WO2019041423A1 - A型肉毒毒素在抗抑郁症中的应用 - Google Patents
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- the invention belongs to the technical field of medicine, and particularly relates to the application of a botulinum toxin type A in antidepressant, in particular to raising brain-derived neurotrophic factor (BDNF).
- BDNF brain-derived neurotrophic factor
- Depression is one of the most common mental illnesses and a major concomitant symptom of other diseases. Depression has a high suicide rate, high self-harm rate, and high recurrence rate, which determines that depression is a serious social health problem. According to the World Health Organization report, as of 2012, there are an estimated 350 million people with depression worldwide. Epidemiological data released in the 2014 Nature show that the prevalence of depression in China is 3.02%. Today's fast-paced, high-pressure lifestyle makes depression a high-risk species with an increasing incidence.
- the main clinical symptoms of depression are: 1) "three lows” - low mood, slow thinking, decreased will activity; 2) psychotic symptoms: “three nos” - helpless, hopeless, useless; 3) "three self "-inferiority, suicide, self-sin; 4" physical symptoms: sleep disorders, fatigue, weight loss, loss of appetite, loss of libido, constipation pain, discomfort, complaints involving autonomic dysfunction; 5) other symptoms: disintegration, reality disintegration, Forced symptoms. If not promptly intervened, it will lead to aggravation of the disease into a vicious circle, resulting in high suicide rate and high self-harm rate, which will bring more and more burdens to patients and their families and society.
- ICD International Classification of Diseases
- ICD-10 diagnostic criteria for depressive episodes are as follows: a) Typical symptoms: 1. Low mood, 2. Loss of interest and pleasure, 3. Insufficient energy or fatigue; 2) Common symptoms: 1. Reduced ability to focus and pay attention, 2. Reduce self-evaluation, 3. Self-criticism and worthlessness, 4. Think of a bleak future Pessimism, 5. Self-injury or suicidal concept/behavior, 6. Sleep disorder, 7.
- the application of drugs for antidepressant treatment mainly includes: monoamine neurotransmitter-based drugs and fast-acting antidepressants.
- Anti-depressant drugs based on monoamine neurotransmitters are mainly: tricyclic antidepressant (TCA), monoamine oxidase inhibitor (MAOI), selective serotonin reuptake inhibitor (selective serotonin reuptake inhibitor, SSRI), selective serotonin and norepinephrine reuptake inhibitor (SNRI), and novel antidepressants based on monoamine.
- SSRI is the drug of choice for the treatment of depression; when other means of treating depression fail TCA and MAOI may be used for treatment; for the optimal therapeutic effect, clomipramine, venlafaxine, escitalopram, sertraline, and amitriptyline should be considered. Or mirtazapine.
- the main antidepressant drugs are: N-methyl-D-aspartic acid receptor (NMDA) receptor antagonist (representative drug: ketamine), selective NR2B subunit An NMDA receptor antagonist, a partial agonist of the NMDA receptor glycine site, and an acetylcholine-muscarinic receptor antagonist.
- NMDA N-methyl-D-aspartic acid receptor
- ketamine selective NR2B subunit
- An NMDA receptor antagonist a partial agonist of the NMDA receptor glycine site
- an acetylcholine-muscarinic receptor antagonist an acetylcholine-muscarinic receptor antagonist.
- Most of the drugs currently available for antidepressants are in clinical trials.
- the neuroplastic hypothesis of depression suggests that the disease occurs because stress destroys the structure and connections of the pathways involved in mood regulation.
- Antidepressants need to repair the connections between synapses and increase synaptic plasticity in order to cure depression.
- the botulinum toxin type A is a white loose body, which is a clear transparent or pale yellow solution after being dissolved in a physiological sodium chloride solution. Applicable to adult patients with eyelids, hemifacial spasm and some strabismus, especially acute paralytic strabismus, common strabismus, strabismus caused by endocrine muscle disease and strabismus patients over 12 years old who can not be corrected or have poor surgical results. Botulinum toxin type A for injection, the indications are eyelids, hemifacial spasm and related focal dystonia. Temporarily improve the moderate to severe frown lines caused by frowning muscles or eyebrow muscle activity in adults aged 65 and under.
