WO2023098273A1 - 一种筛选抑制肠道脂肪酸摄取及防治脂肪肝的药物靶点及其应用 - Google Patents

一种筛选抑制肠道脂肪酸摄取及防治脂肪肝的药物靶点及其应用 Download PDF

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WO2023098273A1
WO2023098273A1 PCT/CN2022/122617 CN2022122617W WO2023098273A1 WO 2023098273 A1 WO2023098273 A1 WO 2023098273A1 CN 2022122617 W CN2022122617 W CN 2022122617W WO 2023098273 A1 WO2023098273 A1 WO 2023098273A1
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soat2
intestinal
screening
drugs
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顾爱华
蒋兆彦
邵文涛
梁静佳
刘倩
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南京医科大学
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Definitions

  • the invention belongs to the field of biomedicine, and discloses a drug target for screening and inhibiting intestinal fatty acid intake and preventing and treating fatty liver and application thereof.
  • Non-alcoholic fatty liver disease is a common clinical disease at present, mainly due to abnormal liver metabolism leading to excessive accumulation of triglycerides, leading to liver cell degeneration and inflammatory response. Fatty liver progression may lead to hepatic fibrosis and become a malignant process that eventually leads to liver cancer.
  • the formation of fatty liver is interrelated and influenced by obesity, diabetes, cardiovascular disease and other diseases.
  • In the mechanism of fatty liver it is related to the increase of intestinal intake of exogenous fatty acids and/or the increase of fatty acid synthesis in the body.
  • the treatment measures for fatty liver are relatively limited, mainly including risk factor intervention and drug treatment for regulating lipid metabolism.
  • NASH non-alcoholic fatty liver disease
  • statins are the most widely used class of lipid-lowering drugs in clinical practice. It reduces intracellular free cholesterol and accelerates the clearance of lipoproteins in the circulation by inhibiting the rate-limiting enzyme in the early synthesis stage of intracellular cholesterol - trihydroxytrimethylglutaryl coenzyme A (HMG-CoA) reductase.
  • statins The effect of statins on lowering circulating lipoprotein cholesterol is dose-dependent, but non-linear. Standard statin doses are often insufficient for therapeutic purposes. The reason may be that while statins suppress cholesterol, intestinal lipid absorption increases compensatoryly, and its lipid-lowering effect is counteracted [1] .
  • fibrate drugs such as fenofibrate and bezafibrate, can induce the expression of lipoprotein esterase by activating peroxisome proliferator-activated receptor (PPARa), and promote the expression of lipoprotein rich in triglycerides. Triglycerides in the particles are hydrolyzed, resulting in a decrease in plasma very low-density lipoprotein (VLDL).
  • VLDL very low-density lipoprotein
  • lipid-lowering drugs promote the clearance of lipoproteins to a certain extent, a large amount of intestinal absorption still exists, which cannot fundamentally inhibit the formation of obesity, and long-term use of drugs themselves has potential side effects that affect liver function.
  • Others such as choline, nitrogen acid, vitamins, and amino acid preparations only play a role in protecting liver cells from further damage and delaying the process of fatty liver, but they do not aim at improving abnormal liver metabolism.
  • Soat2 (Sterol O-acyl transferase, sterol O acyl transferase), as the key enzyme of cholesterol esterification, is known to play an important role in cholesterol metabolism. But in fact, the intestinal absorption mechanism for cholesterol and fatty acids is different. Throughout the digestive tract, cholesterol absorption occurs primarily in the upper midsection. This is related to the high expression of cholesterol absorption key protein Niemann-Pick C1 Like1 (NPC1L1) in this part of the small intestine. NPC1L1 can specifically bind cholesterol, expose endocytic signals, and absorb free cholesterol in the intestinal lumen through vesicle endocytosis. The intestinal absorption of fatty acids is mainly through protein-dependent transport.
  • Leukocyte differentiation antigen 36 CD36
  • FATP4 fatty acid binding protein
  • CD36 is the best transporter of enterocyte fatty acids.
  • SFA saturated fatty acids
  • LDL-C plasma low-density lipoprotein cholesterol
  • Ezetimibe ezetimibe
  • NPC1L1 sterol carrier NPC1L1
  • ezetimibe is currently the only drug found to have a brush border attached to the villous epithelium of the small intestine, targeting and inhibiting the sterol carrier NPC1L1, and inhibiting the absorption of cholesterol, thereby reducing the cholesterol uptake in the intestine and promoting the clearance of blood cholesterol.
  • statins can reduce serum LDL-C levels to a certain extent, it fails to block the progression of subsequent obesity-related diseases [6] .
  • the first object of the present invention is to provide a drug target for inhibiting intestinal fatty acid absorption and a lipid-lowering drug screening model targeting the protein.
  • the second purpose is to provide a method for screening lipid-lowering drugs targeting intestinal Soat2 protein by using a drug screening model.
  • Soat2 as a target in the screening of drugs that inhibit intestinal fatty acid uptake.
  • Soat2 as a target in screening lipid-lowering drugs.
  • Soat2 as a target in screening drugs for the treatment of fatty liver.
  • Soat2 As the key enzyme of cholesterol esterification, Soat2 is only expressed in intestinal absorptive cells and liver hepatocytes. The present invention finds that in addition to inhibiting the absorption of intestinal free cholesterol in previous studies, Soat2 also plays a key role in inhibiting the absorption of intestinal fatty acids.
  • the present invention provides the drug target Soat2 protein.
  • Soat2 as a target in screening drugs for the treatment of fatty liver.
