WO2022089591A1 - 氨基葡萄糖在制备治疗非酒精性脂肪性药物中的应用 - Google Patents

氨基葡萄糖在制备治疗非酒精性脂肪性药物中的应用 Download PDF

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WO2022089591A1
WO2022089591A1 PCT/CN2021/127473 CN2021127473W WO2022089591A1 WO 2022089591 A1 WO2022089591 A1 WO 2022089591A1 CN 2021127473 W CN2021127473 W CN 2021127473W WO 2022089591 A1 WO2022089591 A1 WO 2022089591A1
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glc
mice
glucosamine
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苏政权
曹华
白研
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广东药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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

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  • the invention belongs to the technical field of biomedicine, and particularly relates to the application of glucosamine in the preparation of non-alcoholic fatty drugs.
  • Non-alcoholic fatty liver disease is a metabolic stress-induced liver disease regulated by various factors such as metabolism, genes, environment, and gut microbes.
  • Non-alcoholic simple fatty liver (NAFL) NASH
  • NASH non-alcoholic steatohepatitis
  • the incidence of NAFLD has gradually increased, and it has become the second largest liver disease after viral liver disease.
  • the specific pathogenesis of NAFLD is not very clear, and its formation is mainly due to the accumulation of triglycerides (TG) in hepatocytes due to eating too much fat or lipodystrophy.
  • TG triglycerides
  • the first blow is the accumulation of fat in the liver, especially fatty acids and TG, resulting in simple fatty liver; the second blow is oxidative stress and lipid peroxidation leading to steatosis Inflammation, necrosis, and fibrosis of the liver develops into NASH.
  • Pathogenic pathways such as insulin resistance, lipotoxicity, oxidative stress, endoplasmic reticulum stress, and systemic low-grade inflammatory responses, immune or cytokine or mitochondrial function changes, and apoptosis are involved in the occurrence and development of NAFLD.
  • Targeted intervention for the pathogenesis of NAFLD is a hotspot of current research.
  • the histological improvement of NASH patients, including the improvement of inflammation and liver fibrosis is not very satisfactory.
  • Chinese patent application CN102883721A discloses a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease (NAFLD), which comprises compound 1 represented by formula 1, sitagliptin, vildagliptin, lira Active ingredients of lipins or their pharmaceutically acceptable salts.
  • NFLD non-alcoholic fatty liver disease
  • Chinese patent application CN103800352A discloses a traditional Chinese medicine active ingredient compound preparation and application for resisting non-alcoholic fatty liver disease, and particularly relates to a traditional Chinese medicine active ingredient compound preparation composed of gardeniside and chlorogenic acid and its use.
  • the compound preparation of the invention is composed of two components, geniposide and chlorogenic acid.
  • the compound preparation can be made into commonly used clinical preparations by conventional methods, including oral solid preparations such as granules, tablets and capsules.
  • the purpose of the present invention is to provide an application of glucosamine in the preparation of a drug for treating non-alcoholic fattyness.
  • the present invention has proved through experiments that glucosamine with a daily dose of 300-600 mg/kg can improve insulin resistance, glucose and lipid metabolism disorder, liver lipid accumulation, abnormal liver function, and enhance antioxidant capacity in mice with non-alcoholic fatty liver disease. Reduce the body inflammation, serum endotoxin levels, indicating that glucosamine has a certain therapeutic effect on non-alcoholic fatty liver disease.
  • the invention provides the application of glucosamine in the preparation of a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease and diabetes.
  • glucosamine is one of glucosamine hydrochloride or glucosamine sulfate or a combination thereof.
  • composition is used for: systemic administration or parenteral administration.
  • non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver, non-alcoholic steatohepatitis, liver cirrhosis and hepatocellular carcinoma.
  • the diabetes is hyperglycemia, hyperlipidemia, free fatty acid disorder and insulin resistance in the body caused by a high-fat and high-sugar diet.
  • the pharmaceutical composition is injection, granule, tablet, capsule or oral liquid.
  • the effective amount of the glucosamine is 300-600 mg/kg/day.
  • the effective amount of glucosamine is 600 mg/kg/day.
  • Glucosamine mainly exists in the form of hydrochloride or sulfate, and is the final degradation product of chitin.
  • GLC widely exists in human and animal tendon, cartilage and ligament and other tissues. It can synthesize mucopolysaccharide, collagen and proteoglycan, and has a protective effect on cartilage tissue. GLC easily passes through the biofilm, and after oral absorption, it is distributed to various tissues and organs of the body, especially with high affinity for cartilage tissue and high bioavailability.
  • the human body can also synthesize GLC through the amination of its own glucose, but the amount of synthesis is small, which cannot meet its own needs and easily lead to the occurrence of diseases such as osteoarthritis, so it is necessary to supplement exogenous GLC.
  • the half-life of GLC is 18h, and it is finally decomposed into carbon dioxide, urea and water after being metabolized by the liver.
  • GLC can improve carbon tetrachloride-induced liver damage in mice; it has strong antioxidant capacity and can scavenge free radicals.
