WO2023035867A1 - 枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用 - Google Patents

枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用 Download PDF

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WO2023035867A1
WO2023035867A1 PCT/CN2022/112447 CN2022112447W WO2023035867A1 WO 2023035867 A1 WO2023035867 A1 WO 2023035867A1 CN 2022112447 W CN2022112447 W CN 2022112447W WO 2023035867 A1 WO2023035867 A1 WO 2023035867A1
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glycopeptide
mice
wolfberry
group
transgenic mice
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PCT/CN2022/112447
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WO2023035867A8 (zh
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乐卫东
苏国辉
徐晓兰
徐晓皎
周立兵
张力
于哲雄
王金霞
樊福
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宁夏杞肽科技有限公司
乐卫东
苏国辉
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Priority to CN202280006961.6A priority Critical patent/CN116390740A/zh
Publication of WO2023035867A1 publication Critical patent/WO2023035867A1/zh
Publication of WO2023035867A8 publication Critical patent/WO2023035867A8/zh
Priority to US18/236,085 priority patent/US20240139281A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A61K36/815—Lycium (desert-thorn)
    • 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

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  • the invention relates to the field of biomedicine, in particular to the application of wolfberry glycopeptide in the preparation of drugs for preventing and/or treating amyotrophic lateral sclerosis.
  • Amyotrophic lateral sclerosis is a complex disease involving multiple molecular mechanisms. Its etiology and pathogenesis have not yet been elucidated. Studies have found that its pathogenesis is related to defects in nuclear plasma transporters, abnormalities in RNA metabolism and binding proteins, and abnormal aggregation of proteins. , DNA repair disorders, mitochondrial dysfunction and oxidative stress, oligodendrocyte dysfunction, microglial dysfunction, axonal transport disorders, vesicle transport defects, and the toxic effects of excitatory amino acids.
  • Riluzole belongs to the benzothiazole class and is a glutamate inhibitor that can interfere with NMDA (N ⁇ methyl ⁇ D ⁇ aspartate) receptor-mediated responses, continuously regulate sodium channel currents, block calcium channel currents, Thereby reducing the presynaptic release of glutamate.
  • Riluzole has a certain effect on prolonging the survival period of patients, but has little effect on bulbar function and limb function.
  • Edaravone is a free radical scavenger, which has a certain effect on amyotrophic lateral sclerosis, but it is administered through intravenous injection, cannot be taken orally, and is expensive, so its clinical promotion has limitations.
  • the efficacy and safety of gene therapy and stem cell therapy are still controversial, and they are still in the research stage and have not yet been applied in clinical practice.
  • amyotrophic lateral sclerosis is a difficult point in the field of medicine, and it is also a hot spot in the field of scientific research.
  • Existing treatment methods have many limitations, therefore, it is of great practical significance to provide a drug for the treatment of amyotrophic lateral sclerosis.
  • the present invention provides the application of Lycium barbarum glycopeptide in the preparation of drugs for preventing and/or treating amyotrophic lateral sclerosis.
  • the invention provides the application of wolfberry glycopeptide in the preparation of medicines for preventing and/or treating amyotrophic lateral sclerosis.
  • the wolfberry glycopeptide slows down the weight loss of SOD1G93A transgenic mice.
  • the wolfberry glycopeptide prolongs the survival period of SOD1G93A transgenic mice.
  • the Lycium barbarum glycopeptide prolongs the disease course of SOD1G93A transgenic mice.
  • the wolfberry glycopeptide down-regulates the relative expression levels of inflammatory factors in the lumbar spinal cord of SOD1G93A transgenic mice.
  • the inflammatory factors include one or more of IL-1 ⁇ , IL-6 or TNF- ⁇ .
  • the dose of the wolfberry glycopeptide is 1 mg/kg to 100 mg/kg of animal body weight.
  • the dose of the Lycium barbarum glycopeptide is 20 mg/kg animal body weight.
  • the wolfberry glycopeptide can:
  • the present invention also provides a method for preventing and/or treating amyotrophic lateral sclerosis, using wolfberry glycopeptide.
  • the dose of the wolfberry glycopeptide is 1 mg/kg to 100 mg/kg of animal body weight.
  • the dose of the Lycium barbarum glycopeptide is 20mg/kg animal body weight.
  • the invention provides the application of wolfberry glycopeptide in the preparation of medicines for preventing and/or treating amyotrophic lateral sclerosis.
  • Experimental results show that the wolfberry glycopeptide is used in the preparation of drugs for preventing and/or treating amyotrophic lateral sclerosis.
