CN113521041A - 麝香酮在制备防治骨性关节炎的药物中的用途 - Google Patents
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Abstract
本发明涉及麝香酮在制备防治骨性关节炎的药物中的用途。作为治疗骨性关节炎的药物，麝香酮(Muscone)的有效作用浓度为6.25‑25μM。麝香酮与药物学上可接受的药物辅料混合形成散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。本发明经动物实验证明，麝香酮可降低骨性关节炎小鼠的OARSI评分、滑膜面积和软骨缺损，减轻骨性关节炎小鼠膝关节组织损伤和肿胀。
Description
技术领域
本发明涉及麝香酮在制备预防和治疗骨性关节炎的药物中的用途。
背景技术
骨性关节炎(Osteoarthritis,OA)是临床上常见的负重关节慢性退行性疾病，以进行性关节软骨退变，间隙狭窄、滑膜炎症增生以及软骨下骨骨重塑为主要病理变化，以疼痛、肿胀、关节畸形为主要临床表现的全关节病变。OA关节软骨退变不可逆转，目前仍缺乏有效的治疗药物，胫骨高位截骨术与关节置换手术是目前比较公认的治疗KOA的有效疗法。虽然已经发现了生物力学、代谢和遗传等危险因素，但KOA的确切发病机制仍不清楚，寻找预防和治疗KOA靶点是非常热门的研究领域。
近年来，近年来，越来越多的研究发现免疫系统，尤其是滑膜巨噬细胞在膝骨关节炎退变进程中发挥重要作用。
我们的研究证实滑膜巨噬细胞参与了KOA病理进程，促进抑炎巨噬细胞极化，减轻骨性关节炎，有助于延缓KOA退变。本发明研究的麝香酮可以促进抑炎巨噬细胞极化，降低骨性关节炎小鼠的OARSI评分、滑膜面积和软骨缺损，减轻组织损伤和肿胀，从而减轻骨性关节炎。
发明内容
针对现有技术的上述不足，根据本发明的实施例，希望找到一种疗效确切的治疗骨性关节炎的小分子化合物，并提出其医药用途。
根据实施例，本发明技术方案找到的小分子化合物为麝香酮(Muscone)。
麝香酮(结构简式如图1所示)是从麝科动物林麝成熟雄体香囊中的干燥分泌物麝香经蒸馏提取的有效成分。本发明的研究证实，它可以改善骨性关节炎关节的淋巴回流功能，降低骨性关节炎小鼠的OARSI评分、滑膜面积和软骨缺损，减轻组织损伤和肿胀，从而减轻骨性关节炎。
根据实施例，麝香酮作为骨性关节炎的治疗药物，其有效作用浓度为6.25-25μM。
根据一个实施例，麝香酮可与药物学上可接受的药物辅料混合形成散剂、膏剂、粉剂、针剂、水剂或注射剂。
根据一个实施例，麝香酮作为骨性关节炎治疗药物，为主要有效成分制备的各种形式的药物或保健食品，可以用于预防和治疗上述骨性关节炎。在使用时可以采取皮下、静脉注射或肛肠给药；注射液的使用可以任意选用生理盐水、葡萄糖、稳定剂、防腐剂、悬浮剂或乳化剂等。
本发明进行了动物实验，将40只清洁级3月龄C57BL雄性小鼠按照体重随机分为Sham组(假手术组)、KOA组(骨性关节炎组)、阳性对照组和麝香酮组(后者为本发明药物，也称治疗组)，麝香酮组每天以麝香酮灌胃，SHAM组和KOA组每天以等量生理盐水灌胃，阳性对照组每天以莫比可灌胃。经动物实验的组织形态学分析结果显示麝香酮能降低骨性关节炎小鼠的OARSI评分、滑膜面积和软骨缺损，减轻组织损伤和肿胀，从而减轻骨性关节炎。因此，以麝香酮为主要有效成分制备的各种形式的药物或保健食品，可以用于预防和治疗上述骨性关节炎疾病。
附图说明
图1是麝香酮的结构简式。
图2是动物体内实验的膝关节ABOG染色检测结果图。
图3是动物体内实验的关节OARSI评分结果图。