  • a lipid-lowering drug screening model is colon cancer cell Caco2 stably expressing Soat2 protein.
  • a method for screening lipid-lowering drugs including first selecting intestinal epithelial cells Caco2 stably expressing Soat2 protein, adding compounds 24 hours after inoculating cells in a dish, and cultivating for a certain period of time, using high-throughput NBD-cholesterol screening, PCR and BODIPY TM FL C 16 fluorescence to determine the amount of Soat2 protein.
  • NBD-cholesterol indicates the change of Soat2 activity
  • PCR indicates the relative expression of Soat2
  • BODIPY TM FL C 16 indicates the intake of free fatty acids. If the above results decrease, it indicates that the compound can inhibit the expression and activity of intestinal Soat2.
  • the present invention provides a method for screening lipid-lowering drugs targeting the intestinal Soat2 protein using the above-mentioned drug screening model:
  • BODIPY TM FL C 16 fluorescence after seeding Caco2 intestinal epithelial cells stably expressing Soat2 protein in a confocal dish, add the initially screened drug to the culture medium, and use blank reagent as For the control group, after incubation for a period of time, BODIPY TM FL C 16 was added for incubation. The fluorescence conditions of each group were photographed and compared using a fluorescence microscope.
  • the substance that inhibits intestinal Soat2 can be selected from small molecular compounds that inhibit intestinal Soat2, and can also be selected from biological drugs such as siRNA, shRNA, lentivirus, and adenovirus that inhibit the expression of intestinal Soat2.
  • the substance that inhibits intestinal Soat2 can be selected from small molecular compounds that inhibit intestinal Soat2, and can also be selected from biological drugs such as siRNA, shRNA, lentivirus, and adenovirus that inhibit the expression of intestinal Soat2.
  • the substance that inhibits intestinal Soat2 can be selected from small molecular compounds that inhibit intestinal Soat2, and can also be selected from siRNA, shRNA, lentivirus, adenovirus, plasmids that edit Soat2 gene, gRNA that inhibit intestinal Soat2 expression and other biological drugs.
  • the drug action target Soat2 and the drug screening model provided by the present invention can screen out drug compounds with strong lipid-lowering effect and less toxic and side effects from the level of intestinal fatty acid uptake.
  • the drug screening model has the advantages of high throughput, strong specificity, and convenient operation for compound screening.
  • intestinal Soat2 can be used as a target for screening drugs that inhibit intestinal fatty acid uptake, lipid-lowering drugs, fatty liver drugs, and obesity drugs, and play a role in drug screening.
  • A is the full knockout mouse constructed by CRISPR/Cas9 genome editing technology
  • B is the genotype detection of the full knockout mouse
  • C is the conditional knockout mouse constructed by the embryonic stem cell targeting technology
  • D, E, F are the genotypes of the conditional knockout mouse Detection
  • G, H, I are the detection of knockout efficiency of Soat2 knockout mice
  • FIG. 2 The targeting plasmid map in the construction of Soat2 knockout mice of the present invention
  • Fig. 3 is the situation of hepatic steatosis induced by high-fat diet in the present invention in Soat2 knockout mice.
  • A is the body shape change of Soat2 knockout mice after 12 weeks of high-fat feeding
  • B is the fatty degeneration of the mouse liver
  • C is hematoxylin and eosin (H&E) staining and oil red staining of liver slices
  • D is non-alcoholic liver tissue NAFLD activity score
  • E is the liver TG content of mice.
  • Fig. 4 The situation of hepatic steatosis induced by high-fat diet in the present invention in Soat2 liver knockout mice.
  • A is the body shape change of Soat2 liver knockout mice after 12 weeks of high-fat feeding
  • B is the liver steatosis of mice
  • C is H&E staining and oil red staining of liver sections
  • D is the NAFLD activity score of liver tissue
  • E is TG in mouse liver content.
  • Fig. 5 The high-fat diet in the present invention induces hepatic steatosis in Soat2 intestinal knockout mice.
  • A is the body shape change of Soat2 intestinal knockout mice after 12 weeks of high-fat feeding
  • B is the liver steatosis of mice
  • C is H&E staining and oil red staining of liver sections
  • D is the NAFLD activity score of liver tissue
  • E is TG in mouse liver content.
  • Example 1 Based on various Soat2 conditional knockout mouse model experiments, this study provides a lipid-lowering drug screening target Soat2 that can be used to inhibit intestinal fatty acid uptake.
  • Soat2 knockout mouse (Soat2 -/- ) model was generated by CRISPR/Cas9 genome editing (Fig. 1A). Through in vitro transcription, Cas9mRNA and gRNAs were microinjected into the fertilized eggs of C57BL/6J mice, and then the fertilized eggs were implanted into C57BL/6 pseudopregnant female mice to obtain F0 generation mice. The upstream and downstream sequence information of the knockout site and Giude RNA design information are shown in Table 1. The genotype of the F0 generation mice was identified, and the F0 generation mice positive for the knockout of the target gene were obtained, and the PCR product was confirmed to be knocked out successfully by Sanger sequencing.
  • Soat2 +/- heterozygous mice F0-positive mice were mated with wild-type mice to obtain Soat2 +/- heterozygous mice for maintenance and breeding.
  • Soat2 +/- mice were genotyped with F1:5′-ACAGCCTTTCAAGAACCCTCAG-3′ and R2:5′-AAGACCTGCCTTGCCCACA-3′, amplified to give a 1028bp wild-type allele and a 217 or 193bp knockout alleles (Fig. 1B).
