CN112791080B - 白藜芦醇二聚体tvn在制备治疗骨性关节炎的药物中的应用 - Google Patents
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Abstract
本发明公开白藜芦醇二聚体TVN在制备治疗骨性关节炎药物中的应用。所述TVN通过抑制骨吸收和骨重塑，改善骨流失，保护关节软骨；并且通过改善软骨退化和软骨损伤延缓骨性关节炎的进程；同时，通过维持软骨基质的稳态来抑制软骨基质的降解，可用于开发能够延缓或逆转骨性关节炎进程的药物。
Description
技术领域
本发明涉及中医药技术，具体涉及白藜芦醇二聚体TVN在制备治疗骨性关节炎的药物中的应用。
背景技术
骨性关节炎(osteoarthritis,OA)是最常见的年龄相关性退行性疾病，65岁以上老年人OA的发生率达到37％以上，其中75岁以上老年人OA的发生率达到80％以上，女性OA的发病率是男性的2倍。其特征是滑膜炎症，软骨退化，软骨基质降解、软骨下骨重建和骨赘形成，最终导致关节疼痛和功能障碍。临床常表现为缓慢进展的关节疼痛、肿胀、僵硬、关节活动受限等,严重者表现为关节畸形、关节功能丧失，是社会经济成本的主要来源，严重影响患者的活动功能，降低了生活质量。OA是一个多因素疾病，主要的致病因素包括：年龄、性别、肥胖、创伤、遗传及代谢因素,其发病机制到目前尚未明确，因此对于OA的防治尚无明确的方案。当前治疗OA的药物主要为非甾体抗炎药和类固醇等。关节置换术是有症状的终末期疾病的有效治疗方法，但其效果并不理想，并且假体的寿命有限。因此，对骨性关节炎研究的重点已转移到疾病预防和早期骨关节炎的治疗上。
白藜芦醇二聚体，(7R,8R)-Trans-δ-viniferin，简称TVN，结构式如下：
其是从葡萄中分离得到的一种天然产物，也可用苦瓜过氧化酶催化白藜芦醇进行生物转化得到TVN，据报道，该化合物就有较强的抗氧化生物活性。专利CN 201310238137.6通过药效学实验发现，TVN可以显著降低蔗糖导致的小鼠高血糖。在此基础上，申请人进一步研究TVN抗骨性关节炎的生物活性。
发明内容
发明目的：针对上述现有技术，本申请提供了白藜芦醇二聚体TVN在制备治疗骨性关节炎的药物中的应用。
技术方案：本发明公开了白藜芦醇二聚体TVN在制备治疗骨性关节炎的药物中的应用。
所述白藜芦醇二聚体TVN能够延缓或逆转骨性关节炎进程。
进一步的，所述白藜芦醇二聚体TVN通过抑制骨吸收来抑制骨重塑，缓解骨性关节炎的进程。
进一步的，所述白藜芦醇二聚体TVN通过缓解软骨退化来发挥抗骨性关节炎作用。
进一步的，所述白藜芦醇二聚体TVN通过抑制软骨基质的降解来保护关节软骨，延缓骨性关节炎进程。
有益效果：本申请通过实验验证白藜芦醇二聚体TVN能够通过抑制骨吸收来抑制骨重塑，缓解骨性关节炎的进程；能够缓解软骨退化，发挥抗骨性关节炎作用；能够抑制软骨基质的降解保护关节软骨，延缓骨性关节炎进程，从而白藜芦醇二聚体TVN能够用于开发抗骨性关节炎相关药物。
附图说明
图1为BV/TV定量分析TVN对ACLT造模大鼠骨密度的影响；
图2为Tb.N定量分析TVN对ACLT造模大鼠骨小梁数量的影响；
图3为Tb.Th定量分析TVN对ACLT造模大鼠骨小梁厚度的影响；
图4为Tb.Sp定量分析TVN对ACLT造模大鼠骨小梁分离度的影响；
图5为番红固绿评估TVN对ACLT造模大鼠软骨及软骨下骨结构变化的影响；
图6为甲苯胺蓝评估TVN对ACLT造模大鼠软骨下骨的保护作用；
图7为OARSI定量分析TVN对ACLT造模大鼠软骨退化的影响；