  • the polymer chitosan oligosaccharide of glucosamine has many metabolic benefits, especially a significant anti-obesity effect.
  • There are currently no studies on the metabolic benefit activity of GLC, and investigating the anti-NAFLD activity of GLC will help to further elucidate the metabolic benefits of chitosan oligosaccharides.
  • NAFLD mice were given GLC high, medium and low doses by gavage for 12 weeks.
  • the doses were GLC-H: 600mg/Kg/d, GLC-M: 300mg/Kg/d, GLC-L: 150mg/Kg/d d.
  • GLC can significantly improve the body's hyperglycemia and insulin resistance caused by high-fat and high-sugar diet without affecting appetite, as well as the disturbance of blood lipid four items and free fatty acid levels, indicating that GLC administration can improve the glycolipid in NAFLD mice. Metabolic abnormalities.
  • GLC administration can increase the level of catalase in the serum of mice, enhance the total antioxidant capacity, and reduce the oxidative damage caused by the high-fat and high-sugar diet.
  • GLC had no significant effect on the level of IL-6, but could significantly improve the levels of serum IL-10 and TNF- ⁇ , indicating that GLC had the activity of improving the anti-inflammatory ability of NAFLD mice.
  • GLC administration significantly reduced serum endotoxin levels in NAFLD mice, indicating that GLC may reduce serum endotoxin levels by improving intestinal barrier function, thereby improving NAFLD indicators.
  • GLC administration can significantly reduce liver steatosis in NAFLD mice, indicating that GLC can improve liver lipid accumulation caused by high-fat and high-sugar diet.
  • GLC can improve insulin resistance, glucose and lipid metabolism disorder, liver lipid accumulation, improve liver function abnormality, enhance antioxidant capacity, and reduce inflammation and serum endotoxin levels in NAFLD mice. It shows that GLC has a certain therapeutic effect on NAFLD.
  • FIG 1 Weekly changes in body weight of mice after administration (A); changes in food intake of NAFLD mice during administration (B);
  • FIG. 1 Serum glucose curve of oral glucose tolerance test of mice in each group after administration (A); area under the glucose curve (B); serum glucose level (C) and insulin level (D) of mice in each group;
  • Figure 3 Changes in serum total cholesterol (A), triglyceride (B), high-density lipoprotein cholesterol (C), low-density lipoprotein cholesterol (D) and free fatty acid (E) levels of mice in each group after administration picture;
  • FIG. 4 Serum AST (A) and ALT (B) of mice in each group after administration;
  • Figure 5 Changes in serum antioxidant indexes CAT (A) and T-AOC (B) levels of mice in each group after administration;
  • FIG. 8 H&E staining of the livers of mice in each group after drug treatment (200X).
  • Embodiment 1 Experiment on the effect of glucosamine on non-alcoholic fatty liver disease
  • mice 70 healthy C57BL/6 male mice (7 weeks old) were adaptively fed for 1 week, the animals were fed with free food and water, and fed with common chow, and were randomly divided into 2 groups.
  • the blank group (Control) of 10 mice was fed with ordinary feed; the model group of 60 mice was fed with high-fat and high-sugar diet for 8 weeks.
  • the 60 mice given high-fat and high-sugar diet were randomly divided into six groups (10 mice in each group).
  • NAFLD model group (Model), positive control group (Metformin, Metformin), positive control group (Rosuvastatin, Rosuvastatin), Glucosamine high dose (GLC-H), Glucosamine medium dose (GLC-M) , Glucosamine low dose (GLC-L).
  • Common feed consists of Provided by Guangdong Provincial Medical Laboratory Animal Center. High-fat and high-sugar feed was purchased from Research Diets, USA (Item No.: D1237). Its energy source is: 40% from fat and 17% from sucrose.)
  • mice in each experimental group were given different drugs by gavage, and the mice in the blank group and model group were given the same amount of distilled water by gavage. Each mouse was gavaged with distilled water at 10 mL/kg.
  • the doses of GLC in the experiment were GLC-H: 600mg/Kg/d, GLC-M: 300mg/Kg/d, GLC-L: 150mg/Kg/d; Metformin: 50mg/Kg/d; Rosuvastatin: 40 mg/Kg/day.
  • Corresponding doses of test samples were administered by gavage for 12 consecutive weeks.
  • the amount of feed consumed by the mice was recorded at regular times every day (before and after administration), and the general state of the mice in each experimental group, such as: mental state, liveliness, hair gloss, and defecation volume, were observed or not.
  • mice in each experimental group was weighed every week, and the changes in body weight were recorded.
  • mice were subjected to oral glucose tolerance test with a glucose injection dose of 2.0 g/kg.
  • Five mice in each group of Control, Model, metformin and GLC-H, GLC-M and GLC-L were randomly selected for testing.