  • the knockout effect of Soat2 in the mouse liver was verified at the RNA level, and the knockout efficiency was above 99% ( FIG. 1G ).
  • the flox modification of the Soat2 gene was carried out in the way of embryonic stem cell targeting, and a conditional knockout mouse model was constructed (Fig. 1C).
  • the embryonic stem cell targeting vector (see Figure 2 for the targeting plasmid map), which contains a 5.1 kb 5' homology arm, a 0.8 kb flox region, PGK-Neo-polyA, exon 6, and a 3.6 kb 3' homology arm.
  • Destination vectors were linearized and electroporated into JAM8A3ES cells.
  • Alb-Cre-P1 5'-TGGCAAACATACGCAAGGG-3'
  • Alb-Cre-P2 5'-CGGCAAACGGACAGAAGCA-3'
  • Alb-Cre-P3 5'-GGCAATGGTTCCTCTCTGCT-3' to express Alb-Cre
  • the mice were genotyped, and a target product of about 450 bp and a control product of about 800 bp were obtained ( FIG. 1F ).
  • the knockout effect of Soat2 in the small intestine of intestinal knockout mice and the knockout effect of Soat2 in the liver of liver knockout mice were verified at the RNA level, and the knockout efficiency was above 99% (Fig. 1H, I).
  • Soat2 knockout mouse Soat2 -/-
  • conditional knockout mouse model was entrusted to Shanghai Ncapturing Model Biotechnology Co., Ltd. It can also be constructed according to conventional technical means in this field.
  • mice The constructed Soat2 knockout, liver knockout and intestinal knockout mice were fed with high fat for 12 weeks. The mice were sacrificed at 12 weeks, and serum, liver, intestine and other tissues were collected for subsequent experiments.
  • Liver tissue NAFLD activity score was evaluated according to the NASH clinical research network scoring system [7] .
  • Oil red staining fix frozen liver slices with a thickness of 5 ⁇ m, and stain with oil red for 15 minutes. Nuclei were stained with hematoxylin for 2 min. Sections were imaged with a light microscope.
  • mice Intestinal fatty acid intake: In order to determine the dietary lipid absorption of Soat2 knockout mice under specific conditions, the mice were fasted for 10 h, and then injected with 200 ⁇ l of olive oil containing 5 ⁇ l [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. Mice were sacrificed 2 hours later. Collect contents of small intestine, liver, heart, white fat, brown fat, gallbladder, testis, large intestine and cecum, etc.
  • Intestines were harvested, rinsed with phosphate-buffered saline (PBS), divided into nine equal portions, and the mucosa was removed and frozen in liquid nitrogen. After the samples were homogenized in methanol/water (2:1), the distribution of radioactivity in the tissue was determined by scintillation counting.
  • PBS phosphate-buffered saline
  • the uptake of isotope-labeled fatty acids in intestinal knockout mice further proved that the deletion of Soat2 can effectively reduce the uptake of fatty acids in the intestine and promote the increase of fecal excretion.
  • Soat2 can effectively reduce the uptake of fatty acids in the intestine and promote the increase of fecal excretion.
  • There are several known key links in fatty acid uptake in the small intestine such as cell membrane fat uptake proteins FATP4 and CD36, and intracellular transport protein FABP4, among which CD36 plays a key role in the uptake of fatty acids.
  • the present invention finds that the deletion of Soat2 does not affect other fatty acid uptake-related proteins, but promotes the ubiquitination and degradation of CD36, thereby reducing the uptake of fatty acids.
  • Example 2 Using the Soat2 protein as the target, the lipid-lowering drug screening model and screening method for targeting and inhibiting intestinal fatty acid uptake:
  • Colon cancer cells Caco2 were cultured in MEM complete medium, cultured in a 37°C, 5% CO 2 incubator, and subcultured when they grew to 80% of the area of the culture dish. Cells in the logarithmic growth phase were used for experiments.
  • NBD-cholesterol was dissolved in ethanol to make a stock solution of 1 mg/ml. After the cells in the 96-well plate were cultured in the compound-containing medium for 24 hours, they were replaced with the medium containing 1 ⁇ g/ml NBD-cholesterol ethanol solution, and incubated in a 37° C. incubator for 2 hours. After incubation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. A microplate reader was used to detect the fluorescence intensity (excitation wavelength 485 nm, emission wavelength 535 nm).
  • Control group blank reagent control
  • (2) cDNA was obtained by reverse transcription with Prime Script RT kit (Takara, Japan).
  • the total volume of the reverse transcription reaction system is 20 ⁇ l, including 2 ⁇ L of 10 ⁇ RT Buffer, 0.8 ⁇ L of 25 ⁇ dNTP Mix (100 mM), 2 ⁇ L of 10 ⁇ RT Random Primers, 1 ⁇ L of MultiScribe TM Reverse Transcriptase, 4.2 ⁇ L of DEPC water, and 2 ⁇ g/10 ⁇ L of RNA.
  • Reverse transcription was carried out in Dongshenglong gradient PCR instrument: 10 minutes at 25°C, 120 minutes at 37°C, 5 minutes at 85°C.
  • SYBR-Green dye method (Real-time qPCR Master Mix, Takara, Japan) was used for real-time fluorescent quantitative PCR to detect the expression levels of Soat2 and GAPDH in cells.
  • the reaction system is: SYBR 5 ⁇ L, Forward Primer (10 ⁇ M) 0.2 ⁇ L, Reverse Primer (10 ⁇ M) 0.2 ⁇ L, cDNA (1:10 dilution) 1 ⁇ L, add water to a total volume of 10 ⁇ L.
