CN115919838B - 五味子酯乙的医药用途 - Google Patents
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Abstract
本发明公开了五味子酯乙的医药用途,五味子酯乙在治疗或预防骨质疏松症中的应用。所述五味子酯乙通过调控NF‑κB信号通路抑制破骨细胞的形成;本发明五味子酯乙为从传统中药五味子中提取的天然药物单体,不仅疗效确切,而且毒副作用很低,可以显著提高患者的用药依从性和提高药物的治疗效果;五味子酯乙用于预防或治疗骨质疏松价格低廉,质量控制简单方便,与现有的生物制剂相比成本低,可大幅度降低骨质疏松的治疗费用,同时还可以有效预防骨质疏松的发生。
Description
技术领域
本发明涉及医药技术领域,具体涉及五味子酯乙的医药用途。
背景技术
骨质疏松症是临床常见的老年疾病,发病率逐年增高,约占50岁以上人口的14%,严重危害着人们的健康。该病的发病机制主要是因为骨骼系统调节失衡后出现单位体积内骨量减少,机体容易引起脆性骨折,其发病率和年龄老化密切相关。骨吸收和骨形成是骨骼重建的重要组成部分,一旦打破这个动态平衡,当骨吸收大于骨形成的时候,机体将会出现骨质疏松,因此促进骨形成,抑制骨吸收是治疗骨质疏松的核心。临床根据骨质疏松的发病原因将其分为原发性骨质疏松症和继发性骨质疏松症,原发性骨质疏松症包括绝经后骨质疏松症(I型)和老年性骨质疏松症(II型),继发性骨质疏松症具有明确的病因机制,如营养不良、长期服用药物等。
骨质疏松临床表现主要包括骨强度丧失,骨脆性增加,易发生骨折,常发生在髋部、脊柱和腕部,严重者可导致残疾,严重危害着患者的健康。骨密度测定时是诊断骨质疏诊断的金标准,也是区分骨质疏松和骨量减少的可靠方法。根据世界卫生组织可将二者作如下区别,骨量减少:骨密度低于“年轻正常”成人(T评分在−1.0和−2.5之间)1.0-2.5SD;骨质疏松症:T评分−2.5或更低[18]。世界卫生组织(WHO)推荐双能X射线骨密度仪测量脊柱、髋部或前臂的骨密度更为可靠。目前,FDA批准的预防和治疗骨质疏松症的药物不足十种,但这些药物的疗效因人而异,长期服用存在一定的安全问题,如双磷酸盐和地诺单抗是目前常见的抗骨质疏松药物,但是长期服用可能出现下颌骨骨坏死的风险,而降钙素在治疗骨质疏松的过程中出现肝癌的风险增加,这使得人们对发现能够抑制破骨细胞形成的替代药物越来越感兴趣。
骨质疏松属于祖国医学“骨痿”范畴,其中肾虚是主要的病机,临床治疗肾虚型骨质疏松以滋补肝肾为主要原则。肾主骨,只有肾精充沛,才能维持骨骼的健壮,一旦肾精失常,出现肾虚,将会导致骨代谢的紊乱,最后出现骨质疏松,因此补肾精是治疗骨质疏松的根本方法。五味子为收涩类中药的典型代表,因地域之不同,各地五味子的炮制方法和称谓也不尽相同,其中应用最多的是醋酒五味子、蜜五味子、 炒五味子等,该药味酸、收涩,性温滋润,敛补兼备,入肺、肾、心经,上能敛肺止咳平喘,下能滋肾涩精止泻,内能生精宁心安神,外能固表收敛止汗,临床主要治疗遗尿尿频、梦遗滑精,自汗、盗汗、久嗽虚喘,津伤口干、短气脉虚、久泻不止、心悸失眠等。现代研究发现,五味子主要成分包括木脂素、挥发油和多糖等,这些成分具有抗炎、抗氧化、抗肿瘤和抗溃疡、抗细菌和抗真菌活性作用;此外,它们还具有保护肝脏和解毒特性。
五味子酯乙是五味子木酚素的一种生物活性物质,研究发现其具有预防和治疗抑郁症、阿尔茨海默病作用,但现有技术并没有五味子酯乙治疗骨质疏松的报道。
因此,提供具有治疗或预防骨质疏松症用途的五味子酯乙的医药用途,已是一个值得研究的问题。
发明内容
本发明的目的是提供五味子酯乙的医药用途。
本发明的目的是这样实现的:
五味子酯乙的医药用途,五味子酯乙在治疗或预防骨质疏松症中的应用。
所述五味子酯乙通过调控NF-κB信号通路抑制破骨细胞的形成。