- botulinum toxin type A can inhibit the release of acetylcholine from the presynaptic membrane of peripheral motor nerve terminals, causing relaxation paralysis of the muscle. There is no precedent for the use of botulinum toxin type A in the treatment of depression.
- the object of the present invention is to provide a botulinum toxin type A application for antidepressant in view of the above-mentioned technical problems.
- botulinum toxin type A for the prevention of brain-derived neurotrophic factor (BDNF) in antidepressant.
- BDNF brain-derived neurotrophic factor
- botulinum toxin type A in the treatment of antidepressants, said botulinum toxin type A for the preparation of an antidepressant product.
- the botulinum toxin type A is added to the adjuvant to make any clinical or pharmaceutically acceptable dosage form.
- the dosage form is a powder, a tablet, a capsule, a dropping pill or an injection.
- the excipients are sucrose, dextran and gelatin.
- the botulinum toxin type A of the present invention can effectively control and treat depression, and the technical solution of the invention has less effect and stable therapeutic effect than the chemically synthesized drug. Moreover, the technology of the present invention has a simple process and a few operation steps, and is suitable for industrial scale production.
- Figure 1 shows botulinum toxin type A (BoNT/A or BTX-A) to improve depression
- FIG. 1 shows chronic stress (SRS)-induced depression-like behavior
- Figure 3 shows botulinum toxin type A improving depression-like behavior in SRS mice
- Figure 4 shows that botulinum toxin type A increases the content of 5-HT in the brain
- Figure 5 shows the recovery of NMDA receptor subunits in SRS mice by botulinum toxin type A
- Figure 6 shows that botulinum toxin type A up-regulates the expression of BDNF in the brain regions of SRS mice
- Figure 7 shows that the botulinum toxin type A activates the ERK-CREB pathway.
- botulinum toxin type A for the prevention of brain-derived neurotrophic factor (BDNF) in antidepressant.
- BDNF brain-derived neurotrophic factor
- botulinum toxin type A in the treatment of antidepressants, said botulinum toxin type A for the preparation of an antidepressant product.
- the botulinum toxin type A is added to the adjuvant to make any clinical or pharmaceutically acceptable dosage form.
- the dosage form is a powder, a tablet, a capsule, a dropping pill or an injection.
- the excipients are sucrose, dextran and gelatin.
- HAMD Hamilton Depression Rating Scale
- HAMA Hamiltonian Anxiety Rating Scale
- Each group of botulinum toxin type A was given a one-time facial injection of 100 U of botulinum toxin type A, and the Zuoluo group was orally administered 50-100 mg once a day, with a mean value of 62.5 mg/day.
- the HAMD follow-up score showed that botulinum toxin type A improved the patient's depression and was superior to Zoloft.
- the HAMD scores of the two groups were decreased to some extent.
- the HAMD scores of the botulinum toxin type A group and the ZOLU group were significantly different.
- botulinum toxin type A improved the depression in patients with anxiety and was superior to Zoloft.
- the HAMA scores of the two groups were also decreased to some extent.
- the botulinum toxin type A group was significantly better than the Zolojol group; the two were the same at the beginning of the onset time, at week 4, botulinum toxin type A
- Botulinum toxin type A improves depression-like behavior
- Botulinum toxin type A reduces TST and FST immobility in normal mice.
- ICR mice were injected with a single injection of botulinum toxin type A (0.02 u, 0.06 u and 0.18 u).
- FST(a) and TST(b) are detected at different time points.
- the rotation test detects motion (C) and open field test (d-f).
- SRS Chronic stress
- SRS causes depression-like behavior and has been widely used to establish rodent depression models, and we repeated this model.
- Stressed mice showed significantly increased depression-like behavior of TST and FST compared to control mice.
- our data indicate that mice with SRS depressive phenotypes have been successfully established.
- SRS causes depression-like behavior.
- the experimental process is shown in (a).
- the FST immobility time (c) and TST(d) of stress-induced depression mice were significantly reduced.
- the body weight of the mice was expressed in (c) and was significantly reduced.
- Each group is represented by an average and a standard error, and the number is 10-22.