  • the primer sequences are 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 in triplicate samples using Roche RT-PCR LC480II: 95°C, pre-denaturation for 30s; 95°C, 5s; 60°C, 30s; 40 reaction cycles. Results were normalized to mRNA levels using GAPDH. 2 - ⁇ CT is used to represent the fold change of Soat2 expression in the experimental group relative to the control group.
  • BODIPY TM FL C 16 is dissolved in DMSO.
  • the final concentration of BODIPY TM FL C 16 is 1 ⁇ M, and the DMSO concentration is not higher than 0.1%.
  • 1 ⁇ M BODIPY TM FL C16 was added and incubated in a 37° C. incubator for 30 minutes. After washing with PBS three times, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature. DAPI staining was incubated at room temperature for 5 min. Observed and photographed under a confocal laser microscope LSM700.
  • the screening results of 41 drugs using the screening system are shown in FIG. 6 .

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Abstract

本发明公开了一种筛选抑制肠道脂肪酸摄取及防治脂肪肝的药物靶点及其应用。依据敲除小鼠实验，本发明提供了药物作用靶点Soat2（Sterol O-acyl transferase，甾醇-O-酰基转移酶）及其药物筛选模型。采用高通量NBD-cholesterol筛选、PCR以及BODIPYTM FL C16荧光组合筛选来确定药物化合物对Soat2的抑制效应。以Soat2为靶点的药物筛选模型可以从肠道脂肪酸摄取层面筛选出降脂效果较强、毒副作用较小的药物化合物。

Description

一种筛选抑制肠道脂肪酸摄取及防治脂肪肝的药物靶点及其应用
技术邻域
本发明属于生物医药领域，公开了一种筛选抑制肠道脂肪酸摄取及防治脂肪肝的药物靶点及其应用。
背景技术
非酒精性脂肪肝是目前一种临床常见疾病，主要由于肝脏代谢异常导致过多甘油三酯蓄积致肝细胞变性进而发生炎症反应。脂肪肝进展可能形成肝纤维化，并成为最终导致肝癌的恶性进程。脂肪肝的形成与肥胖、糖尿病、心血管疾病等疾病相互关联和影响。在脂肪肝发生机制上，与肠道摄取外源性脂肪酸增加和/或体内脂肪酸合成增多有关。脂肪肝的治疗措施目前较为局限，主要为危险因素干预治疗和调节脂质代谢的药物治疗。比如肥胖人群中，非酒精性脂肪肝(NAFLD)的发生率高达75％。目前除减重代谢手术外，对肥胖及脂肪肝的治疗主要以降脂药物为主。临床使用的降脂药物大多靶向于肝脏，通过促进脂质清除和代谢来达到降脂的目的。比如目前临床上应用最广泛的一类降脂药-他汀类药物。它通过抑制细胞内胆固醇早期合成阶段的限速酶——三羟基三甲基戊二酰辅酶A(HMG-CoA)还原酶，减少胞内游离胆固醇，加速循环中脂蛋白的清除。他汀类药物降低循环脂蛋白胆固醇的作用虽然与剂量有一定的相关性，但非直线相关。标准的他汀药物剂量往往不足以达到治疗的目的。其原因可能是他汀类在抑制胆固醇的同时，肠道脂质吸收代偿性增加，其调脂作用被抵消 [1]。另外，如贝特类药物主如非诺贝特，苯扎贝特，通过激活过氧化物酶增殖体激活受体(PPARa)，诱导脂蛋白酯酶表达，促进富含甘油三酯的脂蛋白颗粒中甘油三酯水解，导致血浆极低密度脂蛋白(VLDL)减少。并且促进肝脏摄取脂肪酸和抑制肝脏合成甘油三酯，还抑制脂肪组织的激素敏感性酯酶以减少脂肪酸的生成，进一步抑制肝脏合成甘油三酯。虽然降脂药物在一定程度上促进了脂蛋白的清除，但其大量肠道吸收仍然存在，无法从根源上抑制肥胖的形成，且长期使用药物本身也有影响肝功能的潜在副作用。其他如胆碱、氮氨酸、维生素、氨基酸制剂，则只是在保护肝细胞进一步损伤方面起到作用，延缓脂肪肝进程，但并不针对改善肝脏代谢的异常。