  • wolfberry glycopeptide slows down the weight loss of SOD1G93A transgenic mice, prolongs the survival period of SOD1G93A transgenic mice, prolongs the course of disease of SOD1G93A transgenic mice, and down-regulates the relative expression of inflammatory factors in the lumbar spinal cord of SOD1G93A transgenic mice.
  • Fig. 1 shows technical route of the present invention
  • Fig. 2 shows the body weight curve of Tg-LBP, Tg-Con group mice
  • FIG. 3 shows that Lycium barbarum glycopeptide prolongs the survival cycle and course of disease of SOD1 G93A mice; Kaplan-Meier survival analysis curves suggest (A) (B) survival cycle, (C) ( D) Changes in onset time and (E) (F) course of disease; Log-rank test analysis showed that compared with Tg-Con group mice, the average survival period of Tg-LBP group mice was prolonged by 39 days (199.2 ⁇ 10.3 vs 160.5 ⁇ 12.2, *p ⁇ 0.05) disease; at the same time, the average duration of disease in Tg-LBP group mice was prolonged by 22 days (53.3 ⁇ 4.3 vs 31.2 ⁇ 3.0, **p ⁇ 0.01);
  • Figure 4 shows the effect of wolfberry glycopeptide on IL-1 ⁇ (A), IL-6 (B) and TNF- ⁇ (C) in the lumbar spinal cord of SOD1G93A mice;
  • Fig. 5 shows the technical route of embodiment 4.
  • Figure 6 shows the impact of Lycium barbarum glycopeptide treatment on the onset time, survival period and disease course of SOD1 G93A mice;
  • (f) comparison of the disease course between the SOD1 G93A mouse administration group and the control group; data are expressed as mean ⁇ SEM, each Group n 10, **p ⁇ 0.01, ***p ⁇ 0.001;
  • the invention discloses the application of Lycium barbarum glycopeptide in the preparation of medicines for preventing and/or treating amyotrophic lateral sclerosis.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • Lycium barbarum glycopeptide The mixture of several polysaccharide conjugates extracted from Lycium barbarum is called Lycium barbarum glycopeptide, which is referred to as LBP or LbGp in some experiments. Lycium barbarum glycopeptide is separated by column chromatography to obtain five components. Expressed as LbGp1 (LB1), LbGp2 (LB2), LbGp3 (LB3), LbGp4 (LB4), LbGp5 (LB5).
  • the raw materials and reagents used can be purchased from the market.
  • wolfberry glycopeptide was purchased from Ningxia Tianren Lycium Biotechnology Co., Ltd.
  • Lycium barbarum glycopeptide (source: Ningxia Tianren Lycium barbarum Biotechnology Co., Ltd.) was dissolved in normal saline to a final concentration of 2 mg/ml, and administered by intragastric administration at 0.01 ml/g of body weight at a concentration of 20 mg/Kg ⁇ d.
  • mice From the age of 90 days, the body weight of the mice was measured every 3 days to observe the effect of Lycium barbarum glycopeptide on the body weight of SOD1G93A mice. From the body weight curve of the mice in Figure 2, we can observe that the body weight of the mice in the Tg-Con group and the Tg-LBP group decreased significantly after the onset of the disease. Compared with the mice in the Tg-Con group, the body weight of the mice in the Tg-LBP group decreased Later in the day, the rate of decline in the weight curve slows down. The results suggested that Lycium barbarum glycopeptide had a tendency to slow down the weight loss of SOD1G93A transgenic mice, and there was a significant statistical difference (p ⁇ 0.01).
  • the Tg-LBP Tg-Con 90 19.74 ⁇ 0.95 19.60 ⁇ 0.29 93 19.74 ⁇ 0.97 19.35 ⁇ 0.4 96 19.78 ⁇ 0.96 19.88 ⁇ 0.29 99 19.82 ⁇ 0.89 19.80 ⁇ 0.39 102 20.26 ⁇ 1.01 19.90 ⁇ 0.34 105 20.26 ⁇ 0.97 19.83 ⁇ 0.30 108 20.5 ⁇ 1.03 20.00 ⁇ 0.37 111 21.18 ⁇ 1.09 20.8 ⁇ 0.21 114 20.72 ⁇ 1.05 20.18 ⁇ 0.38 117 21.08 ⁇ 1.01 20.05 ⁇ 0.42 120 21.02 ⁇ 1.27 20.6 ⁇ 0.38 123 20.8 ⁇ 1.2 20.35 ⁇ 0.21 126 21 ⁇ 1.22 20.7 ⁇ 0.07 129 20.8 ⁇ 1.02 20.6 ⁇ 0.2 132 20.84 ⁇ 1.13 19.63 ⁇ 0.33 135 20.62 ⁇ 0.87 20.18 ⁇ 0.31 138 21.16 ⁇ 1.05 20.28 ⁇ 0.43 141 21.24 ⁇ 1.17 19.33 ⁇ 0.53 144 21.04 ⁇ 1.11 19.08 ⁇ 0.58 147 20.5 ⁇ 1.22 17.