所述五味子酯乙为从传统中药五味子中提取的天然药物单体。
本发明的有益效果是:本发明五味子酯乙为从传统中药五味子中提取的天然药物单体,不仅疗效确切,而且毒副作用很低,可以显著提高患者的用药依从性和提高药物的治疗效果;五味子酯乙用于预防或治疗骨质疏松价格低廉,质量控制简单方便,与现有的生物制剂相比成本低,可大幅度降低骨质疏松的治疗费用,同时还可以有效预防骨质疏松的发生。
附图说明
图1为本发明实施例1中采用不同浓度的五味子酯乙对骨髓巨噬细胞作用后测得不同OD值示意图;
图2为本发明实施例2中采用不同浓度的五味子酯乙干预破骨细胞形成后破骨细胞数量的示意图;
图3和图4为本发明实施例3中采用不同浓度的五味子酯乙作用于RAW细胞后制得PVDF膜并对PVDF膜采用Odyssey 扫膜仪进行扫膜得到的蛋白种类和蛋白含量图像;
图5为本发明实施例3中采用同一浓度五味子酯乙作用于RAW细胞不同时间后制得PVDF膜并对PVDF膜采用Odyssey 扫膜仪进行扫膜得到的蛋白种类和蛋白含量与时间关系的图像;
图6为本发明实施例4中治疗组和对照组的股骨近段CT扫描图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
五味子酯乙的医药用途,五味子酯乙在治疗或预防骨质疏松症中的应用。所述五味子酯乙通过调控NF-κB信号通路抑制破骨细胞的形成;所述五味子酯乙为从传统中药五味子中提取的天然药物单体。
以下为本发明的实施例,通过以下实施例对本发明进一步说明。
实施例1:细胞毒性试验(CCK8试验)检测
(1)试验材料
α-Minimum Eagle’s Medium,改良α-Minimum Eagle’s Medium,胎牛血清,青霉素/链霉素,Versene,胰酶,小鼠M-CSF,CCK8溶液。
(2)实验方法
(i)骨髓巨噬细胞的分离和培养:取6-8周龄雄性C57BL/6小鼠颈椎脱位法处死,小鼠尸体用70%乙醇喷洒消毒,取完整的股骨、胫骨后放入无菌PBS中,送入细胞培养室,剔除多余的软组织和骨骺后,用5ml的α-MEM 的完全培养基(含10%FBS+1%青霉素/链霉素混合液)反复冲洗骨髓腔直至髓腔发白,完全冲出骨髓并过滤,过滤液用离心机离心5min(1500r/min),去除上清,10ml完全培养基重悬细胞后移入T-75培养瓶中,加入M-CSF使其最终浓度为20ng/ml,放进CO2培养箱中24小时候后贴壁细胞即为骨髓巨噬细胞(BMMs)。去除上清,加入M-CSF(20ng/ml)继续培养1-2天,待培养瓶内的BMMS长满后置于超净台内,去除培养液,加入3ml Versene消化酶放入培养箱中消化5min,观察约半数细胞脱落,加入3ml完全培养基终止消化并轻柔吹打,将吹打下的细胞收集在15ml离心管内离心,弃掉上清,加入4mL完全培养基重悬并进行细胞计数。
(ii)将计数后的细胞种植入96孔板内,种植密度为4×103个/孔。
(iii)放入37°C,5%CO2培养箱内过夜培养。
(iv)第二天待细胞贴壁稳定后,在培养孔中分别加入不同浓度的五味子酯乙(0、0.03、0.1、0.3、1、3、10、30μM),放入37°C,5%CO2培养箱内培养48h后,每孔加入10μL的CCK8溶液,置于37°C培养箱孵育2h。
(v)采用TriStar2多功能酶标仪(490nm波长)测量OD值,结果进行统计学分析。
(3)实验结果:
细胞毒性试验结果表明,五味子酯乙浓度在30μM以内药物均是安全的,实验数据具体如图1所示。
实施例2:TRAP染色试验
(1)试验材料
α-Minimum Eagle’s Medium,改良 α-Minimum Eagle’s Medium,胎牛血清,青霉素/链霉素,Versene,胰酶,小鼠M-CSF,小鼠RANKL,TRAP染色液,甲醛固定液,PBST液。
