WO2018053807A1 - Use of niclosamide ethanolamine salt in preparing medicine for type 1 diabetes - Google Patents

Use of niclosamide ethanolamine salt in preparing medicine for type 1 diabetes Download PDF

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WO2018053807A1
WO2018053807A1 PCT/CN2016/099876 CN2016099876W WO2018053807A1 WO 2018053807 A1 WO2018053807 A1 WO 2018053807A1 CN 2016099876 W CN2016099876 W CN 2016099876W WO 2018053807 A1 WO2018053807 A1 WO 2018053807A1
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nen
effect
mice
shows
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孙惠力
杜蘅
韩鹏勋
邵牧民
郭岚
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深圳市中医院
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Priority to PCT/CN2016/099876 priority Critical patent/WO2018053807A1/en
Priority to CN201611166593.4A priority patent/CN106727472B/en
Priority to PCT/CN2016/110319 priority patent/WO2018053954A1/en
Publication of WO2018053807A1 publication Critical patent/WO2018053807A1/en

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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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol

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  • the present invention relates to a novel application of Niclosamide ethanolamine salt (NEN) and, more particularly, to the use of NEN for the preparation of a medicament for the prevention and treatment of type 1 diabetes and its complications.
  • NEN Niclosamide ethanolamine salt
  • Type 1 diabetes also known as insulin-dependent diabetes, is a type of metabolic disease caused by the destruction of islet beta cells leading to an absolute deficiency of insulin, which in turn causes an increase in blood sugar.
  • the typical type 1 diabetes is mainly characterized by "three more and one less" symptoms, that is, polydipsia, polyphagia, polyuria, weight loss, often spontaneous ketosis tendency, serious complications, high disability and mortality, currently In addition to insulin therapy, there is no effective oral hypoglycemic agent.
  • Niclosamide and its ethanolamine salt are recommended by WHO for snail-killing and anthelmintic drugs. In recent years, studies have shown that these drugs have obvious anti-tumor effects.
  • the invention relates to a new application of NEN, and finds that NEN has a strong preventive effect on type 1 diabetes and its complications. It is an object of the present invention to provide a use of NEN for the preparation of a medicament for the prevention and treatment of type 1 diabetes and its complications.
  • the present invention provides the following technical solutions:
  • the present invention has the following beneficial effects: the present invention aims to investigate the protective effect of NEN on type 1 diabetes and its complications and the mechanism of action of NEN.
  • the results show that NEN can improve the polydipsia, polyphagia, and more of type 1 diabetic mice.
  • Urinary symptoms lower blood sugar, glycosylated hemoglobin, urine sugar levels, increase serum insulin levels, and improve pancreatic pathological damage.
  • it can reduce urinary albumin excretion rate, reduce glomerular filtration rate, reduce glomerular vasospasm area, reduce urine NAG, NGAL excretion, and inhibit Akt/mTOR/ in renal cortex.
  • Activation of the 4E-BP1 signaling pathway Activation of the 4E-BP1 signaling pathway.
  • NEN also has obvious protection for the liver, which has a significant improvement on muscle function. According to the above studies, it can be concluded that NEN has a significant protective effect on type 1 diabetes and its complications, and its protective mechanism of renal target organs is related to the inhibition of Akt/mTOR/4E-BP1 signaling pathway.
  • NEN is prepared into an oral administration form, an injection administration form, a mucosal administration form or a transdermal administration form using an existing preparation process, or a tablet, a capsule, a granule, an oral solution, a patch or a coagulant.
  • Glue medications can be used to prevent and treat type 1 diabetes and its complications.
  • Figure 1 shows the effect of NEN on the physiological indexes of type 1 diabetic mice.
  • Figure 1 (a) shows the effect of NEN on the water consumption of mice
  • Figure 1 (b) shows the effect of NEN on the food intake of mice.
  • (c) the effect of NEN on the urine volume of mice.
  • Figure 1 (d) shows the effect of NEN on the body weight of mice; among them, compared with the control group, ***P ⁇ 0.001; compared with the STZ group, ## P ⁇ 0.01, ### P ⁇ 0.001;
  • Figure 2 shows the effect of NEN on metabolic markers and pancreatic pathology in type 1 diabetic mice.
  • Figure 2(a) shows the effect of NEN on fasting blood glucose in mice
  • Figure 2(b) shows the effect of NEN on glycosylated hemoglobin in mice.
  • Figure 2(c) shows the effect of NEN on serum insulin in mice
  • Figure 2(d) shows the effect of NEN on urine glucose in mice
  • Figure 2(e) shows the effect of NEN on islet area in mice.
  • 2(f) showed the effect of NEN on the islet area of mice by HE staining
  • Fig. 2(g) showed the effect of NEN on islet cells by immunofluorescence staining; among them, compared with the control group, ***P ⁇ 0.001; Comparison of STZ group, ## P ⁇ 0.01, ### P ⁇ 0.001;
  • Figure 3 shows the effect of NEN on urinary albumin excretion rate and creatinine clearance rate in type 1 diabetic mice;
  • Fig. 3(a) shows the effect of NEN on urinary albumin excretion rate in mice, and
  • Fig. 3(b) shows NEN pair. results Effect of creatinine clearance in mice; wherein, compared with the control group, *** P ⁇ 0.001; compared with STZ group, # P ⁇ 0.05, ## P ⁇ 0.01;
  • Figure 4 shows the effect of NEN on kidney weight/body weight and glomerular vasospasm in type 1 diabetic mice;
  • Figure 4(a) shows the effect of NEN on kidney weight/body weight in mice, and
  • Figure 4(b) shows NEN.
  • Figure 4(c) shows the effect of NEN on mouse glomeruli by PAS staining; *P ⁇ 0.05, ***P ⁇ compared with control group 0.001; compared with STZ group, # P ⁇ 0.05, ### P ⁇ 0.001;
  • Figure 5 shows the effect of NEN on renal tubular injury in type 1 diabetic mice.
  • Figure 5 (a) shows the effect of NEN on urine NAG in mice
  • Figure 5 (b) shows the effect of NEN on urine NGAL in mice.
  • Figure 6 shows the effect of NEN on Akt/mTOR/4E-BP1 signaling pathway in renal cortex of type 1 diabetic mice;
  • Figure 6(a) shows the effect of NEN on Akt/mTOR/4E-BP1 signaling pathway protein by immunoblotting
  • Fig. 6(b) shows the effect of NEN on the expression of p-Akt (Ser473) protein in mouse renal cortex
  • Fig. 6(c) shows the effect of NEN on the expression of p-mTOR (Ser2448) protein in mouse renal cortex.
  • 6(d) is the effect of NEN on the expression of p-4E-BP1 (Thr37/46) protein in mouse renal cortex
  • Figure 6(e) shows the expression of p-4E-BP1 (Thr70) protein in mouse renal cortex by NEN.
  • Figure 6 (f) is the effect of NEN on the expression of 4E-BP1 total protein in mouse renal cortex; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 compared with control group; compared with STZ group, # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001;
  • Figure 7 shows the effect of NEN on liver function in type 1 diabetic mice.
  • Figure 7 (a) shows the effect of NEN on serum alanine aminotransferase (ALT)
  • Figure 7 (b) shows NEN on mouse serum aspartate aminotransferase (AST).
  • Figure 7 (c) is the effect of NEN on serum total protein (TP)
  • Figure 7 (d) is the effect of NEN on mouse serum albumin (ALB); among them, compared with the control group, ** P ⁇ 0.01, *** P ⁇ 0.001; compared with STZ group, # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001;
  • Figure 8 shows the effect of NEN on the muscle function of type 1 diabetic mice.
