knockout mice were fed with high fat for 12 weeks. The mice were euthanized at 12 weeks, and tissues such as serum, liver, and intestine were collected for subsequent experiments.
2. Histopathological examination: The liver tissue was fixed overnight in 10% formalin and embedded in paraffin. 5 pm paraffin sections were prepared with hematoxylin and eosin (H&E) after dewaxing and rehydration. An NAFLD activity score (NAS) of the liver tissue was evaluated according to a NASH clinical research network scoring system [71.
3. Staining with oil red: The frozen liver sections having a thickness of 5 pm were fixed and stained with oil red for 15 minutes. Cell nuclei were stained with hematoxylin for 2 minutes. The sections were imaged by an optical microscope.
4. Intestinal fatty acid uptake: In order to determine the dietary lipid absorption of Soat2 knockout mice under specific conditions, the mice were fasted for 10 hours and then injected with 200 pl of olive oil containing 5 pl of [9,10-3H (N)] trioleic acid. Blood was collected from the tail at 0, 30, 60, 90, and 120 minutes, and plasma was separated for liquid scintillation counting. The mice were euthanized after 2 hours. Contents of the small intestine, liver, heart, white fat, brown fat, gallbladder, testes, large intestine, and cecum were collected. The intestine was collected and rinsed with phosphate buffered saline (PBS) and then divided into nine equal parts, and the mucosa was extracted and frozen in liquid nitrogen. After the samples were homogenized in methanol/water (2:1), the radioactive distribution in the tissue was determined by a scintillation counting method.
5. Results: This study found that the degree of liver steatosis in the intestinal knockout mice was significantly alleviated, manifested by a decrease in liver TG and a decrease in NAS score. This indicated that even if liver Soat2 was normally present, the deficiency of small intestine Soat2 was sufficient to resist fatty liver induced by a high-fat diet. It also suggested the importance of small intestine uptake of free fatty acids for the formation of fatty liver. On the contrary, because the uptake of free fatty acids in the small intestine of the liver knockout mice were not inhibited, the uptake of exogenous fatty acids can still promote the occurrence of fatty liver (FIGS. 3, 4, and 5). In addition, the uptake results of isotopic labeled fatty acids in the intestinal knockout mice further demonstrated that the deficiency of Soat2 can effectively reduce intestinal uptake of fatty acids and increase fecal excretion. There were several known key links in the uptake of fatty acids in the small intestine, such as cell membrane fat uptake proteins FATP4 and CD36 and intracellular transport protein FABP4, where CD36 played a crucial role in the uptake of fatty acids. The present invention found that the deficiency of Soat2 did not affect other fatty acid uptake related proteins, but promoted the ubiquitination degradation of CD36, thereby reducing the absorption of fatty acids.
Example 2: A lipid-lowering drug screening model and screening method targeting a Soat2 protein to inhibit intestinal fatty acid uptake: 1. Cell culture Colon cancer cells Caco2 were cultured in an MEM complete medium, placed in a 37°C, 5% CO2 incubator, and passaged after grown to 80% of the area of a culture dish. The cells in a logarithmic growth phase were collected for experiments.
2. Preliminary screening of drugs by high throughput NBD-cholesterol fluorescence technology: (1) After the cells filled the culture dish, 0.25% of trypsin was added for digestion for 5 minutes, then the trypsin was discarded, and a medium was added for blowing and beating to prepare a single-cell suspension. The cells were counted with a counter, and an appropriate amount of cell suspension was inoculated to a black wall and transparent bottom 96-well plate, with consistent cell count in each well. After the cells adhered to the wall, a compound having a final concentration of 10 ftM was added to each well, the cells were continuously cultured for 24 hours, and drugs were preliminarily screened by high throughput NBD-cholesterol fluorescence technology.