  • the specific operation process the experimental mice were changed to clean bedding 12 hours in advance, and the water was fasted; the body weight was first weighed during the experiment; the fasting blood glucose was measured: the mice were taken out, and after disinfecting the mouse tail with alcohol, cut off 0.1 cm of the tail end of the mouse, Blood was collected, and fasting blood glucose was measured with a blood glucose meter, and the measured value was the blood glucose level at 0 min; according to the weight of the mice, when the glucose solution was injected, the 0 min time of each mouse was recorded; the time of 15 min, 30 min, 60 min, 90 min, The blood glucose value at 120 min; the tail of the mice was sterilized and the bleeding was stopped, and a diet was given at the same time.
  • mice were dissected, and the liver, epididymis, perirenal and subcutaneous fat were removed, weighed, and placed in a cassette for fixation. After being fixed for 24 hours, the samples were taken out and soaked in water, and then dehydrated step by step with 75%, 80%, 90%, 95%, 100% ethanol, xylene and high melting point paraffin respectively. Then, it was embedded in an embedding machine, and the embedded paraffin was placed at -20°C for pre-cooling, and then sliced, spread and baked using a microtome. The cut sections were stained with hematoxylin-eosin, dehydrated, and mounted. Finally, the pathological changes of the liver were observed using an electron microscope, and pictures were taken.
  • liver function indicators showed that compared with the Model group, each dose of GLC could significantly improve the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the Model group ( Figure 4A-B); Abnormal liver function indexes caused by high-fat and high-sugar diet; the experimental data are shown in Table 4.
  • Endotoxin is a metabolite of intestinal bacteria. Elevated serum endotoxin level means that the intestinal barrier is blocked, and endotoxin passes through the barrier and enters the blood circulation more.
  • the measurement results of serum LPS content showed that each dose of GLC could significantly reduce the level of serum endotoxin; indicating that GLC may reduce the level of serum endotoxin in mice by improving the intestinal barrier function (Figure 7); the experimental data are shown in Table 7.