- Botulinum toxin type A improves depression-like behavior in SRS mice
- FIG. 3 Antidepressant effect of botulinum toxin type A.
- the FST(a) and TST(b) are detected at different time points, and the immobility time is reduced.
- the weight changes to (c). **p ⁇ 0.01, ***p ⁇ 0.001 vs control mice (Ordinary two-way ANOVA); #p ⁇ 0.05,##p ⁇ 0.01 vs SRS-treated mice (Ordinary two-way ANOVA).
- the number and standard error indicate that the number is 7-10.
- Botulinum toxin type A increases the content of 5-HT in the brain
- FIG. 4 Increased 5-HT in botulinum toxin type A in mice with SRS.
- NMDA receptors NR1, NR2A and NR2B
- NR2A has not changed.
- NMDA receptor system is involved in the antidepressant effect of botulinum toxin type A.
- NR2A was decreased in normal mice after injection of botulinum toxin type A for 1 hour without affecting SNAP25, NR1, NR2B.
- Botulinum toxin type A upregulates NMDA receptors that are reduced in stress.
- SNAP25 (b, g), NR1 (c, h), NR2A (d, i), NR2B (e, j) were detected by Western blot.
- botulinum toxin type A is injected into SRS-treated mice; on the right, botulinum toxin type A is injected in normal mice.
- Each group is represented by an average and a standard error, and the number is 4.
- Botulinum toxin type A up-regulates the expression of BDNF in the brain regions of SRS mice
- Botulinum toxin type A increases BDNF, whereas SRS mice have decreased BDNF.
- Hippocampal brain-derived neurotrophic factor (e-f) was determined by immunoblotting. Protein expression of BDNF at different time points (f) was detected after injection of botulinum toxin in normal mice.
- Botulinum toxin type A activates the ERK-CREB pathway
- the ERK-CREB pathway is an important pathway for neurotrophic factors and nerve regeneration, which is beneficial for anti-depression.
- botulinum toxin type A Phosphorylated ERK and CREB were increased 1 day after injection. However, this effect was not observed at other points in time.
- Botulinum toxin type A activates the ERK CREB signaling pathway.
- the pERK (b) and pCREB (c) proteins were determined by immunoblotting. *p ⁇ 0.05, **p ⁇ 0.01 vs control mice (Unpaired t test); #p ⁇ 0.05,###p ⁇ 0.001 vs SRS-treated mice (Ordinary one-way ANOVA). Average and standard error for each group Indicates that the quantity is 4.
- the botulinum toxin type A may be a botulinum toxin type A for injection of Hengli brand produced by Lanzhou Biological Products Research Institute Co., Ltd., and the standard of Chinese medicine is S10970037, and the specification is 100 units/bottle.
- Beta active ingredient Clostridium botulinum toxin type A, obtained by fermentation of Clostridium botulinum type Hall strain grown in culture medium. Excipients: human albumin and sodium chloride.
- Botulinum toxin type A for injection This product is a white powder. After dilution with physiological saline for injection, it is a clear liquid which is colorless to slightly yellow and contains no impurities.
