WO2018028433A1 - Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途 - Google Patents

Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途 Download PDF

Info

Publication number
WO2018028433A1
WO2018028433A1 PCT/CN2017/094509 CN2017094509W WO2018028433A1 WO 2018028433 A1 WO2018028433 A1 WO 2018028433A1 CN 2017094509 W CN2017094509 W CN 2017094509W WO 2018028433 A1 WO2018028433 A1 WO 2018028433A1
Authority
WO
WIPO (PCT)
Prior art keywords
creg
obesity
protein
creg protein
mice
Prior art date
Application number
PCT/CN2017/094509
Other languages
English (en)
French (fr)
Inventor
韩雅玲
田孝祥
闫承慧
张权宇
张效林
刘丹
Original Assignee
中国人民解放军沈阳军区总医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国人民解放军沈阳军区总医院 filed Critical 中国人民解放军沈阳军区总医院
Priority to US16/323,950 priority Critical patent/US20190216894A1/en
Publication of WO2018028433A1 publication Critical patent/WO2018028433A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • the present invention relates to the pharmaceutical use of the Cellular Repressor of E1A-stimulated Genes (CREG), in particular to the preparation of a CREG protein or an active fragment thereof for the prevention or treatment of overweight, obesity, insulin resistance, hyperlipidemia and The use of drugs for its related diseases.
  • CREG Cellular Repressor of E1A-stimulated Genes
  • BMI body weight index
  • BMI ⁇ 30Kg / m 2 is defined as obesity
  • the domestic standard is: BMI ⁇ 24Kg / m 2 is defined as being overweight
  • BMI ⁇ 28Kg / m 2 is defined as obesity, if overweight is not controlled in time, it can develop into obesity.
  • Overweight and obesity are both independent diseases, and can be associated with or secondary to a variety of obesity-related diseases, including metabolic syndrome, pre-diabetes, type 2 diabetes, dyslipidemia, hypertension, nonalcoholic fatty liver disease, polycystic ovary Syndrome, sleep apnea, osteoarthritis, gastroesophageal reflux disease, etc., are listed by WHO as one of the top ten risk factors for disease burden.
  • obesity-related diseases including metabolic syndrome, pre-diabetes, type 2 diabetes, dyslipidemia, hypertension, nonalcoholic fatty liver disease, polycystic ovary Syndrome, sleep apnea, osteoarthritis, gastroesophageal reflux disease, etc.
  • Orlistat is a lipase inhibitor that prevents the hydrolysis of triglycerides into absorbable free fatty acids and monoglycerides, which are not absorbed, thereby reducing caloric intake and controlling body weight.
  • its weight loss is not strong (in 1-4 years, drug-treated patients lose 2.5-3.2 kg less than the placebo group), and it can cause some adverse reactions such as flatulence, oily spots and emergency.
  • fat cell dysfunction is the initiating factor of obesity, but the key factors regulating the homeostasis of fat cells have not yet been elucidated.
  • CREG is a small molecule glycoprotein that is widely expressed in mature tissues and cells.
  • the CREG protein is mainly localized in the Golgi and lysosomes of the perinuclear nucleus, involved in the transport of lysosomal enzymes and endocytosis of extracellular factors (Schahs P, Exp Cell Res, 2008, 314(16): 3036-3047; Kowalewski - Nimmerfall E, Biochim Biophys Acta, 2014, 1843(12): 2900-2912).
  • CREG protein is involved in the occurrence and progression of various cardiovascular diseases such as hypertension, vascular remodeling, atherosclerosis, myocardial ischemia-reperfusion injury, myocardial infarction, etc., and maintains cardiovascular homeostasis.
  • cardiovascular diseases such as hypertension, vascular remodeling, atherosclerosis, myocardial ischemia-reperfusion injury, myocardial infarction, etc.
  • the role of CREG protein in obesity and its related diseases and its mechanism are still unclear.
  • the inventors of the present invention found through a large number of experiments that the expression of CREG in adipose tissue of obese mice was significantly lowered.
  • Exogenous supplementation of CREG protein into obese mice can significantly reduce body weight, reduce adipose tissue weight, reduce liver tissue steatosis, lower blood lipid levels, improve adipokines secretion, and improve insulin resistance.
  • the above results indicate that exogenous supplemental CREG protein can be used to prevent or treat overweight, obesity, insulin resistance, hyperlipidemia and related diseases.
  • the present invention has been completed based on the above findings.
  • a first aspect of the invention relates to the use of a CREG protein or an active fragment thereof for the preparation of a medicament for the prevention and/or treatment of obesity.
  • the present invention also relates to a nucleic acid molecule encoding a CREG protein or an active fragment thereof, a recombinant vector expressing the CREG protein or an active fragment thereof, or a recombinant cell for use in the preparation of a medicament for preventing and/or treating overweight, obesity, Insulin resistance, hyperlipidemia and related diseases.
  • the recombinant vector comprises a nucleic acid molecule encoding a CREG protein or an active fragment thereof.
  • the invention also relates to the use of an agent for detecting the expression level of a CREG protein or an active fragment thereof for use in a kit for overweight, obesity, insulin resistance, hyperlipidemia prediction and/or therapeutic effect, prognosis Evaluation.
  • the invention also relates to the use of a CREG protein or an active fragment thereof for screening for a medicament for the prevention and/or treatment of overweight, obesity, insulin resistance, hyperlipidemia.
  • the CREG protein or an active fragment thereof can be used as a target protein for screening for a drug for preventing and/or treating overweight, obesity, insulin resistance, hyperlipidemia and related diseases; for example, promoting CREG protein or An agent whose expression of an active fragment is up-regulated can be used as a drug for preventing and/or treating overweight, obesity, insulin resistance, hyperlipidemia, and related diseases.
  • the invention also relates to a composition
  • a composition comprising a CREG protein or an active fragment thereof, a nucleic acid molecule encoding a CREG protein or an active fragment thereof, a recombinant vector or recombinant cell expressing a CREG protein or an active fragment thereof, and optionally a pharmaceutically acceptable A carrier or excipient for preventing and/or treating overweight, obesity, insulin resistance, hyperlipidemia and related diseases.
  • the invention also relates to a kit comprising an agent for detecting the expression level of a CREG protein or an active fragment thereof for use in prediction and/or therapeutic effects of overweight, obesity, insulin resistance, hyperlipidemia and related diseases, Assessment of prognosis.
  • the CREG protein is a recombinant CREG protein derived from a mammal, particularly from a human.
  • the GenBank number of the CREG protein is NP_003842.1.
  • the GenBank number of the CREG gene is NM_003851.2.
  • the active fragment of the CREG protein refers to a fragment having a function of a CREG protein, which may be a part of the CREG protein, or a fragment obtained by deleting, adding or replacing the amino acid sequence of the CREG protein;
  • Methods for obtaining a CREG protein active fragment are known in the art, for example, the active fragment is a fragment comprising a portion of a CREG protein that binds to a ligand or receptor, or a fragment that retains CREG protein function after deletion, addition or substitution of an amino acid.
  • the active fragment is a fragment comprising a portion of a CREG protein that binds to a ligand or receptor, or a fragment that retains CREG protein function after deletion, addition or substitution of an amino acid.
  • some key amino acids on the CREG protein are closely related to the activity, and the mutation may affect the activity of the protein.
  • the lysine at positions 136 and 137 of the CREG protein is mutated to alanine, or the CREG protein is 141.
  • -144 amino acid deletion mutations affect protein activity and function (Sacher M, PNAS, 2005; 102(51): 18326-18331).