  • GLC high, medium and low doses of GLC are administered to NAFLD mice by gavage for 12 weeks, and the doses are respectively GLC-H: 600mg/Kg/d, GLC-M: 300mg/Kg/d, GLC-L: 150mg/Kg/d d.
  • GLC can significantly improve the body's hyperglycemia and insulin resistance caused by high-fat and high-sugar diet, as well as the disturbance of blood lipids and free fatty acid levels without affecting appetite. It shows that GLC administration can improve the abnormal glucose and lipid metabolism in NAFLD mice.
  • High and medium doses of GLC can reduce serum AST and ALT levels in NAFLD mice, indicating that GLC can improve the abnormal liver function indexes caused by high-fat and high-sugar diet.
  • GLC administration can increase the level of catalase in the serum of mice, enhance the total antioxidant capacity, and reduce the oxidative damage caused by the high-fat and high-sugar diet.
  • GLC has no significant effect on the level of IL-6, but can significantly improve the levels of serum IL-10 and TNF- ⁇ , indicating that GLC has the activity of improving the anti-inflammatory ability of NAFLD mice.
  • GLC administration significantly reduced serum endotoxin levels in NAFLD mice, indicating that GLC may reduce serum endotoxin levels by improving intestinal barrier function, thereby improving NAFLD indicators.
  • GLC administration can also significantly reduce liver steatosis in NAFLD mice, indicating that GLC can improve liver lipid accumulation caused by high-fat and high-sugar diet.
  • GLC can improve insulin resistance, glucose and lipid metabolism disorders, liver lipid accumulation, abnormal liver function, enhance antioxidant capacity, and reduce inflammation and serum endotoxin levels in NAFLD mice. It shows that GLC has a certain therapeutic effect on NAFLD.

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Abstract

氨基葡萄糖在制备治疗非酒精性脂肪性肝病和糖尿病药物中的应用。通过实验证明，日剂量为300～600mg/kg的氨基葡萄糖能够改善非酒精性脂肪性肝病小鼠胰岛素抵抗、糖脂代谢紊乱，肝脏脂质堆积，改善肝功能异常，增强抗氧化能力，降低机体炎症、血清内毒素水平，说明氨基葡萄糖对非酒精性脂肪性肝病具有一定治疗作用。

Description

氨基葡萄糖在制备治疗非酒精性脂肪性药物中的应用 技术领域
本发明属于生物医药技术领域，具体涉及氨基葡萄糖在制备治疗非酒精性脂肪性药物中的应用。
背景技术
非酒精性脂肪性肝病(Non-alcoholic fatty liver disease，NAFLD)是一种受代谢、基因、环境和肠道微生物等多种因素调节的代谢应激性肝脏疾病，根据肝脏病理状态的不同分为非酒精性单纯性脂肪肝(Non-alcoholic simple fatty liver，NAFL)、非酒精性脂肪性肝炎(Non-alcoholic steatohepatitis，NASH)及其相关肝硬化和肝细胞癌。随着肥胖、2型糖尿病的流行，NAFLD已成为世界范围内的公共卫生问题，全球25％-30％的人口都患有不同程度的非酒精性脂肪肝。在我国，由于饮食结构和生活方式的改变，NAFLD的发病率也逐渐上升，已成为仅次于病毒性肝病的第二大肝病。一项由武汉大学人民医院研究团队开展的研究显示，1999-2018年，中国非酒精性脂肪性肝病的总患病率为29.6％(95％CI：28.2％-31.0％)，预计到2030年，中国非酒精性脂肪性肝病患者总数将增至3.1458亿。