(2)实验方法
(i)骨髓巨噬细胞的分离和培养:取6-8周龄雄性C57BL/6小鼠颈椎脱位法处死,小鼠尸体用70%乙醇喷洒消毒,取完整的股骨、胫骨后放入无菌PBS中,送入细胞培养室,剔除多余的软组织和骨骺后,用5ml的α-MEM 的完全培养基(含10%FBS+1%青霉素/链霉素混合液)反复冲洗骨髓腔直至髓腔发白,完全冲出骨髓并过滤,过滤液用离心机离心5min(1500r/min),去除上清,10ml完全培养基重悬细胞后移入T-75培养瓶中,加入M-CSF使其最终浓度为20ng/ml,放进CO2培养箱中24小时候后贴壁细胞即为骨髓巨噬细胞(BMMs)。去除上清,加入M-CSF(20ng/ml)继续培养1-2天,待培养瓶内的BMMS长满后置于超净台内,去除培养液,加入3ml Versene消化酶放入培养箱中消化5min,观察约半数细胞脱落,加入3ml完全培养基终止消化并轻柔吹打,将吹打下的细胞收集在15ml离心管内离心,弃掉上清,加入4mL完全培养基重悬并进行细胞计数。
(ii)将计数后的BMMs细胞以6×103个/孔种植入96孔培养板。
(iii)BMMs用α-MEM培养基培养,放进37°C,5%CO2培养箱中。
(iv)第二天BMMs细胞贴壁稳定后加入不同浓度的五味子酯乙(0、0.3、1、3、10μM)和RANKL(100ng/ml)、M-CSF(10 ng/ml)刺激向破骨细胞分化,48h换一次培养基。
(v)细胞培养6-7天后待对照组内大量破骨细胞形成后进行TRAP染色。染色方法如下:吸掉培养基,PBS洗涤3次后加入4%甲醛固定液(覆盖住细胞)室温固定10min;去除固定液,PBS洗涤3次,加入0.1%的TritonX-100液体,100ul/孔,室温通透6min后去除,PBS洗涤3次,加入1×PBST室温处理5min后去除,PBS洗涤2次,避光加入TRAP染色液,50ul/孔,37°C培养箱孵育30min,之后10min观察一次,染色成功后,去除染色液,PBS洗3次;光学显微镜下拍照并统计TRAP染色阳性,细胞核≥3个的BMMs数目;统计软件分析。
(3)实验结果:
经过TRAP染色与对照组比,用药后各组破骨细胞形成均得到抑制且呈浓度依赖性,当五味子酯乙浓度为10μM时抑制效果最明显,具体如图2所示。
实施例3:Western Blot
(1)试验材料
α-Minimum Eagle’s Medium,改良α-Minimum Eagle’s Medium,胎牛血清,青霉素/链霉素,Versene胰酶,小鼠M-CSF,小鼠RANKL,RAPI裂解液,Pro、PMSF、Pho/>酶拮抗剂,巯基乙醇,BCA试剂,PVDF膜, running buffer溶液,TBST溶液,一抗(IκBα, p-IκBα,p65,p-p65,CST公司,1000:1稀释;GAPDH,5000:1稀释),二抗(IgG rabbit)。
(2)试验方法
(i)RAW细胞复苏与传代:取出冷冻的细胞,解冻后放入15ml离心管内,800RPM/min,离心5min,弃去上清液,冲洗吹打后移至6cm培养皿内,放入培养箱内培养,密度达到70%-80%时进行传代,吸掉培养基,PBS洗2-3次,加入600μL胰酶,放进37℃培养箱中消化1-2min,见细胞大部分脱落加入3-4ml培养基终止消化,收集细胞放进15ml离心管内离心5min后弃上清,加入3ml培养基重新吹打均匀后,按1:3比例分到新的培养皿内,放入培养箱内培养。
(ii)将状态良好的RAW264.7细胞用α-MEM培养基重悬后接种在6cm皿内,放入培养箱内过夜培养。
(iii)待细胞长到占皿70%,加入不同浓度的五味子酯乙 (0、5、10μM),4h后除了阴性对照组(control组)外,其余逐个加入RANKL,使其浓度为100ng/ml。