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Abstract
本发明公开了一种A型肉毒毒素在抗抑郁症中的应用。所述的A型肉毒毒素用于制备抗抑郁症产品。本发明所述的A型肉毒毒素能够对抑郁症起到有效的控制和治疗作用。
Description
本发明属于医药技术领域，具体涉及一种A型肉毒毒素在抗抑郁症中的应用，尤其是用于升高脑源性神经营养因子(BDNF)。
抑郁症是最常见的精神疾病之一，也是其他疾病的主要伴随症状。抑郁症的自杀率高、自残率高、复发率高的特点决定了抑郁症是一个严重的社会健康问题。世界卫生组织报告显示，截至2012年，全球估计有超过3.5亿的抑郁症患者。2014年《Nature》上发布的流行病学数据显示，我国的抑郁症患病率为3.02％。现今快节奏、高压的生活方式使得抑郁症成为一个发病率逐年攀升的高发病种。抑郁症的临床核心症状主要有：1)“三低”—心境低落、思维迟缓、意志活动减退；2)精神病性症状:“三无”——无助、无望、无用；3)“三自”——自卑、自杀、自罪；4)躯体症状：睡眠障碍、乏力、体重下降食欲下降、性欲减退、便秘疼痛、不适主诉涉及自主神经功能失调；5)其他症状:人格解体、现实解体、强迫症状。如果不及时干预会导致病情加重进入恶性循环，导致高自杀率、高自残率，给患者和家属以及社会带来越来越沉重的负担。
国际疾病分类(International Classification ofDiseases,ICD),ICD-10关于抑郁发作(不包括双相障碍，即首次发作/复发的抑郁症)的诊断标准如下：一)典型症状：1.心境低落，2.兴趣和愉悦感丧失，3.精力不济或疲劳感；二)常见症状：1.集中注意和注意的能力降低，2.自我评价降低，3.自罪观念和无价值感，4.认为前途暗淡悲观，5.自伤或自杀观念/行为，6.睡眠障碍，7.食欲下降；三)严重程度判定标准——轻度抑郁：至少两条典型症状+至少两条常见症状+日常工作和社交活动有一定困难，社会功能受到影响；中度抑郁：至少两条典型症状+至少三条常见症状+工作、社交或家务活动有相当困难；重度抑郁：具备三条典型症状+至少四条常见症状(某些症状应达到严重程度)+工作、社交或家务活动不能继续；四)病程标准：>＝2周(症状极为严重或起病非常急骤时可以不足2周)；五)排除标准：排除器质性精神障碍，或精神活性物质和非成瘾物质
所致。
应用药物进行抗抑郁治疗主要包括：基于单胺类神经递质的药物和速效抗抑郁药物。基于单胺类神经递质的抗抑郁药物主要有：三环类抗抑郁药物(tricyclic antidepressant,TCA)、单胺氧化酶的抑制剂(MAOI)、选择性5-羟色胺再摄取抑制剂(selective serotonin reuptake inhibitor,SSRI)、选择性5-羟色胺和去甲肾上腺素再摄取抑制剂(selective serotonin and norepinephrine reuptake inhibitor,SNRI)以及基于单胺的新型抗抑郁药等。根据英国精神药理协会发布的2015版的《抗抑郁药治疗抑郁症》指南，病人对SSRI类药物有较高的耐受性，SSRI是治疗抑郁症的首选药物；当其它治疗抑郁症的手段失败时，可采用TCA和MAOI类药物治疗；以取得最优的治疗效果为目标时，则应考虑使用氯丙咪嗪、文拉法辛、艾司西酞普兰、舍曲林、阿米替林或米氮平。速效抗抑郁药物主要有：N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid receptor,NMDA)受体的拮抗剂(代表药物：氯胺酮)、选择性的含有NR2B亚基的NMDA受体拮抗剂、NMDA受体甘氨酸位点的部分激动剂和乙酰胆碱－毒蕈碱受体拮抗剂。目前速效抗抑郁的药物大都处于临床试验阶段。抑郁症的神经可塑性假说认为疾病的发生是由于压力破坏了情绪调节有关通路的结构和联系，抗抑郁药物需要修复突触之间的联系，增加突触的可塑性，才能治愈抑郁症。单胺类的神经递质只作为神经通路的调节分子，不能促进突触的形成，并且在治疗时间上存在延迟，但速效抗抑郁药物可以产生快速的和持续的抗抑郁效果。
