WO2018040627A1 - 梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用 - Google Patents

梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用 Download PDF

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WO2018040627A1
WO2018040627A1 PCT/CN2017/085837 CN2017085837W WO2018040627A1 WO 2018040627 A1 WO2018040627 A1 WO 2018040627A1 CN 2017085837 W CN2017085837 W CN 2017085837W WO 2018040627 A1 WO2018040627 A1 WO 2018040627A1
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muscle
group
caused
mice
sterol
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French (fr)
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江振洲
徐登球
王涛
张陆勇
俞沁玮
张玲
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中国药科大学
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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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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

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  • the invention belongs to the technical field of medicine, and particularly relates to the application of sterol for preparing a drug direction for preventing or delaying muscle weakness and/or muscle atrophy.
  • Muscular atrophy or muscle weakness is a description of the decline in skeletal muscle volume and skeletal muscle strength compared to the previous state of the same age or own muscle. It is mainly associated with motor nerve fiber damage of peripheral nerves and primary lesions of muscle fibers. It is clinically divided into two types: neurogenic (including muscle-induced muscle atrophy) and myogenic (including Duchenne muscular dystrophy). The incidence of diabetes is increasing year by year, and the mortality rate and disability rate are also increasing. According to incomplete statistics, as of 2012, the number of diabetic patients worldwide was 3.71 million, and that of early diabetic patients was 6.42 million. Diabetic proximal muscular atrophy syndrome (Bruns-Garland syndrome) is a classic diabetic myopathy.
  • DMD Duchenne muscular dystrophy
  • muscle weakness or muscle atrophy including muscle weakness or muscle atrophy caused by diabetes or Duchenne muscular dystrophy.
  • researchers in this area are also studying in the world, but so far there is no drug that can be used for clinical treatment of diabetes or muscle atrophy caused by Duchenne muscular dystrophy.
  • it is urgent to develop safer and more effective drugs use skeletal muscle as a therapeutic target, improve skeletal muscle function, prevent or delay muscle weakness and muscle atrophy, including diabetic myasthenia or muscle atrophy and Duchenne muscular dystrophy. Muscle weakness or muscle atrophy caused by the disease.
  • the technical problem to be solved by the present invention is to provide a use of sterols in the preparation of a medicament for preventing or delaying muscle weakness and/or muscle atrophy.
  • the present invention provides the following technical solutions: inducing early diabetes with a high-fat diet, and transgenic diabetic mice simulating type 2 diabetes, and studying the therapeutic effect of sterols on muscle weakness and/or muscle atrophy caused by diabetes.
  • the present invention provides a technical solution for simulating clinical Duchenne muscular dystrophy with a dystrophin-deficient mouse, and studying the therapeutic effect of sterol against muscle weakness and/or muscle atrophy caused by dystrophin deficiency.
  • the invention has the beneficial effects that the present invention discloses the use of sterols in the preparation of a medicament for preventing or delaying muscle weakness and/or muscle atrophy, including muscle weakness and/or muscle atrophy caused by diabetes, and dystrophin deficiency.
  • Sterols can significantly improve muscle weakness caused by diabetes, reverse the loss of type I muscle fibers in skeletal muscle caused by diabetes, improve insulin resistance, reduce fasting blood sugar, and improve the exercise capacity caused by diabetes; sterol can significantly improve Duchenne nutrition Muscle weakness caused by bad signs, lower serum creatine kinase, lactate dehydrogenase levels, improved skeletal muscle inflammation and skeletal muscle cell necrosis. Therefore, sterol can be used to prevent or delay muscle weakness and/or muscle atrophy.
  • the drug can be extracted from traditional Chinese medicine, the drug used is cheap, safe, effective, easy to obtain, and the market prospect is good.
  • Figure 1 shows the body weight curves of db/db mice in different treatment groups.
  • Figure 2 is a graph showing the results of fasting blood glucose and glucose tolerance in db/db mice of different treatment groups.
  • Figure 3 is a graph showing the results of skeletal muscle weight and serum insulin content in db/db mice of different treatment groups.
  • Figure 4 is a graph showing immunohistochemical staining and muscle fiber analysis of skeletal muscle muscle fibers (A: wild type group; B: model group; C: sterol group).
  • Figure 5 is a graph showing glucose tolerance and insulin tolerance in an early diabetic model mouse.
  • Figure 6 is a diagram showing immunohistochemical staining of skeletal muscle fiber in early diabetic model mice.
  • A wild type group
  • B IFG model group
  • C IFG model sterol treatment group
  • D IFG model metformin treatment group
  • E IGT model group
  • F IGT model sterol treatment group
  • G IGT model metformin group therapy group
  • Figure 7 is a graph showing the body weight of DMD mice in different treatment groups.
  • Figure 8 shows the results of HE staining in DMD mice of different treatment groups.
  • Figure 9 shows the results of F4/80 and CD206 staining in DMD mice of different treatment groups (A: wild type group; B: DMD model group; C: sterol group; D: simvastatin group).
  • Figure 10 is an immunohistochemical staining of skeletal muscle fiber (A: wild type group; B: DMD model group; C: sterol group; D: simvastatin group).
  • Figure 11 shows the results of immunofluorescence staining of fibronectin in DMD mice of different treatment groups (A: wild type group; B: DMD model group; C: sterol group; D: simvastatin group).
  • Db/db mice are Leptin receptor point mutations leading to leptin signaling pathway disorders, resulting in obesity, insulin resistance, hyperglycemia, fatty liver and other symptoms in mice.
  • the db/db mice showed obvious obesity and fasting blood glucose increased 6 weeks after birth. The amount of water and urine increased, and the blood glucose increased significantly at 8-12 weeks.
  • Db/db mice not only have typical clinical manifestations of diabetes, but also show cardiomyopathy, diabetic retinopathy, diabetic nephropathy, diabetic foot, peripheral neuropathy and muscle weakness.
  • Dmd mdx refers to a recessive mutation in the mdx of the Dmd gene, and the heterozygous female is visually indistinguishable from wild-type mice.