  • microglia play an important role in the occurrence and development of ALS.
  • M1 microglia promote neuroinflammation and aggravate neuronal damage by secreting pro-inflammatory factors such as IL-1 ⁇ , IL-6 and TNF- ⁇ .
  • the relative expression of pro-inflammatory factors (IL-1 ⁇ , IL-6, TNF- ⁇ ) in the lumbar spinal cord was detected by RT-PCR method.
  • the experimental results shown in Figure 4 suggest that, compared with the mice in the Tg-Con group, the relative expression levels of IL-1 ⁇ and IL-6 mRNA in the lumbar spinal cord of mice in the Tg-LBP group decreased significantly, and the difference was statistically significant.
  • Lycium barbarum glycopeptide can inhibit the expression of pro-inflammatory factors in the lumbar spinal cord of SOD1G93A transgenic mice, and play a neuroprotective role by inhibiting neuroinflammation.
  • Lycium barbarum glycopeptide (source: Ningxia Tianren Lycium barbarum Biotechnology Co., Ltd.) was dissolved in normal saline to a final concentration of 2 mg/ml, and administered by intragastric administration at 0.01 ml/g body weight at a concentration of 20 mg/Kg ⁇ d.
  • mice 32 female transgenic mice (transgenic mice, TG) were reared in mixed cages, 4 in each cage. Using random grouping, 32 transgenic female mice were randomly divided into a wolfberry glycopeptide treatment group (TG-LbGp) and a normal saline control group (TG-NS), 16 in each group. At the same time, 32 wild-type (WT) female mice with matched age and gender were randomly divided into a wolfberry glycopeptide treatment group (WT-LbGp) and a normal saline control group (WT-NS), 16 in each group.
  • WT wild-type
  • mice in the TG-LbGp and WT-LbGp groups were orally given Lycium barbarum glycopeptide solution at a dose of 20 mg/kg until the transgenic mice died, and the mice in the TG-NS and WT-NS groups The transgenic mice were gavaged with an equal volume of normal saline until they died.
  • 6 mice in each group were randomly sacrificed for detection of pathological and biochemical indicators, and the remaining 10 mice in each group were used to observe the onset time and survival period.
  • mice in the TG-NS group By comparing the disease course of the mice in the TG-NS group and the mice in the TG-LbGp group, we found that after treatment with Lycium barbarum glycopeptide, the disease course of the mice in the TG-LbGp group was prolonged by about 35.1% (37.60 ⁇ 2.566 vs 50.60 ⁇ 1.572, P ⁇ 0.001), (see Fig. 6e, Fig. 6f), the results suggest that treatment with Lycium barbarum glycopeptide can delay the disease process. The mice in the TG-NS group were prolonged by about 35.1% (37.60 ⁇ 2.566 vs 50.60 ⁇ 1.572, P ⁇ 0.001), (see Figure 6e, Figure 6f). This result suggested that treatment with Lycium barbarum glycopeptide can delay the disease process.
  • Astrocytes serve multiple functions and are the most common cells in the central nervous system, as well as a key factor in maintaining and supporting the survival of CNS motor neurons.
  • Enlarged astrocyte somata, increased synapses, and upregulated GFAP expression were found in the spinal cord and motor regions of the brain in both SOD1 mice and human ALS patients.
  • Activated astrocytes have been found to play a key role in the pathology of ALS through multiple mechanisms in animal models of ALS and patients with ALS, so regulating the activation of astrocytes may be a potential therapeutic target for ALS.
  • Lycium barbarum glycopeptide used anti-GFAP antibody to perform immunofluorescent staining on frozen sections of lumbar spinal cord of SOD1 G93A transgenic mice to evaluate the number of astrocytes.
  • qPCR technology was used to detect the relative mRNA expression of M1 microglial markers CD86 and iNOS and M2 microglial marker CD206 in the lumbar spinal cord tissue of the SOD1 G93A transgenic mouse model .
  • the experimental results showed (see Figure 9) that compared with wild-type mice, the relative expression of CD86 mRNA in the lumbar spinal cord tissue of the SOD1G 93A transgenic mouse model was significantly increased.
  • the relative mRNA expression of iNOS in mice in TG-LbGp group was 54% lower than that in TG-NS group.