Soat2(Sterol O-acyl transferase,甾醇O酰基转移酶),作为胆固醇酯化的关键酶,已知其在胆固醇代谢中发挥重要作用。但是事实上，肠道对于胆固醇与脂肪酸的吸收作用机理是不同的。在整个消化道中，胆固醇的吸收主要发生在上中段。这与胆固醇吸收关键蛋白Niemann-Pick C1 Like1(NPC1L1)在小肠该部位的高表达有关。NPC1L1能特异性结合胆固醇，暴露内吞信号， 通过囊泡内吞作用，吸收肠腔内游离胆固醇。而脂肪酸的肠道吸收主要通过蛋白依赖转运，白细胞分化抗原36(CD36)、脂肪酸结合蛋白(FATP4)被认为参与肠道脂肪酸摄取的脂肪酸转运相关蛋白。其中，CD36是肠细胞脂肪酸的最佳转运体。此外，研究发现，抑制肠道对胆固醇的吸收，对肠道脂肪酸摄取影响很小 [2,3]。而饮食中的饱和脂肪酸(saturated fatty acids,SFA)会促进肠道胆固醇转运相关蛋白的表达，促进肠道对胆固醇的吸收，增加血浆低密度脂蛋白胆固醇(LDL-C) [4,5]。因此，若只限制肠道胆固醇的吸收，往往达不到预期的降脂效果。近年来提出的抑制肠道吸收的降脂药物，大多通过抑制肠道对饮食和胆汁中胆固醇的吸收，来达到降脂的目的，且此类药物上市很少。依折麦布(ezetimibe)是目前唯一发现具有附着在小肠绒毛上皮的刷状缘，靶向抑制甾醇载体NPC1L1，抑制胆固醇的吸收的药物，从而减少肠道中的胆固醇摄取，促进血液胆固醇的清除。但临床研究发现，依折麦布与他汀类药物联用，虽然能在一定程度上降低血清LDL-C水平，但未能达到阻断后续肥胖相关疾病的进展 [6]。若能发现通过抑制肠道脂肪酸摄取从而缓解肥胖及脂肪肝的靶点，研制出以相应蛋白为靶点的降脂药筛选模型及方法，脂肪肝、肥胖等脂质代谢相关疾病的治疗将是一次强有力的推动。
发明内容
为解决上述技术问题，本发明的第一个目的是，提供抑制肠道脂肪酸吸收的药物靶点和以该蛋白为靶点的降脂药物筛选模型。
第二个目的是，提供利用药物筛选模型筛选以肠道Soat2蛋白为靶点的降脂药物的方法。
本发明的目的可通过以下技术方案实现：
Soat2作为靶点在筛选抑制肠道脂肪酸摄取的药物中的应用。
Soat2作为靶点在筛选降脂药物中的应用。
Soat2作为靶点在筛选治疗脂肪肝的药物中的应用。
Soat2作为胆固醇酯化的关键酶，仅在肠道吸收性细胞及肝脏肝细胞表达。本发明发现除了以往研究的抑制肠道游离胆固醇的吸收外，Soat2在抑制肠道脂肪酸吸收方面也具有关键作用。本研究提供的各型Soat2条件敲除小鼠肝脏脂肪变性情况指出，小肠Soat2缺失小鼠肝脏脂肪变性较轻(肠敲与全敲小鼠)。而，单纯肝脏丧失Soat2(肝敲小鼠)，虽然降低胆固醇酯，但依旧带来甚至加重甘油三酯(TG)蓄积(图3，4，5)。表明即使肝脏Soat2正常存在，小 肠Soat2丧失足以抵抗饮食导致的脂肪肝。其关键性机制可能是由于肠道Soat2的缺失促进了脂肪酸转运蛋白CD36的泛素化降解，从而降低了外源性脂肪酸的摄入。因此，本发明提供了所述药物靶点Soat2蛋白。
Soat2作为靶点在筛选治疗脂肪肝的药物中的应用。
一种降脂药物筛选模型，为稳定表达Soat2蛋白的结肠癌细胞Caco2。
一种筛选降脂药物的方法，包括首先选择稳定表达Soat2蛋白的肠上皮细胞Caco2，皿中接种细胞后24h，添加化合物，培养一定时间后，采用高通量NBD-cholesterol筛选、PCR以及BODIPY TM FL C 16荧光来确定Soat2蛋白的量。NBD-cholesterol提示Soat2活性改变，PCR提示Soat2相对表达量，BODIPY TM FL C 16提示游离脂肪酸摄取情况，若上述结果出现降低，则表明化合物能抑制肠道Soat2的表达及活性。
本发明提供利用上述药物筛选模型筛选以肠道Soat2蛋白为靶点的降脂药物的方法：
A.利用高通量筛选分析检测NBD-cholesterol荧光强度，对所有目标药物进行初筛：细胞接种于96孔板后，向培养液中加入初筛选出的药物，同时以空白试剂作为对照组，Soat2特异性抑制剂PPPA，完全抑制细胞中Soat2活性后，作为背景值，孵育一段时间后，加入NBD-cholesterol进行孵育。弃掉培养液，冷PBS洗涤2遍，使用酶标仪检测荧光强度(激发波长485nm，发射波长535nm)。将与各种天然化合物一起孵育后的酶标仪读数减去背景值，作为Soat2活性的依据。
B.采用qRT-PCR方法，扩大样本量进行验证。进一步对初筛药物进行Soat2表达情况的筛选。
C.利用BODIPY TM FL C 16荧光进行游离脂肪酸摄取情况的检测：稳定表达Soat2蛋白的肠上皮细胞Caco2接种于共聚焦皿后，向培养液中加入初筛选出的药物，同时以空白试剂作为对照组，孵育一段时间后，加入BODIPY TM FL C 16孵育。利用荧光显微镜拍摄并比较各组荧光情况。
抑制肠道Soat2的物质在制备抑制肠道脂肪酸摄取的药物中的应用。
所述的抑制肠道Soat2的物质可以选自抑制肠道Soat2的小分子化合物，也可以选自抑制肠道Soat2表达的siRNA、shRNA、慢病毒、腺病毒等生物药物。
抑制肠道Soat2的物质在制备降脂药物中的应用。
所述的抑制肠道Soat2的物质可以选自抑制肠道Soat2的小分子化合物，也可以选自抑制肠道Soat2表达的siRNA、shRNA、慢病毒、腺病毒等生物药物。