目前NAFLD的具体发病机制还不十分清楚，其形成主要由于进食过多脂肪或脂肪代谢障碍，导致甘油三酯(triglycerides,TG)在肝细胞内积聚。Day等提出的以氧应激和脂质过氧化为轴心的“二次打击”学说似乎可以解释其复杂的发病机理，在学术界较为流行。在NAFLD形成的“二次打击”中，第一次打击是肝脏内脂肪的积聚，尤其是脂肪酸和TG，形成单纯性脂肪肝；第二次打击氧化应激和脂质过氧化导致脂肪变的肝脏发生炎症、坏死和纤维化，发展为NASH。胰岛素抵抗、脂肪毒性、氧化应激、内质网应激以及系统性低度炎症反应、免疫或细胞因子或线粒体功能改变，以及细胞凋亡等致病途径共同参与了NAFLD的发生和发展。
针对NAFLD发病机制的靶向干预是当前研究的热点，截止到2019年，目前进入临床三期的靶点药物有8个，处于二期的靶点药物7个，处于一期的靶点药物2个。临床迫切需要NAFLD治疗药物，全球在NAFLD的新药研发领域投 入巨大，但从目前临床试验结果来看，对NASH患者的组织学改善，包括炎症和肝纤维化的改善效果并不十分理想。
中国专利申请CN102883721A公开了一种用于预防和治疗非酒精性脂肪性肝病(NAFLD)的药物组合物，其包含选自式1表示的化合物1、西格列汀、维格列汀、利拉利汀或它们的药学可接受的盐的活性成分。中国专利申请CN103800352A公开了一种抗非酒精性脂肪性肝病的中药有效成分复方制剂及应用，尤其涉及一种由栀子苷和绿原酸组成的中药有效成分复方制剂及其用途。本发明的复方制剂由栀子苷和绿原酸2种成分组成。本复方制剂可按常规方法制成临床常用制剂，包括颗粒剂、片剂、胶囊剂等口服固体制剂。
虽然上述专利中公布的药物可调节脂代谢、抗氧化、改善IR等，或多或少显示出一定治疗作用，但尚无完全有效、作用肯定的药物可供临床使用，且这些药物长期服用存在一定毒性，因此患者很难长期坚持。
因此，有必要提供一种疗效确切、安全、无毒副作用的药物，以满足在制备预防和治疗非酒精性脂肪性药物应用。
发明内容
为了解决现有技术中存在的问题，本发明的目的在于提供一种氨基葡萄糖在制备治疗非酒精性脂肪性药物中的应用。本发明通过实验证明，日剂量为300～600mg/kg的氨基葡萄糖能够改善非酒精性脂肪性肝病小鼠胰岛素抵抗、糖脂代谢紊乱，肝脏脂质堆积，改善肝功能异常，增强抗氧化能力，降低机体炎症、血清内毒素水平，说明氨基葡萄糖对非酒精性脂肪性肝病具有一定治疗作用。
本发明的技术方案是：
本发明提供了一种氨基葡萄糖在制备预防和治疗非酒精性脂肪性肝病和糖尿病药物组合物中的应用。
进一步地，所述氨基葡萄糖是氨基葡萄糖盐酸盐或氨基葡萄糖硫酸盐其中的一种或其组合。
进一步地，所述的药物组合物用于：全身给药或肠胃外给药。
进一步地，所述的非酒精性脂肪性肝病包括非酒精性单纯性脂肪肝、非酒精性脂肪性肝炎、肝硬化及肝细胞癌。
进一步地，所述的糖尿病为高脂高糖饮食引起的机体高血糖、高血脂、游离脂肪酸紊乱和胰岛素抵抗。
进一步地，所述的药物组合物为注射液、颗粒剂、片剂、胶囊剂或口服液。
进一步地，所述氨基葡萄糖的有效量为300～600mg/kg/日。
更进一步地，所述氨基葡萄糖的有效量为600mg/kg/日。
氨基葡萄糖(Glucosamine，GLC)主要以盐酸盐或硫酸盐的形式存在，是甲壳素的最终降解产物。GLC广泛存在于人和动物的肌腱、软骨和韧带等组织中，它可合成黏多糖、胶原蛋白和蛋白聚糖，对软骨组织具有保护作用。GLC极易通过生物膜，口服吸收后分布到全身各组织和器官中，尤其对软骨组织具有较高的亲和力，生物利用度高。人体也能够通过自身葡萄糖的氨基化合成GLC，但是合成量少，不能满足自身需要，易导致骨性关节炎等疾病的发生，所以需要补充外源性GLC。GLC半衰期为18h，经肝脏代谢后最终分解为二氧化碳、尿素和水。
本申请发明人研究发现GLC能改善四氯化碳诱导的小鼠肝损伤；具有较强的抗氧化能力，能够清除自由基。氨基葡萄糖的聚合物壳寡糖具有许多代谢益处，尤其是拥有显著的抗肥胖作用。目前没有关于GLC代谢益处活性的研究，研究GLC的抗NAFLD活性将有助于进一步阐述壳寡糖的代谢益处。
本实验通过灌胃给予NAFLD小鼠GLC高、中和低剂量12周，剂量分别为GLC-H：600mg/Kg/d，GLC-M：300mg/Kg/d，GLC-L：150mg/Kg/d。GLC能在不影响食欲的情况下，显著的改善高脂高糖饮食引起的机体高血糖和胰岛素抵抗，以及血脂四项和游离脂肪酸水平的紊乱，说明GLC给药能够改善NAFLD小鼠的糖脂代谢异常。
GLC高、中剂量能降低NAFLD小鼠血清AST和ALT水平，说明GLC能改善高脂高糖饮食引起的肝功能指标异常。GLC给药能够增加小鼠血清中过氧化氢酶水平，增强总抗氧化能力，减少机体受高脂高糖饮食带来的氧化损伤。GLC对IL-6水平无显著性影响，但能显著改善血清IL-10和TNF-α水平，说明GLC具有一定改善NAFLD小鼠抗炎能力的活性。GLC给药显著降低NAFLD小鼠血清内毒素水平，说明GLC可能通过改善肠道屏障功能从而减轻血清内毒素的水平，从而起到改善NAFLD指标的作用。此外，通过小鼠肝组织切片发现， GLC给药还能显著降低NAFLD小鼠肝脏脂肪变性的情况，说明GLC能改善高脂高糖饮食带来的肝脂质堆积。
综上所述，GLC能够改善NAFLD小鼠胰岛素抵抗、糖脂代谢紊乱，肝脏脂质堆积，改善肝功能异常，增强抗氧化能力，降低机体炎症、血清内毒素水平。说明GLC对NAFLD具有一定治疗作用。