  • Lycium barbarum glycopeptide may be able to regulate the activation state of microglia, inhibit its activation to M1 type, and promote its ability to activate to M2 type, thereby increasing the proportion of M2 type microglia cells and alleviating the disease of ALS progress.
  • microglia when there is neuronal injury or other damage, depending on the type and intensity of the stimulus, microglia will be activated to secrete pro-inflammatory factors that enhance cytotoxicity (such as IL-1 ⁇ , IL-6 and TNF- ⁇ ) or anti-inflammatory neuroprotective factors (eg IL-10).
  • pro-inflammatory factors such as IL-1 ⁇ , IL-6 and TNF- ⁇
  • anti-inflammatory neuroprotective factors eg IL-10
  • the neuroinflammatory response mediated by activated microglia in the SOD1 G93A mouse model plays an important role in the occurrence and progression of ALS.
  • the relative expression of inflammatory factor mRNA in the L4-5 segment of the spinal cord of the SOD1 G93A mouse model was detected by qPCR. The experimental results are shown in Figure 10.

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Abstract

本发明涉及生物医药领域，特别涉及枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。实验结果表明，枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。具体的，枸杞糖肽减缓SOD1G93A转基因小鼠体重下降，延长SOD1G93A转基因小鼠的生存周期，延长SOD1G93A转基因小鼠的病程，下调SOD1G93A转基因小鼠腰段脊髓炎症因子的相对表达量。

Description

枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用
本申请要求于2021年09月08日提交中国专利局、申请号为202111047838.2、发明名称为“枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，特别涉及枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。
背景技术
肌萎缩侧索硬化是一种涉及多种分子机制的复杂性疾病，其病因和发病机制尚未阐明，有研究发现其发病与核浆转运体缺陷、RNA代谢及结合蛋白的异常、蛋白的异常聚集、DNA修复障碍、线粒体功能障碍和氧化应激、少突胶质细胞功能缺陷、小胶质细胞功能异常、轴突运输障碍、囊泡运输缺陷、兴奋氨基酸毒性作用等相关。
随着科学技术的进步，对肌萎缩侧索硬化的病因学及发病机制有了更深入的了解，科学家根据其发病机制开发了众多的治疗方式，包括药物治疗，基因治疗，干细胞治疗等。其中，唯有利鲁唑和依达拉奉通过批准，在临床上用于肌萎缩侧索硬化的治疗。利鲁唑属于苯并噻唑类，是一种谷氨酸抑制剂，它可以干扰NMDA(N‐methyl‐D‐aspartate)受体介导的反应，持续调节钠通道电流，阻滞钙通道电流，从而减少谷氨酸的突触前释放。利鲁唑在延长患者的生存期方面具有一定作用，但对延髓功能和肢体功能的作用甚微。依达拉奉是一种自由基清除剂，对肌萎缩侧索硬化有一定的疗效，但其通过静脉注射给药，不能口服，且价格昂贵，因此临床推广有局限性。而基因治疗和干细胞治疗的疗效和安全性仍存在很大的争议，目前仍在研究阶段，尚未应用于临床。