  • Those skilled in the art can circumvent these above-mentioned sites which may affect the activity as needed, and perform modifications such as deletion, addition or substitution on other sites, so that the modified CREG protein still has the activity or function of the CREG protein.
  • the overweight and obesity have the meanings well known in the art with or without obesity related diseases.
  • the overweight or obesity-related disease has a meaning well-known in the art and refers to accompanying or secondary A series of diseases that are overweight or obese, including metabolic syndrome, pre-diabetes, type 2 diabetes, and dyslipidemia.
  • the prevention and/or treatment of overweight or obesity means inhibiting or slowing the occurrence of overweight or obesity, inhibiting or slowing the occurrence of an overweight or obesity-related disease.
  • the detection of the expression level of the CREG protein or its active fragment for prediction and/or evaluation means that when the expression level of the CREG protein or its active fragment in blood, tissue or cells is lower than the reference value, Overweight or obesity is predicted to occur, or its treatment effect or prognosis is assessed.
  • the mammal may be, for example, a rat, a mouse, a dog, a miniature pig, a monkey, a human or the like.
  • the expression level of the CREG protein or an active fragment thereof can be detected by a method known in the art, for example, amplification of CREG mRNA by a polymerase chain reaction and quantitative reaction, or detection of CREG protein expression level by Western Blot.
  • the expression level of the protein refers to the level of mRNA or the level of protein.
  • the up-regulating/down-regulating the expression of a protein in a tissue/cell means increasing or decreasing at least 20%, 30%, 40%, 50%, 60%, 70% of the protein level or mRNA level in the tissue/cell. , 80%, 90%, 100%, or increase by more than 100%.
  • the up- or down-regulation described therein is compared to uninterrupted tissues/cells (e.g., tissues/cells of the transfected control vector group).
  • Figure 1 Preparation of obesity model of high-fat fed C57BL/6J mice and detection of CREG protein expression in adipose tissue.
  • mice Three groups of mice were treated with normal feeding, high-fat feeding and high-fat feeding + CREG protein. Glucose tolerance test and insulin tolerance test were performed at 16 weeks to evaluate insulin resistance. The results suggest that CREG protein treatment can significantly improve insulin resistance in obese mice.
  • the experimental data of the present invention are all percentages.
  • the two-sample rate was compared using the chi-square test, and the statistical processing was performed using the SPSS 19.0 software package. There was a statistical difference at P ⁇ 0.05.
  • Example 1 Preparation of obesity model of C57BL/6J mice fed with high fat and detection of CREG protein expression in adipose tissue.
  • mice 40 male 8-week-old C57BL/6 mice [body weight (22.3 ⁇ 1.2) g] were divided into the following two groups by random table method: normal feeding group and high fat feeding group, 20 rats in each group.
  • the normal feeding group was fed with normal diet feed (3.85kcal/g, fat provided 10% calories, American Research Diet Company), and the high fat diet group was fed with high fat diet (5.24kcal/g, fat provided 60% calories, American Research Diet) the company). Continue feeding for 16 weeks.
  • the mice were fed with standard conditions and received 12 hours of light without restriction on diet and water intake.
  • mice Each group of mice was weighed every 2 weeks. Fasting and water for 12h before weighing. Each mouse was weighed 3 times, recorded and averaged.
  • the electrophoresis termination time was determined by SDS-PAGE electrophoresis on 12% separation gel.
  • the sample was transferred to a PVDF membrane at a voltage of 21 V for 45 min; it was blocked at room temperature for 1.5 h in TBS-T diluted 5% skim milk powder, and then added to the primary antibody at 4 ° C overnight.
  • Example 2 CREG protein treatment significantly attenuated obesity in high fat fed C57BL/6J mice.
  • 1CREG protein treatment can alleviate obesity in mice caused by high fat feeding.
  • mice Sixty male 8-week-old C57BL/6 mice were divided into the following three groups by random table method: normal feeding group, high fat feeding group, high fat feeding + CREG protein treatment group. 20 in each group.
  • the normal feeding and high fat feeding methods were the same as in Example 1.
  • the high-fat feeding + CREG protein treatment group was treated with high-fat feeding, and the mouse CREG protein (Abcam, 150 ⁇ g/kg ⁇ d) was treated with subcutaneous embedding micro-osmotic pump (Alzet, USA).
  • the treatment time was 16 week.
  • the standard of feeding conditions in mice was 12 hours of light, and the diet and water intake were not limited. The general morphology of the mice was observed after 16 weeks.
  • mice developed significant obesity after high-fat feeding. After treatment with CREG protein, the mice were obese. Significant improvement indicates that CREG has an effect on the treatment of obesity (see Figure 2A for results).
  • mice Each group of mice was weighed every two weeks. Fasting and water for 12 hours before weighing. Each mouse was weighed 3 times, recorded and averaged.
  • the total feed quality of each mouse in 2 weeks was weighed, and the caloric intake of the normal feeding group was calculated at 3.85 kcal/g of normal feed.
  • the caloric intake of the high fat fed group and the CREG protein treated group was calculated at 5.24 kcal/g of high fat diet. And divide by 14 to get the average daily caloric intake per mouse.
  • testis white adipose tissue was taken for HE staining analysis.
  • mice were weighed and statistically analyzed.
  • Example 3 CREG protein treatment significantly attenuated the steatosis of the liver of high fat fed C57BL/6J mice.
  • mice liver tissue HE staining Three groups of mice liver tissue HE staining.
  • liver tissues of each group of mice were taken for HE staining, and the specific method was the same as in Example 2.
  • mice were sacrificed and liver tissue was taken. After washing with PBS, the filter paper is removed as much as possible. The liver was weighed and each mouse was measured 3 times for averaging, recorded and statistically analyzed.
  • mice liver CREG protein immunohistochemical staining Three groups of mice liver CREG protein immunohistochemical staining.
  • Example 4 CREG protein treatment can reduce blood lipid levels and improve adipokines expression in obese mice.
  • mice Three groups of mice were measured for adipokines leptin and adiponectin levels.
  • Plasma samples were obtained in the same manner as in the present Example 1 for the determination of leptin and adiponectin levels.
  • the assay method was performed by ELISA, and the leptin and adiponectin ELISA assay kits were purchased from RD Corporation of the United States.
  • Example 5 CREG protein treatment improves insulin resistance in obese mice.
  • mice were intraperitoneally injected with a total volume of 0.1 ml of glucose aqueous solution at a dose of 2 g/kg.
  • the blood glucose levels of the tail vein blood were measured at 15 min, 30 min, 45 min, 60 min and 120 min after injection to determine the function of islet cells.
  • mice were injected intraperitoneally with human insulin at a dose of 0.75 U/kg.
  • the blood glucose levels of the tail vein blood were measured at 15 min, 30 min, 45 min, 60 min and 120 min after injection to determine insulin resistance.
  • CREG protein is expected to be an effective drug for the prevention and treatment of overweight, obesity, insulin resistance, hyperlipidemia and related diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Diabetes (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Emergency Medicine (AREA)
  • Child & Adolescent Psychology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

