CN109200047A - 一种治疗类风湿性关节炎的药物及其用途 - Google Patents
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Abstract
本发明涉及一种治疗类风湿性关节炎的药物，其有效成分为蛇床子素，有效浓度为1‑100μM。蛇床子素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，蛇床子素能减轻胶原诱导类风湿性关节炎小鼠的关节损伤和炎症浸润面积、骨量缺损降低，减轻踝关节炎症损伤，能够改善类风湿性关节炎症状。本发明还公开了蛇床子素在制备防治类风湿性关节炎的药物或保健品中的用途。
Description
技术领域
本发明涉及一种治疗类风湿性关节炎的药物，并提出该药物在制备各种形式的预防和治疗类风湿性关节炎的药物或保健食品中的应用。
背景技术
现有技术公开了类风湿性关节炎(Rheumatoid Arthritis，RA)是一种慢性、系统性、进行性、自身免疫性疾病，以广泛的持续存在的关节滑膜炎及对称性、破坏性的关节病变为特征。RA的治疗却非常困难，到目前为止，仍然缺乏安全有效的治疗药物。现代医学主要是抗炎及减轻后遗症，TNFα抑制剂Etanercept、Infliximab和Adalimumab等药物能够将一部分RA患者肿胀、触痛关节计数等客观症状指标减少到50％，但仍然有许多患者在初次治疗时对其不应答，或二次治疗失败，平均每年约有10％的患者因此停药。寻找安全有效的治疗药物成为RA研究亟待解决的重大问题。中医学在治疗RA方面历史悠久，积累了丰富的临床经验，在缓解和改善临床症状方面常能取得较好疗效。
滑膜炎症是RA的主要病理表现，抑制滑膜炎症因子表达是重要的治疗靶点。炎症因子IL-6和IL-8在RA滑液的炎症反应中起关键作用。IL-6是由T、B淋巴细胞等多种细胞分泌的一类具有广泛生物学活性的小分子蛋白，它能够激活补体、诱导促炎性细胞因子释放和刺激中性粒细胞趋化，加强炎性反应。IL-6还可诱导B淋巴细胞分化和产生抗体，介导RA患者产生自身抗体和类风湿因子。在关节囊局部，IL-6活化内皮细胞使之释放IL-8和单核细胞趋化因子，并高表达黏附分子，加强白细胞向炎性部位聚集。IL-6还可刺激滑膜细胞增殖，加强破骨细胞活性，最终导致关节血管翳形成。IL-6与IL-1共同作用使基质金属蛋白酶产量增加，导致关节软骨的破坏。这是IL-6在RA发病中的重要机制。人为阻断可溶性IL-6可减轻RA症状，这些为抑制IL-6治疗RA提供了理论基础。
发明内容
针对现有技术的上述不足，根据本发明的实施例，希望提供一种疗效确切的治疗类风湿性关节炎的药物。
根据实施例，本发明技术方案提出的一种治疗类风湿性关节炎的药物，其有效成分为蛇床子素，有效浓度为1-100μM。蛇床子素是伞形科植物蛇床Cnidium monnieri(L.)Cusson的果实中提取的有效成分，它可以减轻类风湿性关节炎小鼠的炎症浸润面积，减轻关节炎症损伤。
根据实施例，本发明技术方案的提出是基于蛇床子素具有治疗类风湿性关节炎的作用。
根据一个实施例，本发明技术方案的提出是基于蛇床子素作为类风湿性关节炎治疗药物，其在患者体液中的有效作用浓度为1-100μM。
根据一个实施例，本发明技术方案中，蛇床子素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，蛇床子素作为类风湿性关节炎治疗药物，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类风湿性关节炎。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。
本发明随后的实验例进行了动物实验，建立DBA/1小鼠CIA模型。DBA/1雌鼠6-8周，适应性饲养1周后，采用牛二型胶原(2mg/mL)和完全弗氏佐剂(卡介苗浓度为4mg/ml)，完全乳化后，尾部皮下注射100μL(含100μg牛二型胶原)，21天后进行二次免疫，采用牛二型胶原(2mg/mL)和不完全弗氏佐剂，完全乳化后，尾部皮下注射100μL(含100μg牛二型胶原)。DBA/1小鼠二次免疫第1天(D20)后，将造模小鼠随机分为组，分为蛇床子素组和模型组，每组10只以上。蛇床子素组中蛇床子素溶解在0.5％的羧甲基纤维素钠(CMC-Na)制备成混悬液，模型组为0.5％的CMC-Na。蛇床子素用量为50mg/kg。灌胃给药，每天一次，连续治疗20天(D40)。