可见，肌萎缩侧索硬化的治疗是医学领域的难点，也是科研领域的热点。现有的治疗方法具有诸多局限性，因此，提供一种治疗肌萎缩侧索硬化的 药物具有重要的现实意义。
发明内容
有鉴于此，本发明提供了枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。
在本发明的一些具体实施方案中，所述枸杞糖肽减缓SOD1G93A转基因小鼠体重下降。
在本发明的一些具体实施方案中，所述枸杞糖肽延长SOD1G93A转基因小鼠的生存周期。
在本发明的一些具体实施方案中，所述枸杞糖肽延长SOD1G93A转基因小鼠的病程。
在本发明的一些具体实施方案中，所述枸杞糖肽下调SOD1G93A转基因小鼠腰段脊髓炎症因子的相对表达量。
在本发明的一些具体实施方案中，所述炎症因子包括IL-1β，IL-6或TNF-α中的一个或多个。
在本发明的一些具体实施方案中，所述枸杞糖肽的剂量为1mg/kg动物体重～100mg/kg动物体重。
在本发明的一些具体实施方案中，所述枸杞糖肽的剂量为20mg/kg动物体重。
在本发明的一些具体实施方案中，所述枸杞糖肽能够：
(1)促进小胶质细胞向M2型活化和/或抑制小胶质细胞向M1型活化；和/或
(2)所述枸杞糖肽上调IL-10的表达。
本发明还提供了预防和/或治疗肌萎缩侧索硬化的方法，施用枸杞糖肽。在本发明的一些具体实施方案中，所述枸杞糖肽的剂量为1mg/kg动物体重～100mg/kg动物体重。在本发明的一些具体实施方案中，所述枸杞 糖肽的剂量为20mg/kg动物体重。
本发明提供了枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。实验结果表明，枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。具体的，枸杞糖肽减缓SOD1G93A转基因小鼠体重下降，延长SOD1G93A转基因小鼠的生存周期，延长SOD1G93A转基因小鼠的病程，下调SOD1G93A转基因小鼠腰段脊髓炎症因子的相对表达量。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示本发明技术路线；
图2示Tg-LBP、Tg-Con组小鼠的体重曲线；
图3示枸杞糖肽延长SOD1 G93A小鼠的生存周期和病程；Kaplan-Meier生存分析曲线提示枸杞糖肽组小鼠和生理盐水组小鼠的(A)(B)生存周期、(C)(D)发病时间和(E)(F)病程的变化；Log-rank test分析显示，与Tg-Con组小鼠相比，Tg-LBP组小鼠的平均生存周期延长39天(199.2±10.3 vs 160.5±12.2,*p<0.05)病；与此同时，Tg-LBP组小鼠的平均病程延长22天(53.3±4.3 vs 31.2±3.0,**p<0.01)；
图4示枸杞糖肽对SOD1G93A小鼠腰段脊髓IL-1β(A)、IL-6(B)和TNF-α(C)的影响；
图5示实施例4的技术路线；
图6示枸杞糖肽治疗对SOD1 G93A小鼠的发病时间、生存期和疾病病程的影响；其中；(a)SOD1 G93A小鼠给药组和对照组发病时间的kaplan-Meier统计分析；(b)SOD1 G93A小鼠给药组和对照组发病年龄比较；(c)SOD1 G93A小鼠给药组和对照组生存期的kaplan-Meier统计分析；(d)SOD1 G93A小鼠给药组和对照组发病年龄比较；(e)SOD1 G93A小鼠给药组和对照组疾病病程的kaplan-Meier统计分析；(f)SOD1 G93A小鼠给药组和对照组病程比较；数据以mean±SEM表示，每组n＝10，**p<0.01，***p <0.001；
图7示枸杞糖肽对SOD1 G93A小鼠星型胶质细胞的影响；其中(a)WT-NS，WT-LbGp，TG-NS，TG-LbGp组小鼠脊髓星形胶质细胞GFAP的免疫荧光染色图片；(b)GFAP免疫荧光染色的单位面积平均荧光密度定量分析；数据以mean±SEM表示，每组n＝3；与WT-NS相比，***p<0.001，与WT-LbGP相比，###p<0.001，与TG-NS相比，&&p<0.01；
图8示枸杞糖肽对SOD1 G93A小鼠小胶质细胞的影响；其中，(a)四组小鼠脊髓小胶质细胞Iba-1的免疫荧光染色(b)Iba-1免疫荧光染色的单位面积平均荧光密度定量分析；数据以mean±SEM表示，每组n＝3；与WT-NS相比，****p<0.0001，与WT-LbGP相比，####p<0.0001，与TG-NS相比，&&p<0.01；