E1A激活基因阻遏子CREG蛋白的用途，具体涉及CREG蛋白或其活性片段在制备用于预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的药物中的用途。还涉及表达CREG蛋白或其活性片段的重组载体或重组细胞在制备用于预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病中的用途。

Description

CREG蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途 技术领域
本发明涉及E1A激活基因阻遏子(Cellular Repressor of E1A-stimulated Genes,CREG)的医药用途，具体涉及CREG蛋白或其活性片段用于制备预防或治疗体重超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的药物的用途。
背景技术
超重和肥胖是一种以脂肪堆积过多或分布异常为特征的慢性代谢性疾病。根据体重指数(body weight index,BMI＝体重/身高2)，世界卫生组织将BMI≥25Kg/m2定义为超重，将BMI≥30Kg/m2定义为肥胖，国内标准为：BMI≥24Kg/m2定义为超重，BMI≥28Kg/m2定义为肥胖，体重超重如不能及时得到控制，可发展为肥胖。超重和肥胖既是一个独立的疾病，又可以伴发或继发多种肥胖相关疾病，包括代谢综合征，糖尿病前期，2型糖尿病，血脂异常，高血压，非酒精性脂肪性肝病，多囊卵巢综合征，睡眠呼吸暂停，骨关节炎，胃食管返流病等，被WHO列为导致疾病负担的十大危险因素之一。在中国，肥胖人群呈持续增长趋势。根据2002年卫生部发布的《中国居民营养与卫生状况调查》显示，我国成人超重率高达22.8％(2亿)，肥胖率7.1％(六千万)，较1980年翻了一倍。预计到2020年，我国肥胖率将接近10％。因此，预防和控制肥胖刻不容缓。
超重和肥胖发生率的持续增长一方面归因于人们生活方式的改变，另一方面也反映出肥胖治疗手段的缺乏。药物治疗是控制慢性代谢性疾病的利器。目前对于糖尿病及高脂血症等慢性代谢性疾病，均已开发出多种安全有效的药物供临床医生选择。而对于超重和肥胖患者，目前经美国食品药品管理局(FDA)批准的药物仅有5种，其中除奥利司他外，其余4种均在近3年才被批准，其长期的疗效及副作用尚不明确。在中国，仅有奥利司他一种药物被批准用于肥胖的治疗。奥利司他是一种脂肪酶抑制剂，它能阻止甘油三酯水解为可吸收的游离脂肪酸和单酯酰甘油，使其不被吸收，从而减少热量摄入，控制体重。但它的减重作用不强(1-4年内，药物治疗患者较安慰剂组体重减少2.5-3.2kg)，且会导致一些不良反应如胃肠胀气、油性斑点和便紧急感。总之，目前缺乏理想的安全有效的针对超重和肥胖的治疗药物。
肥胖的发生机制非常复杂，受遗传、环境等多种因素影响。研究发现，脂肪组织中脂肪细胞功能障碍、免疫细胞浸润、脂肪组织慢性低水平炎症反应是肥胖发生的主要病理改变。其中，脂肪细胞功能障碍是肥胖发生的始动因素，但调控脂肪细胞稳态平衡的关键因子仍未阐明。
CREG是一个在成熟组织和细胞中广泛表达的小分子糖蛋白。CREG蛋白主要定位于细胞核周的高尔基体和溶酶体中，参与溶酶体酶的转运和细胞外因子的内吞(Schahs P,Exp Cell Res,2008,314(16):3036-3047；Kowalewski-Nimmerfall E,Biochim Biophys Acta,2014,1843(12):2900-2912)。并且，已有大量研究表明CREG蛋白参与高血压、血管重塑、动脉粥样硬化、心肌缺血再灌注损伤、心肌梗死等多种心血管系统疾病的发生与进展，是维持心血管稳态及胚胎发育的重要因子。但是，CREG蛋白对肥胖及其相关疾病的作用及其机制尚不清楚。
发明内容
本发明的发明人通过大量实验发现，肥胖小鼠脂肪组织中CREG的表达显著降低。向发生肥胖的小鼠外源性补充CREG蛋白，能明显降低小鼠体重、减轻脂肪组织重量、减轻肝脏组织脂肪变性、降低血脂水平、改善脂肪因子分泌、改善胰岛素抵抗。以上结果表明，外源性补充CREG蛋白可用于预防或治疗体重超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病。本发明基于以上发现而完成。
本发明第一方面涉及CREG蛋白或其活性片段在制备药物中的用途，所述药物用于预防和/或治疗肥胖。
本发明还涉及编码CREG蛋白或其活性片段的核酸分子、表达CREG蛋白或其活性片段的重组载体或重组细胞在制备药物中的用途，所述药物用于预防和/或治疗体重超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病。
在本发明的实施方案中，所述重组载体含有编码CREG蛋白或其活性片段的核酸分子。
本发明还涉及检测CREG蛋白或其活性片段表达水平的试剂在制备试剂盒中的用途，所述试剂盒用于超重、肥胖、胰岛素抵抗、高脂血症的预测和/或治疗效果、预后的评估。
本发明还涉及CREG蛋白或其活性片段用于筛选预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症的药物的用途。
在本发明的实施方案中，CREG蛋白或其活性片段可以作为靶蛋白用于筛选预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的药物；例如促进CREG蛋白或其活性片段表达上调的试剂可以作为预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的药物。
本发明还涉及组合物，其含有CREG蛋白或其活性片段、编码CREG蛋白或其活性片段的核酸分子、表达CREG蛋白或其活性片段的重组载体或重组细胞，以及任选的药学上可接受的载体或赋形剂，所述组合物用于预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病。
本发明还涉及试剂盒，其含有检测CREG蛋白或其活性片段表达水平的试剂，所述试剂盒用于超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的预测和/或治疗效果、预后的评估。