  • the female homozygous and male hemizygous Dmd mdx alleles have normal lifespan and can survive for 2 years.
  • the Dmd mdx mutant does not express dystrophin and is therefore routinely used as an animal model for this disease, although it causes muscle pathology to be much milder than human disease.
  • DMD Duchenne muscular dystrophy
  • the muscles of the Dmd mdx mutant were histologically normal in the early postnatal development, but muscle gangrene began to appear around 3 weeks, showing some muscle weakness.
  • Biochemical analysis of related pathologies including elevated levels of serum creatine kinase and lactate dehydrogenase, and accumulation of macrophages are early signs of muscle degeneration. This model was therefore chosen for the evaluation of the therapeutic effects of sterols on muscle weakness and/or muscle atrophy.
  • mice The experimental animals used in the present invention are db/db transgenic mice (named BKS.Cg-Dock7m+/+Leprdb/Jnju) and C57BL/6NCrlVr mice, all purchased from Nanjing Biomedical Research Institute of Nanjing University.
  • the experimental animals used in the present invention are DMD model mice (named C57BL/10ScSn-Dmd mdx ) and C57BL/10ScSnJ normal control mice, all of which are purchased from Nanjing Biomedical Research Institute of Nanjing University.
  • the drug in the present invention is sterol, and its structural formula is as follows:
  • bismuth hydroxybenzoate which is an iridoid glycoside compound and is one of the main active constituents of Dioscorea zingiberensis.
  • the positive control drug used in the blood glucose lowering of the present invention is metformin which is a first-line drug for clinical treatment of diabetes.
  • the positive control drug used in the present invention is simvastatin, which is a first-line drug for clinical use in lowering blood fat, but recent research shows that it can significantly improve muscle weakness, resistance to fibrosis and inflammation in DMD model rats (Proc Natl Acad Sci.2015 Oct 13; 112 (41): 12864–12869. doi: 10.1073/pnas.1509536112.).
  • mice with db/db C57BL/KsJ
  • 6 C57BL/6NCrlVr mice wild type C57BL/6NCrlVr mice
  • the specific grouping and administration methods are shown in Table 1:
  • the drug is administered from 9:00 am to 10:00 every day, and the body weight is weighed from 10:00 to 11:00 every Monday.
  • the weighing range of the electronic scale is between 0.1 and 300 g, and the results are shown in Fig. 1. It was shown that sterol has a slight increase in body weight in db/db mice.
  • Suspension time measurement On the 55th day after administration, the pre- and hind limb grip strength of the mice was measured, and the neuromuscular exercise intensity was measured.
  • the grip-sensing device was composed of parallel wires (diameter 5 mm), which was inclined to the ground 45°. The mice were placed in the susceptor and slowly rotated by 180°. The device was placed 20 cm away from the ground to prevent the mice from falling down to cause injury. The stopwatch recorded the time during which the mice were grasped by the wire, and the average was measured 3 times. The results are shown in Table 2. Shown.
  • mice After 56 days (8 weeks) of administration, the mice were sacrificed by anesthesia on the 57th day (12 h overnight), 20% urethane was anesthetized, blood was taken from the inguinal vein, centrifuged at 2500 rpm for 5 min, and the upper serum was taken to determine the insulin content. Subsequently, the total muscles of the hind limbs were dissected and frozen in liquid nitrogen, and transferred to a negative 80 refrigerator for use the next day. The results of skeletal muscle weight and insulin content are shown in Figure 3.
  • Immunohistochemically labeled skeletal muscle type I muscle fibers and type II muscle fibers skeletal muscle sections were diluted with fast-shrinking myosin antibody (MY-32, 1:500, Abcam, USA) at 4 ° C overnight, after washing with biotin The mouse secondary antibody was incubated for 1 h at room temperature, washed and washed, and photographed under laser confocal. Subsequently The muscle fiber ratio was statistically analyzed by image-Plus, and the results are shown in Fig. 4.
  • NC control group 1 and the NC control group 2 were fed with normal feed RD (Nantong Trophy Feed Technology Co., Ltd.), and the other groups were fed with high fat feed HFD (TP24200, Nantong Trophi Feed Technology Co., Ltd.).
  • HFD high fat feed
  • TP24200 Nantong Trophi Feed Technology Co., Ltd.
  • the specific grouping and administration methods are shown in Table 3:
  • Suspension time measurement The pre- and hind limb grip strength of the mice was measured on the 26th day of administration, and the neuromuscular exercise intensity was measured.
  • the grip-sensing device was composed of parallel wires (diameter 5 mm), which was inclined to the ground 45°. The mice were placed in the susceptor and slowly rotated by 180°. The device was placed 20 cm off the ground to prevent the mice from falling down to cause injury. The stopwatch recorded the time the mouse was gripped by the wire. The results are shown in Table 4.
  • NC control group 1 107.76 ⁇ 27.52 IFG model group 19.85 ⁇ 5.99 * Sterol treatment group 1 38.43 ⁇ 12.28 *# Metformin treatment group 1 32.33 ⁇ 10.29 * NC control group 2 124.17 ⁇ 29.24 IGT model group 21.5 ⁇ 7.24 * Sterol treatment group 2 48.5 ⁇ 19.16 *# Metformin treatment group 2 30 ⁇ 11.45 *
  • Muscle grip measurement The anterior and hind limb grip strength of the mice was measured on the 26th day of administration, and each mouse was measured 3 times, and the average value was the maximum muscle grip of the mouse. The results are shown in Table 5.
  • Sterol treatment group 1 88.75 ⁇ 5.96 Metformin treatment group 1 86.25 ⁇ 8.45 NC control group 2 96.67 ⁇ 3.60 IGT model group 73.54 ⁇ 6.63 * Sterol treatment group 2 90.83 ⁇ 6.67 # Metformin treatment group 2 77.71 ⁇ 6.90 *
  • Depletion experiment adaptive treadmill training for each group of mice from the 24th day after administration, the speed was 16m/min, continuous training for 3 days, and the depletion experiment was carried out on the 27th day of administration.
  • the running speed started at 10m/min, every time.