图9示枸杞糖肽对SOD1 G93A小鼠腰段脊髓M1/M2型小胶质细胞标志物mRNA相对表达量的影响；其中，(a)四组小鼠脊髓组织CD86的mRNA相对表达量；(b)四组小鼠脊髓组织iNOS的mRNA相对表达量；(c)四组小鼠脊髓组织CD206的mRNA相对表达量；数据以mean±SEM表示，每组n＝3；与WT-NS相比，**p<0.01，***p<0.001；与WT-LbGP相比，#p<0.05，##p<0.01，###p<0.001；与TG-NS相比，&p<0.05，&&p<0.01；
图10示枸杞糖肽对SOD1 G93A小鼠腰段脊髓炎症因子mRNA相对表达量的影响；其中，(a)四组小鼠脊髓IL-6的mRNA相对表达量；(b)四组小鼠脊髓TNF-α的mRNA相对表达量；(c)四组小鼠脊髓IL-1β的mRNA相对表达量；(c)四组小鼠脊髓IL-10的mRNA相对表达量；数据以mean±SEM表示，每组n＝3；与WT-NS相比，****p<0.0001，***p<0.001；与WT-LbGP相比，##p<0.01，###p<0.001，####p<0.0001；与TG-NS相比，&p<0.05，&&p<0.01。
具体实施方式
本发明公开了枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。 特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
术语解释：枸杞中提取的几个多糖缀合物的混合物称之为枸杞糖肽，部分实验中以LBP或LbGp指代，枸杞糖肽经柱层析分离获得其中的五种组分，文章中常以LbGp1(LB1)、LbGp2(LB2)、LbGp3(LB3)、LbGp4(LB4)、LbGp5(LB5)表示。
本发明提供的枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用中，所用原料及试剂均可由市场购得。其中，枸杞糖肽购自宁夏天仁枸杞生物科技股份有限公司。
实验动物分组及给药
使用生理盐水溶解枸杞糖肽(来源：宁夏天仁枸杞生物科技股份有限公司)使其终浓度2mg/ml，按0.01ml/g体重灌胃给药，给药浓度为20mg/Kg·d。
10只SOD1G93A雌性小鼠：
Tg-LBP组(n＝5)，枸杞糖肽20mg/Kg·d灌胃；
Tg-Con组(n＝5)，0.01ml/g·d生理盐水灌胃。
下面结合实施例，进一步阐述本发明：
实施例1 枸杞糖肽对SOD1 G93A转基因小鼠体重的影响
从小鼠90天龄开始，每3天测一次体重，以观察枸杞糖肽对SOD1G93A小鼠体重的影响。从图2小鼠的体重曲线中，我们可以观察到Tg-Con组和Tg-LBP组小鼠在发病后体重显著下降，与Tg-Con组小鼠相比，Tg-LBP组小鼠体重下降时间稍晚，体重曲线下降速度减缓。该结果提示枸杞糖肽有减缓SOD1G93A转基因小鼠体重下降速度的趋势，且具有显著的统计学差异(p<0.01)。
表1枸杞糖肽对SOD1 G93A转基因小鼠体重的影响
  Tg-LBP Tg-Con
90 19.74±0.95 19.60±0.29
93 19.74±0.97 19.35±0.4
96 19.78±0.96 19.88±0.29
99 19.82±0.89 19.80±0.39
102 20.26±1.01 19.90±0.34
105 20.26±0.97 19.83±0.30
108 20.5±1.03 20.00±0.37
111 21.18±1.09 20.8±0.21
114 20.72±1.05 20.18±0.38
117 21.08±1.01 20.05±0.42
120 21.02±1.27 20.6±0.38
123 20.8±1.2 20.35±0.21
126 21±1.22 20.7±0.07
129 20.8±1.02 20.6±0.2
132 20.84±1.13 19.63±0.33
135 20.62±0.87 20.18±0.31
138 21.16±1.05 20.28±0.43
141 21.24±1.17 19.33±0.53
144 21.04±1.11 19.08±0.58
147 20.5±1.22 17.98±0.65
150 21.02±1.24 17.5±0.23*
153 21.22±1.3 14.08±0.5**
所有数据都以mean±SEM表示，使用GraphPad 8.0进行统计分析,*p<0.05,**p<0.01。
实施例2 枸杞糖肽对SOD1G93A转基因小鼠发病时间和生存周期的影 响
从小鼠90天龄开始，每3天测一次转棒，根据小鼠的转棒成绩和体重变化评估小鼠的发病时间。根据疾病终末期小鼠的翻正反射判断小鼠的死亡时间，以评估小鼠的生存周期。由图3实验结果提示：枸杞糖肽延长SOD1G93A转基因小鼠生存周期(n＝5,*p<0.05)和病程(n＝5，**p<0.01)。