在本发明中，所述CREG蛋白为重组CREG蛋白，来源于哺乳动物，特别是来源于人。在本发明的优选实施方案中，所述CREG蛋白的GenBank号为NP_003842.1。在本发明的优选实施方案中，所述CREG基因的GenBank号为NM_003851.2。
在本发明中，所述CREG蛋白的活性片段是指具有CREG蛋白功能的片段，其可以为CREG蛋白的一部分，也可以为CREG蛋白的氨基酸序列经过缺失、添加或替换后得到的片段；制备或得到CREG蛋白活性片段的方法为本领域所公知，例如该活性片段为包含CREG蛋白与配体或受体结合的部分的片段，或者经过氨基酸的缺失、添加或替换后仍保留CREG蛋白功能的片段。本领域技术人员公知，CREG蛋白上有一些关键的氨基酸和活性密切相关，突变后会影响蛋白的活性，例如，CREG蛋白第136及137位赖氨酸突变为丙氨酸，或者CREG蛋白第141-144位氨基酸缺失突变后，都会影响蛋白的活性和功能(Sacher M,PNAS,2005；102(51):18326-18331)。本领域技术人员可以根据需要避开上述这些可能影响活性的位点，对其它位点进行缺失、添加或替换等改造，使得改造后的CREG蛋白仍具有CREG蛋白的活性或功能。
在本发明中，所述超重及肥胖具有本领域公知的含义，伴或不伴有肥胖相关疾病。
在本发明中，所述超重或肥胖相关疾病具有本领域公知的含义，是指伴随或继发 于超重或肥胖的一系列疾病，包括代谢综合征，糖尿病前期，2型糖尿病，血脂异常。
在本发明中，所述预防和/或治疗超重或肥胖，是指抑制或减缓超重或肥胖的发生、抑制或减缓超重或肥胖相关疾病的发生。
在本发明中，所述通过检测CREG蛋白或其活性片段表达水平用于预测和/或评估是指当血液、组织或细胞中的CREG蛋白或其活性片段表达水平低于参考值时，即可以预测超重或肥胖发生，或者评估其治疗效果或预后。
在本发明中，所述哺乳动物例如可以为大鼠、小鼠、犬、小型猪、猴、人等。
在本发明中，可以通过本领域公知的方法检测CREG蛋白或其活性片段的表达水平，例如通过聚合酶链式反应扩增CREG的mRNA并进行定量反应，或者用Western Blot检测CREG蛋白表达水平。
在本发明中，所述蛋白的表达水平是指mRNA的水平或者蛋白的水平。
在本发明中，所述上调/下调组织/细胞中蛋白的表达是指提高或降低组织/细胞中蛋白水平或mRNA水平的至少20％、30％、40％、50％、60％、70％、80％、90％、100％，或者提高大于100％。其中所述的上调或下调是与未干预的组织/细胞(例如转染对照载体组的组织/细胞)进行比较。
附图说明
图1.高脂喂养C57BL/6J小鼠肥胖模型制备及脂肪组织中CREG蛋白表达检测。
(A)高脂喂养C57BL/6J小鼠16周后，发生明显的肥胖。
(B)高脂喂养与正常喂养C57BL/6J小鼠不同周数体重。*p<0.05，**p<0.01，***p<0.001(与正常喂养组相比)。
(C)免疫组织化学染色方法检测高脂喂养的肥胖小鼠与对照组小鼠脂肪组织中CREG蛋白的表达。
(D)Western Blot方法检测高脂喂养的肥胖小鼠与对照组小鼠脂肪组织中CREG蛋白的表达。**p<0.01(与正常喂养组相比)。
图2.CREG蛋白治疗显著减轻高脂喂养C57BL/6J小鼠肥胖。
(A)给予高脂喂养的C57BL/6J小鼠CREG蛋白治疗(150μg/kg·d)，发现CREG 蛋白治疗可显著减轻小鼠肥胖。
(B)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠不同周数的体重，结果提示CREG蛋白治疗可显著降低肥胖小鼠体重。*p<0.05，**p<0.01(与高脂喂养组相比)。
(C)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠不同周数的食物摄入量，结果提示CREG蛋白不影响小鼠的热量摄入。
(D)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点附睾脂肪组织HE染色结果，发现CREG蛋白治疗可显著降低脂肪细胞大小。
(E)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点附睾脂肪组织脂肪细胞面积的统计分析。**p<0.01(与高脂喂养组相比)。
(F)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点腹股沟与附睾白色脂肪组织重量，结果提示CREG治疗可显著降低肥胖小鼠白色脂肪重量。**p<0.01(与高脂喂养组相比)。
(G)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点附睾脂肪组织CREG免疫组织化学染色，结果提示CREG蛋白治疗后可增加脂肪组织CREG的量。
图3.CREG蛋白治疗显著减轻高脂喂养C57BL/6J小鼠肝脏脂肪变性。
(A)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点肝脏组织HE染色，结果提示CREG治疗可明显减轻肥胖小鼠肝脏的脂肪变性。
(B)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点肝脏重量。*p<0.05(与高脂喂养组相比)。
(C)正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点肝脏组织CREG蛋白免疫组织化学染色，结果提示CREG蛋白治疗可增加肥胖小鼠肝脏组织CREG蛋白的量。
图4.CREG蛋白治疗可降低肥胖小鼠的血脂中水平，改善脂肪因子的表达。
(A)：正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点小鼠 血脂水平测定，结果提示CREG蛋白治疗可显著降低肥胖小鼠的总胆固醇、甘油三酯及低密度脂蛋白胆固醇水平。*p<0.05(与高脂喂养组相比)。
(B)和(C)：正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠16周时间点小鼠脂肪因子瘦素和脂联素水平测定，结果提示CREG蛋白治疗可抑制肥胖小鼠瘦素的表达，促进肥胖小鼠脂联素的表达。
图5.CREG蛋白治疗可改善肥胖小鼠的胰岛素抵抗。
正常喂养、高脂喂养与高脂喂养+CREG蛋白治疗3组小鼠，在16周时间点时进行葡萄糖耐量试验和胰岛素耐量试验，评价胰岛素抵抗情况。结果提示CREG蛋白治疗可显著改善肥胖小鼠的胰岛素抵抗情况。