随着经典抗抑郁药物的耐药性、副作用的显现，急切需要寻找新的抗抑郁药物。
A型肉毒毒素为白色疏松体，生理氯化钠溶液溶解后为澄清透明或淡黄色溶液。适用于眼睑痉挛，面肌痉挛等成人患者及某些斜视，特别是急性麻痹性斜视、共同性斜视、内分泌肌病引起的斜视及无法手术矫正或手术效果不佳的12岁以上的斜视患者。注射用A型肉毒毒素，适应症为眼睑痉挛、面肌痉挛及相关局灶性肌张力障碍。暂时改善65岁及65岁以下成人因皱眉肌/或降眉间肌活动引起的中度至重度皱眉纹。治疗用A型肉毒毒素能抑制周围运动神经末梢突触前膜乙酰胆碱释放，引起肌肉的松弛性麻痹。A型肉毒毒素应用于治疗抑郁症尚无先例。
发明内容
本发明的目的是针对上述存在的技术问题提供了一种A型肉毒毒素在抗抑郁症中的应用。
本发明的目的可以通过以下技术方案实现：
一种A型肉毒毒素在抗抑郁症中的应用，所述的A型肉毒毒素用于升高脑源性神经营养因子(BDNF)。
一种A型肉毒毒素在抗抑郁症中的应用，所述的A型肉毒毒素用于制备抗抑郁症产品。
所述的A型肉毒毒素加入辅料后制成临床或者药学上可接受的任一剂型。
所述的剂型为粉末剂、片剂、胶囊、滴丸或者注射剂。
所述的辅料为蔗糖、右旋糖苷和明胶。
本发明的有益效果：
本发明所述的A型肉毒毒素能够对抑郁症起到有效的控制和治疗，相对于化学合成的药物而言，本发明的技术方案的效果更小且疗效稳定。且本发明技术发难工艺简单，操作步骤较少，适于工业化规模生产。
图1为A型肉毒毒素(BoNT/A或BTX-A)改善抑郁行为；
图2为慢性应激(SRS)诱发抑郁样行为；
图3为A型肉毒毒素改善SRS小鼠抑郁样行为；
图4为A型肉毒毒素增加脑区内5-HT的含量；
图5为A型肉毒毒素对SRS小鼠NMDA受体亚基下降的恢复作用；
图6为A型肉毒毒素上调SRS小鼠脑区中BDNF的表达；
图7为A型肉毒毒素激活了ERK-CREB通路。
下面结合实施例对本发明做进一步说明，但本发明的保护范围不限于此：
一种A型肉毒毒素在抗抑郁症中的应用，所述的A型肉毒毒素用于升高脑源性神经营养因子(BDNF)。
一种A型肉毒毒素在抗抑郁症中的应用，所述的A型肉毒毒素用于制备抗抑郁症产品。
所述的A型肉毒毒素加入辅料后制成临床或者药学上可接受的任一剂型。
所述的剂型为粉末剂、片剂、胶囊、滴丸或者注射剂。
所述的辅料为蔗糖、右旋糖苷和明胶。
A型肉毒毒素对于治疗抑郁症的治疗效果
表1
入组病例影响因素([Mean(SEM)])
图注：HAMD，哈密顿抑郁评分量表；HAMA，哈密顿焦虑评分量表。数据均以平均值和标准误表示。
经过HAMA和HAMD两种量表的评分，共有43例病人入组。这43例患者满足了HAMA和HAMD评分需大于等于7分的标准。其中入组A型肉毒毒素的患者有29例(伴有帕金森病的患者有11例)，入组左洛复的有14例(伴有帕金森病的患者有4例)。两组患者的年龄均值接近,A型肉毒毒素治疗组年龄均值为60岁，左洛复为59.5岁，2者对照的年龄相近。其中A型肉毒毒素组有27名(93％)女性患者，左洛复组有10名(71％)女性患者。A型肉毒毒素组每人都给与一次性面部注射100U的A型肉毒毒素，左洛复组每天口服一次50-100mg，给予均值为62.5mg/天。
表2
HAMD随访评分结果
图注：在HAMD评分量表中，A型肉毒毒素与左洛复治疗后随访8周的结果。#p<0.05,表示最初起效时间(One-way ANOVA)，*p<0.05，表示该时间点疗效更好(Unpaired t test)。数据均以平均值和标准误表示。N＝14-29。
HAMD随访评分显示A型肉毒毒素对患者的抑郁症起到改善作用，并且药效优于左洛复。
经过8周的随访，两组患者HAMD评分均有不同程度的下降，其中在第8周，A型肉毒毒素组与左洛复组HAMD评分有显著性差异，A型肉毒毒素组疗效明显优于左洛复组(P＝0.0355，t＝2.346)；但两者在开始起效时间上有所不同，在第2周，A型肉毒毒素组HAMD评分较基线数据相比较下降明显[F(3，112)＝17.7，P<0.0001]；在第4周，左洛复组HAMD评分较基线数据下降显著[F(3，52)＝3.527，P＝0.0211]。从以上数据可知，A型肉毒毒素对患者抑郁症的改善作用起效时间早于左洛复，且改善作用强于左洛复。