(iv)RANKL刺激30min,提取总蛋白,并采用BCA定量法进行蛋白测定。
(v)10%的SDS-PAGE制胶并进行电泳实验,设置电压100 V,按蛋白质1 KD需转膜1min计算转膜时间,转膜结束后用5%的BSA对PVDF膜进行封闭处理,室温封闭1 h,加入一抗孵育过夜;
(vi)洗膜,并加入二抗孵育1小时,然后采用Odyssey 扫膜仪进行扫膜。
(3)实验结果:
通过图分析可知,五味子酯乙通过促进NF-κB信号通路中IκBα本体蛋白的表达,抑制IκBα和P65磷酸化表达来调控破骨细胞形成,实验结果如图3、4所示;在时间梯度上,五味子酯乙干预后随着时间增加,P-IκBα在15min后表达明显受到抑制,而IκBα、P-P65在30min后表达均明显受到抑制,如图5。
实施例4:五味子酯乙改善去卵巢小鼠骨质疏松
(1)试验材料
C57BL/6雌性小鼠,镊子,戊巴比妥钠,碘伏,剃毛器,缝线,多聚甲醛,Micro-CT。
(2)试验方法
(i)去卵巢小鼠(OVX模型)模型构建:按小鼠体重的0.1%给予腹腔注射戊巴比妥钠进行麻醉,手术部位消毒,两侧肋骨下约一横指处分别做一长约0.5cm纵行切口,钝性分离肌肉和腹膜,小心分离腹膜间的黄白色脂肪,清晰可见一暗红色且呈桑葚样的卵巢,直径约3-4mm,沿着输卵管逆向找到子宫角,将输卵管从子宫角处结扎并完整切除,术毕,分层缝合筋膜和皮肤。
(ii)本实验共有C57BL/6品系小鼠24只,共分3组;卵巢切除五味子酯乙治疗组(OVX+五味子酯乙组)8只、卵巢切除模型组(OVX)8只、假手术组(Sham组)8只,其中OVX组术后第二天死亡一只。术后4W开始给予腹腔注射,五味子酯乙治疗组注射剂量为20mg/kg(含1%的DMSO),隔天给药一次;OVX组和Sham组注射同等剂量的生理盐水(含1%的DMSO)。每周称重一次,根据体重调整注射剂量,8周后处死小鼠并取下肢骨。
(iii)Micro-CT检测标本收集和保存:完全剔除软组织,处理好的胫骨和股骨放进4%的多聚甲醛固定2-3天,水下冲洗3-4h,至置于1%PBS中保存。
(iv)Micro-CT扫描:固定标本,放进Micro-CT扫描仪进行扫描,扫描仪参数设置:扫描电压70kV,扫描时间间隔300ms,扫描层厚度14.8nm,扫描结束后进行组织三维重建。
(3)实验结果:
通过Micro-CT扫描分析,由三维重建图我们可以清楚看出OVX小鼠股骨远端骨小梁数量明显减少,而五味子酯乙治疗组的骨小梁明显密集,实验结果具体如图6所示。
Claims (3)
1.五味子酯乙在制备治疗或预防骨质疏松症药物中的用途。
2.根据权利要求1所述的五味子酯乙在制备治疗或预防骨质疏松症药物中的用途,其特征在于:所述五味子酯乙通过调控NF-κB信号通路抑制破骨细胞的形成。
3.根据权利要求1所述的五味子酯乙在制备治疗或预防骨质疏松症药物中的用途,其特征在于:所述五味子酯乙为从传统中药五味子中提取的天然药物单体。
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Schisantherin A suppresses osteoclast formation and wear particle-induced osteolysis via modulating RANKL signaling pathways;Yi He等;Biochemical and Biophysical Research Communications;第449卷(第3期);344–350 * |
五味子木脂素研究进展;魏雪苗;侯建成;刘洋;来永巍;范红艳;任旷;;吉林医药学院学报(02);115-118 * |
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