图4是动物体内实验的关节滑液超声检测结果图。
图5是动物体内实验的膝关节COLX免疫组化染色检测结果图。
图6是细胞体外实验的麝香酮促进抑炎巨噬细胞极化表型因子mRNA表达的检测结果图。
具体实施方式
下面结合附图和具体实施例，进一步阐述本发明。这些实施例应理解为仅用于说明本发明而不用于限制本发明的保护范围。在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等效变化和修改同样落入本发明权利要求所限定的范围。
本发明以下实施例中，麝香酮(结构简式如图1所示)采用市售品或按照中药领域常用提取方法从麝科动物林麝成熟雄体香囊中的干燥分泌物麝香经蒸馏提取。
本发明以下实施例中，M为摩尔浓度，即mol/L；μM为微摩尔每升。
实验例
将40只清洁级3月龄C57BL雄性小鼠按照体重随机分为Sham组、KOA组、阳性对照组和麝香酮组，麝香酮组每天以麝香酮10mg/kg/d灌胃，SHAM组和KOA组每天以等量生理盐水灌胃，阳性对照组每天以莫比可灌胃。连续灌胃治疗12周后检测膝关节肿胀程度及关节淋巴回流功能，然后取材膝关节，制作石蜡标本,4μm连续切片，进行阿尔辛兰，橙红G(ABOG)染色观察组织损伤、滑膜增生和关节软骨破坏情况，超声检测膝关节腔滑液体积，免疫组化染色观察软骨COLX，Q-PCR检测抑炎巨噬细胞表型基因Arg1和Fizz1。
1.小鼠关节标本ABOG染色检测。
小鼠膝关节取材后，4％多聚甲醛固定24h，PBS浸泡清洗后，脱钙18天，每2-3天换脱钙液，PBS浸泡清洗后，组织脱水机中18h脱水，石蜡包埋机中浸蜡包埋，矢状位切片(4μm)，载玻片平置于烤片机进行贴片5h，垂直置于60℃烤箱8h流蜡后，于通风橱中常规脱蜡至水；1％盐酸乙醇30sec；阿尔新蓝染-苏木素染液(AB)中40min；水洗3遍；1％盐酸乙醇分化3sec；水洗三遍；2％氨水返蓝15sec；水洗3遍；95％乙醇2min；橙红染液(OG)中2min30sec；95％乙醇2min；无水乙醇5min；二甲苯5minX3；中性树胶封片，放置于纸板上；通风橱中放置3-4h，使剩余二甲苯挥发；置于60℃烤箱烘干2h；VS120全片扫描仪扫描。
如图2所示，艾尔新蓝可将关节软骨蓝染，在SHAM小鼠，可见完整的蓝染的踝关节软骨。但是在KOA小鼠膝踝关节，蓝染的软骨出现明显缺损，关节结构破坏。与KOA组比较，麝香酮组关节软骨破坏明显减轻。在SHAM小鼠的膝关节，兰色的关节软骨平整饱满，在KOA小鼠的膝关节，出现比较严重的软骨缺失与滑膜炎症。与KOA组比较，麝香酮组软骨损伤明显减少。
麝香酮研究结果提示麝香酮可以减轻骨性关节炎的炎症程度,降低OARSI评分，如图3所示。
2.小动物超声检测小鼠膝关节滑液量。
开启Matrx麻醉机，小鼠放入麻醉盒，异氟烷吸入麻醉(浓度1％-1.5％)，将小鼠放置于恒温加热板上(28℃)，面罩维持麻醉，使用脱毛膏常规备皮，温水擦净脱毛膏，保护小鼠皮肤；将小鼠膝关节置于vevo2100小动物超声的探头下，通过B-mode与3D-mode完成对膝关节液体积的扫描，并通过本机软件完成3D重建。
如图4所示，与Sham组相比，KOA组关节滑液量明显增加，麝香酮组可显著降低KOA组关节滑液量。结果提示，麝香酮通过降低OA关节积液肿胀减轻骨性关节炎，说明麝香酮是一个可用于防治骨性关节炎的药物。
3.小鼠关节标本COLX免疫组化检测软骨退变。