表3
HAMA随访评分结果
图注：在HAMA评分量表中，A型肉毒毒素与左洛复治疗后随访8周的结果。#p<0.05,表示最初起效时间(One-way ANOVA)，**p<0.01，表示该时间点
疗效更好(Unpaired t test)。数据均以平均值和标准误表示。N＝14-29。
HAMA随访评分显示A型肉毒毒素对患者的抑郁症伴随焦虑起到改善作用，并且药效优于左洛复。
经过8周的随访，两组患者HAMA评分也有不同程度的下降，其中在第4、8周，肉毒组与左洛复组HAMA评分之间有显著差异(4周：P＝0.0092，t＝3.053；8周：P＝0.0032，t＝3.611)，A型肉毒毒素组疗效明显优于左洛复组；而两者在开始起效时间上相同，在第4周，A型肉毒毒素组与左洛复组HAMA评分较各自基线数据下降显著[BTX-A:F(3，112)＝10.63，P<0.0001；ZOLOFT:F(3，52)＝4.447，P＝0.0074]，两组均在4周开始起效。从以上数据可知，A型肉毒毒素对患者抑郁症伴随焦虑的改善作用强于左洛复。
A型肉毒毒素改善抑郁样行为
从图1，我们研究了A型肉毒毒素注射行为正常的小鼠。首先，我们采用TST(悬尾实验)和FST(强迫游泳)，两无助绝望的测量，评估抑郁样行为。与对照组相比，小鼠单次腹腔注射A型肉毒毒素在0.06u和0.18u，在注射后1h到3天，TST显著降低(图1bFInteraction(20,140)＝1.787,P＝0.0274,FTime(5,140)＝9.354,P<0.0001,FColumnFactor(4,28)＝5.215,P＝0.0029,FSubjects(28,140)＝2.146,P＝0.0020).在FST发现了类似的结果，观察0.18uA型肉毒毒素注射后不动时间降低持续到第十四天(图1a,a:FInteraction(20,190)＝4.415,P<0.0001,FTime(5,190)＝32.02,P<0.0001,FColumnFactor(4,38)＝6.860,P＝0.0003,FSubjects(38,190)＝4.525,P<0.0001).我们发现自发的活动，由旋转试验和矿场试验中观察，并没有改变(图1c-f)。这些数据表明，A型肉毒毒素也许有利于缓解抑郁样行为。此外，我们用丙咪嗪为阳性对照。结果表明，丙米嗪在FST和TST两个指标，在第一次注射后分别在1-7天和1-3天起作用。
图1，A型肉毒毒素降低正常小鼠TST和FST不动时间。ICR小鼠单次注射A型肉毒毒素(0.02u，0.06u和0.18u)。FST(a)、TST(b)在不同时间点检测。旋转试验检测运动(C)和旷场试验(d-f)。*p<0.05,**p<0.01,***p<0.001vs control mice(Ordinary two-way ANOVA)每组以平均数和标准误表示，数量为5-10。
慢性应激(SRS)诱发抑郁样行为
SRS引起抑郁样行为，已被广泛用于建立啮齿动物抑郁模型，我们重复了这个模型。如图2b所示，与对照组相比，SRS导致体重显著下降(图2b，T＝3.972，p＝0.0004)。与对照小鼠相比，应激小鼠显示显著增加TST和FST的抑郁样行为。(图2C，t＝3.435，P＝0.0020；2D，t＝2.093，P＝0.0449)。总之，我们的数据表明，已成功地建立了SRS抑郁表型的小鼠。
图2，SRS引起抑郁样行为。实验过程显示在(a)。应激的抑郁小鼠FST不动时间(c)、TST(d)明显降低。小鼠体重表现在(c)，并明显降低。*p<0.05,**p<0.01,***p<0.001vs control mice(Unpaired t test)。每组以平均数和标准误表示，数量为10-22。
A型肉毒毒素改善SRS小鼠抑郁样行为