  • the running speed of 2 minutes was increased by 2m/min, and the maximum speed was 36m/min.
  • the maximum distance and running time of each mouse were recorded. The results are shown in Table 6.
  • NC control group 1 1052 ⁇ 67.26 2850 ⁇ 138 IFG model group 503.8 ⁇ 59.24 1728.7+167.7 * Sterol treatment group 1 751.4 ⁇ 41.15 2361 ⁇ 83.07 # # Metformin treatment group 1 640 ⁇ 35.0 2147.4 ⁇ 71.2 * NC control group 2 900.5 ⁇ 52.5 3346.7 ⁇ 201.3 IGT model group 341.3 ⁇ 47,2 1347.5 ⁇ 118.6 * Sterol treatment group 2 490.8 ⁇ 41,2 1734.5 ⁇ 142.7 *# Metformin treatment group 2 306.5 ⁇ 107.7 1215.7 ⁇ 339.8 *
  • the high-fat diet can significantly reduce the distance and time of the mice, and the sterol treatment can restore the body's physical ability and increase the exercise distance and exercise time to some extent after 4 weeks of treatment. Metformin does not have this effect. Therefore, sterols can improve the muscle weakness caused by HFD-induced early diabetes.
  • the glucose tolerance test protocol was on the 24th day of administration, fasted for 12 hours overnight, and intraperitoneally injected with glucose (2g/kg, 0.1ml/10kg), measured at 0, 30, 60, 90, 120 minutes. Blood sugar (Wittman Biotech Nanjing Co., Ltd.). Insulin tolerance program was administered on the 26th day, fasted for 12 hours overnight, intraperitoneal injection of insulin (0.75U / kg, 0.1ml / 10kg), blood glucose was measured at 0, 15, 30, 60, 120 minutes (Wittman Biotechnology Nanjing Limited) the company). The results of glucose tolerance and insulin tolerance are shown in Figure 5.
  • mice After administration for 28 days (4 weeks), the mice were sacrificed by anesthesia on the 29th day (12 h overnight) by 20% urethane anesthesia, blood was taken from the inguinal vein, centrifuged at 2500 rpm for 5 min, and the upper serum was taken to measure blood glucose. Subsequently, the hindlimb was dissected and the gastrocnemius muscle and the soleus muscle were weighed. The total skeletal muscle was quickly frozen in liquid nitrogen, and the next day, it was transferred to a negative 80 refrigerator for use. The results of skeletal muscle weight and fasting blood glucose levels are shown in Table 7.
  • Immunohistochemically labeled skeletal muscle type I muscle fibers and type II muscle fibers skeletal muscle sections were diluted with fast-shrinking myosin antibody (MY-32, 1:500, Abcam, USA) at 4 ° C overnight, after washing with biotin The mouse secondary antibody was incubated for 1 h at room temperature, washed and washed, and photographed under laser confocal. The result is shown in Figure 6.
  • Suspension time measurement On the 14th day, the 28th day and the 42nd day of administration, the anterior and hind limb grip strength of the mice was measured, and the nerve muscle exercise intensity was measured.
  • the gripper feeling device was composed of parallel wires (diameter 5 mm). , so that it is inclined to the ground 45 °, the mouse is placed in the sensor slowly rotated 180 °, the device 20 cm away from the ground to prevent the mouse from falling enough to cause injury, the stopwatch records the mouse in the wire grip time, each test 3 times The average value is shown in Table 9.
  • Muscle grip measurement On the 14th day, the 28th day, and the 42nd day of the administration, the anterior and hind limb grip strength of the mice was measured, and each mouse was measured 3 times, and the average value was the maximum muscle grip of the mouse. The results are shown in Table 10.
  • Serum creatine phosphokinase (CK) assay On the 14th day, the 28th day, the 42nd day of administration, the anesthetized venous plexus was taken for about 100 ul, centrifuged at 2500 rpm for 10 min, and the upper serum was taken according to the CK test kit (Nanjing Jian Biotechnology) Ltd.) The CK level was measured and the results are shown in Table 11.
  • serum CK in the DMD model group was 10-20 times higher than that in the NC control group.
  • Serum CK levels in the 14-day continuous administration of sterol and simvastatin showed a certain downward trend. After 28 days of continuous administration, sterol lowered serum CK than simvastatin. The statin is stronger.
  • LDH serum lactate dehydrogenase
  • Hydroxyproline assay in skeletal muscle Hydroxyproline levels were used to assess total collagen content and reflect skeletal muscle fibrosis levels. Accurately weigh 30 mg of skeletal muscle and measure according to the hydroxyproline assay kit (Nanjing Jian Biotechnology Co., Ltd.). The results are shown in Table 13:
  • the sterol has the same anti-skeletal muscle fibrosis effect as simvastatin.
  • Gastrointestinal HE staining After 42 days of administration, the animals were sacrificed by anesthesia, and the bilateral gastrocnemius muscles were quickly dissected. One side of the gastrocnemius muscle was fixed with formalin fixative for paraffin section, and the other side was quickly frozen in -20 refrigerator. For immunofluorescence staining. Gastrocnemius muscle sections were prepared by gastrocnemius paraffin section for HE staining. After a series of elution, fixation, staining, and photographing, the images were photographed under laser confocal (FV1000). The results are shown in Fig. 8.
  • Immunohistochemical labeling of skeletal muscle type I muscle fibers and type II muscle fibers such as gastrocnemius HE staining, taking gastrocnemius paraffin sections to make gastrocnemius sections, using diluted fast-shrinking myosin antibody (MY-32, 1:500, Abcam, USA) After overnight washing at 4 ° C, the cells were incubated with biotinylated murine secondary antibody for 1 h at room temperature, washed and washed, and photographed under laser confocal (FV1000). The muscle fiber ratio was then statistically analyzed using image-Plus, and the results are shown in FIG.
  • fibronectin protein expression is significantly increased in skeletal muscle fibrosis, so fibronectin can evaluate the degree of fibrosis.
