CN110269861A - D-甘露糖在制备预防和治疗骨质疏松药物中的应用 - Google Patents
D-甘露糖在制备预防和治疗骨质疏松药物中的应用 Download PDFInfo
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Abstract
本发明提供了D‑甘露糖在制备预防和治疗骨质疏松的药物、保健品或饮食补充剂的应用。从自然界植物中提取或利用生物发酵法产生的D‑甘露糖能够上调调节性T细胞与CD4+T细胞比例，抑制免疫炎症，抑制破骨细胞的活性及分化，增加增龄性骨质疏松小鼠及雌激素缺乏性骨质疏松小鼠的骨密度、骨体积分数、骨小梁厚度及数量，降低骨表面积与骨体积之比及骨小梁分离度，从而达到预防和治疗增龄性及雌激素缺乏性骨质疏松的效果。本发明产品D‑甘露糖，天然存在于植物及水果果皮中，具有来源广、易于产业化、安全有效、副作用小等优点，在预防和治疗增龄性及雌激素缺乏性骨质疏松中具有广阔的应用前景。
Description
本发明涉及一种药物的应用，具体涉及D-甘露糖在制备预防和治疗骨质疏松的药物、保健品或饮食补充剂的应用。
背景技术
骨质疏松是一种以骨量低下、骨的微结构损坏、导致骨脆性增加、易发生骨折为特征的全身性代谢性骨病，其临床表现为疼痛、脊柱变形及发生脆性骨折。2006年全国流行病学调查显示，50岁以上人群以椎骨和股骨颈骨密度值为基础的骨质疏松症总患病率为女性20.7％，男性14.4％。女性一生中发生骨质疏松的危险性(40％)高于乳腺癌、子宫内膜癌及卵巢癌的总和，男性一生发生骨质疏松的危险性(13％)高于前列腺癌。骨质疏松最严重的并发症是髋部骨折，一年内死于各种合并症者达20％，而存活者中约50％致残，生活不能自理。随着人类平均寿命的延长及老龄化社会的到来，骨质疏松症已成为人类日益重要的健康问题，其治疗花费高，并发症多，死亡率高，给家庭和社会带来沉重负担。
目前预防和治疗骨质疏松的药物主要分为抑制骨吸收类、促进骨合成类及骨矿化物类。抑制骨吸收类的药物有雌激素、双磷酸盐及降钙素等；促进骨合成类的药物有氟化物及甲状旁腺激素等；骨矿化物主要包括钙剂及维生素D等。雌激素能抑制骨吸收，但其除了作用于骨外，还作用于全身其他器官，增加了乳腺及子宫癌的风险；双磷酸盐长期应用会导致骨转换受到抑制，还会引起恶心、呕吐、腹痛、腹泻及造成颌骨骨髓炎等不良反应；降钙素能抑制骨吸收，但其不良反应包括面部或躯体皮肤潮红、恶心、呕吐等。氟化物的应用可以促进新骨形成，但长期应用会导致钙化缺陷；甲状旁腺素的不良反应包括恶心、头痛、关节痛等不适。钙剂的单纯应用效果不明显，其常见的不良反应有胃肠道刺激症状、便秘等；对于维生素D，过量补充可能引起维生素D中毒。由于目前抗骨质疏松药物的副作用限制了骨质疏松患者的长期使用。
因此，研发副作用小、能同时抑制骨吸收且促进骨合成的治疗骨质疏松药物具有深切的意义。
发明内容
针对现有预防和治疗骨质疏松药物存在的上述副作用及不足之处，本发明要解决的技术问题是提供一种有助于预防和治疗骨质疏松且副作用小的药物。旨在解决因现有预防和治疗骨质疏松药物副作用而限制了骨质疏松患者长期使用这些药物的问题。
为了解决上述技术问题，本申请采用如下技术方案予以实现：D-甘露糖在制备预防和治疗增龄性及雌激素缺乏性骨质疏松的药物、保健品或饮食补充剂的应用。
D-甘露糖通过上调调节性T细胞与CD4+ T细胞比例，抑制免疫炎症，抑制破骨细胞的活性及分化，增加增龄性骨质疏松小鼠及雌激素缺乏性骨质疏松小鼠的骨密度、骨体积分数、骨小梁厚度及数量，降低骨表面积与骨体积之比及骨小梁分离度，从而达到预防和治疗增龄性及雌激素缺乏性骨质疏松的效果。
