CN111686102B - 连翘脂素在制备用于预防或治疗骨质疏松的药物中的用途 - Google Patents
连翘脂素在制备用于预防或治疗骨质疏松的药物中的用途 Download PDFInfo
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Abstract
本发明涉及连翘脂素在制备用于预防和/或治疗骨质疏松的药物中的用途。实验证明,连翘脂素能够抑制破骨细胞形成,因而能够预防和/或治疗骨质疏松。
Description
技术领域
本发明涉及连翘脂素在制备用于预防和/或治疗骨质疏松的药物中的用途。
背景技术
连翘脂素(phillygenin),一种双环氧型木脂体化合物,是木犀科植物连翘Forsythiasuspense(Thunb.)Vahl的主要成分之一,结构如下所示:
连翘脂素在一些研究中被用于中药连翘的质量控制标记物。近年来,由于连翘脂素重要的药理活性,如抗氧化、降血脂、酪氨酸酶活性的抑制、抗炎、抗高血压及治疗肝损伤等,它受到了广泛关注(张炜等,连翘的药理学研究[J],中国现代应用药学,2000,017(001):7-10;汤韵秋等,连翘脂素对LPS诱导RAW264.7细胞炎症反应的影响[J],天然产物研究与开发,2019(7);宋蔚,桂花源连翘脂素的护肝作用及其机制研究[D],2015.4)。然而它在破骨细胞上以及破骨细胞过度活跃导致的骨质疏松中的作用还没有被研究过。
骨质疏松症(Osteoporosis,OP)是一种以骨量低下、骨微结构破坏、骨脆性增加、易发生骨折为特征的全身性骨病,多发于绝经后妇女和老年男性,严重影响老年人的身体健康及生活质量,甚至缩短寿命,增加家庭及国家财力与人力负担,已经引起全社会的高度重视。
骨组织始终处于旧骨被新骨替代的骨改建/骨转换过程中。破骨细胞(Osteoclast,OC)的骨吸收与成骨细胞(Osteoblast,OB)的骨形成功能间的“耦联”,构成了骨改建/骨转换的细胞学基础,而骨质疏松症则是骨改建/骨转换的“平衡”过程向骨吸收倾斜的结果(代晓霞等,骨质疏松症与破骨细胞性骨吸收[J],中国地方病学杂志,2002(01):74-76;王想福等,破骨细胞与骨质疏松症的关系研究进展%Research progress in therelationship between osteoclasts and osteoporosis[J],中国骨质疏松杂志,2015,021(011):1420-1424)。基于这样的认识,骨质疏松的药物防治药物的研发以及目前临床的一线药物均为破骨细胞活性抑制剂(林灵敏等,靶向破骨细胞的骨质疏松治疗药物研究进展[J],中国民康医学,2015(14):200),如双膦酸盐类及单抗药物狄诺塞麦。尽管临床上有多种抑制破骨细胞形成的骨质疏松治疗药物,但多数药物对骨质疏松的作用依然有限,而且部分药物毒副作用较大,不良反应较多。如双膦酸盐类生物利用度低,易发生胃肠道反应,长期使用易导致食道癌、下颌骨坏死等;狄诺塞麦则由于价格原因在使用上也受到一定限制。
破骨细胞来源于骨髓源巨噬细胞(BMMs),在细胞因子如巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)的调节下分化活化形成成熟破骨细胞,行使骨吸收的功能。鉴于天然产物连翘脂素的多靶点多重药理作用及破骨细胞在骨质疏松症中的关键作用,研究连翘脂素对破骨细胞前体细胞分化与功能的抑制作用,以及其对骨质疏松症的预防和/或治疗作用。
发明内容
本发明的发明人经研究后发现,连翘脂素可抑制破骨细胞的形成,可用于新型抗骨质疏松药物。
根据本发明的一个方面,提供了连翘脂素在制备用于抑制破骨细胞前体细胞的分化的药物的用途。
根据前述用途,所述破骨细胞前体细胞为骨髓源巨噬细胞(BMMs)。
根据本发明的另一方面,提供了连翘脂素在制备用于抑制破骨细胞形成的药物的用途。
根据本发明的另一方面,提供了连翘脂素在制备用于预防和/或治疗骨质疏松的药物中的用途。
根据前述用途,所述连翘脂素用于抑制破骨细胞前体细胞的分化或破骨细胞的形成。
根据前述用途,所述破骨细胞前体细胞为骨髓源巨噬细胞(BMMs)。
根据前述用途,所述骨质疏松为***缺乏引起的骨质疏松症。
根据本发明,连翘脂素能够抑制破骨细胞前体细胞,如BMMs的分化,或抑制破骨细胞的形成,因而能够预防和/或治疗骨质疏松。另外,经OVX造模实验验证显示,连翘脂素也能保护OVX引起的骨丢失,不仅能够预防***缺乏引起的骨质疏松症,而且能够在一定程度上治疗***缺乏引起的骨质疏松症。
附图说明
图1示出了连翘脂素对BMMs分化的影响(TRAP染色),图1A显示了破骨细胞前体细胞BMMs经药物处理后TRAP染色后的显微图片,图1B显示了破骨细胞前体细胞BMMs经药物处理后形成的破骨细胞数,*p<0.01,***p<0.001,如图连线所示的两组之间比较具有显著性差异。