  • Figure 8 (a) shows the effect of NEN on the grip of mice, and
  • Figure 8 (b) shows the number of shocks per minute by NEN on the treadmill. The results were affected; among them, ***P ⁇ 0.001 compared with the control group; ## P ⁇ 0.01 compared with the STZ group.
  • Type 1 diabetes mainly causes insulin deficiency due to destruction of islet ⁇ cells, which leads to high blood sugar, serious complications, high disability and high mortality.
  • the current pathogenesis is still unclear.
  • Sugar drugs There is no effective oral drop in addition to insulin treatment.
  • the present invention aims to observe the preventive and therapeutic effects of NEN on type 1 diabetes and its complications.
  • Animal model 8 week old male C57BL/6 mice (body weight 18-22g) were purchased from Guangdong Medical Laboratory Animal Center. Animal experiments were carried out in strict accordance with the relevant guidelines and regulations for animal ethics at Guangzhou University of Traditional Chinese Medicine. The experimental animals were controlled to freely ingest and drink at a constant room temperature of 20 ⁇ 1 ° C, 12 hours light and 12 hours dark cycle. The mouse model of type 1 diabetes was induced by a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ, purchased from Sigma, USA). The fasting blood glucose ⁇ 16.7 mmol/L after 7 days of STZ injection confirmed the successful modeling.
  • STZ streptozotocin
  • mice were randomly assigned to the following groups (8-10 per group): the normal control group (ie, the control group in the chart of the present invention), the type 1 diabetic mouse model group (ie, the STZ group), and the NEN treatment group (ie, In the STZ+NEN group, the control group and the STZ group were fed with regular food, and the STZ+NEN group was fed with NEN-added food.
  • NEN was purchased from Hubei Shengtian Hengchuang Biotechnology Co., Ltd., and was added to the food of mice at a standard rate of 10g/kg. The above experimental treatment lasted for 8 weeks.
  • mice At 8 weeks of treatment, the body weight of the mice was weighed, and urine was collected using a mouse metabolic cage (Tenibus, Italy), urine volume was recorded, and water intake and food intake were recorded.
  • the blood glucose of each group of mice was measured using a blood glucose meter (Roche, Switzerland), and after 8 weeks of administration, the mice were sacrificed and blood samples, pancreas and kidney tissue samples were collected.
  • the glycated hemoglobin (HbA 1C ) content was measured using an Ultra2 glycated hemoglobin analyzer (Primus, USA).
  • Tissue preparation Immediately after the mice were sacrificed, the pancreas was removed and fixed with 10% formalin. The kidneys were then removed, weighed, rinsed in phosphate buffer, and a certain amount of kidney tissue was cut along the longitudinal section and fixed with 10% formalin. The remaining kidney tissue was immediately frozen in liquid nitrogen and stored at -80 °C. Subsequent experimental studies.
  • pancreatic paraffin section (3 ⁇ m thick) and kidney paraffin section (2 ⁇ m thick)
  • HE staining and PAS staining were used to evaluate the pathological damage of islets and glomeruli. Areas of islet area and glomerular vasospasm were counted using the 4.10 version of NIS-Elements image processing software (Nikon, Japan) for image processing analysis. Each pancreatic tissue section measures 4-10 islet area, and each kidney tissue section is measured. 40-50 glomerular vasospasm area, the length of the scale in the picture is 50 ⁇ m.
  • pancreatic immunofluorescence staining pancreatic tissue paraffin section (3 ⁇ m thick), dewaxing, hydration, the slice was placed in boiling sodium citrate buffer (pH 6) for 20 minutes for antigen retrieval, and then cooled to room temperature, PBS Wash 3 times, each time 5 min, then add mouse glucagon primary antibody (Abcam, UK) and rabbit-derived insulin primary antibody (CST, USA) overnight at 4 ° C, wash PBS 3 times, each time Anti-mouse fluorescent secondary antibody (Abcam, UK) and anti-rabbit fluorescent secondary antibody (Jackson ImmunoResearch, USA) were added at intervals of 5 min, respectively, and incubated at 37 ° C for 1 hour, followed by water-soluble sealing tablets containing DAPI (Southern Biotech, The United States) covers (blue, representing the nucleus), and finally uses laser confocal microscopy (Zeiss, Germany) to observe the content of alpha cells (glucagon, green) and beta cells (insulin, red).
  • ELISA ELISA kits for detecting serum insulin, urinary albumin and urinary NGAL in mice were purchased from Merck, Germany, Bethyl, USA and R&D systems. ELISA experiments were performed according to the manufacturer's instructions to detect serum insulin, urinary albumin, and urinary NGAL levels in each group of mice.
  • p-Akt Ser473
  • p-mTOR Ser2448
  • p-4E-BP1 Thr37/46
  • p-4E-BP1 Thr70
  • 4E-BP1 antibody was purchased from CST Company of the United States; ⁇ -actin antibody was purchased. In the United States sigma company.
  • Measurement data are expressed as mean ⁇ standard deviation. Statistical differences between the two groups of samples were analyzed by independent sample t-test. Comparisons between groups of samples were analyzed by one-way analysis of variance, and statistical analysis was performed using SPSS16.0 statistical software. A statistical difference was considered to be significant at P ⁇ 0.05.
  • NEN can improve the symptoms of polydipsia, polyphagia, and polyuria in type 1 diabetic mice.
  • Figure 1 shows the effect of NEN on the physiological indexes of mice.
  • Figure 1 (a) shows the effect of NEN on the 24-hour water consumption of mice
  • Figure 1 (b) shows the effect of NEN on the 24-hour intake of mice.
  • 1(c) is the effect of NEN on the 24-hour urine volume of mice.
  • the group showed symptoms of polydipsia, polyphagia, polyuria and weight loss (Fig. 1a, b, c, d); compared with the STZ group, the symptoms of polydipsia, polyphagia, and polyuria were significantly improved in the STZ+NEN group (Fig. 1a). , b, c).
  • NEN can reduce blood sugar, glycosylated hemoglobin, urine sugar in type 1 diabetic mice, increase serum insulin levels, and improve pancreatic pathological damage.
  • Figure 2 shows the effect of NEN on metabolic parameters and pancreatic pathology in mice.
  • Figure 2(a) shows the effect of NEN on blood glucose in mice
  • Figure 2(b) shows the effect of NEN on glycosylated hemoglobin in mice.
  • Figure 2(c) shows the effect of NEN on serum insulin in mice
  • Figure 2(d) shows the effect of NEN on urine glucose in mice
  • Figure 2(e) shows the effect of NEN on islet area in mice
  • Figure 2 (f) The results of HE staining showed the effect of NEN on mouse islet area
  • Figure 2 (g) shows the effect of NEN on mouse islet cells by confocal imaging
  • n 6-8 per group, 8 weeks, with Compared with the control group, the blood glucose, glycated hemoglobin, and urine sugar of the STZ group increased significantly (Fig.
  • NEN can reduce the excretion rate of albuminuria and reduce the creatinine clearance rate in type 1 diabetic mice.
  • Figure 3 shows the effect of NEN on urinary albumin excretion rate and creatinine clearance in mice
  • NEN can reduce the kidney weight/body weight ratio and glomerular vasospasm area in type 1 diabetic mice.
  • Figure 4 shows the effect of NEN on kidney weight/body weight and glomerular vasospasm in mice
  • Figure 4(c) shows the effect of NEN on the glomerulus of mice by PAS staining; compared with control group In the STZ group, the kidney weight/body weight and glomerular vasospasm area increased significantly (Fig. 4a, b, c). After 8 weeks of NEN treatment, the kidney weight/body weight and glomerular vasospasm area were significantly smaller (Fig. 4a, b, c).
  • NEN can improve renal tubular damage in type 1 diabetic mice.