(2) NBD-cholesterol was dissolved in ethanol to prepare a 1 mg/m1 stock solution. After the cells in the 96-well plate were cultured in the compound-containing medium for 24 hours, the cells were cultured in a medium containing 1 pg/m1 of NBD-cholesterol ethanol solution and incubated in a 37°C incubator for 2 hours. The incubated cells were washed 3 times with PBS and fixed at room temperature with 4% paraformaldehyde for 20 minutes. The fluorescence intensity was tested using an ELISA (excitation wavelength 485 nm, emission wavelength 535 nm).
(3) Analysis on test results Control group: blank reagent control; Background value: A Soat2 specific inhibitor PPPA served as the background value after completely inhibiting the activity of Soat2 in cells; Calculation on Soat2 activity: Relative activity of Soat2 = (fluorescence value of the experimental group -background value)/ (fluorescence value of the control group background value) 3. Re-screening of the preliminarily screened drugs by PCR technology for Soat2 expression (1) RNA was extracted using a TRIzol reagent. 1-5x107 cells were scraped into a 1.5m1 centrifuge tube, 1 ml of TRIzol was added, and the cells were stood at room temperature for 3 minutes. 200 pl of chloroform was added, and the cells were shaken up and down for 8 seconds and stood for 2 minutes. The solution was centrifuged at 4°C, 12000 gx15 min, and the supernatant was collected. 0.5 ml of isopropanol was added, followed by gentle mixing and standing on ice for 10 minutes. The solution was centrifuged at 4°C, 12000 gx10 min, and the supernatant was collected. 1 ml of 75% ethanol was added, and the precipitate was gently washed. The solution was centrifuged at 4°C, 7500 g x 5 min, the supernatant was discarded. The precipitate was dried and dissolved in an appropriate amount of DEPC water.
(2) Reverse transcription was performed using Prime Script RT kit (Takara, Japan) to obtain cDNA. The total volume of the reverse transcription reaction system was 20 gl, including 2 pi" of 10/RT Buffer, 0.8 p1_, of 25 x dNTP Mix (100 mM), 2 fiL of 10/11T Random Primers, 1 [IL of Multi ScribeTm Reverse Transcriptase, 4.2 pl., of DEPC water, and 2 pg/10 1.11_, of RNA. The reverse transcription was performed in a Dongshenglong gradient PCR instrument: reaction at 25°C for 10 minutes, reaction at 37°C for 120 minutes, and reaction at 85°C for 5 minutes.
(3) Real-time fluorescence quantitative PCR was performed using a SYBR-Green staining method (Real-time qPCR Master Mix, Takara, Japan) to test the expression levels of Soat2 and GAPDH in cells. The reaction system was: SYBR 5 gL, Forward Primer (10 pM) 0.2 pt., Reverse Primer (10 pM) 0.2 pL, cDNA (1:10 dilution) 1 pL, and water added to a total volume of 10 ftL. Primer sequences were as follows: Soat2, Forward: 5'-ACGTTGCCAGGCATCTTCAT-3', Reverse: 5'-AGTCATGGACCACCACGTTC-3'; GAPDH, Forward: 5'-AGGTCGGTGTGAACGGATTTG-3', Reverse: 5'-GGGGTCGTTGATGGCAACA-3'. Real-time quantitative PCR was performed using Roche RT-PCR LC480II in three-time repeated samples: 95°C, pre-denaturation for 30 seconds; 95°C, 5 seconds; 60°C, 30 seconds; 40 reaction cycles. The results were standardized at the mRNA level using GAPDH. 2' represented the multiple of changes in the expression of Soat2 in the experimental group compared to the control group.
4. Test on free fatty acid uptake using BODLPY" FL C16 fluorescence.
(1) After the cells filled the culture dish, 0.25% of trypsin was added for digestion for 5 minutes, then the trypsin was discarded, and a medium was added for blowing and beating to prepare a single-cell suspension. An appropriate amount of cell suspension was inoculated into a confocal dish. After the cells adhered to the wall, the preliminarily screened compound having a final concentration of 10!TM was added to each well, and the cells were continuously cultured for 24 hours.