石蜡组织切片常规脱蜡至水，水洗3遍；无尘纸吸干组织周围水珠，组化笔画圈并放入湿盒内；滴加3％的双氧水，室温10min；水洗3遍，滴加胰蛋白酶37℃恒温修复10min；水洗3遍，4％BSA/PBS溶液封闭1h；滴加一抗稀释液(anti-Collagen X，1:1000比例，0.4％BSA/PBS溶液稀释)并置于4℃冰箱中孵育过夜；PBS洗3遍，PBS浸泡30min；二抗Goat Anti-Rabbit(1:200比例，0.4％BSA/PBS溶液稀释)室温30min，同时配制A+B稀释液(A两滴+B两滴+5ml 0.4％BSA/PBS溶液稀释，室温30min)；PBS洗3遍，滴加A+B混合稀释液，室温30min；加DAB显色剂3min(Buffer两滴+H2O2两滴+DAB四滴+5ml蒸馏水，震荡混匀，现配现用)；水洗3遍，苏木素染液1min30sec；水洗3遍，2％氨水返蓝15sec；水洗3遍，脱水至二甲苯；中性树胶封片；通风橱中放置3-4h后置于60℃烤箱烘干；VS120全片扫描仪扫描。
如图5所示，与Sham组相比，KOA组关节软骨COLX明显升高，麝香酮组可显著降低KOA组关节软骨COLX表达的作用。
4.麝香酮对抑炎巨噬细胞表型基因Arg1和Fizz1的mRNA表达检测。
RNA提取(Trizol法)：PBS对6孔板洗涤后，加入Trizol 1ml/孔，反复吹打，室温放置5min，使其充分裂解，将裂解液移入1.5ml无酶EP管，做好标记；加入200μl的氯仿，充分震荡混匀15sec，室温下静置3min；4℃12000rpm离心15min；离心后溶液分三层，取200μl上层水相，至另1.5ml EP中；加入200μl异丙醇混匀，室温放置10min；4℃12000rpm离心15min，弃去上清；加入1ml 75％乙醇，悬浮沉淀；4℃7500rpm离心5min，弃去乙醇，用20μl枪头吸去剩余上清；室温晾干5min；加入20μl DEPC水溶解RNA样品。
反转录：配制20μl反应体系：将1.5ml EP管置于冰上，按takara说明书配制：5XPrimeScript 4μl、Oligo dT Primer 1μl、PrimeScript RT Enzyme Mix I 1μl、Random6mers 1μl、DEPC水+RNA样品13μl，将上述混合液充分振荡混匀后离心。设置逆转录程序，37℃15min，85℃5sec，4℃保存。将反转录后的cDNA样品稀释5倍后备用。
PCR反应：引物高速离心后，加入相应DEPC水配成100μM浓度，然后将引物稀释至10μM，按takara说明书配制PCR反应体系20μl：SYBR 10μl，dd水7μl，cDNA 1μl，上下游引物各1μl。95℃5min，95℃30sec→58℃30sec→72℃40sec，30个循环，72℃7min，4℃保存。同一样本同时作beta-actin内参PCR反应。待反应结束后，用Rotor Gene6.0软件自动进行绝对定量分析，并计算结果，以每一样体所含各基因的拷贝数和其beta-actin内参基因的拷贝数的比值进行比较。
如图6所示，6.25μM、25μM的麝香酮显著升高抑炎巨噬细胞表型基因Arg1和Fizz1的表达，说明麝香酮可能通过促进抑炎巨噬细胞极化，降低关节炎症，增强OA关节的自我修复功能。
Claims (3)
1.麝香酮在制备防治骨性关节炎的药物中的用途。
2.根据权利要求1所述的用途，其特征是，麝香酮的有效作用浓度为6.25-25μM。
3.根据权利要求1或2所述的用途，其特征是，麝香酮与药物学上可接受的药物辅料混合形成散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。
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