抑制肠道Soat2的物质在制备治疗脂肪肝的药物中的应用。
所述的抑制肠道Soat2的物质可以选自抑制肠道Soat2的小分子化合物，也可以选自抑制肠道Soat2表达的siRNA、shRNA、慢病毒、腺病毒、对Soat2基因进行编辑的质粒、gRNA等生物药物。
本发明的有益效果如下：
1)本发明提供的药物作用靶点Soat2及药物筛选模型，可以从肠道脂肪酸摄取层面筛选出降脂效果较强、毒副作用较小的药物化合物。
2)在96孔板中，采用此药物筛选模型对化合物的筛选具有高通量、特异性较强、操作便捷等优点。
3)本发明实验证明肠道Soat2可以作为筛选抑制肠道脂肪酸摄取的药物、降脂药物、脂肪肝治疗药物、肥胖症治疗药物的靶点，在药物的筛选中发挥作用。
附图说明
图1本发明中Soat2敲除小鼠构建及验证
A为CRISPR/Cas9基因组编辑技术构建全敲小鼠，B为全敲小鼠基因型检测，C为胚胎干细胞打靶技术构建条件敲除小鼠，D、E、F为条件敲除小鼠基因型检测，G、H、I为Soat2敲除小鼠敲除效率检测
图2本发明Soat2敲除小鼠构建中打靶质粒图谱
图3本发明中高脂饮食诱导Soat2全敲小鼠肝脏脂肪变性情况。
A为高脂喂养12周后Soat2全敲小鼠体型改变，B为小鼠肝脏脂肪变性情况，C为肝脏切片苏木精和伊红(H&E)染色及油红染色，D为肝脏组织非酒精性脂肪肝(NAFLD)活性评分，E为小鼠肝脏TG含量。
图4本发明中高脂饮食诱导Soat2肝敲小鼠肝脏脂肪变性情况。
A为高脂喂养12周后Soat2肝敲小鼠体型改变，B为小鼠肝脏脂肪变性情况，C为肝脏切片H&E染色及油红染色，D为肝脏组织NAFLD活性评分，E为小鼠肝脏TG含量。
图5本发明中高脂饮食诱导Soat2肠敲小鼠肝脏脂肪变性情况。
A为高脂喂养12周后Soat2肠敲小鼠体型改变，B为小鼠肝脏脂肪变性情况，C为肝脏切片H&E染色及油红染色，D为肝脏组织NAFLD活性评分，E为小鼠肝脏TG含量。
图6本发明实施例1中利用筛选系统对41个药物的筛选结果。
具体实施方式
下列实施例用于本发明，但不限制本发明的范围。除非另有说明，实施例中使用的技术手段是该领域技术人员熟悉的常规手段，所用的原材料是商业上可获得的商品。
实施例1：本研究依据各类Soat2条件敲除小鼠模型实验，提供了可用于抑制肠道脂肪酸摄取的降脂药物筛选靶点Soat2。
1、小鼠饲养与处理：Soat2全敲小鼠(Soat2 -/-)模型通过CRISPR/Cas9基因组编辑生成(图1A)。通过体外转录的方式，将Cas9mRNA和gRNAs微量注射到C57BL/6J小鼠的受精卵中，再将受精卵植入C57BL/6假孕雌鼠体，获得F0代小鼠。敲除位点上下游序列信息和Giude RNA设计信息见表1。对F0代小鼠的基因型进行鉴定，，获得目的基因敲除阳性的F0代小鼠,PCR产物经Sanger测序确认敲除成功。F0代阳性小鼠与野生型小鼠交配得到Soat2 +/-杂合子小鼠，用于维持和育种。用F1:5′-ACAGCCTTTCAAGAACCCTCAG-3′和R2:5′-AAGACCTGCCTTGCCCACA-3′对Soat2 +/-小鼠进行基因分型，扩增可得到1028bp的野生型等位基因和217或193bp的敲除等位基因(图1B)。在RNA水平验证小鼠肝脏中Soat2敲除效果，敲除效率在99％以上(图1G)。利用同源重组原理，采用胚胎干细胞打靶的方式，对Soat2基因进行flox修饰，构建条件敲除小鼠模型(图1C)。首先构建胚胎干细胞打靶载体(打靶质粒图谱见图2)，该载体包含5.1kb的5′同源臂、0.8kb的flox区、PGK-Neo-polyA、6号外显子、3.6kb的3′同源臂和MC1-TK-polyA阴性筛选标记(质粒)。将目标载体线性化并电穿孔入JAM8A3ES细胞。经筛选，共获得144个G418和Ganc抗性无性系。通过长片段PCR分析克隆的正确整合。将正确的靶向克隆注入C57BL/6囊胚，获得嵌合小鼠。嵌合小鼠与Flp小鼠交配获得阳性de-Neo Soat2 flox/flox小鼠。Soat2 flox/flox小鼠分别与Vil1-Cre小鼠和Alb-Cre小鼠杂交，获得肠道特异性Soat2敲除小鼠(Soat2 I-KO)和肝脏特异性Soat2敲除小鼠(Soat2 L-KO)。采用引物F1:5'-TCGTCCCAGCCCAGTCTTT-3'和R2:5'-CTGCCTTGCCCACAGTTTCT-3'对携带Soat2 flox等位基因的小鼠进行基因分型，扩增可得到289bp的野生型等位基因和344bp的敲除等位基因(图1D)。用引物Vil1-Cre-F1:5’-TCGATGCAACGAGTGATGAG-3’, Vil1-Cre-R1:5’-TCCATGAGTGAACGAACCTG-3’、Control-F1:5’-CAAATGTTGCTTGTCTGGTG-3’and Control-R1:5’-GTCAGTCGAGTGCACAGTTT-3’对表达Vil1-Cre的小鼠进行基因分型，得到约400bp的目标产物和约200bp的对照产物(图1E)。用引物Alb-Cre-P1:5’-TGGCAAACATACGCAAGGG-3’，Alb-Cre-P2:5’-CGGCAAACGGACAGAAGCA-3’，Alb-Cre-P3:5’-GGCAATGGTTCCTCTCTGCT-3’对表达Alb-Cre的小鼠进行基因分型，得到约450bp的目标产物和约800bp的对照产物(图1F)。在RNA水平验证肠敲小鼠小肠中Soat2敲除效果和肝敲小鼠肝脏中Soat2敲除效果，敲除效率均在99％以上(图1H，I)。上述Soat2全敲小鼠(Soat2 -/-)模型和条件敲除小鼠模型的构建均委托上海南方模式生物科技股份有限公司进行。也可以按照本领域的常规技术手段构建。