(2) BODIPY" FL C16 was dissolved in DMSO. During use, the final concentration of BODIPY' FL C16 was 1 pM, and the concentration of DMSO was not more than 0.1%. After the cells were treated with the compound for 24 hours, 1 04 of BODIPY' FL C16 was added, and the cells were incubated in the 37°C incubator for 30 minutes. The cells were washed 3 times with PBS and fixed at room temperature with 4% paraformaldehyde for 20 minutes. The cells were stained with DAPI and incubated at room temperature for 5 minutes. The cells were observed and photos were taken under a confocal laser microscope LSM700.
5. Results By screening the inhibition of Soat2 protein in 41 drug compounds, it was found that the lipid-lowering drug berberine can effectively inhibit the activity and expression of Soat2 enzyme and reduce the uptake of free fatty acids by cells. The results suggested that the inhibition of Soat2 on intestinal fatty acid uptake can serve as a target for screening lipid-lowering drugs, with some clinical application significance.
The screening results of 41 drugs using the screening system were shown in FIG. 6.
The above implementations are specific examples of the present invention. In practical applications, any changes in form or details made without deviating from the spirit of the present invention fall within the scope of protection of the present invention.

Claims (11)

  1. CLAIMS1. A use of Soat2 as a target in screening a drug for nhibiting intestinal fatty acid uptake.
  2. 2. Anse of Soat2 as a target in screening a lipid-lowering drug.
  3. 3. A use of Soat2 as a target in screening a drug for treating fatty liver.
  4. 4. A use of Soat2 as a target in screening a drug for treating obesity.
  5. 5. A lipid-lowering drug screening model, wherein the model is a colon cancer cell Caco2 that stably expresses a Soat2 protein.
  6. 6. A method for screening a lipid-lowering drug, comprising: first selecting intestinal epithelial cells Caco2 that stably express a Soat2 protein, inoculating the cells to a dish for 20 to 26 hours, adding a material to be screened, culturing for a period of time, and then determining a quantity of the Soat2 protein by high throughput NBD-cholesterol screening, PCR, and BODIPYTM FL C16 fluorescence, wherein the NBD-cholesterol indicates changes in Soat2 activity, the PCR indicates a relative expression quantity of Soat2, the BODIPYTM FL C16 indicates uptake of free fatty acids, and if the above results show a decrease, it indicates that the material can inhibit the expression of Soat2 in the intestine.
  7. 7. The method according to claim 6, comprising the following steps: (1) preliminarily screening all target drugs using high throughput screening analysis test on fluorescence intensity of NBD-cholesterol: inoculating cells to a 96-well plate, adding each preliminarily screened drug to a medium, using a blank reagent as a control group, using a Soat2 specific inhibitor PPPA to completely inhibit the activity of Soat2 in the cells as a background value, incubating for a period of time, then adding NBD-cholesterol for incubation, discarding the medium, washing twice with cold PBS, and testing the fluorescence intensity using an ELISA with an excitation wavelength of 485 nm and an emission wavelength of 535 nm, and subtracting the background value from ELISA readings after the incubation with various drugs as the basis for Soat2 activity; (2) using a qRT-PCR method to expand the sample size for verification, and further screening the preliminarily screened drugs for the expression of Soat2; and (3) testing free fatty acid uptake using BODIPYTM FL C16 fluorescence: inoculating cells to a confocal dish, then adding each preliminarily screened drug to a medium, using a blank reagent as a control group, incubating for a period of time, then adding BODIPYTM FL C16 for incubation, using a fluorescence microscope for capture and comparing the fluorescence levels of various groups.
  8. 8. A use of a material for inhibiting intestinal Soat2 in preparation of a drug for inhibiting intestinal fatty acid uptake.
  9. 9 A use of a material for inhibiting intestinal Soat2 in preparation of a lipid-lowering drug.
  10. 10. A use of a material for inhibiting intestinal Soat2 in preparation of a drug for treating fatty liver.
  11. 11. A use of a material for inhibiting intestinal Soat2 in preparation of a drug for treating obesity.
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