图2示出了连翘脂素对***缺乏引起的骨质疏松症的预防作用(pq-CT)**p<0.01,如图连线所示的两组之间比较具有显著性差异。
图3示出了连翘脂素对***缺乏引起的骨质疏松症的治疗作用(pq-CT)*p<0.05,***p<0.001,如图连线所示的两组之间比较具有显著性差异。
具体实施方式
以下实施例对本发明做进一步的描述,但该实施例并非用于限制本发明的保护范围。
实施例
抗骨质疏松作用
连翘脂素抑制破骨细胞的分化及对***缺乏引起的骨质疏松症的预防和治疗作用。
1、主要实验材料与仪器
材料:胎牛血清、ɑ-MEM培养基、0.25%胰酶和青霉素/链霉素购自Gibco,抗酒石酸酸性磷酸酶(TRAP)酶染色试剂盒,DMSO,细胞因子如M-CSF和RANKL购自novoprotein,PBS(磷酸盐缓冲液)购自WISENT公司,连翘脂素(Phi)购自MCE,4周龄C57BL/6小鼠购自斯莱克,骨髓源巨噬细胞(BMMs)取自C57BL/6小鼠股骨与胫骨髓腔。
仪器:Thermo scientific公司CO2培养箱;Olympus倒置显微镜。
2、实验方法:
1)受试细胞的制备:
C57BL/6小鼠断颈处死,75%酒精浸泡消毒,无菌条件下剥离四肢长骨(股骨、胫骨),去除附着的软组织后用完全培养基反复冲洗骨髓腔,使髓腔的细胞彻底脱落,细胞悬液用细胞筛过滤后,培养于10cm细胞培养皿。次日取上清细胞离心后,用添加有30ng/mL的M-CSF的完全培养基将细胞悬起,重新再培养于新的10cm细胞培养皿中。两天后用细胞刮刀将收集贴壁的BMMs,重悬并定量接种于细胞培养板内,于5%CO2和饱和湿度条件下培养过夜,次日按不同实验要求进行处理。
2)连翘脂素对破骨细胞形成影响的测试(TRAP染色法):
破骨细胞前体细胞BMMs以5000个/孔的浓度接种到96孔板,用增殖培养基(含有30ng/mL的M-CSF的完全培养基)培养过夜。用诱导培养基(含有M-CSF(30ng/mL)+RANKL(50ng/mL)的完全培养基)配制不同浓度的连翘脂素(0,1,5和10uM)处理细胞作为实验组。细胞每2天更换一次新鲜培养液,约4-5天后BMMs融合形成泡状的成熟破骨细胞。取出细胞,PBS清洗,4%的多聚甲醛固定细胞10分钟,再用PBS清洗。按照TRAP(抗酒石酸酸性磷酸酶)染色试剂盒配制TRAP染色液,将染色液加入细胞内,置于37℃反应1h左右,倒置显微镜观察并拍照,结果示于图1A,以≥3个细胞核的破骨细胞样细胞计数,结果示于图1B。图1显示连翘脂素抑制了骨髓破骨细胞前体细胞BMMs向破骨细胞的分化过程,显示为破骨细胞的个数大大减少。
3)连翘脂素对***缺乏引起的骨质疏松症的防治作用:
***缺乏引起的骨质疏松症(OVX)小鼠造模方法:无菌条件下,8周龄雌性小鼠侧卧位,在脊柱两侧2cm、肋弓下1cm拔出长毛,取背部纵行切开皮肤,在Y型的子宫末端可见粉红色绿豆大小菜花状卵巢,结扎切除卵巢缝合腹部。对于假手术组(SHAM)小鼠,切开同样位置之后,不结扎切除卵巢即缝合上。
预防性和治疗性药物的药效研究区别在于给药起始时间上有所不同。对于预防性药物干预,术后待动物体能恢复即开始连续6周给药;对于治疗性药物干预,术后8周等动物形成明显的骨质疏松之后,才开始连续6周给药。SHAM组与OVX组小鼠给予溶剂对照(2.5%DMSO+2.5%TW80+95%生理盐水),Phi组给予30mg/kg药物,每组6只小鼠,给药方式为口服用药。给药结束后,取动物股骨,4%的多聚甲醛固定后,检测其骨密度。
图2示出了连翘脂素对***缺乏引起的骨质疏松症的预防作用。
图3示出了连翘脂素对***缺乏引起的骨质疏松症的治疗作用。
3、结果:
(1)本发明的连翘脂素对破骨细胞前体细胞分化的抑制作用研究
TRAP反映了破骨细胞的代谢活性,因此TRAP染色成为了破骨细胞鉴定的一个重要方法。为了评价连翘脂素的抗破骨细胞形成能力,发明人将不同剂量的连翘脂素与BMMs一起孵育(同时利用M-CSF和RANKL诱导其分化为破骨细胞),待破骨细胞分化成熟采用TRAP染色鉴定破骨细胞形成数量。结果表明:本发明的连翘脂素以剂量依赖性方式抑制破骨细胞的形成,具有着非常好的抑制作用。
(2)本发明的连翘脂素对***缺乏引起的骨质疏松症的防治作用
***缺乏引起的骨质疏松症是最常见的骨质疏松症之一,小鼠的卵巢切除手术(OVX)能够很好地模拟女性的***缺乏引起的骨质疏松症,也是最常用的动物模型。因此发明人在小鼠体内进一步研究了口服连翘脂素对***缺乏引起的骨质疏松症的影响。结果显示,连翘脂素也保护OVX引起的骨丢失。不仅能够预防***缺乏导致的骨质疏松(图2),而且能够在一定程度上治疗***缺乏导致的骨质疏松(图3)。
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