  • Frozen sections of the gastrocnemius muscle were fixed with pre-cooled acetone, PBS rinsed, incubated with diluted Fibronectin antibody, overnight at 4 ° C, washed with biotinylated rabbit secondary antibody for 1 h at room temperature after washing for the next day, and then stained with DAPI for nucleus at room temperature. After 10 min incubation, the photographs were washed and the results are shown in FIG.
  • the present invention adopts an internationally recognized db/db transgenic mouse model of type 2 diabetes as a model mouse and a high-fat diet-fed C57 mouse as an early diabetes model, and an internationally recognized study of muscle weakness and/or Or muscle atrophy Dmd mdx gene-deficient mice are model mice, and the therapeutic effect of sterol on muscle weakness and muscle atrophy is investigated.
  • the effects of sterols on muscle weakness and muscle atrophy caused by diabetes or Duchenne muscular dystrophy were systematically studied in pharmacodynamics, behavioral and molecular biology. The results showed that sterols can effectively improve diabetic mice. Hyperglycemia increases the muscle grip of diabetic rats, increases the weight of skeletal muscles, and improves their exercise capacity.
  • sterol can effectively improve the muscle weakness and/or muscle atrophy in the DMD model, which can significantly increase the muscle grip of the model rats and resist bones.
  • Muscle fibrosis has the effect of relieving skeletal muscle inflammation and reducing skeletal muscle damage, providing a theoretical basis for the development of sterols for the treatment of muscle weakness and/or muscle atrophy.

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Abstract

梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用，包括糖尿病引起的肌无力和/或肌萎缩，抗肌萎缩蛋白缺陷引起的肌无力和/或肌萎缩。梓醇可以显著改善糖尿病引起的肌无力，逆转糖尿病引起的骨骼肌中I型肌纤维的缺失，改善胰岛素抵抗，降低空腹血糖，并且能够改善糖尿病引起的运动能力下降；梓醇可以显著改善杜氏肌营养不良征引起的肌无力，降低血清肌酸激酶，乳酸脱氢酶水平，改善骨骼肌炎症和骨骼肌细胞坏死。

Description

梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用 技术领域
本发明属于医药技术领域，特别涉及梓醇用于制备防治或延缓肌无力和/或肌萎缩的药物方向的应用。
背景技术
肌萎缩或肌无力是骨骼肌容积和骨骼肌力量较同龄人或自身肌肉以前的状态出现下降状态的描述。多与周围神经的运动神经纤维损害以及肌纤维原发病变有关，从临床上分为神经源性(包括糖尿病引起的肌萎缩)和肌源性(包括杜氏肌营养不良)两种类型。糖尿病发病率逐年升高，致死率、致残率也不断增加。据不完全统计，截止2012年全球糖尿病患者为371万人，早期糖尿病患者为642万人。糖尿病近端肌萎缩综合征(Bruns-Garland综合征)是经典的糖尿病性肌病，该症患病率约占所有糖尿病患者的0.8％，其特点是：主要累及下肢近端肌群，但远端也可受累，手部肌萎缩也较常见。临床呈亚急性、疼痛性、非对称性，常发生于2型糖尿病，并常伴肌无力、肌萎缩。杜氏肌营养不良症(DMD)是一种严重的神经肌肉遗传性疾病，是抗肌萎缩蛋白dystrophin基因发生突变所致，属于进行性肌营养不良症常见类型。显微镜下可以看到DMD患者骨骼肌纤维数量明显变少，能观察到大的纤维坏死和小群变性肌纤维；HE染色能看到明显的炎性细胞浸润，ATP酶染色能观察到DMD组织中肌纤维大小不一，肌纤维圆形化。该病发病率约为：男婴1/3500，女性为致病基因的携带者。中国内地患者及携带者总数近10万人(含BMD)。患儿普遍在2-3岁阶段发病，骨骼肌进行性萎缩，丧失活动能力。若不治疗干预，病人通常在12岁前失去行走能力，与轮椅为伴，最终因心肺功能衰竭在20至30岁期间死亡。