所述D-甘露糖可以由植物水解、化学方法或者生物发酵法制备，所述植物水解是通过牙棕榈子、椰子壳，进行酸水解，所述的化学方法为用葡萄糖进行化学合成，所述的生物发酵法为微生物通过生物转化来制备D-甘露糖。
本发明产品D-甘露糖治疗骨质疏松与现有治疗骨质疏松的药物相比，具有以下优点：
本发明产品D-甘露糖，天然存在于植物及水果果皮中，具有来源广、易于产业化等优点，而且，D-甘露糖在国外已被广泛用作营养补充剂，表明其安全性和副作用小已得到肯定，故甘露糖在预防和治疗增龄性及雌激素缺乏性骨质疏松中具有广阔的开发应用前景。
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，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1.D-甘露糖显著增加增龄性骨质疏松小鼠及雌激素缺乏性骨质疏松小鼠的骨密度及改善骨质疏松小鼠的骨微结构。
具体实施方式
下面结合附图和实施例对本发明进行进一步说明。
实验过程包括如下几个方面：
(1)建立小鼠增龄性及雌激素缺乏性骨质疏松模型；
(2)实验组小鼠在饮水中额外加入甘露糖，对照组小鼠正常饮水；
(3)Micro CT分析补充甘露糖2月后，小鼠股骨、椎骨、下颌骨的变化；
(4)流式细胞术(FCM)、酶联免疫吸附测定(ELISA)、破骨细胞TRAP染色及小鼠肠道菌群多样性检测甘露糖预防和治疗骨质疏松的体内机制；
(5)培养骨髓间充质干细胞，探索甘露糖对干细胞直接促成骨作用。
具体实施步骤如下：
(1)预防和治疗骨质疏松的甘露糖溶液的制备：
称取19.8g甘露糖粉末，溶于100mL无菌蒸馏水中，制备成1.1M/L甘露糖溶液，然后进行高温高压蒸汽(102kPa，121℃，30分钟)灭菌。
(2)两种骨质疏松模型的建立：
12只代表增龄性骨质疏松6月龄的老年C57BL/6J小鼠，平均分为对照组和老年甘露糖组；12只代表雌激素缺乏性骨质疏松6周龄OVX后1个月的C57BL/6J小鼠，平均分为OVX组和OVX+甘露糖组；
(3)取各组小鼠的右侧股骨、椎骨及下颌骨去除软组织后，固定于4％多聚甲醛溶液24 小时，进行Micro CT扫描，检测甘露糖对小鼠股骨、椎骨及下颌骨的骨密度及骨微结构参数的影响，从而检测甘露糖对两种骨质疏松的预防和治疗效果，扫描参数为：电流为220μA，电压为60kV，曝光时间为1500ms；Micro CT扫描结果如下：
表1各组骨密度及骨微结构参数
注：*P＜0.05，**P＜0.01，与老年对照组相比；#P＜0.05，##P＜0.01，与VOX组相比；(n＝6；X±SD)
表1结果表明，增龄性骨质疏松(老年组)及雌激素缺乏性骨质疏松(OVX组)小鼠摄入甘露糖2个月后，股骨、椎骨及下颌骨的骨密度(BMD)显著增加；股骨和椎骨的骨体积与总体积之比(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N)显著增加；骨表面积与体积之比(BS/BV)及骨小梁分离度(Tb.SP)显著下降，均有统计学差异。
(4)甘露糖摄入2个月后，小鼠股骨去除软组织后，放入10％EDTA脱钙液中脱钙2周，切取5μm厚的组织，用HE及TRAP染色试剂盒进行HE染色(从组织切片中检测甘露糖对小鼠骨组织微结构的改变)和TRAP染色(检测甘露糖对破骨细胞的抑制作用)，TRAP染色破骨细胞计数结果如下：
表2各组破骨细胞计数分析
注：**P＜0.01，与老年对照组相比；(n＝6；X±SD)
表2结果表明，摄入甘露糖后，破骨细胞面积与骨表面积之比(OC.S/BS)及单位面积破骨细胞数量显著下降，进一步表明甘露糖预防和治疗骨质疏松是通过抑制可以破骨细胞的生成来实现。