具体实施方式：
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明的实验数据均为百分数。两样本率的比较应用卡方检验，统计学处理均应用SPSS 19.0软件包处理。以P<0.05为有统计学差异。
实施例1.高脂喂养C57BL/6J小鼠肥胖模型制备及脂肪组织中CREG蛋白表达检测。
①C57BL/6J小鼠肥胖模型的建立。
采用随机表法，将40只雄性8周龄C57BL/6小鼠[体重(22.3±1.2)g]分为以下2组：正常喂养组与高脂喂养组，每组20只。正常喂养组给予正常饮食饲料喂养(3.85kcal/g，脂肪提供10％热量，美国Research Diet公司)，高脂喂养组给予高脂饲料喂养(5.24kcal/g，脂肪提供60％热量，美国Research Diet公司)。持续喂养16周。小鼠喂养条件标准，接受12小时光照，不限制饮食和饮水量。
结果显示：喂养16周后，观察小鼠大体形态，发现高脂喂养组小鼠发生明显的肥胖， 表明肥胖模型建立成功(结果见图1A)。
②高脂喂养与正常喂养C57BL/6J小鼠不同周数体重。
每2周对各组小鼠进行称重。称重前禁食禁水12h。每只小鼠称重3次，记录并取平均值。
结果显示：高脂喂养的C57BL/6J小鼠的体重在喂养第4周时即明显高于正常喂养对照组。之后随喂养时间增加，高脂喂养组小鼠体重增加更加显著，在第16周时体重接近50g，而对照组只有30g左右。上述结果表明我们成功建立了高脂喂养诱导的小鼠肥胖模型(结果见图1B)。
③免疫组织化学染色方法检测高脂喂养的肥胖小鼠与对照组小鼠脂肪组织中CREG蛋白的表达。
1)喂养16周时，取附睾组织白色脂肪，经4％多聚甲醛固定后，常规石蜡包埋，5μm切片；
2)切片常规用二甲苯脱蜡，经各级乙醇至水洗：二甲苯(I)5min→二甲苯(II)5min→100％乙醇2min→95％的乙醇1min→80％乙醇1min→75％乙醇1min→蒸馏水洗2min；
3)将石蜡切片浸泡入合适PH值的抗原修复液中，100℃煮沸40min，自然冷却降至室温；
4)每张切片加一滴3％过氧化氢，室温孵育10min，以消除内源性过氧化物酶活性；PBS冲洗3次，每次5min；
5)10％山羊血清封闭，室温孵育10min；
6)弃除血清，加入1:100稀释的抗CREG(美国Abcam公司)抗体，4℃过夜，次日于室温复温30min，PBS冲洗3次，每次5min；
7)弃除PBS，加入生物素标记的二抗，室温孵育30min，PBS冲洗3次，每次5min；
8)弃除PBS，加链霉菌抗生物素-过氧化物酶溶液，室温孵育10min，PBS冲洗3次，每次5min；
9)弃除PBS，滴加DAB溶液室温孵育5-10min，根据情况中止显色；
10)自来水充分冲洗干净，苏木素复染，封片。
结果显示：高脂喂养组小鼠附睾白色脂肪组织中的脂肪细胞体积明显增大，同时CREG 表达与正常喂养对照组相比显著降低，提示CREG表达与肥胖呈负相关关系(结果见图1C)。
④Western Blot方法检测高脂喂养的肥胖小鼠与对照组小鼠脂肪组织中CREG蛋白的表达。
为检测CREG在小鼠脂肪组织中的表达情况，分别提取高脂喂养与正常喂养16周小鼠附睾部位的白色脂肪组织，用Western Blot方法检测两组小鼠CREG蛋白表达情况。首先对所取的组织进行称重，然后按照1mg:10μl的比例加入蛋白裂解液，冰上放置20min。4℃离心机13000rpm离心收集蛋白上清。采用BCA比色法试剂盒测定裂解液中的蛋白质浓度。将50μg蛋白在95℃煮沸5min后，经12％分离胶行SDS-PAGE电泳，判断电泳终止时间。以21V的电压将样品转印到PVDF膜上，时间为45min；在TBS-T稀释的5％脱脂奶粉中常温封闭1.5h后加入一抗4℃孵育过夜。分别以1:1 000抗CREG(美国Abcam公司)抗体、1：1 000抗beta-actin(美国Santa cruz公司)抗体作为一抗，以辣根过氧化物酶标记羊抗小鼠抗体(美国Cell signalling公司)作为二抗，行Western Blot检测，用ECL试剂盒(美国Amersham公司)发光显影。用CREG抗体和beta-actin抗体可分别检测到大小约为24KD和43KD的蛋白表达条带。采用Quantity One软件进行条带的灰度值测量并进行统计学分析。
结果显示：高脂喂养的肥胖小鼠与对照组小鼠相比，脂肪组织中CREG蛋白表达显著降低(结果见图1C)，提示脂肪组织中CREG的表达与肥胖呈负相关关系，CREG可能具有对抗肥胖的作用。
实施例2：CREG蛋白治疗显著减轻了高脂喂养C57BL/6J小鼠肥胖。
①CREG蛋白治疗可减轻高脂喂养引起的小鼠肥胖。
采用随机表法，将60只雄性8周龄C57BL/6小鼠分为以下3组：正常喂养组、高脂喂养组、高脂喂养+CREG蛋白治疗组。每组20只。正常喂养与高脂喂养方法同实施例1。高脂喂养+CREG蛋白治疗组在高脂喂养同时，采用皮下埋置微渗透泵(美国Alzet公司)的方法，给予小鼠CREG蛋白(美国Abcam公司，150μg/kg·d)治疗，治疗时间16周。小鼠喂养条件标准，接受12h光照，不限制饮食和饮水量。16周后观察小鼠大体形态。
结果显示：高脂喂养后小鼠发生明显的肥胖，给予CREG蛋白治疗后，小鼠肥胖得到 明显改善，说明CREG具有治疗肥胖的作用(结果见图2A)。
②三组小鼠体重的比较。
每两周对各组小鼠进行称重。称重前禁食禁水12小时。每只小鼠称重3次，记录并取平均值。
结果显示：CREG蛋白治疗组小鼠体重较高脂喂养组显著降低，表明CREG具有治疗肥胖的作用(结果见图2B)。
③三组小鼠热量摄入量比较。
称量2周内每只小鼠的饲料总质量，以正常饲料3.85kcal/g计算正常喂养组热量摄入，以高脂饲料5.24kcal/g计算高脂喂养组与CREG蛋白治疗组热量摄入，再除以14获得每只小鼠平均每天的热量摄入。
结果显示：高脂喂养组与高脂喂养+CREG蛋白治疗组由于都摄入的是高脂饲料，因此其热量摄入显著高于正常喂养对照组。但是高脂喂养组与高脂喂养+CREG蛋白治疗组之间热量摄入没有差别，表明CREG蛋白治疗不影响小鼠的食欲和热量摄入(结果见图2C)。
④三组小鼠脂肪细胞大小及脂肪组织重量比较。
取附睾白色脂肪组织，进行HE染色分析。
1)取材附睾白色脂肪组织，经10％甲醛固定后，常规石蜡包埋，5μm切片；