进一步研究A型肉毒毒素对SRS小鼠抑郁样行为的治疗作用，SRS的小鼠单次注射A型肉毒毒素0.18u。TST和FST不动时间的在不同的时间点记录。幸运的是，注射A型肉毒毒素1h到14天后，明显降低TST和FST的不动时间(图3a:FInteraction(15,160)＝5.504,P<0.0001,FTime(5,160)＝8.300,P<0.0001,FColumn
Factor(3,32)＝13.43,P<0.0001,FSubjects(32,160)＝9.056,P<0.0001；3b:FInteraction(15,160)＝5.504,P<0.0001,FTime(5,160)＝8.300,P<0.0001,FColumn Factor(3,32)＝13.43,P<0.0001,FSubjects(32,160)＝9.056,P<0.0001).同时，以氟西汀作为阳性抗抑郁药物治疗SRS小鼠。我们发现氟西汀连续注射14天后降低TST和FST的不动时间(图3A，B，P＜0.05)。我们还发现，受激的抑郁样行为可以持续至少14天。此外，A型肉毒毒素可以恢复因应激而降低的体重(Fig.3c:FInteraction(15,140)＝3.947,P<0.0001,FTime(5,140)＝114.0,P<0.0001,FColumn Factor(3,28)＝11.69,P<0.0001,FSubjects(28,140)＝13.75,P<0.0001)。
图3，A型肉毒毒素抗抑郁作用。在不同的时间点检测FST(a)、TST(b)，不动时间减少。体重改变为(c)。**p<0.01,***p<0.001vs control mice(Ordinary two-way ANOVA)；#p<0.05,##p<0.01vs SRS-treated mice(Ordinary two-way ANOVA).每组以平均数和标准误表示，数量为7-10。
A型肉毒毒素增加脑区内5-HT的含量
抑郁症动物模型和患者都表现出5-HT缺乏。本研究采用高效液相色谱法测
定5-羟色胺。我们发现海马和下丘脑的5-HT含量显著降低(Fig.4a：t＝3.383，P＝0.0277；b：t＝6.596，P＝0.0027)，而前额皮质没有。然而，A型肉毒毒素增加5-HT在海马、下丘脑和前额叶皮层的含量(Fig.4a：F(4,10)＝21.46，P<0.0001；b:F(4,10)＝12.29,P＝0.0007；c:F(4,10)＝7.101,P＝0.0056).提示A型肉毒毒素可以恢复抑郁状态下5-HT能神经递质的平衡。
图4，在SRS的小鼠A型肉毒毒素增加5-HT。采用高效液相色谱法检测不同脑区海马(a)、下丘脑(b)和前额叶皮层(c)的5-HT含量。*p<0.05,**p<0.01vs control mice(Unpaired t test)；#p<0.05,##p<0.01,###p<0.01vs SRS-treated mice(Ordinary one-wayANOVA).每组以平均数和标准误表示，数量为3。
A型肉毒毒素对SRS小鼠NMDA受体亚基下降的恢复作用
NMDA受体(NR1，NR2A和NR2B)在抑郁中的作用不明确。在本研究中，我们发现与对照组相比，NR1和NR2B在SRS小鼠海马显著下降，(Fig.5c,t＝6.725,P＝0.0025；5e,t＝5.416,P＝0.0056).相比之下，A型肉毒毒素显著增加NR1和NR2B在SRS小鼠海马(Fig.5c,F(5,12)＝43.20,P<0.0001,5e,F(5,13)＝10.69,P＝0.0003).此外，NR2A没有改变。结果表明NMDA受体系统参与了A型肉毒毒素的抗抑郁作用。正常小鼠注射A型肉毒毒素1hour后NR2A降低，而不影响SNAP25，NR1，NR2B。
图5，A型肉毒毒素上调在应激中减少的NMDA受体。SNAP25(b,g),NR1(c,h),NR2A(d,i),NR2B(e,j)用Western blot检测。在左，A型肉毒毒素是SRS治疗的小鼠注射；在右，A型肉毒毒素在正常小鼠注射。*p<0.05,**p<0.01,***p<0.001vs control mice(Unpaired t test)；#p<0.05,##p<0.01,###p<0.001vs SRS-treated mice(Ordinary one-wayANOVA).每组以平均数和标准误表示，数量为4。