对构建的Soat2全敲、肝敲及肠敲小鼠进行12周的高脂喂养。12周时处死小鼠，取血清、肝脏、肠等组织，进行后续的实验。
2、组织病理学检查：肝组织在10％福尔马林中固定过夜，石蜡包埋。苏木精和伊红(H&E)经脱蜡和复水后制备5μm石蜡切片。依据NASH临床研究网络评分系统评估肝脏组织NAFLD活性评分(NAS) [7]。
3、油红染色：将厚度为5μm的冰冻肝脏切片固定，油红染色15min。细胞核苏木素染色2min。切片用光学显微镜成像。
4、肠道脂肪酸摄取：为了确定特定条件下Soat2敲除小鼠的膳食脂质吸收，小鼠禁食10h，然后注射200μl含有5μl[9,10-3H(N)]三油酸的橄榄油。分别于0、30、60、90和120分钟从尾部采集血液，分离血浆进行液体闪烁计数。2h后处死小鼠。收集小肠、肝脏、心脏、白色脂肪、棕色脂肪、胆囊、睾丸、大肠和盲肠内容物等。取肠道，用磷酸盐缓冲盐水(PBS)冲洗后，分成九等份，取出粘膜，在液氮中冷冻。样品在甲醇/水(2:1)中均质后，用闪烁计数法测定组织中放射性分布。
5、结果：本研究发现肠敲小鼠的肝脏脂肪变性程度明显缓解，表现为肝脏TG减少，NAS评分降低。表明即使肝脏Soat2正常存在，小肠Soat2的缺失足以抵抗高脂饮食导致的脂肪肝。也提示了小肠摄入游离脂肪酸对于脂肪肝形成的重要性。相反的，肝敲小鼠由于其小肠游离脂肪酸摄入未受抑制，外源性脂肪酸的摄入依旧可以促进其脂肪肝的发生(图3，4，5)。此外，肠敲小鼠对同位素标记脂肪酸的摄取结果进一步证明了Soat2的缺失能有效减少肠道对脂肪酸的摄取，促进粪便排泄增加。小肠脂肪酸摄取主要有几个已知的关键环 节，如细胞膜脂肪摄取蛋白FATP4、CD36，细胞内转运蛋白FABP4，其中，CD36对脂肪酸的摄取在其中起关键的作用。本发明发现，Soat2的缺失不影响其他脂肪酸摄取相关蛋白，而促进CD36的泛素化降解，从而减少对脂肪酸的吸收。
实施例2以Soat2蛋白为靶点，靶向抑制肠道脂肪酸摄取的降脂药物筛选模型及筛选方法：
1、细胞培养
将结肠癌细胞Caco2培养于MEM完全培养基中，置于37℃、5％CO 2培养箱培养，待生长至培养皿的80％面积时，传代培养。取对数生长期的细胞用于实验。
2、高通量NBD-cholesterol荧光技术进行药物初筛：
(1)待细胞长满培养皿后，加入0.25％胰酶消化5min，弃去胰酶，加入培养基进行吹打，制成单细胞悬液。用计数仪进行计数，取适量细胞悬液接种于黑壁透明底的96孔板，确保每孔细胞数一致。待细胞贴壁后，每孔加入终浓度为10μM的化合物，继续培养24h，进行高通量NBD-cholesterol荧光技术进行药物初筛。
(2)NBD-cholesterol溶于乙醇，配成1mg/ml的原液。96孔板中的细胞经24h的含化合物培养基培养后，换用含1μg/ml的NBD-cholesterol乙醇溶液的培养基，于37℃培养箱中孵育2h。孵育后的细胞，用PBS洗涤3次，4％多聚甲醛室温固定20min。使用酶标仪检测荧光强度(激发波长485nm，发射波长535nm)。
(3)检测结果分析
对照组：空白试剂对照；
背景值：Soat2特异性抑制剂PPPA，完全抑制细胞中Soat2活性后，作为背景值；
Soat2活性计算：Soat2相对活性＝(实验组荧光值-背景值)/(对照组荧光值-背景值)
3、PCR技术对初筛所得药物进行Soat2表达的复筛：
(1)使用TRIzol试剂提取RNA。刮取细胞1-5×10 7于1.5ml离心管，加入1mlTRIzol，室温静置3min。加入200μl氯仿，上下震荡8s，静置2min。4℃离心，12000g×15min，取上清。加入0.5ml异丙醇，轻轻混匀，冰上静置10min。4℃离心，12000g×10min，弃上清。加入1ml 75％乙醇，轻轻洗涤沉淀。4℃，7500g×5min，弃上清。晾干，加入适量的DEPC水溶解。
(2)用Prime Script RT kit(Takara,Japan)逆转录得到cDNA。其中逆转录反应体系总体积为20μl，包括10×RT Buffer 2μL，25×dNTP Mix(100mM)0.8μL，10×RT Random  Primers 2μL，MultiScribe TM Reverse Transcriptase 1μL，DEPC水4.2μL，RNA 2μg/10μL。逆转录在东胜龙梯度PCR仪进行：25℃反应10min，37℃反应120min，85℃反应5min。