2)切片常规用二甲苯脱蜡，经各级乙醇至水洗：二甲苯(I)5min→二甲苯(II)5min→100％乙醇2min→95％的乙醇1min→80％乙醇1min→75％乙醇1min→蒸馏水洗2min；
3)苏木素染色5min，自来水冲洗；
4)盐酸乙醇分化30s；
5)自来水浸泡15min；
6)置伊红液2min；
7)常规脱水，透明，封片：95％乙醇1min→95％乙醇1min→100％乙醇(I)1min→100％乙醇(II)1min→二甲苯(I)1min→二甲苯(II)1min→中性树脂封固；
8)显微镜下观察形态并照相保存用于统计分析。
另外对各组小鼠腹股沟及附睾白色脂肪组织进行称重，并进行统计学分析。
结果显示：与高脂喂养组相比，CREG治疗组脂肪细胞明显减小(结果见图2D、2E)，腹股沟及附睾白色脂肪组织重量均显著降低(结果见图2F)，表明CREG蛋白具有治疗肥胖的作用。
⑤三组小鼠附睾白色脂肪组织CREG免疫组织化学染色。
取三组小鼠附睾白色脂肪组织，行CREG免疫组织化学染色，具体方法同实施例1。
结果显示：与高脂喂养组相比，CREG蛋白治疗后，脂肪组织CREG染色明显增强(结果见图2G)，表明CREG蛋白量增加，提示CREG蛋白可能通过提高脂肪组织CREG蛋白表达，逆转高脂喂养引起的肥胖。
实施例3：CREG蛋白治疗显著减轻了高脂喂养C57BL/6J小鼠肝脏的脂肪变性。
①三组小鼠肝脏组织HE染色。
喂养16周时，取各组小鼠肝脏组织，进行HE染色，具体方法同实施例2。
结果显示：与正常喂养对照组小鼠相比，高脂喂养后小鼠的肝脏发生了显著的脂肪变性，肝细胞被大量脂滴占据。与单纯高脂喂养组小鼠相比，给予高脂喂养组小鼠CREG蛋白治疗后，其肝细胞脂肪变性程度显著减轻(结果见图3A)，表明CREG治疗可对抗小鼠肥胖相关的脂肪肝。
②三组小鼠肝脏组织重量的比较。
喂养16周时，牺牲小鼠，取肝脏组织。用PBS清洗后蘸滤纸尽量除去多余液体。对肝脏进行称重，每只小鼠测量3次取平均值，记录并进行统计学分析。
结果显示：与正常喂养对照组相比，高脂喂养后小鼠肝脏重量明显增加。与单纯高脂喂养组小鼠相比，给予高脂喂养组小鼠CREG蛋白治疗后，其肝脏重量显著降低(结果见图3B)，表明CREG蛋白治疗可减轻小鼠肥胖时肝脏重量的异常增加。
③三组小鼠肝脏CREG蛋白免疫组织化学染色。
取16周时三组小鼠肝脏组织，进行CREG蛋白免疫组织化学染色，具体方法同实施例1。
结果显示：与单纯高脂喂养组相比，给予高脂喂养组小鼠CREG蛋白治疗后，其肝脏组织CREG染色明显增强(结果见图3C)，表明CREG蛋白量增加，提示外源性CREG蛋白可能通过提高肝脏组织CREG蛋白表达起到对抗小鼠肥胖时肝脏脂肪变性的作用。
实施例4：CREG蛋白治疗可降低肥胖小鼠的血脂水平，改善脂肪因子的表达。
①三组小鼠血脂水平测定。
喂养16周时，采用颈动脉取血法采集各组小鼠动脉血1-2ml，3000rpm离心后收集血清，一部分分装冻存于超低温冰箱用于后续检测。一部分进行血脂水平的检测。血脂检测采用日立全自动生化分析仪进行。
结果显示：与正常喂养组相比，用高脂食物喂养小鼠后，其血浆总胆固醇、甘油三脂与低密度脂蛋白胆固醇均显著增高，表明肥胖发生时，伴随明显的血脂异常。给予小鼠CREG蛋白治疗后，与高脂喂养组相比，血浆总胆固醇、甘油三脂与低密度脂蛋白胆固醇水平均显著降低(结果见图4A)，表明CREG蛋白对肥胖伴发的高脂血症具有治疗作用。
②三组小鼠脂肪因子瘦素和脂联素水平测定。
采用与本实施①中相同的方法获得血浆标本，用于瘦素和脂联素水平的测定。测定方法采用ELISA法，瘦素和脂联素ELISA测定试剂盒购自美国RD公司。
1)准备样品及标准品，取出微板条，每孔加入50μl Diluent Buffer；
2)每孔加入50μl标准品、对照品及样品，每个样本设置2个复孔，粘性贴覆盖。轻轻敲打1min使其混合均匀；
3)室温孵育2h。吸弃液体，每孔加入400μl Wash Buffer洗板5次。每一步都尽量将液体全部移除。最后一步清洗时尽量移除所有残存的Wash Buffer；
4)每孔加入100μl Mouse leptin(瘦素)/Adiponectin(脂联素)Conjugate，换一张新的粘性贴，室温孵育2h；
5)重复洗板5次。每孔加入100μl Substrate Solution，避光室温孵育30min；
6)每孔加入100μl Stop Solution，轻轻敲打板子以混合均匀；
7)30min内酶标仪读取结果，450nm波长读数，540nm或570nm波长校正。每次实验读数取3个，此实验重复3次；
8)绘制标准曲线并计算各组样本瘦素与脂联素的水平，最后进行统计学分析。
结果显示：与正常喂养组相比，小鼠高脂喂养后，血浆中有害的脂肪因子瘦素水平明显增高，而有益的脂肪因子脂联素水平明显降低。对高脂喂养组小鼠外源性给予CREG蛋白治疗后，与单纯高脂喂养组相比，瘦素水平显著下降，而脂联素水平显著升高(结果见图4B和4C)，表明CREG蛋白可改善小鼠肥胖时伴发的脂肪因子表达。
实施例5：CREG蛋白治疗可改善肥胖小鼠的胰岛素抵抗。
①葡萄糖耐量试验。
以2g/kg剂量向小鼠腹腔注射总体积0.1ml的葡萄糖水溶液，测定注射后15min、30min、45min、60min及120min鼠尾静脉血的血糖水平，判断胰岛细胞功能。
结果显示：与正常喂养组相比，在相同时间点，高脂喂养组小鼠血糖水平显著升高，提示高脂喂养后引起葡萄糖耐量受损。对高脂喂养组小鼠外源性给予CREG蛋白治疗后，在相同时间点其血糖水平均较高脂喂养组小鼠显著下降(结果见图5)，表明CREG蛋白可改善肥胖时伴发的葡萄糖耐量受损。
②胰岛素耐量试验。
以0.75U/kg剂量向小鼠腹腔注射人胰岛素，测定注射后15min、30min、45min、60min及120min鼠尾静脉血的血糖水平，判断胰岛素抵抗情况。
结果显示：与正常喂养组小鼠相比，在相同时间点，高脂喂养组小鼠血糖水平显著升高，提示高脂喂养后小鼠发生胰岛素抵抗。对高脂喂养组小鼠外源性给予CREG蛋白治疗后，在相同时间点其血糖水平均较高脂喂养组小鼠显著下降(结果见图5)，表明CREG蛋白可改善小鼠肥胖时伴发的胰岛素抵抗。
上述研究结果提示，CREG蛋白有望成为预防和治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的有效药物。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (7)