  • Figure 5 shows the effect of NEN on renal tubular injury in mice.
  • Figure 5(a) shows the effect of NEN on urine NAG in mice
  • Figure 5(b) shows the effect of NEN on urine NGAL in mice
  • n In each group of 6-8, compared with the control group, the urine NAG and NGAL excretion of the STZ group were significantly increased (Fig. 5a, b), and the NAG and NGAL excretion rates were significantly reduced after 8 weeks of NEN treatment (Fig. 5a, b).
  • NEN can inhibit the overactivation of Akt/mTOR/4E-BP1 signaling pathway in the renal cortex of type 1 diabetic mice.
  • Figure 6 shows the effect of NEN on the renal cortex Akt/mTOR/4E-BP1 signaling pathway in mice;
  • Figure 6(a) shows the effect of NEN on the Akt/mTOR/4E-BP1 signaling pathway protein by immunoblotting.
  • 6(b) is the statistical result of the effect of NEN on mouse renal cortex p-Akt (Ser473) protein
  • Figure 6 (c) is the statistical result of the effect of NEN on mouse renal cortex p-mTOR (Ser2448) protein
  • Figure 6 ( d) is the statistical result of the effect of NEN on mouse renal cortex p-4E-BP1 (Thr37/46) protein
  • Figure 6(e) is the statistical result of the effect of NEN on mouse renal cortex p-4E-BP1 (Thr70) protein.
  • -mTOR(Ser2448), p-4E-BP1 (Thr37/46), p-4E-BP1 (Thr70) and 4E-BP1 protein were significantly increased, and NEN treatment significantly reduced its expression.
  • NEN has obvious liver protection effect on type 1 diabetic mice.
  • Figure 7 shows the effect of NEN on liver function in mice.
  • Figure 7(a) shows the effect of NEN on serum alanine aminotransferase (ALT)
  • Figure 7(b) shows the effect of NEN on serum aspartate aminotransferase (AST) in mice.
  • Fig. 7(c) shows the effect of NEN on serum total protein (TP)
  • serum ALT and AST in the STZ group were significantly increased (Fig. 7a, b), serum TP, ALB decreased significantly (Fig. 7c, d), NEN treatment can significantly reduce ALT, AST ( Figure 7a, b), TP, ALB increased significantly (Fig. 7c, d).
  • NEN has a significant improvement in muscle function in type 1 diabetic mice.
  • Figure 8 shows the effect of NEN on muscle function in mice.
  • Figure 8(a) shows the effect of NEN on the grip of mice
  • Figure 8(b) shows the effect of NEN on the number of shocks per 30 minutes on the treadmill.
  • n 6-8 in each group, at 8 weeks, compared with the control group, the grip strength of the STZ group was significantly reduced (Fig. 8a), and the number of shocks was significantly increased (Fig. 8b).
  • the NEN treatment significantly increased the grip of the mice. (Fig. 8a), reduce the number of shocks (Fig. 8b).
  • NEN as an anthelmintic and anti-tumor drug, can improve the symptoms of polydipsia, polyphagia and polyuria in type 1 diabetic mice, and lower blood sugar, glycated hemoglobin and urine sugar levels.
  • High serum insulin levels improve pathological damage in the pancreas. It has obvious protective effects on kidney, liver and muscle damage complicated by type 1 diabetes.
  • NEN has a significant preventive effect on type 1 diabetes and its complications.

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Abstract

The invention provides a use of niclosamide ethanolamine salt in preparing a medicine for preventing and treating type 1 diabetes and complications thereof. The niclosamide ethanolamine salt can improve symptoms of polydipsia, polyphagia, and polyuria in a type I diabetic mice, reduce blood glucose, glycosylated hemoglobin and urine glucose level, increase serum insulin level, improve pancreatic pathological injury, reduce urinary albumin excretion rate, creatinine clearance, glomerular tuft area and urinary excretion of NAG and NGAL, and inhibit activation of Akt/mTOR/4E-BP1 signaling pathway in renal tissue, thereby protecting the liver and improving muscle function.

Description

氯硝柳胺乙醇胺盐在制备1型糖尿病药物中的应用Application of niclosamide ethanolamine salt in the preparation of type 1 diabetes drugs 技术领域Technical field
本发明涉及氯硝柳胺乙醇胺盐(Niclosamide ethanolamine salt，NEN)的新应用，更具体的说，涉及NEN在制备预防和治疗1型糖尿病及其并发症药物中的应用。The present invention relates to a novel application of Niclosamide ethanolamine salt (NEN) and, more particularly, to the use of NEN for the preparation of a medicament for the prevention and treatment of type 1 diabetes and its complications.
背景技术Background technique
1型糖尿病又称为胰岛素依赖性糖尿病，是以胰岛β细胞破坏导致胰岛素绝对缺乏，进而引起血糖升高的一类代谢性疾病。典型的1型糖尿病主要表现为“三多一少”症状，即多饮，多食，多尿，体重下降，常有自发酮症倾向，其并发症严重，致残和致死率高，目前除胰岛素治疗外，尚无有效的口服降糖药物。 Type 1 diabetes, also known as insulin-dependent diabetes, is a type of metabolic disease caused by the destruction of islet beta cells leading to an absolute deficiency of insulin, which in turn causes an increase in blood sugar. The typical type 1 diabetes is mainly characterized by "three more and one less" symptoms, that is, polydipsia, polyphagia, polyuria, weight loss, often spontaneous ketosis tendency, serious complications, high disability and mortality, currently In addition to insulin therapy, there is no effective oral hypoglycemic agent.
氯硝柳胺及其乙醇胺盐是WHO推荐使用的灭螺药及驱虫药，近年研究显示该类药物具有明显的抗肿瘤作用。Niclosamide and its ethanolamine salt are recommended by WHO for snail-killing and anthelmintic drugs. In recent years, studies have shown that these drugs have obvious anti-tumor effects.
发明内容Summary of the invention
本发明涉及NEN的新应用，研究发现NEN对1型糖尿病及其并发症具有较强的防治作用。本发明的目的在于提供一种NEN在制备预防和治疗1型糖尿病及其并发症药物中的应用。The invention relates to a new application of NEN, and finds that NEN has a strong preventive effect on type 1 diabetes and its complications. It is an object of the present invention to provide a use of NEN for the preparation of a medicament for the prevention and treatment of type 1 diabetes and its complications.
本发明为了达到上述目的，提供如下技术方案：In order to achieve the above object, the present invention provides the following technical solutions:
1、研究NEN治疗对1型糖尿病小鼠多饮、多食、多尿症状的影响；1. To study the effects of NEN treatment on polydipsia, polyphagia and polyuria in type 1 diabetic mice;
2、研究NEN治疗对1型糖尿病小鼠血糖、糖化血红蛋白、血清胰岛素、尿糖、胰腺病理损伤的影响；2. To study the effects of NEN treatment on blood glucose, glycosylated hemoglobin, serum insulin, urine glucose and pancreatic pathological damage in type 1 diabetic mice;
3、研究NEN治疗对1型糖尿病小鼠尿白蛋白***率、肌酐清除率的影响； 3. To study the effect of NEN treatment on urinary albumin excretion rate and creatinine clearance rate in type 1 diabetic mice;
4、研究NEN治疗对1型糖尿病小鼠肾小球病变的影响；4. To study the effect of NEN treatment on glomerular lesions in type 1 diabetic mice;
5、研究NEN治疗对1型糖尿病小鼠肾小管损伤的影响；5. To study the effect of NEN treatment on renal tubular injury in type 1 diabetic mice;
6、研究NEN治疗对1型糖尿病小鼠肾皮质Akt/mTOR/4E-BP1信号通路活化的影响；6. To study the effect of NEN treatment on the activation of Akt/mTOR/4E-BP1 signaling pathway in renal cortex of type 1 diabetic mice;
7、研究NEN治疗对1型糖尿病小鼠肝功能的影响；7. To study the effect of NEN treatment on liver function in type 1 diabetic mice;
8、研究NEN治疗对1型糖尿病小鼠肌肉功能的影响。8. To study the effect of NEN treatment on muscle function in type 1 diabetic mice.