图8为MMP13，collagenⅡ免疫组化评估TVN对ACLT造模大鼠软骨基质降解的影响。
具体实施方式
下面结合具体实施例对本申请作出详细说明。
实施例所用TVN参照专利CN 201310238137.6中的方法制备所得。
实施例1前交叉韧带切除术(ACLT)动物模型的建立
所有的动物实验程序均符合中国药科大学动物伦理委员会的批准。从上海必凯购买180-200g的SPF级SD雄性大鼠50只，自由饮食饮水适应性饲养一周。之后将40只大鼠进行ACLT造模模拟OA，具体方案：将大鼠麻醉后固定，将大鼠右腿膝关节附近毛剔除干净，酒精擦拭，尽量保证无菌环境，用手术刀在大鼠髌骨旁内侧皮肤做一长约1cm的纵行切口，进而暴露膝关节，通过轻柔地将髌骨及髌韧带向外侧进行脱位，将膝关节囊打开，以便肉眼清楚观察前交叉韧带，用显微镊轻轻拨开脂肪垫，剪开前交叉韧带。在关节软骨表面滴加无菌生理盐水，以避免关节软骨表面附着的关节液在手术过程中完全干涸。然后将髌骨复位，用4-0可吸收缝合线缝合肌肉，碘伏消毒，然后用无菌针线缝合即可。造模后连续三天，每天一次，给予青霉素10万U/次。
实施例2TVN预防治疗骨性关节炎
造模一周后将50只大鼠随机分组分别为：正常组，ACLT手术造模组，玻璃酸钠阳性对照组(2.5mL：25mg)，TVN(0.5mg/kg)，TVN(1mg/kg)，每组10只。TVN用40％的PEG400溶解，膝关节注射TVN，按剂量为0.5mg/kg和1mg/kg给药，每次注射50μL，连续注射5周，每周2次。玻璃酸钠组每次注射50μL，连续注射5周，每次1次。模型组注射相同体积的40％PEG400。给药5周后，大鼠安乐处死，将右腿膝关节固定在4％多聚甲醛中，用于之后的组织学评价。
实施例3Micro-CT评估TVN对造模大鼠骨密度及软骨下骨微结构的影响
使用Scanco viva CT 80仪器扫描标本。设置参数：分辨率，21μm；电压，70kV；电流，113μA。检测的参数有：骨密度(BV/TV)、骨小梁分离度(Tb.Sp)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)。
BV/TV结果如图1所示，模型组(0.5072±0.03585)比正常组(0.6720±0.01821)显著性降低(P<0.001)，比玻璃酸钠组(0.6264±0.04524)显著性降低(P<0.001)，比TVN(0.5mg/kg)组(0.5767±0.03377)和TVN(1mg/kg)组(0.6229±0.03187)也显著性降低(P<0.001)。Tb.N和Tb.Th得到结果与BV/TV结果一致(图2，3)。Tb.Sp结果如图4所示，模型组(0.3102±0.02452)比正常组(0.2838±0.02157)显著性升高(P<0.001)，玻璃酸钠组(0.3836±0.02649)和TVN(0.5mg/kg)组(0.3320±0.03286)和(1mg/kg)组(0.3059±0.02456)较模型组显著性降低(P<0.001)。这些结果说明ACLT造模后导致膝关节不稳，早期软骨下骨微结构发生变化，以软骨破坏为主，表现在软骨下骨出现骨重塑。TVN(0.5,1mg/kg)能发挥抗骨吸收的作用来抑制软骨下骨的骨重塑，缓解骨质破坏和吸收，保护软骨下骨的骨结构，从而发挥保护关节软骨的作用。
实施例4番红固绿和甲苯胺蓝染色评估TVN对软骨下骨的保护作用