所以急需要找到激素以外的药物来治疗肌无力或肌萎缩，包括糖尿病或者杜氏肌营养不良症引起的肌无力或肌萎缩。这方面的药物全世界的研究者也在研究当中，但是至今仍没有一种药物能用于临床治疗糖尿病或者杜氏肌营养不良症引起的肌无力或肌萎缩。基于以上原因，急需开发更为安全、有效的药物，以骨骼肌为治疗靶点，改善骨骼肌功能，防治或者延缓肌无力和或肌萎缩，包括糖尿病性肌无力或肌萎缩以及杜氏肌营养不良症引起的肌无力或肌萎缩。
发明内容
本发明要解决的技术问题在于提供梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用。
为解决上述问题，本发明提供如下技术方案：用高脂饮食诱导早期糖尿病，转基因糖尿病鼠模拟2型糖尿病，研究梓醇对糖尿病引起的肌无力和/或肌萎缩的治疗作用。
为解决上述问题，本发明提供如下技术方案：用抗肌萎缩蛋白基因缺陷小鼠模拟临床杜氏肌营养不良征，研究梓醇对抗肌萎缩蛋白缺陷引起的肌无力和/或肌萎缩的治疗作用。
本发明的有益效果在于：本发明公开了梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用，包括糖尿病引起的肌无力和/或肌萎缩，抗肌萎缩蛋白缺陷引起的肌无力和/或肌萎缩。梓醇可以显著改善糖尿病引起的肌无力，逆转糖尿病引起的骨骼肌中I型肌纤维的缺失，改善胰岛素抵抗，降低空腹血糖，并且能够改善糖尿病引起的运动能力下降；梓醇可以显著改善杜氏肌营养不良征引起的肌无力，降低血清肌酸激酶，乳酸脱氢酶水平，改善骨骼肌炎症和骨骼肌细胞坏死。因此梓醇可用于防治或延缓肌无力和/或肌萎缩。并且该药物可以从传统中药中提取，所用药物价格便宜、安全有效、容易获得，市场前景大好。
附图说明
图1为不同处理组db/db小鼠体重曲线。
图2为不同处理组db/db小鼠空腹血糖及糖耐量结果图。
图3为不同处理组db/db小鼠骨骼肌重量及血清胰岛素含量结果图。
图4为骨骼肌肌纤维免疫组化染色及肌纤维分析图(A：野生型组；B：模型组；C：梓醇组)。
图5为早期糖尿病模型小鼠糖耐量和胰岛素耐量曲线图。
图6为早期糖尿病模型小鼠骨骼肌肌纤维免疫组化染色图。(A：野生型组；B：IFG模型组；C：IFG模型梓醇治疗组；D：IFG模型二甲双胍治疗组；E：IGT模型组；F：IGT模型梓醇治疗组；G：IGT模型二甲双胍治疗组)。
图7为不同处理组DMD小鼠体重图。
图8为不同处理组DMD小鼠HE染色结果。
图9为不同处理组DMD小鼠F4/80和CD206染色结果(A：野生型组； B：DMD模型组；C：梓醇组；D：辛伐他汀组)。
图10为骨骼肌肌纤维免疫组化染色(A：野生型组；B：DMD模型组；C：梓醇组；D：辛伐他汀组)。
图11为不同处理组DMD小鼠fibronectin免疫荧光染色结果(A：野生型组；B：DMD模型组；C：梓醇组；D：辛伐他汀组)。
具体实施方式
下面将对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
db/db(C57BL/KsJ)小鼠是Leptin受体点突变导致leptin信号通路障碍，从而导致小鼠出现肥胖、胰岛素抵抗、高血糖、脂肪肝等症状。db/db小鼠出生后6周即可出现明显的肥胖和空腹血糖增加，饮水量、尿量增加，8-12周时血糖显著升高。db/db小鼠不仅有典型的糖尿病临床表现，也表现出心肌病、糖尿病视网膜病变、糖尿病肾病、糖尿病足、周围神经病变及肌无力等。
Dmdmdx模型鼠：Dmdmdx是指Dmd基因的mdx发生隐性突变，杂合体雌性在视觉上是不能从野生型小鼠中辨认出来的。Dmdmdx等位基因的雌性纯合体和雄性半合子寿命正常，能够存活2年。就像患了最普通的神经肌肉疾病的病人一样，Dmdmdx突变体不表达抗肌萎缩蛋白，因此被常规的用作此疾病的动物模型，虽然它导致的肌肉病理比人类的病情要轻微许多，但是其是全世界公认的杜氏肌营养不良症(DMD)的最好模型。Dmdmdx突变体的肌肉在出生后的早期发展中组织学上是正常的，但是在3周左右开始出现肌肉坏疽，表现出一些肌无力。相关病理的生化分析包括血清肌酸激酶和乳酸脱氢酶水平升高，巨噬细胞的积聚，都是肌肉退化的早期标志。因此选择该模型用于评价梓醇对肌无力和/或肌萎缩的治疗作用。
本发明所用实验动物为db/db转基因小鼠(命名为BKS.Cg-Dock7m+/+Leprdb/Jnju)和C57BL/6NCrlVr小鼠，均购自南京大学南京生物医药研究院。
本发明所用实验动物为DMD模型鼠(命名为C57BL/10ScSn－Dmdmdx)和C57BL/10ScSnJ正常对照鼠，均购自南京大学南京生物医药研究院。
本发明中的药物为梓醇，其结构式如下所示：
Figure PCTCN2017085837-appb-000001
全名脱对羟基苯甲酸梓苷，属环烯醚萜苷类化合物，是玄参科植物地黄中主要活性成分之一。
本发明降血糖所用阳性对照药为二甲双胍是临床上治疗糖尿病的一线药物。
针对杜氏肌营养不良症，本发明所用阳性对照药为辛伐他汀是临床上用于降血脂的一线药物，但最新研究表明其能显著改善DMD模型鼠肌无力，抵抗纤维化，减轻炎症(Proc Natl Acad Sci.2015Oct 13；112(41):12864–12869.doi:10.1073/pnas.1509536112.)。
实施例1
梓醇治疗糖尿病后期引起的肌无力的药效学评价，具体方法如下：购买db/db(C57BL/KsJ)小鼠12只，C57BL/6NCrlVr小鼠6只，野生型的C57BL/6NCrlVr小鼠作为正常对照NC组(n＝6)，db/db(C57BL/KsJ)小鼠按体重分层随机分为2组，分别命名为db/db模型组(n＝6)和梓醇治疗组(200mg/kg，n＝6)具体分组及给药方式如表1所示：