实施本发明，具有如下有益效果：本发明旨在探讨NEN对1型糖尿病及其并发症的保护作用及NEN的作用机制，结果显示：NEN可改善1型糖尿病小鼠多饮、多食、多尿症状，降低血糖、糖化血红蛋白、尿糖水平，升高血清胰岛素水平，改善胰腺病理损伤。在对肾脏靶器官的保护方面，可减少尿白蛋白***率，降低肾小球滤过率，减小肾小球血管襻面积，减少尿液NAG、NGAL***，抑制肾皮质中Akt/mTOR/4E-BP1信号通路的活化。另外，NEN对肝脏也有明显的保护，对肌肉功能有明显的改善作用。根据上述研究可以得出结论：NEN对1型糖尿病及其并发症具有明显的保护作用，其肾脏靶器官保护作用机制与抑制Akt/mTOR/4E-BP1信号通路有关。使用现有的制备工艺将NEN制成口服给药剂型、注射给药剂型、粘膜给药剂型或者经皮给药剂型的药物，或者片剂、胶囊剂、颗粒剂、口服液、贴剂或者凝胶剂的药物，可以用于预防和治疗1型糖尿病及其并发症。The present invention has the following beneficial effects: the present invention aims to investigate the protective effect of NEN on type 1 diabetes and its complications and the mechanism of action of NEN. The results show that NEN can improve the polydipsia, polyphagia, and more of type 1 diabetic mice. Urinary symptoms, lower blood sugar, glycosylated hemoglobin, urine sugar levels, increase serum insulin levels, and improve pancreatic pathological damage. In the protection of kidney target organs, it can reduce urinary albumin excretion rate, reduce glomerular filtration rate, reduce glomerular vasospasm area, reduce urine NAG, NGAL excretion, and inhibit Akt/mTOR/ in renal cortex. Activation of the 4E-BP1 signaling pathway. In addition, NEN also has obvious protection for the liver, which has a significant improvement on muscle function. According to the above studies, it can be concluded that NEN has a significant protective effect on type 1 diabetes and its complications, and its protective mechanism of renal target organs is related to the inhibition of Akt/mTOR/4E-BP1 signaling pathway. NEN is prepared into an oral administration form, an injection administration form, a mucosal administration form or a transdermal administration form using an existing preparation process, or a tablet, a capsule, a granule, an oral solution, a patch or a coagulant. Glue medications can be used to prevent and treat type 1 diabetes and its complications.
附图说明DRAWINGS
下面将结合附图及实施例对本发明作进一步说明，附图中：The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:
图1为NEN对1型糖尿病小鼠生理指标的影响结果，图1(a)为NEN对小鼠饮水量的影响结果，图1(b)为NEN对小鼠进食量的影响结果，图1(c)为NEN对小鼠尿量的影响结果，图1(d)为NEN对小鼠体重的影响结果；其中，与control组比较，***P<0.001；与STZ组比较，﹟﹟P<0.01，﹟﹟﹟P<0.001；Figure 1 shows the effect of NEN on the physiological indexes of type 1 diabetic mice. Figure 1 (a) shows the effect of NEN on the water consumption of mice, and Figure 1 (b) shows the effect of NEN on the food intake of mice. (c) the effect of NEN on the urine volume of mice. Figure 1 (d) shows the effect of NEN on the body weight of mice; among them, compared with the control group, ***P<0.001; compared with the STZ group, ## P<0.01, ### P<0.001;
图2为NEN对1型糖尿病小鼠代谢指标及胰腺病理的影响结果，图2(a)为NEN对小鼠空腹血糖的影响结果，图2(b)为NEN对小鼠糖化血红蛋白的影响结果，图2(c)为NEN对小鼠血清胰岛素的影响结果，图2(d)为 NEN对小鼠尿糖的影响结果，图2(e)为NEN对小鼠胰岛面积的影响结果，图2(f)为HE染色显示NEN对小鼠胰岛面积影响结果，图2(g)为免疫荧光染色显示NEN对胰岛细胞的影响结果；其中，与control组比较，***P<0.001；与STZ组比较，﹟﹟P<0.01，﹟﹟﹟P<0.001；Figure 2 shows the effect of NEN on metabolic markers and pancreatic pathology in type 1 diabetic mice. Figure 2(a) shows the effect of NEN on fasting blood glucose in mice, and Figure 2(b) shows the effect of NEN on glycosylated hemoglobin in mice. Figure 2(c) shows the effect of NEN on serum insulin in mice, Figure 2(d) shows the effect of NEN on urine glucose in mice, and Figure 2(e) shows the effect of NEN on islet area in mice. 2(f) showed the effect of NEN on the islet area of mice by HE staining, and Fig. 2(g) showed the effect of NEN on islet cells by immunofluorescence staining; among them, compared with the control group, ***P<0.001; Comparison of STZ group, ## P<0.01, ### P<0.001;
图3为NEN对1型糖尿病小鼠尿白蛋白***率、肌酐清除率的影响结果；图3(a)为NEN对小鼠尿白蛋白***率的影响结果，图3(b)为NEN对小鼠肌酐清除率的影响结果；其中，与control组比较，***P<0.001；与STZ组比较，﹟P<0.05，﹟﹟P<0.01；Figure 3 shows the effect of NEN on urinary albumin excretion rate and creatinine clearance rate in type 1 diabetic mice; Fig. 3(a) shows the effect of NEN on urinary albumin excretion rate in mice, and Fig. 3(b) shows NEN pair. results Effect of creatinine clearance in mice; wherein, compared with the control group, *** P <0.001; compared with STZ group, # P <0.05, ## P <0.01;
图4为NEN对1型糖尿病小鼠肾重/体重、肾小球血管襻面积的影响结果；图4(a)为NEN对小鼠肾重/体重的影响结果，图4(b)为NEN对小鼠肾小球血管襻面积的影响结果，图4(c)为PAS染色显示NEN对小鼠肾小球的影响结果；其中，与control组比较，*P<0.05，***P<0.001；与STZ组比较，﹟P<0.05，﹟﹟﹟P<0.001；Figure 4 shows the effect of NEN on kidney weight/body weight and glomerular vasospasm in type 1 diabetic mice; Figure 4(a) shows the effect of NEN on kidney weight/body weight in mice, and Figure 4(b) shows NEN. The effect of glomerular vasospasm on mouse, Figure 4(c) shows the effect of NEN on mouse glomeruli by PAS staining; *P<0.05, ***P< compared with control group 0.001; compared with STZ group, # P <0.05, ### P <0.001;
图5为NEN对1型糖尿病小鼠肾小管损伤的影响结果，图5(a)为NEN对小鼠尿液NAG的影响结果，图5(b)为NEN对小鼠尿液NGAL的影响结果；其中，与control组比较，***P<0.001；与STZ组比较，﹟﹟﹟P<0.001；Figure 5 shows the effect of NEN on renal tubular injury in type 1 diabetic mice. Figure 5 (a) shows the effect of NEN on urine NAG in mice, and Figure 5 (b) shows the effect of NEN on urine NGAL in mice. Wherein, compared with the control group, ***P<0.001; compared with the STZ group, ### P<0.001;