附图说明
图1：给药处理后小鼠体重每周变化(A)；给药期间NAFLD小鼠摄食量变化(B)；
图2：给药处理后各组小鼠口服糖耐量试验血清葡萄糖曲线图(A)；葡萄糖曲线下面积(B)；各组小鼠血清葡萄糖水平(C)与胰岛素水平(D)；
图3：给药处理后各组小鼠血清总胆固醇(A)、甘油三酯(B)、高密度脂蛋白胆固醇(C)、低密度脂蛋白胆固醇(D)和游离脂肪酸(E)水平变化图；
图4：给药处理后各组小鼠血清AST(A)、ALT(B)；
图5：给药处理后各组小鼠血清抗氧化指标CAT(A)和T-AOC(B)水平变化图；
图6.给药处理后各组小鼠血清炎症因子IL-6(A)、IL-10(B)和TNF-α (C)水平变化图；
图7：给药处理后各组小鼠血清内毒素水平变化图；
图8：给药处理后各组小鼠肝脏H&E染色图(200X)。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的保护范围之内。
实施例1、氨基葡萄糖对非酒精性脂肪性肝病的影响实验
1.实验方案
1.1实验主要仪器和试剂
本发明实验中所用到的主要仪器和试剂如下表所示：
部分设备和试剂 生产厂家
JJ500型电子天平 常熟市双杰测试仪器厂
BT224S型万分之一电子天平 北京赛多利斯科学仪器有限公司
HVE-50全自动高压灭菌锅 日本平山制作所
AF103AS雪花制冰机 上海斯科茨曼制冰系统有限公司
DHG-9030A型电热恒温鼓风干燥箱 上海一恒科学仪器有限公司
Master-Q15型超纯水机 山东博科生物产业有限公司
Scieuitife-702型超低温冰箱 美国Thermo科技公司
Centrifuge5418小型高速冷冻离心机 德国Eppendorf公司
BioTek Eon酶标仪 美国伯腾仪器有限公司
正置显微镜 奥林巴斯(中国)有限公司
PerkinElmer倒置显微镜 珀金埃尔默企业管理有限公司
YGQ-300L生物组织冷冻切片机 湖北省孝感市亚光医用电子技术有限公司
ZT-12M型生物组织脱水机 湖北省孝感市亚光医用电子技术有限公司
YB-6LF生物组织石蜡包埋机 湖北省孝感市亚光医用电子技术有限公司
3126F型石蜡切片机 湖北省孝感市亚光医用电子技术有限公司
YT-7FB型生物组织摊烤片机 湖北省孝感市亚光医用电子技术有限公司
Insulin ELISA试剂盒 江苏酶免实业有限公司
LPS ELISA试剂盒 江苏酶免实业有限公司
TNF-αELISA试剂盒 江苏酶免实业有限公司
IL-6 ELISA试剂盒 江苏酶免实业有限公司
IL-10 ELISA试剂盒 江苏酶免实业有限公司
TG试剂盒 南京建成生物工程研究所
TC试剂盒 南京建成生物工程研究所
HDL-C试剂盒 南京建成生物工程研究所
LDL-C试剂盒 南京建成生物工程研究所
ALT试剂盒 南京建成生物工程研究所
AST试剂盒 南京建成生物工程研究所
T-AOC试剂盒 南京建成生物工程研究所
CAT试剂盒 南京建成生物工程研究所
FFA试剂盒 南京建成生物工程研究所
苏木素-伊红染液 北京雷根生物技术有限公司
氨基葡萄糖 上海麦克林生化科技有限公司
二甲双胍 中美上海施贵宝制药有限公司
瑞舒伐他汀 阿斯利康药业(中国)有限公司
1.2实验动物：C57BL/6雄性小鼠，7周龄，SPF级，购买于湖南斯莱克景达实验动物有限公司，动物生产许可证号：SCXK(粤)2018-0002。
1.3饲养环境：实验期动物房环境温度为24±2℃，相对湿度为50％-60％，换气 次数＞15次/小时，光照节律为12小时的SPF级环境。
1.4实验方法：将70只健康的C57BL/6雄性小鼠(7周龄)，适应性喂养1周，动物自由饮食和饮水，喂养普通饲料，随机分为2组。空白组(Control)10只，普通饲料喂养；模型组60只，高脂高糖饲料喂养8周后，将给予高脂高糖饲料的60只小鼠随机分成六组(每组10只)，分别为：NAFLD模型组(Model)、阳性对照组(二甲双胍，Metformin)、阳性对照组(瑞舒伐他汀，Rosuvastatin)、氨基葡萄糖高剂量(GLC-H)、氨基葡萄糖中剂量(GLC-M)、氨基葡萄糖低剂量(GLC-L)。(普通饲料配比：20％粗蛋白，4.8％粗纤维，4.3％粗脂肪，9.7％水分，1.19％钙，0.77％磷，钙/磷比值为1.55％，粗灰分为6.6％。普通饲料由广东省医学实验动物中心提供。高脂高糖饲料购自美国Research Diets公司(货号：D1237)，其能量来源为：40％来自脂肪，17％来自蔗糖。)
每天上午，采用灌胃的方式给予各实验组小鼠不同的药物，空白组和模型组小鼠给予等量的蒸馏水灌胃。每只小鼠按10mL/kg量予蒸馏水灌胃。GLC在实验中的给药剂量分别GLC-H：600mg/Kg/d，GLC-M：300mg/Kg/d，GLC-L：150mg/Kg/d；二甲双胍(Metformin)：50mg/Kg/天；瑞舒伐他汀(Rosuvastatin)：40mg/Kg/天。分别通过灌胃给予对应剂量的试验样品，连续12周。
实验结束采用眼球取血，留取血清、肝脏等把脂肪组织标本对TG、TC、ALT、AST、HDL-C、LDL-C、FFA、T-AOC、CAT、GLU、Insulin、LPS、TNF-α、IL-6和IL-10含量进行检测，观察肝脏的病理组织学病变情况。
2.检测指标
2.1日摄食量
每天定时(给药前后)记录小鼠摄入的饲料量，观察各实验组小鼠一般状态如：精神状况、活泼度、毛发光泽度、排便量等有无变化。
2.2周体重增重量
每周一对各实验组小鼠进行称量体重，记录体重变化。
2.3血清、肝脏指标检测