(5)分别于小鼠摄入甘露糖2周及2月后，手术摘取小鼠脾脏，将脾脏在70μm细胞筛网上研磨成细胞悬液，裂解红细胞，用含5％血清的1640洗涤细胞，制备脾脏单细胞；加入CD4、CD25抗体，避光孵育20分钟，用含1％血清的PBS洗涤细胞后，加入核内抗原专用固定破膜液，孵育30分钟，再用核内抗原专用破膜洗液洗涤细胞两次，加入Foxp3抗体，孵育30分钟，再用核内抗原专用破膜洗液洗涤细胞，再用含1％血清的PBS洗涤细胞后，重悬后用流式细胞仪获取分析调节性T(Treg)细胞，结果如下：
表3 Treg细胞与CD4+ T细胞比例
注：*P＜0.05，与老年对照组相比；#P＜0.05，与OVX组相比；(n＝6；X±SD)
表3表明摄入甘露糖2周后，调节性T细胞(Treg细胞)与CD4+ T细胞比例上调；2个月后调节性T细胞比例趋于稳定，表明甘露糖可以上调Treg细胞比例，进一步抑制免疫炎症和破骨细胞。
可以理解的是，以上关于本发明的具体描述，仅用于说明本发明而并非受限于本发明实施所描述的技术方案，本领域的普通技术人员应当理解，仍然可以对本发明进行局部修改或等同替换，以达到相同的技术效果；只要满足使用需要，都在本发明的保护范围之内。
Claims (7)
1.D-甘露糖在制备预防和治疗骨质疏松的药物、保健品或饮食补充剂的应用。
2.如权利要求1所述的应用，其特征在于：D-甘露糖能够抑制破骨细胞的生成。
3.如权利要求1所述的应用，其特征在于：D-甘露糖能够上调脾脏及骨髓中调节性T细胞与CD4+T细胞比例，抑制白细胞介素4(IL4)、白细胞介素6(IL6)、白细胞介素17(IL17)、肿瘤坏死因子及(TNFα)、核因子κB受体活化因子配体(RANKL)相关炎症因子的表达。
4.如权利要求1所述的应用，其特征在于：D-甘露糖能够抑制破骨细胞的生成。
5.如权利要求1所述的应用，其特征在于：D-甘露糖能够增加骨密度、骨体积分数、骨小梁厚度及数量，降低骨表面积与骨体积之比及骨小梁分离度，从而达到预防和治疗增龄性及雌激素缺乏性骨质疏松的作用。
6.如权利要求1所述的应用，其特征在于：所述D-甘露糖可以由植物水解、化学方法或者生物发酵法制备。
7.如权利要求6所述的应用，其特征在于：所述植物水解是通过牙棕榈子、椰子壳，进行酸水解，所述的化学方法为用葡萄糖进行化学合成，所述的生物发酵法为微生物通过生物转化来制备D-甘露糖。
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| US20070072244A1 (en) * | 2003-05-19 | 2007-03-29 | Quark Biotech, Inc. | Use of the endo-180 gene and polypeptide for diagnosis and treatment |
| CN107921083A (zh) * | 2015-05-27 | 2018-04-17 | 皮埃蒙特阿米阿伏伽德罗东方大学 | 在治疗骨质减少症和骨质疏松症中的B7h受体配体 |
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| US20070072244A1 (en) * | 2003-05-19 | 2007-03-29 | Quark Biotech, Inc. | Use of the endo-180 gene and polypeptide for diagnosis and treatment |
| CN107921083A (zh) * | 2015-05-27 | 2018-04-17 | 皮埃蒙特阿米阿伏伽德罗东方大学 | 在治疗骨质减少症和骨质疏松症中的B7h受体配体 |
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