图6为NEN对1型糖尿病小鼠肾皮质Akt/mTOR/4E-BP1信号通路的影响结果；图6(a)为免疫印迹实验显示NEN对Akt/mTOR/4E-BP1信号通路蛋白的影响结果，图6(b)为NEN对小鼠肾皮质p-Akt(Ser473)蛋白表达的影响结果，图6(c)为NEN对小鼠肾皮质p-mTOR(Ser2448)蛋白表达的影响结果，图6(d)为NEN对小鼠肾皮质p-4E-BP1(Thr37/46)蛋白表达的影响结果，图6(e)为NEN对小鼠肾皮质p-4E-BP1(Thr70)蛋白表达的影响结果，图6(f)为NEN对小鼠肾皮质4E-BP1总蛋白表达的影响结果；其中，与control组比较，*P<0.05，**P<0.01，***P<0.001；与STZ组比较，﹟P<0.05，﹟﹟P<0.01，﹟﹟﹟P<0.001；Figure 6 shows the effect of NEN on Akt/mTOR/4E-BP1 signaling pathway in renal cortex of type 1 diabetic mice; Figure 6(a) shows the effect of NEN on Akt/mTOR/4E-BP1 signaling pathway protein by immunoblotting Fig. 6(b) shows the effect of NEN on the expression of p-Akt (Ser473) protein in mouse renal cortex, and Fig. 6(c) shows the effect of NEN on the expression of p-mTOR (Ser2448) protein in mouse renal cortex. 6(d) is the effect of NEN on the expression of p-4E-BP1 (Thr37/46) protein in mouse renal cortex, and Figure 6(e) shows the expression of p-4E-BP1 (Thr70) protein in mouse renal cortex by NEN. The results of the effect, Figure 6 (f) is the effect of NEN on the expression of 4E-BP1 total protein in mouse renal cortex; *P<0.05, **P<0.01, ***P<0.001 compared with control group; compared with STZ group, # P <0.05, ## P <0.01, ### P <0.001;
图7为NEN对1型糖尿病小鼠肝功能的影响结果，图7(a)为NEN对血清谷丙转氨酶(ALT)的影响结果，图7(b)为NEN对小鼠血清谷草转氨酶(AST)的影响结果，图7(c)为NEN对血清总蛋白(TP)的影响结果， 图7(d)为NEN对小鼠血清白蛋白(ALB)的影响结果；其中，与control组比较，**P<0.01，***P<0.001；与STZ组比较，﹟P<0.05，﹟﹟P<0.01，﹟﹟﹟P<0.001；Figure 7 shows the effect of NEN on liver function in type 1 diabetic mice. Figure 7 (a) shows the effect of NEN on serum alanine aminotransferase (ALT), and Figure 7 (b) shows NEN on mouse serum aspartate aminotransferase (AST). The results of the effect, Figure 7 (c) is the effect of NEN on serum total protein (TP), Figure 7 (d) is the effect of NEN on mouse serum albumin (ALB); among them, compared with the control group, ** P <0.01, *** P <0.001; compared with STZ group, # P <0.05, ## P <0.01, ### P <0.001;
图8为NEN对1型糖尿病小鼠肌肉功能的影响结果，图8(a)为NEN对小鼠抓力的影响结果，图8(b)为NEN对跑步机上小鼠每30分钟电击次数的影响结果；其中，与control组比较，***P<0.001；与STZ组比较，﹟﹟P<0.01。Figure 8 shows the effect of NEN on the muscle function of type 1 diabetic mice. Figure 8 (a) shows the effect of NEN on the grip of mice, and Figure 8 (b) shows the number of shocks per minute by NEN on the treadmill. The results were affected; among them, ***P<0.001 compared with the control group; ## P<0.01 compared with the STZ group.
具体实施方式detailed description
下面将结合附图对本发明的实施例进行具体描述。The embodiments of the present invention will be specifically described below with reference to the accompanying drawings.
1型糖尿病主要由于胰岛β细胞破坏导致胰岛素绝对缺乏，进而导致血糖升高，并发症严重，致残和致死率高，目前发病机制尚不明确，治疗上除胰岛素外，尚无有效的口服降糖药物。 Type 1 diabetes mainly causes insulin deficiency due to destruction of islet β cells, which leads to high blood sugar, serious complications, high disability and high mortality. The current pathogenesis is still unclear. There is no effective oral drop in addition to insulin treatment. Sugar drugs.
本发明旨在观察NEN对1型糖尿病及其并发症的防治作用。The present invention aims to observe the preventive and therapeutic effects of NEN on type 1 diabetes and its complications.
一、实验方法First, the experimental method
1、动物模型：8周龄雄性C57BL/6小鼠(体重18-22g)购买于广东省医学实验动物中心。动物实验严格按照广州中医药大学动物伦理相关准则和条例进行。实验动物受控在恒定室温20±1℃，12小时光照和12小时黑暗循环的条件下，同时自由摄食和饮水。1型糖尿病小鼠模型由一次性腹腔注射200mg/kg链脲佐菌素(streptozotocin，STZ，购自美国sigma公司)诱导而成，STZ注射7天后空腹血糖≥16.7mmol/L确定造模成功。1. Animal model: 8 week old male C57BL/6 mice (body weight 18-22g) were purchased from Guangdong Medical Laboratory Animal Center. Animal experiments were carried out in strict accordance with the relevant guidelines and regulations for animal ethics at Guangzhou University of Traditional Chinese Medicine. The experimental animals were controlled to freely ingest and drink at a constant room temperature of 20 ± 1 ° C, 12 hours light and 12 hours dark cycle. The mouse model of type 1 diabetes was induced by a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ, purchased from Sigma, USA). The fasting blood glucose ≥16.7 mmol/L after 7 days of STZ injection confirmed the successful modeling.
将实验小鼠随机分配到以下几组(每组8-10只)：正常对照组(即本发明图表中control组)、1型糖尿病小鼠模型组(即STZ组)、NEN治疗组(即STZ+NEN组)，其中control组和STZ组小鼠喂养常规食物，STZ+NEN组喂养添加NEN的食物。NEN购买于中国湖北盛天恒创生物科技有限公司，以10g/kg标准比例添加到小鼠的食物中。以上实验处理持续8周。The experimental mice were randomly assigned to the following groups (8-10 per group): the normal control group (ie, the control group in the chart of the present invention), the type 1 diabetic mouse model group (ie, the STZ group), and the NEN treatment group (ie, In the STZ+NEN group, the control group and the STZ group were fed with regular food, and the STZ+NEN group was fed with NEN-added food. NEN was purchased from Hubei Shengtian Hengchuang Biotechnology Co., Ltd., and was added to the food of mice at a standard rate of 10g/kg. The above experimental treatment lasted for 8 weeks.
2、生理和代谢参数：用药8周时，称量小鼠体重，应用小鼠代谢笼(泰尼百斯公司，意大利)收集尿液，记录尿量，记录进水量和进食量。采用血糖仪(罗氏公司，瑞士)测量各组小鼠的血糖，用药8周后，处死小鼠，并 采集血样、胰腺和肾脏组织样本。使用Ultra2糖化血红蛋白分析仪(Primus公司，美国)测量糖化血红蛋白(HbA1C)的含量。采用全自动生化分析仪(罗氏公司，瑞士)测量尿液和血液的生化指标，包括尿肌酐、尿葡萄糖、NAG(尿N-乙酰-β-葡萄糖苷酶)，血清肌酐、ALT、AST、TP、ALB。肌酐清除率(Ccr)是通过尿肌酐×尿液体积×1000/血肌酐/1440/体重计算得出，并用微升/每分钟/克表示。2. Physiological and metabolic parameters: At 8 weeks of treatment, the body weight of the mice was weighed, and urine was collected using a mouse metabolic cage (Tenibus, Italy), urine volume was recorded, and water intake and food intake were recorded. The blood glucose of each group of mice was measured using a blood glucose meter (Roche, Switzerland), and after 8 weeks of administration, the mice were sacrificed and blood samples, pancreas and kidney tissue samples were collected. The glycated hemoglobin (HbA 1C ) content was measured using an Ultra2 glycated hemoglobin analyzer (Primus, USA). Measurement of biochemical indicators of urine and blood using an automatic biochemical analyzer (Roche, Switzerland), including urinary creatinine, urinary glucose, NAG (urinary N-acetyl-β-glucosidase), serum creatinine, ALT, AST, TP , ALB. Creatinine clearance (Ccr) was calculated by urine creatinine x urine volume x 1000 / serum creatinine / 1440 / body weight and expressed in microliters per minute per gram.