A型肉毒毒素上调SRS小鼠脑区中BDNF的表达
有研究指出BDNF有利于抑郁症的治疗，抗抑郁药的长期治疗增加BDNF的表达。图6a-d显示的是：在应激抑郁模型上，海马和杏仁核中BDNF的mRNA水平是降低的(Fig.6a,t＝4.117,P＝0.0146；6d,t＝4.129,P＝0.0145)，并且可以被A型肉毒毒素上调表达(Fig.6a,1-7days post injection in hippocampus,F(4,10)＝8.700,P＝0.0027；Fig.6d,1-14days post injection in amygdala,F(4,10)＝
7.423,P＝0.0048).但是在应激抑郁模型上，前额皮质和下丘脑中BDNF的mRNA水平没有改变，注射A型肉毒毒素后可上调BDNF的mRNA表达一天(Fig.6f,F(4,15)＝4.415,P＝0.0148)。在蛋白表达水平上也做出了相似的结果。在应激之后BDNF蛋白表达降低(Fig.6e,t＝7.594,P＝0.0003)，而给予A型肉毒毒素后BDNF蛋白表达增高F(5,18)＝10.68,P<0.0001)。这些提示，A型肉毒毒素参与了BDNF的抗抑郁途径。
图6，A型肉毒毒素增加BDNF，而SRS的小鼠BDNF降低。BDNF mRNA的定量测定，海马(a)、前额叶皮层(b)、下丘脑(c)和杏仁核(d)。免疫印迹法测定海马脑源性神经营养因子(e-f)。在正常小鼠注射肉毒毒素后检测在不同时间点BDNF的蛋白表达(f)。*p<0.05,***p<0.001vs control mice(Unpaired t test)；#p<0.05,##p<0.01,###p<0.001vs SRS-treated mice(Ordinary one-wayANOVA)。每组以平均数和标准误表示，数量为4。
A型肉毒毒素激活了ERK-CREB通路
ERK-CREB通路是神经营养因子和神经再生的重要通路，有利于抗抑郁。如图7，ERK和CREB的磷酸化在SRS小鼠海马显著抑制(Fig,7b,t＝6.012,P＝0.0039；7c,t＝4.235,P＝0.0133).幸运的是，A型肉毒毒素注射1天后增加磷酸化的ERK和CREB。然而，这种效应在其他时间点没有被观察到。
图7，A型肉毒毒素激活ERK CREB信号通路。pERK(b)和pCREB(c)蛋白用免疫印迹法测定。*p<0.05,**p<0.01vs control mice(Unpaired t test)；#p<0.05,###p<0.001vs SRS-treated mice(Ordinary one-wayANOVA).每组以平均数和标准误表示，数量为4。
上述实施例中，所述A型肉毒毒素可采用兰州生物制品研究所有限公司生产的衡力牌注射用A型肉毒毒素，国药准字S10970037，规格为100单位/瓶。
β活性成分：A型肉毒梭菌毒素，由生长在培养基的A型肉毒梭菌Hall株经发酵制备而得。辅料：人血白蛋白和氯化钠。
注射用A型肉毒毒素性状：本品为白色粉末。注射用生理盐水稀释后为无色至略显黄色的、不含杂质的澄明液体。
Claims (5)
- 一种A型肉毒毒素在抗抑郁症中的应用，其特征在于：所述的A型肉毒毒素用于升高脑源性神经营养因子(BDNF)。
- 一种A型肉毒毒素在抗抑郁症中的应用，其特征在于：所述的A型肉毒毒素用于制备抗抑郁症产品。
- 根据权利要求2所述的A型肉毒毒素在抗抑郁症中的应用，其特征在于：所述的A型肉毒毒素加入辅料后制成临床或者药学上可接受的任一剂型。
- 根据权利要求3所述的A型肉毒毒素在抗抑郁症中的应用，其特征在于：所述的剂型为粉末剂、片剂、胶囊、滴丸或者注射剂。
- 根据权利要求4所述的A型肉毒毒素在抗抑郁症中的应用，其特征在于：所述的辅料为蔗糖、右旋糖苷和明胶。
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