(3)采用SYBR-Green染料法(Real-time qPCR Master Mix，Takara,Japan)进行实时荧光定量PCR，检测细胞Soat2及GAPDH表达水平。反应体系为：SYBR 5μL，Forward Primer(10μM)0.2μL，Reverse Primer(10μM)0.2μL，cDNA(1:10稀释)1μL，加水至总体积为10μL。引物序列如下：Soat2，Forward:5’-ACGTTGCCAGGCATCTTCAT-3’，Reverse:5’-AGTCATGGACCACCACGTTC-3’；GAPDH，Forward:5’-AGGTCGGTGTGAACGGATTTG-3’，Reverse:5’-GGGGTCGTTGATGGCAACA-3’。使用罗氏RT-PCR LC480II在三次重复样本中进行实时定量PCR：95℃，预变性30s；95℃，5s；60℃，30s；40个反应循环。结果用GAPDH进行mRNA水平标准化。采用2 -ΔΔCT表示实验组Soat2表达相对于对照组变化的倍数。
4、利用BODIPY TM FL C 16荧光进行游离脂肪酸摄取情况的检测。
(1)待细胞长满培养皿后，加入0.25％胰酶消化5min，弃去胰酶，加入培养基进行吹打，制成单细胞悬液。取适量的细胞悬液接种于共聚焦皿。待细胞贴壁后，每孔加入终浓度为10μM的初筛得到的化合物，继续培养24h。
(2)BODIPY TM FL C 16溶于DMSO，使用时BODIPY TM FL C16终浓度为1μM，DMSO浓度不高于0.1％。细胞经化合物24h处理后，加入1μM的BODIPY TM FL C16于37℃培养箱孵育30min。PBS洗涤3次后，用4％多聚甲醛室温固定20min。DAPI染色室温孵育5min。在共聚焦激光显微镜LSM700下观察并拍照。
5、结果
通过对41种药物化合物进行Soat2蛋白抑制情况的筛选，发现其中降脂药小檗碱可以有效的抑制Soat2酶活性及其表达，且减少了细胞对游离脂肪酸的摄取。结果提示Soat2抑制对肠道脂肪酸的摄取可作为降脂药筛选的靶点，具有一定的临床应用意义。
利用所述筛选系统对41个药物的筛选结果见图6。
上述各实施方式为实现本发明的具体实施例，而在实际应用中，在不偏离本发明精神的基础上所做的形式或细节上的改变，均属于本发明要求保护的范围。

Claims (11)

  1. Soat2作为靶点在筛选抑制肠道脂肪酸摄取的药物中的应用。
  2. Soat2作为靶点在筛选降脂药物中的应用。
  3. Soat2作为靶点在筛选治疗脂肪肝的药物中的应用。
  4. Soat2作为靶点在筛选治疗肥胖的药物中的应用。
  5. 一种细胞在制备抑制肠道脂肪酸摄取的药物筛选模型中的应用，其特征在于所述的细胞为稳定表达Soat2蛋白的结肠癌细胞Caco2。
  6. 一种筛选抑制肠道脂肪酸摄取的药物的方法，其特征在于包括：首先选择稳定表达Soat2蛋白的肠上皮细胞Caco2，皿中接种细胞后20～26h，添加待筛选物质，培养一定时间后，采用高通量NBD-cholesterol筛选、PCR以及BODIPY TMFL C 16荧光来确定Soat2蛋白的量；NBD-cholesterol提示Soat2活性改变，PCR提示Soat2相对表达量，BODIPY TMFL C 16提示游离脂肪酸摄取情况，若上述结果出现降低，则表明该物质能抑制肠道脂肪酸摄取。
  7. 根据权利要求6所述的方法，其特征在于包含以下步骤：
    (1)利用高通量筛选分析检测NBD-cholesterol荧光强度，对所有目标药物进行初筛：细胞接种于96孔板后，向培养液中加入初筛选出的药物，同时以空白试剂作为对照组，Soat2特异性抑制剂PPPA，完全抑制细胞中Soat2活性后，作为背景值，孵育一段时间后，加入NBD-cholesterol进行孵育，弃掉培养液，冷PBS洗涤，，使用酶标仪检测荧光强度，激发波长485nm，发射波长535nm，将与各种药物一起孵育后的酶标仪读数减去背景值，作为Soat2活性的依据；
    (2)采用qRT-PCR方法，扩大样本量进行验证，进一步对初筛药物进行Soat2表达情况的筛选；
    (3)利用BODIPY TMFL C 16荧光进行游离脂肪酸摄取情况的检测：细胞接种于共聚焦皿后，向培养液中加入初筛选出的药物，同时以空白试剂作为对照组，孵育一段时间后，加入BODIPY TMFL C 16孵育。利用荧光显微镜拍摄并比较各组荧光情况。
  8. 抑制肠道Soat2的物质在制备抑制肠道脂肪酸摄取的药物中的应用；所述的抑制肠道Soat2的物质为抑制肠道Soat2的小分子化合物、抑制肠道Soat2表达的siRNA、shRNA、慢病毒、腺病毒、CRISPR/Cas9基因组编辑系统。
  9. 抑制肠道Soat2的物质在制备降脂药物中的应用。
  10. 抑制肠道Soat2的物质在制备治疗脂肪肝的药物中的应用。
  11. 抑制肠道Soat2的物质在制备治疗肥胖的药物中的应用。
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