3、组织准备：处死小鼠后，立即取出胰腺并用10％***固定。然后取出肾脏，称重，在磷酸盐缓冲液中冲洗，沿纵切面切取一定量肾脏组织用10％***固定，剩余的肾脏组织立即在液氮中冷冻并储存在-80℃用于后续实验研究。3. Tissue preparation: Immediately after the mice were sacrificed, the pancreas was removed and fixed with 10% formalin. The kidneys were then removed, weighed, rinsed in phosphate buffer, and a certain amount of kidney tissue was cut along the longitudinal section and fixed with 10% formalin. The remaining kidney tissue was immediately frozen in liquid nitrogen and stored at -80 °C. Subsequent experimental studies.
4、光镜病理：胰腺石蜡切片(3μm厚)和肾脏石蜡切片(2μm厚)分别采用HE染色和PAS染色法评价胰岛和肾小球的病理损伤情况。胰岛面积和肾小球血管襻的面积统计使用4.10版本的NIS-Elements图像处理软件(尼康公司，日本)进行图片处理分析，每个胰腺组织切片测量4-10胰岛面积，每个肾组织切片测量40-50肾小球血管襻面积，图片中标尺长度为50μm。4, light pathology: pancreatic paraffin section (3μm thick) and kidney paraffin section (2μm thick) HE staining and PAS staining were used to evaluate the pathological damage of islets and glomeruli. Areas of islet area and glomerular vasospasm were counted using the 4.10 version of NIS-Elements image processing software (Nikon, Japan) for image processing analysis. Each pancreatic tissue section measures 4-10 islet area, and each kidney tissue section is measured. 40-50 glomerular vasospasm area, the length of the scale in the picture is 50μm.
5、胰腺免疫荧光染色：胰腺组织石蜡切片(3μm厚)，脱蜡，水化，将切片放入煮沸的柠檬酸钠缓冲液(pH 6)中20分钟进行抗原修复，后冷却至常温，PBS洗3遍，每次间隔5min，随后加入鼠源胰高血糖素一抗(Abcam公司，英国)和兔源胰岛素一抗(CST公司，美国)在4℃孵育过夜，PBS洗3遍，每次间隔5min，分别加入抗鼠荧光二抗(Abcam公司，英国)和抗兔荧光二抗(Jackson ImmunoResearch公司，美国)，37℃孵育1小时，随后使用含DAPI的水溶性封片剂(SouthernBiotech公司，美国)封片(蓝色，代表细胞核)，最后使用激光共聚焦显微镜(Zeiss公司，德国)观察α细胞(胰高血糖素，绿色)及β细胞(胰岛素，红色)含量，图片中标尺长度为50μm。5, pancreatic immunofluorescence staining: pancreatic tissue paraffin section (3μm thick), dewaxing, hydration, the slice was placed in boiling sodium citrate buffer (pH 6) for 20 minutes for antigen retrieval, and then cooled to room temperature, PBS Wash 3 times, each time 5 min, then add mouse glucagon primary antibody (Abcam, UK) and rabbit-derived insulin primary antibody (CST, USA) overnight at 4 ° C, wash PBS 3 times, each time Anti-mouse fluorescent secondary antibody (Abcam, UK) and anti-rabbit fluorescent secondary antibody (Jackson ImmunoResearch, USA) were added at intervals of 5 min, respectively, and incubated at 37 ° C for 1 hour, followed by water-soluble sealing tablets containing DAPI (Southern Biotech, The United States) covers (blue, representing the nucleus), and finally uses laser confocal microscopy (Zeiss, Germany) to observe the content of alpha cells (glucagon, green) and beta cells (insulin, red). The length of the scale in the picture is 50 μm.
6、ELISA：检测小鼠血清胰岛素，尿白蛋白和尿NGAL的ELISA试剂盒分别购自德国默克公司，美国Bethyl公司和美国R&D systems公司。按照厂家说明书进行ELISA实验，检测各组小鼠血清胰岛素、尿白蛋白，和尿NGAL，水平。 6. ELISA: ELISA kits for detecting serum insulin, urinary albumin and urinary NGAL in mice were purchased from Merck, Germany, Bethyl, USA and R&D systems. ELISA experiments were performed according to the manufacturer's instructions to detect serum insulin, urinary albumin, and urinary NGAL levels in each group of mice.
7、免疫印迹实验(Western blot)：将冷冻的肾皮质组织在裂解缓冲液中匀浆，平衡总蛋白浓度后通过SDS-PAGE凝胶电泳进行分离，并将蛋白转移到PVDF膜上，将PVDF膜放入用含0.5g/l脱脂奶粉的TBS缓冲液中，室温反应1小时，封闭膜上的非特异性位点，然后加入一抗，在4℃摇晃孵育过夜。洗涤后，采用ChemiDocTMMP成像***(Bio-Rad公司，美国)对蛋白条带进行检测和分析，结果使用β-actin作为内参进行比较。p-Akt(Ser473)、p-mTOR(Ser2448)、p-4E-BP1(Thr37/46)、p-4E-BP1(Thr70)、4E-BP1抗体均购于美国CST公司；β-actin抗体购于美国sigma公司。7. Western blot: The frozen renal cortex tissue was homogenized in lysis buffer, the total protein concentration was balanced, and then separated by SDS-PAGE gel electrophoresis, and the protein was transferred to the PVDF membrane to PVDF. The membrane was placed in TBS buffer containing 0.5 g/l skim milk powder, and reacted at room temperature for 1 hour to block a non-specific site on the membrane, then a primary antibody was added, and the mixture was incubated at 4 ° C overnight. After washing, the imaging system using the ChemiDoc TM MP (Bio-Rad, USA) protein bands were detected and analyzed, the results of using β-actin as an internal control for comparison. p-Akt (Ser473), p-mTOR (Ser2448), p-4E-BP1 (Thr37/46), p-4E-BP1 (Thr70), 4E-BP1 antibody were purchased from CST Company of the United States; β-actin antibody was purchased. In the United States sigma company.
8、小鼠抓力(g)使用抓力测定仪(中国安徽正华生物仪器设备有限公司)进行测定，电击次数(次/30分钟)使用动物实验跑台(中国安徽正华生物仪器设备有限公司)进行测定。8. Mouse grip force (g) using the grip force measuring instrument (China Anhui Zhenghua Biological Instrument Equipment Co., Ltd.) for the measurement, the number of electric shocks (times / 30 minutes) using the animal experiment running platform (China Anhui Zhenghua Biological Instrument Equipment Co., Ltd. Company) to carry out the measurement.
9、统计分析9, statistical analysis
计量资料使用均数±标准差表示。两组样本间的统计差异采用独立样本t检验进行分析，多组样本之间的比较使用单因素方差分析，统计分析采用SPSS16.0统计软件处理。P<0.05时视为在统计学上差异具有显著性。Measurement data are expressed as mean ± standard deviation. Statistical differences between the two groups of samples were analyzed by independent sample t-test. Comparisons between groups of samples were analyzed by one-way analysis of variance, and statistical analysis was performed using SPSS16.0 statistical software. A statistical difference was considered to be significant at P < 0.05.