表1、实验小鼠具体分组及给药剂量和给药方式
Figure PCTCN2017085837-appb-000002
每天上午9时至10时给药，每周一上午10时至11时称量体重，电子称的称重范围是0.1至300g之间，其结果如图1所示。表明梓醇对db/db小鼠具有轻微增加体重的作用。
悬吊时间测定：给药第55天进行小鼠前、后肢抓力测定，测量神经肌肉运动强度，抓力感受装置是由平行的金属丝(直径5mm)构成的，使其倾斜于地面45°，将小鼠置于感受器缓慢旋转180°，装置离地面20cm预防小鼠掉落不足以造成伤害，秒表记录小鼠在钢丝抓握的时间，每只测3次取平均值，结果如表2所示。
表2、db/db小鼠悬杆实验时间统计结果
分组 NC组 db/db模型组 梓醇治疗组
时间(seconds) 304±64.4 7.57±4.04* 23.83±9.66*#
*P<0.01vs NC组；#P<0.01vs模型组
结果表明，给药55天后梓醇治疗组较db/db模型组实验小鼠在吊前后肢抓力方面优于模型组，表明梓醇能够改善db/db小鼠高血糖引起的肌无力。
空腹血糖及糖耐量测定：每周二上午8时(隔夜禁食12h)麻醉眼眶静脉丛取血约100ul，2500rpm离心5min，取上层血清按葡萄糖检测试剂盒(威特曼生物科技南京有限公司)测定血糖含量。给药第50天进行糖耐量测定，隔夜禁食12h，腹腔注射葡萄糖(2g/kg，0.1ml/10kg)，分别于0,30,60,90,120分钟测定血糖。空腹血糖及糖耐量结果如图2所示。
结果表明，梓醇能够显著降低db/db模型小鼠空腹血糖，并且能明显改善糖耐量受损。
给药56天(8周)后，于第57天(隔夜禁食12h)20％乌拉坦麻醉处死小鼠，腹股沟静脉取血，2500rpm离心5min，取上层血清测定胰岛素含量。随后解剖后肢总肌肉称重并快速液氮冷冻，第二天转移至负80冰箱备用。骨骼肌重量及胰岛素含量结果如图3所示。
结果表明，梓醇给药能增加db/db鼠骨骼肌重量，并且能显著改善胰岛素抵抗。
免疫组化标记骨骼肌I型肌纤维和Ⅱ型肌纤维：骨骼肌切片用稀释的快收缩肌肌球蛋白抗体(MY-32,1:500，Abcam公司，美国)4℃过夜，洗涤后用生物素化得鼠二抗室温孵育1h，染色洗涤后封片，于激光共聚焦下拍照。随后 用image-Plus统计分析肌纤维比例，结果如图4所示。
结果表明，db/db模型组小鼠骨骼肌Ⅰ型肌纤维较NC组显著缺失，梓醇治疗后能显著逆转Ⅰ型肌纤维的缺失。
实施例2
梓醇治疗HFD诱导的早期糖尿病引起的肌无力的药效学评价，具体方法如下：购买C57BL/6NCrlVr小鼠64只，小鼠按体重分层随机分为8组，分别为NC对照组1(n＝8)，IFG模型组(n＝8)，梓醇治疗组1(n＝8)，二甲双胍治疗组1(n＝8)，NC对照组2(n＝8)，IGT模型组(n＝8)，梓醇治疗组2(n＝8)，二甲双胍治疗组2(n＝8)。除NC对照组1和NC对照组2用正常饲料RD(南通特洛菲饲料科技有限公司)喂养，其他各组用高脂饲料HFD(TP24200，南通特洛菲饲料科技有限公司)喂养。HFD造模6周后验证空腹血糖调节受损(IFG)模型成功，NC对照组1(n＝8)，IFG模型组(n＝8)，梓醇治疗组1(n＝8)，二甲双胍治疗组1(n＝8)开始给药，各组给药4周；HFD造模12周后验证胰岛素抵抗(IGT)模型成功，NC对照组2(n＝8)，IGT模型组(n＝8)，梓醇治疗组2(n＝8)，二甲双胍治疗组2(n＝8)开始给药，各组给药4周。具体分组及给药方式如表3所示：
表3、实验小鼠具体分组及给药剂量和给药方式
Figure PCTCN2017085837-appb-000003
Figure PCTCN2017085837-appb-000004
悬吊时间测定：给药第26天进行小鼠前、后肢抓力测定，测量神经肌肉运动强度，抓力感受装置是由平行的金属丝(直径5mm)构成的，使其倾斜于地面45°，将小鼠置于感受器缓慢旋转180°，装置离地面20cm预防小鼠掉落不足以造成伤害，秒表记录小鼠在钢丝抓握的时间，结果如表4所示。
表4、小鼠前后肢悬吊时间
分组 时间(seconds)
NC对照组1 107.76±27.52
IFG模型组 19.85±5.99*
梓醇治疗组1 38.43±12.28*#
二甲双胍治疗组1 32.33±10.29*
NC对照组2 124.17±29.24
IGT模型组 21.5±7.24*
梓醇治疗组2 48.5±19.16*#
二甲双胍治疗组2 30±11.45*
*P<0.01vs NC组；#P<0.01vs模型组
结果表明，给药26天后梓醇治疗组较IFG模型组和IGT模型组实验小鼠在吊前后肢抓力方面明显优于模型组，表明梓醇能够改善HFD诱导的早期糖尿病引起的肌无力，但是二甲双胍给药26天并不能改善HFD诱导的早期糖尿病引起的肌无力。
肌抓力测定：给药第26天进行小鼠前、后肢抓力测定，每只小鼠测3次，取平均值为该小鼠的最大肌抓力。结果如表5所示。
表5、小鼠肌抓力测定
分组 肌抓力(g)
NC对照组1 93.89±6.02
IFG模型组 83.33±7.51
梓醇治疗组1 88.75±5.96
二甲双胍治疗组1 86.25±8.45
NC对照组2 96.67±3.60
IGT模型组 73.54±6.63*
梓醇治疗组2 90.83±6.67#
二甲双胍治疗组2 77.71±6.90*
*P<0.01vs NC组；#P<0.01vs模型组
结果显示，IGT模型组肌抓力较NC对照组2明显下降，梓醇治疗4周明显改善HFD诱导的糖尿病早期的肌无力，但是二甲双胍并不能改善糖尿病引起的肌无力。