  1. CREG蛋白或其活性片段，其用于预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病。
  2. 编码CREG蛋白或其活性片段的核酸分子、表达CREG蛋白或其活性片段的重组载体或重组细胞，其用于预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病。
  3. 检测CREG蛋白或其活性片段表达水平的试剂在制备试剂盒中的用途，所述试剂盒用于超重、肥胖、胰岛素抵抗、高脂血症的预测和/或治疗效果、预后的评估。
  4. CREG蛋白或其活性片段用于筛选预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的药物的用途。
  5. 组合物，其含有CREG蛋白或其活性片段、编码CREG蛋白或其活性片段的核酸分子、表达CREG蛋白或其活性片段的重组载体或重组细胞，以及任选的药学上可接受的载体或赋形剂，所述组合物用于预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病。
  6. 试剂盒，其含有检测CREG蛋白或其活性片段表达水平的试剂，所述试剂盒用于肥胖的预测和/或治疗效果、预后的评估。
  7. 预防和/或治疗超重、肥胖、胰岛素抵抗、高脂血症及其相关疾病的方法，包括向有需要的个体施用CREG蛋白或其活性片段、编码CREG蛋白或其活性片段的核酸分子、表达CREG蛋白或其活性片段的重组载体或重组细胞。
PCT/CN2017/094509 2016-08-08 2017-07-26 Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途 WO2018028433A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/323,950 US20190216894A1 (en) 2016-08-08 2017-07-26 Medical Use of Creg Protein for Prevention or Treatment of Overweight, Obesity and Related Diseases Thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610644971.9 2016-08-08
CN201610644971.9A CN107693775A (zh) 2016-08-08 2016-08-08 Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途