二、结果Second, the results
1、NEN可改善1型糖尿病小鼠多饮、多食、多尿症状。1, NEN can improve the symptoms of polydipsia, polyphagia, and polyuria in type 1 diabetic mice.
图1为NEN对小鼠生理指标的影响结果，图1(a)为NEN对小鼠24小时饮水量的影响结果，图1(b)为NEN对小鼠24小时进食量的影响结果，图1(c)为NEN对小鼠24小时尿量的影响结果，图1(d)为NEN对小鼠体重的影响结果；n＝6-8每组，8周时，STZ组小鼠较control组表现出多饮、多食、多尿及体重减轻症状(图1a，b，c，d)；与STZ组相比，STZ+NEN组多饮、多食、多尿症状明显改善(图1a，b，c)。Figure 1 shows the effect of NEN on the physiological indexes of mice. Figure 1 (a) shows the effect of NEN on the 24-hour water consumption of mice, and Figure 1 (b) shows the effect of NEN on the 24-hour intake of mice. 1(c) is the effect of NEN on the 24-hour urine volume of mice. Figure 1(d) shows the effect of NEN on the body weight of mice; n=6-8 per group, 8 weeks, the STZ group is more control The group showed symptoms of polydipsia, polyphagia, polyuria and weight loss (Fig. 1a, b, c, d); compared with the STZ group, the symptoms of polydipsia, polyphagia, and polyuria were significantly improved in the STZ+NEN group (Fig. 1a). , b, c).
2、NEN可降低1型糖尿病小鼠血糖、糖化血红蛋白、尿糖，升高血清胰岛素水平，改善胰腺病理损伤。2, NEN can reduce blood sugar, glycosylated hemoglobin, urine sugar in type 1 diabetic mice, increase serum insulin levels, and improve pancreatic pathological damage.
图2为NEN对小鼠代谢指标及胰腺病理的影响结果，图2(a)为NEN对小鼠血糖的影响结果，图2(b)为NEN对小鼠糖化血红蛋白的影响结果， 图2(c)为NEN对小鼠血清胰岛素的影响结果，图2(d)为NEN对小鼠尿糖的影响结果，图2(e)为NEN对小鼠胰岛面积的影响结果，图2(f)为HE染色显示NEN对小鼠胰岛面积的影响结果；图2(g)为共聚焦成像显示NEN对小鼠胰岛细胞的影响结果；n＝6-8每组，8周时，与control组小鼠比较，STZ组小鼠血糖、糖化血红蛋白、尿糖明显增多(图2a，b，d)，血清胰岛素明显降低(图2c)，胰岛明显变小(图2e，f)，胰岛中β细胞(红色)明显减少，α细胞(绿色)相对增多(图2g)；与STZ组比较，STZ+NEN组小鼠血糖、糖化血红蛋白、尿糖明显降低(图2a，b，d)，血清胰岛素明显升高(图2c)，胰岛明显变大(图2e，f)，胰岛中β细胞明显增多，α细胞相对减少(图2g)。Figure 2 shows the effect of NEN on metabolic parameters and pancreatic pathology in mice. Figure 2(a) shows the effect of NEN on blood glucose in mice, and Figure 2(b) shows the effect of NEN on glycosylated hemoglobin in mice. Figure 2(c) shows the effect of NEN on serum insulin in mice, Figure 2(d) shows the effect of NEN on urine glucose in mice, and Figure 2(e) shows the effect of NEN on islet area in mice, Figure 2 (f) The results of HE staining showed the effect of NEN on mouse islet area; Figure 2 (g) shows the effect of NEN on mouse islet cells by confocal imaging; n=6-8 per group, 8 weeks, with Compared with the control group, the blood glucose, glycated hemoglobin, and urine sugar of the STZ group increased significantly (Fig. 2a, b, d), serum insulin decreased significantly (Fig. 2c), and the islets became significantly smaller (Fig. 2e, f). The β-cell (red) was significantly reduced, and the α-cell (green) was relatively increased (Fig. 2g). Compared with the STZ group, the blood glucose, glycated hemoglobin, and urine glucose of the STZ+NEN group were significantly decreased (Fig. 2a, b, d), serum. Insulin was significantly elevated (Fig. 2c), and islets were significantly larger (Fig. 2e, f). The number of β cells in the islets increased significantly, and the α cells were relatively reduced (Fig. 2g).
3、NEN可减少1型糖尿病小鼠白蛋白尿的***率，降低肌酐清除率。3, NEN can reduce the excretion rate of albuminuria and reduce the creatinine clearance rate in type 1 diabetic mice.
图3为NEN对小鼠尿白蛋白***率、肌酐清除率的影响结果；图3(a)为NEN对小鼠尿白蛋白***率的影响结果(n＝6-8每组)，图3(b)为NEN对小鼠肌酐清除率的影响结果(n＝5-8每组)；8周时，与control组比较，STZ小鼠尿白蛋白***率、肌酐清除率均明显升高(图3a，b)，NEN治疗8周后，小鼠尿白蛋白***率和肌酐清除率均有明显下降(图3a，b)。Figure 3 shows the effect of NEN on urinary albumin excretion rate and creatinine clearance in mice; Figure 3(a) shows the effect of NEN on urinary albumin excretion rate in mice (n=6-8 per group), Figure 3 (b) The effect of NEN on the creatinine clearance rate in mice (n=5-8 per group); at 8 weeks, the urinary albumin excretion rate and creatinine clearance rate of STZ mice were significantly higher than that of the control group ( Figure 3a, b), after 8 weeks of NEN treatment, the urinary albumin excretion rate and creatinine clearance rate of the mice were significantly decreased (Fig. 3a, b).
4、NEN可减小1型糖尿病小鼠肾重/体重比例和肾小球血管襻面积。4. NEN can reduce the kidney weight/body weight ratio and glomerular vasospasm area in type 1 diabetic mice.
图4为NEN对小鼠肾重/体重、肾小球血管襻面积的影响结果；图4(a)为NEN对小鼠肾重/体重的影响结果(n＝6-8每组)，图4(b)为NEN对小鼠肾小球血管襻面积的影响结果(n＝6每组)，图4(c)为PAS染色显示NEN对小鼠肾小球的影响结果；与control组比较，STZ组小鼠肾重/体重、肾小球血管襻面积明显增大(图4a，b，c)，NEN治疗8周后肾重/体重、肾小球血管襻面积均明显变小(图4a，b，c)。Figure 4 shows the effect of NEN on kidney weight/body weight and glomerular vasospasm in mice; Figure 4(a) shows the effect of NEN on kidney weight/body weight in mice (n=6-8 per group), 4(b) is the effect of NEN on the area of glomerular vasospasm in mice (n=6 per group), and Figure 4(c) shows the effect of NEN on the glomerulus of mice by PAS staining; compared with control group In the STZ group, the kidney weight/body weight and glomerular vasospasm area increased significantly (Fig. 4a, b, c). After 8 weeks of NEN treatment, the kidney weight/body weight and glomerular vasospasm area were significantly smaller (Fig. 4a, b, c).
5、NEN可改善1型糖尿病小鼠肾小管损伤。5, NEN can improve renal tubular damage in type 1 diabetic mice.