血清指标测定：小鼠眼眶取血，将血液收集入血常规管中，静置1h，放入低温高速离心机中，离心(3000r/min，15min，4℃)。离心后取上层清液，分装3管。血清中TC、TG、HDL-C、LDL-C、ALT、AST、GLU和FFA的含量 采用商业试剂盒检测，按照说明书操作。血清中TNF-α、IL-6、LPS和IL-10的含量采用ELISA试剂盒检测，按照说明书操作。
2.4葡萄糖耐受试验
小鼠进行口服葡萄糖耐受试验，葡萄糖注射剂量为2.0g/kg，Control、Model、二甲双胍和GLC-H、GLC-M和GLC-L每组随机选择5只进行测试。具体操作过程:实验小鼠提前12h换干净垫料，禁食不禁水；实验时先称量体重；测定空腹血糖：取出小鼠，用酒精消毒鼠尾后，剪去小鼠尾巴末梢0.1cm，收集血液，用血糖仪测定空腹血糖，测定值为0min血糖值；根据小鼠体重，注射葡萄糖溶液时，并记录每只小鼠0min时间；测定记录实验小鼠在15min、30min、60min、90min、120min点的血糖值；消毒小鼠尾部并止血，同时给予饮食。
2.5组织病理切片
解剖小鼠，取出肝脏，附睾、肾周和皮下脂肪，称重后放入包埋盒固定。固定24h，取出用水浸泡，接着分别用75％、80％、90％、95％、100％的乙醇，二甲苯，高熔点石蜡逐级脱水。接着在包埋机中进行包埋，将包埋好的石蜡放入-20℃进行预冷，再使用切片机进行切片、摊片和烤片。将切好的片使用苏木素-伊红染色，脱水，封片。最后，使用电子显微镜观察肝脏的病理改变，并拍摄图片。
3.实验结果
3.1 GLC对NAFLD小鼠摄食量及体重的影响
各组小鼠在实验期间体重组间差异无统计学意义(P＞0.05)。说明GLC对高脂高糖饲料诱导的NAFLD小鼠体重无明显影响(图1A)。同时给予高脂高糖饲料的各组小鼠摄食量无显著性差异(P＞0.05)，且均低于普通饲料组小鼠摄食量；说明高脂高糖饲料使小鼠食欲降低。由GLC与模型对照组、二甲双胍组和瑞舒伐他汀组对照可知，GLC不影响NAFLD小鼠食欲(图1B)；实验数据如表1所示。
表1 GLC对NAFLD小鼠摄食量的影响(means±SEM，n＝8-10)
组别 Food intake(g)
空白对照组 259.7±3.521****
模型对照组 166.7±3.397
二甲双胍组 184±3.191
瑞舒伐他汀组 183.1±2.277
高剂量组 178.1±3.148
中剂量组 162.7±3.11
低剂量组 170.5±3.395
3.2 GLC对NAFLD小鼠胰岛素抵抗的影响
口服糖耐量试验与血清葡萄糖含量结果表明，GLC各剂量能显著改善高脂高糖饮食小鼠的高血糖水平(图2A-C)；GLC各剂量均可显著改善高脂高糖饲料诱导的机体高胰岛素水平(图2D)；以上结果说明GLC可能通过改善机体胰岛素抵抗，改善高血糖水平；实验数据如表2所示。
表2 GLC对NAFLD小鼠胰岛素抵抗的影响(means±SEM，n＝8-10)
Figure PCTCN2021127473-appb-000001
注:与Model组对比，*p<0.05，**p<0.01，***p＜0.001，****p＜0.0001(n＝8-10,mean±SEM)
3.3 GLC对NAFLD小鼠血脂的影响
对血脂指标测定结果表明，相对于Model组，GLC各剂量均能显著改善NAFLD小鼠高血清总胆固醇(TC)和甘油三酯(TG)水平(图3A-B)，且高剂量与阳性药效果相当；GLC各剂量均可改善改善NAFLD小鼠机体高密度脂蛋白胆固醇(HDL-C)与低密度脂蛋白胆固醇(LDL-C)水平(图3C-D)；GLC各剂量均可降低NAFLD小鼠的游离脂肪酸(FFA)水平(图3E)；以上结果说明GLC能够改善高脂高糖饮食引起的脂代谢异常；实验数据如表3所示。
表3 GLC对NAFLD小鼠血脂的影响(means±SEM，n＝8-10)
Figure PCTCN2021127473-appb-000002
注:与Model组对比，*p<0.05，**p<0.01，***p＜0.001(n＝8-10,mean±SEM)
3.4 GLC对NAFLD小鼠肝功能的影响
对肝功能指标测定结果表明，相对于Model组，GLC各剂量均能显著改善Model组小鼠血清谷草转氨酶(AST)和谷丙转氨酶(ALT)水平(图4A-B)；说明GLC能够改善高脂高糖饮食引起的肝功能指标异常；实验数据如表4所示。
表4 GLC对NAFLD小鼠肝功能的影响(means±SEM，n＝8-10)
组别 Serum AST(U/L) Serum ALT(U/L)
空白对照组 13.24±0.9216*** 18.51±0.8445***
模型对照组 27.69±1.199 35.53±0.7899
二甲双胍组 19.44±1.263** 26.75±1.672*
瑞舒伐他汀组 20.06±1.730** 22.29±2.067**
高剂量组 20.9±1.780** 23.69±2.067**
中剂量组 23.69±1.863* 31.05±1.542*
低剂量组 25.83±2.322 38.32±2.734
注：与Model组对比，*p<0.05，**p<0.01，***p＜0.001(n＝8-10,mean±SEM)
3.5 GLC对NAFLD小鼠血清抗氧化功能的影响
血清抗氧化因子测定结果表明，相对于Model组，GLC各剂量均能显著增加Model组小鼠过氧化氢酶(CAT)水平，增强总抗氧化能力(T-AOC)(图 5A-B)；说明GLC能够强NAFLD小鼠的抗氧化功能；实验数据如表5所示。
表5 GLC对NAFLD小鼠抗氧化功能的影响(means±SEM，n＝8-10)