表2
组别 生存周期(天) 发病时间(天) 病程(天)
Tg-Con组 160.5±12.2 129.8±7.3 31.2±3.0
Tg-LBP组 199.2±10.3* 132.6±6.9 53.3±4.3**
所有数据都以mean±SEM表示，使用t-tests(GraphPad 8.0)进行统计分析,*p<0.05,**p<0.01。
实施例3 枸杞糖肽对SOD1G93A转基因小鼠腰段脊髓炎症因子的影响
研究表明，小胶质细胞在ALS的发生发展中发挥重要作用。M1型小胶质细胞通过分泌IL-1β、IL-6和TNF-α等促炎因子促进神经炎症反应并加剧神经元的损伤。使用RT-PCR方法对腰段脊髓促炎因子(IL-1β，IL-6,TNF-α)的mRNA的相对表达量进行检测。如图4的实验结果提示，与Tg-Con组小鼠比较，Tg-LBP组小鼠腰段脊髓IL-1β和IL-6的mRNA的相对表达量有显著的下降，差异有统计学意义，但TNF-α的mRNA的相对表达量仅有下降趋势，差异无统计学意义。由此可见，枸杞糖肽可以抑制SOD1G93A转基因小鼠腰段脊髓促炎因子的表达，通过抑制神经炎症发挥神经保护作用。
表3
组别 IL-1β IL-6 TNF-α
Tg-Con 1.0±0.18 1.0±0.08 1.0±0.16
Tg-LBP 0.4±0.11* 0.62±0.07* 0.74±0.09
所有数据都以mean±SEM表示，使用t-tests(GraphPad 8.0)进行统计分析,*p<0.05,**p<0.01。
实施例4
1.实验动物分组及给药：
使用生理盐水溶解枸杞糖肽(来源：宁夏天仁枸杞生物科技公司)使其终浓度2mg/ml，按0.01ml/g体重灌胃给药，给药浓度为20mg/Kg·d。
将32只雌性转基因鼠(transgenic mouse，TG)混笼饲养，每笼4只。使用随机分组将32只转基因雌鼠随机分为枸杞糖肽治疗组(TG-LbGp)和生理盐水对照组(TG-NS)，每组16只。同时将年龄性别相匹配的同窝野生型(Wildtype，WT)雌鼠32只随机分为枸杞糖肽治疗组(WT-LbGp)和生理盐水对照组(WT-NS)，每组16只。具体方案如下：从90天开始，TG-LbGp和WT-LbGp组小鼠给与枸杞糖肽溶液灌胃，剂量为20mg/kg，直到转基因小鼠死亡，TG-NS和WT-NS组小鼠给与等体积生理盐水灌胃至转基因小鼠死亡。在170天龄时每组随机取6只小鼠处死取材用于病理和生化相关指标的检测，每组中剩余的10只小鼠用来于观察发病时间和生存期。
2.技术路线：如图5所示。
3.实验结果：
3.1枸杞糖肽对发病时间生存期和疾病病程的影响
本研究使用转棒实验评估小鼠的发病时间，如图6所示，给予枸杞糖肽治疗后，与TG-NS组小鼠相比，TG-LbGp组小鼠发病时间有延迟(见图6a)，但无统计学差异(133.5±4.801 vs 148.5±6.712，P＝0.0858)(图6b)。在疾病终末期使用小鼠的翻正反射判定小鼠的死亡时间以统计生存期，研究结果如图6所示，与TG-NS组小鼠比较，TG-LbGp组小鼠的平均生存期延长了约30天(171.1±5.896 vs 201.1±5.305，P<0.01)，(见图6c，图6d)该研究结果提示，枸杞糖肽治疗可延长SOD1 G93A转基因小鼠的寿命约17.5％。病程是指转基因小鼠从发病到死亡所持续的时间，该指标一定程度上反应了疾病的进展速度。通过比较TG-NS组小鼠和TG-LbGp组小鼠病程，我们发现给与枸杞糖肽治疗后，TG-LbGp组小鼠的病程较TG-NS组小鼠延长约35.1％(37.60±2.566 vs 50.60±1.572，P<0.001)，(见图6e，图6f)该结果提示，枸杞糖肽治疗可以延缓疾病进程。TG-NS组小鼠延 长约35.1％(37.60±2.566 vs 50.60±1.572，P<0.001)，(见图6e，图6f)该结果提示，枸杞糖肽治疗可以延缓疾病进程。
3.2枸杞糖肽对星型胶质细胞的影响
星形胶质细胞具有多种功能，是中枢神经系统中最常见的细胞，也是维持和支持中枢神经系统运动神经元存活的关键因素。SOD1小鼠和人类ALS患者的脊髓和大脑运动区域都发现了星形胶质细胞的胞体增大，突触增加，GFAP表达上调。在ALS的动物模型和ALS疾病患者中发现活化的星形胶质细胞通过多种机制在ALS的病理学中起关键作用，因此调控星形胶质细胞的活化可能是ALS的潜在治疗靶点。为观察枸杞糖肽对星形胶质细胞的影响，我们使用抗GFAP抗体对SOD1 G93A转基因小鼠腰段脊髓冰冻切片进行免疫荧光染色，以评估星形胶质细胞的数量。结果如图3-4示，SOD1 G93A转基因小鼠腰段脊髓组织内GFAP染色阳性的星形胶质细胞明显多于野生型小鼠；与TG-NS组小鼠相比，TG-LbGp组GFAP染色阳性的星形胶质细胞明显更少(见图7a)，TG-LbGp组GFAP单位面积平均荧光密度减少了约27.1％(见图7b)也进一步证明该结果，提示枸杞糖肽治疗可以有效抑制ALS转基因模型小鼠体内星形胶质细胞的增生。