将4％多聚甲醛固定的标本换成10％的EDTA进行脱钙4周。脱钙后用不同浓度的乙醇逐级脱水，然后进行石蜡包埋，连续纵向切片取样，切片厚度约为5μm。每组随机选择6个标本进行切片，每个标本切片为6张。将得到的切片用二甲苯脱蜡，然后采用梯度乙醇水化，出去残留的二甲苯。利用番红固绿和甲苯胺蓝进行染色处理，之后将切片用梯度乙醇脱水，然后二甲苯透化，最后用中性树胶进行封片。利用切片扫描仪进行全景扫描。番红能与蛋白多糖结合而呈现红色，番红固绿染色能直观的反映关节软骨及软骨下骨结构的变化，结果如图5所示，正常组软骨下骨表面平整，软骨与软骨下骨界限清楚，潮线完整，关节间隙较大。而模型组番红固绿颜色变浅，表面出现溃烂、不平整，且出现裂缝，细胞簇不规则的出现，关节间隙明显变窄。与模型组相比，TVN(0.5,1mg/kg)给药组能显著性的改善这些变化，而玻璃酸钠组与TVN(0.5,1mg/kg)给药组之间无显著性差异。甲苯胺蓝染色主要反映软骨基质中糖胺聚糖的变化，与番红固绿得到的结果一致(图6)。最后根据OARSI(Osteoarthritis Research Society International)软骨组织病理学评分系统对图像进行评分，分级评估损伤程度，评分等级为：
0分：正常软骨
0.5分：完整软骨但蛋白多糖损失
1分：没有软骨损失但有纤维颤动
2分：垂直裂缝和表面层板损失
3分：钙化层病变占整个关节面1-25％
4分：钙化层病变占整个关节面25-50％
5分：钙化层病变占整个关节面50-75％
6分：钙化层病变>75％
结果如图7所示：正常组OARSI评分为0.5±0.4472，模型组为4.833±0.7528，与正常组相比，模型组评分显著性升高(P<0.001)，与玻璃酸钠组(3.000±0.8944)相比，模型组评分也显著性升高。TVN(0.5mg/kg)组评分为3.167±0.7528，TVN(1mg/kg)组评分为2.833±0.7528，较模型组评分显著性降低，而与玻璃酸钠组之间无显著性差异。这些结果说明关节腔注射TVN能显著性的改善ACLT造模大鼠软骨退化，减轻软骨的损伤，从而延缓骨性关节炎的进展。
实施例5免疫组化评估TVN对软骨基质降解的抑制作用
将上面的切片进行免疫组化染色，先用3％过氧化氢孵育5min，然后用PBS浸泡，再用10％山羊血清室温下孵育10min，加入MMP13(DF6494，1:100)，collagenⅡ(ab34712，1:100)一抗4℃孵育过夜。第二天用PBS清洗3次，然后滴加生物素标记的二抗，在室温下孵育30min。PBS洗过之后用辣根过氧化酶标记30min，再用PBS清洗，DAB显色，之后进行清洗、苏木素复染、封片。MMP13的染色结果如图8所示，模型组MMP13的蛋白表达较正常组显著性升高，较玻璃酸钠组和TVN(0.5，1mg/kg)给药组也显著性升高。CollagenⅡ的染色结果如图8所示，模型组CollagenⅡ的蛋白表达较正常组显著性的降低，较玻璃酸钠组和TVN(0.5，1mg/kg)给药组也显著性的降低。CollagenⅡ是软骨基质(ECM)组成的主要成分，MMP13是降解CollagenⅡ最主要的金属基质酶。这些结果显示，关节腔注射TVN能有效抑制ECM的降解，从而有效抑制骨性关节炎进展。
Claims (4)
1.白藜芦醇二聚体TVN在制备治疗骨性关节炎的药物中的应用。
2.根据权利要求1所述应用，其特征在于，所述白藜芦醇二聚体TVN通过抑制骨吸收来抑制骨重塑，缓解骨性关节炎的进程。
3.根据权利要求1所述应用，其特征在于，所述白藜芦醇二聚体TVN通过缓解软骨退化来发挥抗骨性关节炎作用。
4.根据权利要求1所述应用，其特征在于，所述白藜芦醇二聚体TVN通过抑制软骨基质的降解来保护关节软骨，延缓骨性关节炎进程。
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