Publications (1)

Publication Number Publication Date
WO2018028433A1 true WO2018028433A1 (zh) 2018-02-15

Family

ID=61162745

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/094509 WO2018028433A1 (zh) 2016-08-08 2017-07-26 Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途

Country Status (3)

Country Link
US (1) US20190216894A1 (zh)
CN (1) CN107693775A (zh)
WO (1) WO2018028433A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112915196A (zh) * 2021-03-15 2021-06-08 中国人民解放军北部战区总医院 Creg1蛋白用于预防或治疗索拉非尼诱导的心肌损伤的医药用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2886650A1 (en) * 2012-08-17 2015-06-24 Chubu University Educational Foundation Brown adipocyte differentiation-inducing agent
CN105056208A (zh) * 2015-07-30 2015-11-18 中国人民解放军沈阳军区总医院 Creg蛋白用于预防或治疗心肌梗死的医药用途
CN105169370A (zh) * 2015-08-04 2015-12-23 中国人民解放军沈阳军区总医院 Creg蛋白抑制炎症反应的医药用途
CN105194651A (zh) * 2015-07-30 2015-12-30 中国人民解放军沈阳军区总医院 Creg蛋白用于保护心肌缺血再灌注损伤的医药用途
CN105327335A (zh) * 2014-07-21 2016-02-17 中国人民解放军沈阳军区总医院 Creg蛋白的医药用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW201138808A (en) * 2010-05-03 2011-11-16 Bristol Myers Squibb Co Serum albumin binding molecules
CN105457015B (zh) * 2014-07-21 2019-09-06 中国人民解放军沈阳军区总医院 重组creg蛋白抑制高血压血管重塑的医药用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2886650A1 (en) * 2012-08-17 2015-06-24 Chubu University Educational Foundation Brown adipocyte differentiation-inducing agent
CN105327335A (zh) * 2014-07-21 2016-02-17 中国人民解放军沈阳军区总医院 Creg蛋白的医药用途
CN105056208A (zh) * 2015-07-30 2015-11-18 中国人民解放军沈阳军区总医院 Creg蛋白用于预防或治疗心肌梗死的医药用途
CN105194651A (zh) * 2015-07-30 2015-12-30 中国人民解放军沈阳军区总医院 Creg蛋白用于保护心肌缺血再灌注损伤的医药用途
CN105169370A (zh) * 2015-08-04 2015-12-23 中国人民解放军沈阳军区总医院 Creg蛋白抑制炎症反应的医药用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG, N.: "Overexpression of CREG attenuates atherosclerotic endothelium apoptosis via VEGF/PI3K/AKT pathway", ATHEROSCLEROSIS, vol. 218, no. 2, 3 August 2011 (2011-08-03), pages 543 - 551, XP028301392 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112915196A (zh) * 2021-03-15 2021-06-08 中国人民解放军北部战区总医院 Creg1蛋白用于预防或治疗索拉非尼诱导的心肌损伤的医药用途
CN112915196B (zh) * 2021-03-15 2024-01-09 中国人民解放军北部战区总医院 Creg1蛋白用于预防或治疗索拉非尼诱导的心肌损伤的医药用途

Also Published As

Publication number Publication date
CN107693775A (zh) 2018-02-16
US20190216894A1 (en) 2019-07-18

Similar Documents

Publication Publication Date Title
Badmus et al. Molecular mechanisms of metabolic associated fatty liver disease (MAFLD): Functional analysis of lipid metabolism pathways
Tutunchi et al. A systematic review of the association of neuregulin 4, a brown fat–enriched secreted factor, with obesity and related metabolic disturbances
Yang et al. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance
Tian et al. Efficacy of liraglutide in treating type 2 diabetes mellitus complicated with non-alcoholic fatty liver disease
Duan et al. Obesity, adipokines and hepatocellular carcinoma
Chen et al. Adiponectin protects against cerebral ischemia–reperfusion injury through anti-inflammatory action
Cannataro et al. Ketogenic diet acts on body remodeling and microRNAs expression profile
Nobili et al. Laparoscopic sleeve gastrectomy improves nonalcoholic fatty liver disease–related liver damage in adolescents by reshaping cellular interactions and hepatic adipocytokine production
Cao et al. Recent progress in research on the distribution and function of NUCB2/nesfatin-1 in peripheral tissues
Sujishi et al. Sitagliptin can inhibit the development of hepatic steatosis in high-fructose diet-fed ob/ob mice
Nakamura et al. A long-lasting dipeptidyl peptidase-4 inhibitor, teneligliptin, as a preventive drug for the development of hepatic steatosis in high-fructose diet-fed ob/ob mice
Ariyama et al. Chop‐deficient mice showed increased adiposity but no glucose intolerance
Lv et al. QiShenYiQi pill improves the reparative myocardial fibrosis by regulating autophagy
Kong et al. Loss of C3a and C5a receptors promotes adipocyte browning and attenuates diet-induced obesity via activating inosine/A2aR pathway
WO2018028433A1 (zh) Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途
Zuo et al. Canagliflozin attenuates hepatic steatosis and atherosclerosis progression in Western Diet-Fed ApoE-Knockout Mice
Iizuka et al. Impact of discontinuation of fish oil after pioglitazone–fish oil combination therapy in diabetic KK mice
TWI531650B (zh) 用於預防及治療非酒精性脂肪肝疾病的腺病毒ａｄ３６ ｅ４ｏｒｆ１蛋白
Kou et al. Sesamin activates skeletal muscle FNDC5 expression and increases irisin secretion via the SIRT1 signaling pathway
Guo et al. A novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of nonalcoholic steatohepatitis
EP2808031A1 (en) Sex hormone-binding globulin for use as a medicament
Zhao et al. Sodium glucose cotransporter-2-inhibitor dapagliflozin improves nonalcoholic fatty liver disease by ameliorating dipeptidyl-peptidase-4 protein expression in diabetic mice
US20190226002A1 (en) Obesity-related disease therapeutic agent by hepatic secretory metabolic regulator inhibitory action
Chimienti et al. The Mitochondrial Trigger in an Animal Model of Nonalcoholic Fatty Liver Disease. Genes 2021, 12, 1439
EP2603096A1 (en) Method for treating fatty liver diseases, in particular non-alcoholic steatohepatitis.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17838563

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17838563

Country of ref document: EP

Kind code of ref document: A1