耗竭实验：给药第24天起对各组小鼠适应性跑台训练，速度为16m/min，连续训练3天，给药第27天进行耗竭实验，跑速开始为10m/min，每隔4分钟跑速增加2m/min，最大速度为36m/min，记录每只小鼠跑动的最大距离和跑动的时间，结果如表6所示。
表6、耗竭实验
分组 最大距离(m) 时间(seconds)
NC对照组1 1052±67.26 2850±138
IFG模型组 503.8±59.24 1728.7+167.7*
梓醇治疗组1 751.4±41.15 2361±83.07#
二甲双胍治疗组1 640±35.0 2147.4±71.2*
NC对照组2 900.5±52.5 3346.7±201.3
IGT模型组 341.3±47,2 1347.5±118.6*
梓醇治疗组2 490.8±41,2 1734.5±142.7*#
二甲双胍治疗组2 306.5±107.7 1215.7±339.8*
*P<0.01vs NC组；#P<0.01vs模型组
结果显示，高脂饮食能显著减少小鼠的运动距离和时间，而梓醇治疗4周后能在一定程度上恢复小鼠的体能，增加运动距离和运动时间，而二甲双胍并不能够有此疗效，因此梓醇能够改善HFD诱导的早期糖尿病引起的肌无力。
糖耐量和胰岛素耐量测定：糖耐量测定方案为给药第24天，隔夜禁食12h，腹腔注射葡萄糖(2g/kg，0.1ml/10kg)，分别于0、30、60、90、120分钟测定 血糖(威特曼生物科技南京有限公司)。胰岛素耐量方案为给药第26天，隔夜禁食12h，腹腔注射胰岛素(0.75U/kg，0.1ml/10kg)，分别于0,15,30,60,120分钟测定血糖(威特曼生物科技南京有限公司)。糖耐量和胰岛素耐量结果如图5所示。
结果显示，梓醇和二甲双胍均能够显著改善糖耐量受损和胰岛素抵抗。
给药28天(4周)后，于第29天(隔夜禁食12h)20％乌拉坦麻醉处死小鼠，腹股沟静脉取血，2500rpm离心5min，取上层血清测定血糖。随后解剖后肢取腓肠肌和比目鱼肌称重，总骨骼肌快速液氮冷冻，第二天转移至负80冰箱备用。骨骼肌重量及空腹血糖含量结果如表7所示。
表7、腓肠肌，比目鱼肌重量以及空腹血糖含量
分组 空腹血糖(mmol) 腓肠肌重量(mg) 比目鱼肌重量(mg)
NC对照组1 4.64±0.26 199.5±20.87 18.95±5.33
IFG模型组 6.18±0.78* 156±24.24* 19.75±1.67
梓醇治疗组1 5.45±0.78# 192.25±27.98# 21.78±6.06#
二甲双胍治疗组1 5.94±0.59# 132.25±19.05* 19.71±1.38
NC对照组2 5.05±0.64 188.75±36.06 18.51±3.58
IGT模型组 6.21±0.99* 157.85±37.53* 16.72±2.99*
梓醇治疗组2 3.05±1.02# 242.23±51.26# 20.85±3.36#
二甲双胍治疗组2 4.09±0.64# 178.85±20.39 14.4±2.59
*P<0.01vs NC组；#P<0.01vs模型组
结果显示，梓醇治疗4周降低空腹血糖作用比二甲双胍更明显，此外梓醇能够增加小鼠腓肠肌和比目鱼肌重量。
免疫组化标记骨骼肌I型肌纤维和Ⅱ型肌纤维：骨骼肌切片用稀释的快收缩肌肌球蛋白抗体(MY-32，1:500，Abcam公司，美国)4℃过夜，洗涤后用生物素化得鼠二抗室温孵育1h，染色洗涤后封片，于激光共聚焦下拍照。结果如图6所示。
结果表明，IFG模型组和IGT模型组小鼠骨骼肌Ⅰ型肌纤维较NC组显著缺失，梓醇治疗后能显著逆转Ⅰ型肌纤维的缺失，而二甲双胍无此疗效。
实施例3
梓醇治疗杜氏肌营养不良症引起的肌无力的药效学评价，具体方法如下： 购买C57BL/10ScSn－Dmdmdx小鼠9只，C57BL/10ScSnJ小鼠4只。野生型的C57BL/10ScSnJr小鼠作为正常对照NC组(n＝6)，C57BL/10ScSn－Dmdmd小鼠按体重分层随机分为3组，分别命名为DMD模型组(n＝3)，梓醇治疗组(200mg/kg，n＝3)和辛伐他汀治疗组(20mg/kg，n＝3)具体分组及给药方式如表8所示：
表8、实验小鼠具体分组及给药剂量和给药方式
组别 动物数(只) 给药方式 给药剂量 给药时间(周)
NC组 4 灌喂等体积蒸馏水 0.1ml/10g 6
DMD模型组 3 灌喂等体积蒸馏水 0.1ml/10g 6
梓醇治疗组 3 灌喂等体积梓醇 200mg/kg 6
辛伐他汀治疗组 3 灌喂等体积辛伐他汀 20mg/kg 6
每天上午9时至10时给药，每两天称量体重1次，电子称的称重范围是0.1至300g之间，其结果如图7所示。表明梓醇对DMD小鼠具有轻微增加体重的作用，但无统计学差异；而辛伐他汀能轻微降低体重。
悬吊时间测定：给药第14天，第28天，第42天进行小鼠前、后肢抓力测定，测量神经肌肉运动强度，抓力感受装置是由平行的金属丝(直径5mm)构成的，使其倾斜于地面45°，将小鼠置于感受器缓慢旋转180°，装置离地面20cm预防小鼠掉落不足以造成伤害，秒表记录小鼠在钢丝抓握的时间，每只测3次取平均值，结果如表9所示。
表9、db/db小鼠悬杆实验时间统计结果
Figure PCTCN2017085837-appb-000005
*P<0.01vs NC组；#P<0.01vs模型组
结果表明，DMD模型鼠悬吊时间明显少于NC组，而给药28天起，梓醇治疗组较DMD模型组小鼠在悬吊前后肢抓力方面优于模型组，表明梓醇能够改善DMD鼠的肌无力。
肌抓力测定：给药第14天，第28天，第42天进行小鼠前、后肢抓力测定，每只小鼠测3次，取平均值为该小鼠的最大肌抓力。结果如表10所示。
表10、小鼠肌抓力测定
Figure PCTCN2017085837-appb-000006
*P<0.01vs NC组；#P<0.01vs模型组
结果显示，DMD模型组小鼠肌抓力第28天开始小于NC对照组，而辛伐他汀连续给药28天起能明显提高肌抓力；梓醇治疗42天后较模型组小鼠肌抓力也有一定恢复。