图5为NEN对小鼠肾小管损伤的影响结果，图5(a)为NEN对小鼠尿液NAG的影响结果，图5(b)为NEN对小鼠尿液NGAL的影响结果；n＝6-8每组，与control组比较，STZ组小鼠尿液NAG、NGAL***明显增加(图5a，b)，NEN治疗8周后NAG、NGAL***率明显减少(图5a，b)。 Figure 5 shows the effect of NEN on renal tubular injury in mice. Figure 5(a) shows the effect of NEN on urine NAG in mice, and Figure 5(b) shows the effect of NEN on urine NGAL in mice; n= In each group of 6-8, compared with the control group, the urine NAG and NGAL excretion of the STZ group were significantly increased (Fig. 5a, b), and the NAG and NGAL excretion rates were significantly reduced after 8 weeks of NEN treatment (Fig. 5a, b).
6、NEN可抑制1型糖尿病小鼠肾皮质中Akt/mTOR/4E-BP1信号通路的过度激活。6. NEN can inhibit the overactivation of Akt/mTOR/4E-BP1 signaling pathway in the renal cortex of type 1 diabetic mice.
图6为NEN对小鼠肾皮质Akt/mTOR/4E-BP1信号通路的影响结果；图6(a)为免疫印迹实验显示NEN对Akt/mTOR/4E-BP1信号通路蛋白影响的实物图，图6(b)为NEN对小鼠肾皮质p-Akt(Ser473)蛋白影响的统计结果，图6(c)为NEN对小鼠肾皮质p-mTOR(Ser2448)蛋白影响的统计结果，图6(d)为NEN对小鼠肾皮质p-4E-BP1(Thr37/46)蛋白影响的统计结果，图6(e)为NEN对小鼠肾皮质p-4E-BP1(Thr70)蛋白影响的统计结果，图6(f)为NEN对小鼠肾皮质4E-BP1总蛋白影响的统计结果；n＝4每组，与control组相比，STZ组小鼠肾皮质中p-Akt(Ser473)、p-mTOR(Ser2448)、p-4E-BP1(Thr37/46)、p-4E-BP1(Thr70)、4E-BP1蛋白表达均明显增加，NEN治疗可明显减少其表达。Figure 6 shows the effect of NEN on the renal cortex Akt/mTOR/4E-BP1 signaling pathway in mice; Figure 6(a) shows the effect of NEN on the Akt/mTOR/4E-BP1 signaling pathway protein by immunoblotting. 6(b) is the statistical result of the effect of NEN on mouse renal cortex p-Akt (Ser473) protein, and Figure 6 (c) is the statistical result of the effect of NEN on mouse renal cortex p-mTOR (Ser2448) protein, Figure 6 ( d) is the statistical result of the effect of NEN on mouse renal cortex p-4E-BP1 (Thr37/46) protein, and Figure 6(e) is the statistical result of the effect of NEN on mouse renal cortex p-4E-BP1 (Thr70) protein. Fig. 6(f) is the statistical result of the effect of NEN on the total protein of mouse kidney cortex 4E-BP1; n=4 per group, p-Akt (Ser473), p in the renal cortex of STZ group compared with the control group The expression of -mTOR(Ser2448), p-4E-BP1 (Thr37/46), p-4E-BP1 (Thr70) and 4E-BP1 protein were significantly increased, and NEN treatment significantly reduced its expression.
7、NEN对1型糖尿病小鼠具有明显的肝脏保护作用。7. NEN has obvious liver protection effect on type 1 diabetic mice.
图7为NEN对小鼠肝功能的影响结果，图7(a)为NEN对血清谷丙转氨酶(ALT)的影响结果，图7(b)为NEN对小鼠血清谷草转氨酶(AST)的影响结果，图7(c)为NEN对血清总蛋白(TP)的影响结果，图7(d)为NEN对小鼠血清白蛋白(ALB)的影响结果；n＝6-8每组，用药8周后，与control组比较，STZ组小鼠血清ALT、AST明显升高(图7a，b)，血清TP、ALB明显下降(图7c，d)，NEN治疗可使ALT、AST明显下降(图7a，b)，TP、ALB明显升高(图7c，d)。Figure 7 shows the effect of NEN on liver function in mice. Figure 7(a) shows the effect of NEN on serum alanine aminotransferase (ALT), and Figure 7(b) shows the effect of NEN on serum aspartate aminotransferase (AST) in mice. As a result, Fig. 7(c) shows the effect of NEN on serum total protein (TP), and Fig. 7(d) shows the effect of NEN on serum albumin (ALB) in mice; n=6-8 per group, medication 8 After week, compared with the control group, serum ALT and AST in the STZ group were significantly increased (Fig. 7a, b), serum TP, ALB decreased significantly (Fig. 7c, d), NEN treatment can significantly reduce ALT, AST (Figure 7a, b), TP, ALB increased significantly (Fig. 7c, d).
8、NEN对1型糖尿病小鼠肌肉功能具有明显的改善作用。8, NEN has a significant improvement in muscle function in type 1 diabetic mice.
图8为NEN对小鼠肌肉功能的影响结果，图8(a)为NEN对小鼠抓力的影响结果，图8(b)为NEN对跑步机上小鼠每30分钟电击次数的影响结果；n＝6-8每组，8周时，与control组比较，STZ组小鼠抓力明显减小(图8a)，电击次数明显增加(图8b)，NEN治疗后可显著增加小鼠抓力(图8a)，减少电击次数(图8b)。Figure 8 shows the effect of NEN on muscle function in mice. Figure 8(a) shows the effect of NEN on the grip of mice, and Figure 8(b) shows the effect of NEN on the number of shocks per 30 minutes on the treadmill. n=6-8 in each group, at 8 weeks, compared with the control group, the grip strength of the STZ group was significantly reduced (Fig. 8a), and the number of shocks was significantly increased (Fig. 8b). The NEN treatment significantly increased the grip of the mice. (Fig. 8a), reduce the number of shocks (Fig. 8b).
上述实验结果表明，NEN作为一种驱虫药及抗肿瘤药物，可改善1型糖尿病小鼠多饮、多食、多尿症状，降低血糖、糖化血红蛋白和尿糖水平，升 高血清胰岛素水平，改善胰腺病理损伤。对1型糖尿病并发的肾脏、肝脏及肌肉损伤均有明显的保护作用。综上，NEN对1型糖尿病及其并发症具有明显的防治作用。The above experimental results show that NEN, as an anthelmintic and anti-tumor drug, can improve the symptoms of polydipsia, polyphagia and polyuria in type 1 diabetic mice, and lower blood sugar, glycated hemoglobin and urine sugar levels. High serum insulin levels improve pathological damage in the pancreas. It has obvious protective effects on kidney, liver and muscle damage complicated by type 1 diabetes. In summary, NEN has a significant preventive effect on type 1 diabetes and its complications.
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护范围之内。 The embodiments of the present invention have been described above with reference to the drawings, but the present invention is not limited to the specific embodiments described above, and the specific embodiments described above are merely illustrative and not restrictive, and those skilled in the art In the light of the present invention, many forms may be made without departing from the scope of the invention and the scope of the invention.

Claims (3)

  1. 氯硝柳胺乙醇胺盐在制备预防和治疗1型糖尿病及其并发症药物中的应用。The application of niclosamide ethanolamine salt in the preparation of a medicament for preventing and treating type 1 diabetes and its complications.
  2. 根据权利要求1所述的应用，其特征在于，所述药物为口服给药剂型、注射给药剂型、粘膜给药剂型或者经皮给药剂型。The use according to claim 1, wherein the drug is an oral administration form, an injection administration form, a mucosal administration form or a transdermal administration form.
  3. 根据权利要求1所述的应用，其特征在于，所述药物为片剂、胶囊剂、颗粒剂、口服液、贴剂或者凝胶剂。 The use according to claim 1, wherein the drug is a tablet, a capsule, a granule, an oral solution, a patch or a gel.
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