3.3枸杞糖肽对小胶质细胞的影响
为评估枸杞糖肽治疗对小胶质细胞的作用，本研究使用抗Iba-1抗体免疫荧光染色对SOD1 G93A小鼠脊髓组织冰冻切片进行观察。研究结果如图8所示，SOD1 G93A转基因小鼠腰段脊髓组织内小胶质细胞激活程度较野生型小鼠明显增加，给予枸杞糖肽治疗后，与TG-NS组小鼠相比，TG-LbGp组小鼠腰段脊髓内Iba-1染色阳性的细胞减少(见图8a)，经过定量分析TG-LbGp组Iba-1单位面积平均荧光密度下降了28.5％，(见图8b)提示小胶质细胞的激活程度显著降低。该结果提示，枸杞糖肽治疗可以有效抑制ALS转基因小鼠体内小胶质细胞的激活。
3.4本实验使用qPCR技术对SOD1 G93A转基因小鼠模型腰段脊髓组织的M1型小胶质细胞的标记物CD86和iNOS，以及M2型小胶质细胞的标记物CD206的mRNA相对表达量进行了检测。实验结果表明(见图9)，与野生型小鼠比较，SOD1G 93A转基因小鼠模型腰段脊髓组织中CD86的mRNA相对表达量明显增加，给予枸杞糖肽治疗后，TG-LbGp组小鼠CD86的mRNA相对表达量较TG-NS组小鼠降低54％；给予枸杞糖肽治疗后，TG-LbGp组小鼠iNOS的mRNA相对表达量较Tg-NS组小鼠也有下降趋势。此外，与TG-NS，组小鼠相比较，TG-LbGp组小鼠腰段脊髓组织中CD206的mRNA相对表达量增加约66.1％。以上实验结果提示，枸杞糖肽可能能够调节小胶质细胞的活化状态，抑制其向M1型活化，促进其向M2型活化的能力，从而提高M2型小胶质细胞的比例，缓解ALS的疾病进展。
3.5枸杞糖肽对脊髓炎症因子的影响
目前的研究表明，当存在神经元损伤或其他损伤时，根据刺激的类型和强度，小胶质细胞将被激活以分泌增强细胞毒性的促炎因子(例如IL-1β，IL-6和TNF-α)或抗炎神经保护因子(例如IL-10)。SOD1 G93A小鼠模型中活化的小胶质细胞所介导的神经炎症反应在ALS疾病的发生及进展过程中发挥重要作用。在本次研究中，使用qPCR技术检测了SOD1 G93A小鼠模型L4-5段脊髓炎症因子mRNA的相对表达量。实验结果如图10所示，与野生型小鼠相比较，SOD1 G93A转基因小鼠L4-5段脊髓组织的促炎因子IL-6，IL-1β和TNF-α的mRNA的相对表达量显著增加；给予枸杞糖肽治疗后，TG-LbGp组小鼠IL-6和TNF-α的mRNA相对表达量较TG-NS组小鼠分别降低64.8％和25.9％，IL-1βmRNA相对表达量也出现了下降趋势，但差异无统计学意义。给予枸杞糖肽治疗后，TG-LbGp组小鼠IL-10的mRNA相对表达量较TG-NS组小鼠增加68.2％。以上结果提示，枸杞糖肽治疗可能抑制促炎因子的产生，从而发挥治疗作用。
以上对本发明所提供的枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用进行了详细介绍。本文应用了具体个例对本发明的原理 及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述枸杞糖肽减缓SOD1G93A转基因小鼠体重下降。
  3. 如权利要求1所述的应用，其特征在于，所述枸杞糖肽延长SOD1G93A转基因小鼠的生存周期。
  4. 如权利要求1所述的应用，其特征在于，所述枸杞糖肽延长SOD1G93A转基因小鼠的病程。
  5. 如权利要求1所述的应用，其特征在于，所述枸杞糖肽下调SOD1G93A转基因小鼠腰段脊髓炎症因子的相对表达量。
  6. 如权利要求5所述的应用，其特征在于，所述炎症因子包括IL-1β，IL-6或TNF-α中的一个或多个。
  7. 如权利要求1至6任一项所述的应用，其特征在于，所述枸杞糖肽的剂量为1mg/kg动物体重～100mg/kg动物体重。
  8. 如权利要求7所述的应用，其特征在于，所述枸杞糖肽的剂量为20mg/kg动物体重。
  9. 如权利要求1至8任一项所述的应用，其特征在于，所述枸杞糖肽：
    (1)促进小胶质细胞向M2型活化和/或抑制小胶质细胞向M1型活化；和/或
    (2)所述枸杞糖肽上调IL-10的表达。
  10. 预防和/或治疗肌萎缩侧索硬化的方法，其特征在于，施用枸杞糖肽。
PCT/CN2022/112447 2021-09-08 2022-08-15 枸杞糖肽在制备预防和/或治疗肌萎缩侧索硬化的药物中的应用 WO2023035867A1 (zh)

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