血清肌酸磷酸激酶(CK)测定：给药第14天，第28天，第42天进行麻醉眼眶静脉丛取血约100ul，2500rpm离心10min，取上层血清按CK检测试剂盒(南京建成生物科技有限公司)测定CK水平，结果如表11所示。
表11、血清肌酸磷酸激酶(CK)测定
Figure PCTCN2017085837-appb-000007
结果显示，DMD模型组血清CK是NC对照组10-20倍，梓醇和辛伐他汀连续给药14天血清CK水平即有一定下降趋势，连续给药28天后梓醇降低血清CK作用比辛伐他汀更强。
血清乳酸脱氢酶(LDH)测定：给药第14天，第28天，第42天进行麻醉眼眶静脉丛取血约100ul，2500rpm离心10min，取上层血清按LDH检测试剂盒(南京建成生物科技有限公司)测定LDH水平。结果如表12所示。
表12、血清乳酸脱氢酶(LDH)测定
Figure PCTCN2017085837-appb-000008
结果显示，DMD模型组血清LDH是NC对照组10-20倍，梓醇和辛伐他汀连续给药14天血清LDH水平即有一定下降趋势，连续给药42天后梓醇和辛伐他汀都能明显降低血清LDH水平。
骨骼肌中羟脯氨酸测定：羟脯氨酸水平用于评价总胶原含量，反应骨骼肌纤维化水平。精确称取骨骼肌30mg，按照羟脯氨酸测定试剂盒(南京建成生物科技有限公司)测定，结果如表13所示：
表13、骨骼肌中羟脯氨酸测定
Figure PCTCN2017085837-appb-000009
*P<0.01vs NC组；#P<0.01vs模型组
结果显示，DMD模型组鼠骨骼肌中羟脯氨酸较NC组显著增加，表明DMD模型鼠骨骼肌可能发生纤维化，而梓醇和辛伐他汀连续给药42天后骨骼肌中羟脯氨酸水平较DMD模型组明显降低，因此推测梓醇和辛伐他汀一样有抗骨骼肌纤维化的作用。
腓肠肌HE染色：给药42天后，麻醉处死动物，快速解剖双侧腓肠肌，一侧腓肠肌用福尔马林固定液固定用于制作石蜡切片，另一侧迅速冻于-20冰箱，当天制作冰冻切片用于免疫荧光染色。用腓肠肌石蜡切片制作腓肠肌切片用于HE染色，经过一系列洗脱，固定，染色，封片后于激光共聚焦(FV1000)下拍照，结果如图8所示。
结果显示：DMD模型组腓肠肌肌纤维圆形化、大小不一，有明显炎性细胞浸润；梓醇和辛伐他汀能够明显减少腓肠肌炎性细胞浸润。
腓肠肌F4/80和CD206免疫荧光：通过HE染色发现明显的炎性细胞浸润， 而F4/80和CD206是用于标记巨噬细胞反应炎症情况，因此取腓肠肌冰冻切片，用预冷丙酮固定，PBS润洗后用生物素化的F4/80一抗(BioLegend Lot:123140，美国)和生物素化的CD206一抗(BioLegend Lot:141710，美国)双染，4℃过夜，第二天洗涤后用DAPI染细胞核，室温孵育10min后洗涤拍照，结果如图9所示。
结果表明：DMD模型鼠肌纤维附近巨噬细胞显著增多，炎症明显；而梓醇和辛伐他汀能明显降低F4/80和CD206的表达，减轻炎症。
免疫组化标记骨骼肌I型肌纤维和Ⅱ型肌纤维：如腓肠肌HE染色，取腓肠肌石蜡切片制作腓肠肌切片，用稀释的快收缩肌肌球蛋白抗体(MY-32,1:500，Abcam公司，美国)4℃过夜，洗涤后用生物素化的鼠二抗室温孵育1h，染色洗涤后封片，于激光共聚焦(FV1000)下拍照。随后用image-Plus统计分析肌纤维比例，结果如图10所示。
结果表明，DMD模型组肌纤维明显大小不一、排列无序，能观察到大的纤维坏死和小群变性肌纤维；而梓醇和辛伐他汀能够明显改善肌纤维形态和排列；此外，梓醇和辛伐他汀都能够逆转DMD小鼠腓肠肌Ⅰ型肌纤维的缺失。
腓肠肌纤连蛋白fibronectin免疫荧光：骨骼肌发生纤维化时fibronectin蛋白表达明显增加，因此fibronectin可以评价纤维化程度。取腓肠肌冰冻切片，用预冷丙酮固定，PBS润洗后用稀释的Fibronectin抗体孵育，4℃过夜，第二天洗涤后用生物素化的兔二抗室温孵育1h，再用DAPI染细胞核，室温孵育10min后洗涤拍照，结果如图11所示。
结果显示，DMD模型鼠腓肠肌fibronectin蛋白较NC组表达明显增多，梓醇和辛伐他汀都能完全逆转骨骼肌纤维化。
综上所述，本发明通过以国际公认的研究Ⅱ型糖尿病的db/db转基因小鼠为模型小鼠以及高脂饮食喂养的C57小鼠为早期糖尿病模型，以及国际公认的研究肌无力和/或肌萎缩的Dmdmdx基因缺陷小鼠为模型小鼠，考察梓醇对肌无力和肌萎缩的治疗效果。从药效学、行为学和分子生物学等方面对梓醇对糖尿病或杜氏肌营养不良症引起的肌无力和肌萎缩的影响做了系统研究，结果显示：梓醇能有效改善糖尿病小鼠的高血糖，增加糖尿病鼠肌抓力，增加骨骼肌重量，改善其运动能力；此外，梓醇能有效改善DMD模型鼠肌无力和/或肌萎缩症状，能明显增加模型鼠肌抓力，抵抗骨骼肌纤维化；有缓解骨骼肌炎症， 降低骨骼肌损伤的作用，为将梓醇开发治疗肌无力和/或肌萎缩提供了理论研究基础。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

Claims (6)

  1. 梓醇在制备防治或延缓肌无力和/或肌萎缩的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述的肌无力和/或肌萎缩是由糖尿病所引起的。
  3. 根据权利要求1所述的应用，其特征在于：所述的肌无力和/或肌萎缩是由杜氏肌营养不良症所引起的。
  4. 根据权利要求1所述的应用，其特征在于：所述的肌无力和/或肌萎缩包括重症肌无力。
  5. 根据权利要求1或2所述的应用，其特征在于：所述的肌无力包括糖尿病早期和糖尿病晚期骨骼肌神经受损引起的肌无力。
  6. 根据权利要求1或2所述的应用，其特征在于：所述的肌萎缩包括糖尿病近端肌萎缩综合征。
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