组别 Serum CAT(U/L) T-AOC(U/mg prot)
空白对照组 56.77±4.556** 0.4912±0.007251****
模型对照组 30.92±4.189 0.3732±0.009951
二甲双胍组 49.68±1.907** 0.395±0.01635
瑞舒伐他汀组 54.55±3.201** 0.3785±0.01174
高剂量组 48.22±2.070** 0.4203±0.01001*
中剂量组 46.92±4.477** 0.4333±0.006171**
低剂量组 46.83±4.447** 0.4193±0.006834*
注:与Model组对比，*p<0.05，**p<0.01，***p＜0.001，****p＜0.0001(n＝8-10,mean±SEM)
3.6 GLC对NAFLD小鼠血清炎症因子的影响
血清炎症因子测定结果表明，相对于Model组，GLC各剂量能显著增加血清抗炎因子白介素-10(IL-10)水平(图6B)；但GLC各剂量对血清促炎因子白介素-6(IL-6)水平的影响不显著(图6A)；GLC-H和GLC-M能显著降低血清肿瘤坏死因子-α(TNF-α)水平，而GLC-L作用不显著(图6C)；以上结果表明，GLC具有一定改善NAFLD小鼠抗炎能力的活性；实验数据如表6所示。
表6 GLC对NAFLD小鼠血清炎症因子的影响(means±SEM，n＝8-10)
Figure PCTCN2021127473-appb-000003
注:与Model组对比，*p<0.05，**p<0.01，***p＜0.001(n＝8-10,mean±SEM)
3.7 GLC对NAFLD小鼠血清内毒素的影响
内毒素(LPS)是肠道细菌的代谢产物，血清内毒素水平升高意味着肠道屏障发生障碍，内毒素透过屏障进入血循环增多。血清LPS含量测定结果表明，GLC各剂量能够显著降低血清内毒素水平；说明GLC可能通过改善肠道屏障功能从而减少小鼠血清内毒素水平(图7)；实验数据如表7所示。
表7 GLC对NAFLD小鼠血清内毒素水平的影响(means±SEM，n＝8-10)
组别 LPS(U/mL)
空白对照组 408.9±13.86**
模型对照组 481±16.19
二甲双胍组 361.2±14.47***
瑞舒伐他汀组 356.3±18.10***
高剂量组 371.2±12.72***
中剂量组 416.2±10.50**
低剂量组 413±15.53**
注:与Model组对比，*p<0.05，**p<0.01，***p＜0.001(n＝8-10,mean±SEM)
3.8 GLC对NAFLD小鼠肝脏脂质堆积的影响
根据小鼠肝脏石蜡切片苏木素-伊红染色结果表明，Control组小鼠肝脏基本没有脂质堆积，Model组肝脏有大量脂滴空泡，有严重的脂肪变性。二甲双胍组以及GLC各剂量组的小鼠肝脏与Model组相比明显减少，脂质堆积得到改善。实验结果如图8所示。
4.结论
本发明通过灌胃给予NAFLD小鼠GLC高、中和低剂量12周，剂量分别为GLC-H：600mg/Kg/d，GLC-M：300mg/Kg/d，GLC-L：150mg/Kg/d。GLC能在不影响食欲的情况下，显著的改善高脂高糖饮食引起的机体高血糖和胰岛素抵抗，以及血脂四项和游离脂肪酸水平的紊乱。说明GLC给药能够改善NAFLD小鼠的糖脂代谢异常。
GLC高、中剂量能降低NAFLD小鼠血清AST和ALT水平，说明GLC能改善高脂高糖饮食引起的肝功能指标异常。GLC给药能够增加小鼠血清中过氧化氢酶水平，增强总抗氧化能力，减少机体受高脂高糖饮食带来的氧化损伤。 GLC对IL-6水平无显著性影响，但能显著改善血清IL-10和TNF-α水平，说明GLC具有一定改善NAFLD小鼠抗炎能力的活性。GLC给药显著降低NAFLD小鼠血清内毒素水平，说明GLC可能通过改善肠道屏障功能从而减轻血清内毒素的水平，从而起到改善NAFLD指标的作用。GLC给药还能显著降低NAFLD小鼠肝脏脂肪变性的情况，说明GLC能改善高脂高糖饮食带来的肝脂质堆积。
综上所述，GLC能够改善NAFLD小鼠胰岛素抵抗、糖脂代谢紊乱，肝脏脂质堆积，改善肝功能异常，增强抗氧化能力，降低机体炎症、血清内毒素水平。说明GLC对NAFLD具有一定治疗作用。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (8)

  1. 氨基葡萄糖在制备预防和治疗非酒精性脂肪性肝病和糖尿病药物组合物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述氨基葡萄糖是氨基葡萄糖盐酸盐或氨基葡萄糖硫酸盐其中的一种或其组合。
  3. 如权利要求1或2所述的应用，其特征在于，所述的药物组合物用于：全身给药或肠胃外给药。
  4. 如权利要求1所述的应用，其特征在于，所述的非酒精性脂肪性肝病包括非酒精性单纯性脂肪肝、非酒精性脂肪性肝炎、肝硬化及肝细胞癌。
  5. 如权利要求1所述的应用，其特征在于，所述的糖尿病为高脂高糖饮食引起的机体高血糖、高血脂、游离脂肪酸紊乱和胰岛素抵抗。
  6. 如权利要求1或2所述的应用，其特征在于，所述的药物组合物为注射液、颗粒剂、片剂、胶囊剂或口服液。
  7. 如权利要求1所述的应用，其特征在于，所述氨基葡萄糖的有效量为300～600mg/kg/日。
  8. 如权利要求7所述的应用，其特征在于，所述氨基葡萄糖的有效量为600mg/kg/日。
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