进行关节炎症状评分，取踝关节，关节切片HE染色观察踝关节炎性浸润情况。经动物实验关节炎症状评分，蛇床子素可以减轻关节炎评分，体内B超检测结果表明，蛇床子素能减轻CIA小鼠踝关节滑液体积和膝关节的滑液体积；HE染色结果显示蛇床子素能减轻CIA小鼠踝关节炎症。本发明还进行了人滑膜细胞体外实验，用TNFα(10ng/mL)加不同浓度蛇床子素(0，1，100μM)处理人滑膜细胞，24小时后，抽提细胞RNA，用real time PCR方法检测炎症因子IL-6和IL-8的mRNA表达量，发现TNFα(10ng/mL)显著刺激滑膜细胞表达IL-6和IL-8，蛇床子素呈剂量依赖性降低上述IL-6和IL-8的表达量。因此，以蛇床子素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类风湿关节炎。
附图说明
图1为蛇床子素的结构简式。
图2动物关节炎症状评分图。
图3是动物实验体内的HE染色检测结果图。
图4是人滑膜细胞体外实验qPCR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。
本发明以下实验例中，蛇床子素(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伞形科植物蛇床Cnidium monnieri(L.)Cusson的果实中提取。
本发明以下实验例中，M为摩尔浓度，即mol/L；μM为微摩尔每升。
实验例
建立DBA/1小鼠CIA模型。DBA/1雌鼠6-8周，适应性饲养1周后，采用牛二型胶原(2mg/mL)和完全弗氏佐剂(卡介苗浓度为4mg/ml)，完全乳化后，尾部皮下注射100μL(含100μg牛二型胶原)，21天后进行二次免疫，采用牛二型胶原(2mg/mL)和不完全弗氏佐剂，完全乳化后，尾部皮下注射100μL(含100μg牛二型胶原)。DBA/1小鼠二次免疫第1天(D20)后，将造模小鼠随机分为组，分为蛇床子素组和模型组，每组10只以上。蛇床子素组中蛇床子素溶解在0.5％的羧甲基纤维素钠(CMC-Na)制备成混悬液，模型组为0.5％的CMC-Na。蛇床子素用量为50mg/kg。灌胃给药，每天一次，连续治疗20天(D40)。进行关节炎症状评分，取踝关节，关节切片HE染色观察踝关节炎性浸润情况。本发明还进行了人滑膜细胞体外实验，用TNFα(10ng/mL)加不同浓度蛇床子素(0，1，100μM)处理人滑膜细胞，24小时后，抽提细胞RNA，用real time PCR方法检测炎症因子IL-6和IL-8的mRNA表达量。
4.1进行动物实验关节炎症状评分。
关节炎症状评分标准如下，每一周观察一次，统计评分结果。
每只小鼠最低评分0分，最高评分为16分。
如图2所示，实验结果发现，在治疗第30、35、40天，蛇床子素组的症状评分低于CIA模型组(Osthole，蛇床子素)，效果与甲氨蝶呤类似(MTX，甲氨蝶呤)。
4.2进行动物实验的体内HE染色检测。
小鼠踝关节取材后，10％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(二甲苯4minX3→无水乙醇2min→95％乙醇2min→85％乙醇2min→75％乙醇2min→水洗)→苏木素染液1.5min→水洗→2％盐酸乙醇分化5sec→水洗→0.5％氨水返蓝15sec→水洗→伊红染液1.5min→蒸馏水洗→脱水透明(95％乙醇30sec→95％乙醇30sec→95％乙醇3min→无水乙醇30sec→二甲苯4minX3)→中性树胶封片。通风橱中放置4h后显微镜下观察。
如图3所示，在control组小鼠的踝关节，可见伊红染色的骨组织和滑膜组织组成，未见苏木素细胞核染的炎症组织出现。在CIA组小鼠的踝关节，出现大面积苏木素细胞核染的炎症组织浸润。与CIA组比较，蛇床子素组炎症面积明显减少，效果与阳性对照药甲氨蝶呤类似。实验结果提示蛇床子素可以减轻类风湿关节炎的炎症程度。
4.3进行细胞学体外实验，人滑膜细胞炎症因子mRNA表达的检测。
选用人来源的滑膜细胞株，用TNFα(10ng/mL)加不同浓度蛇床子素(0，1，100μM)处理人滑膜细胞，24小时后，抽提细胞RNA，用real time PCR方法检测炎症因子IL-6和IL-8的mRNA表达量，发现TNFα(10ng/mL)显著刺激滑膜细胞表达炎症因子，蛇床子素呈剂量依赖性降低炎症因子的表达量(如图4所示)。
以上实验结果提示，蛇床子素抑制人滑膜细胞炎症因子表达，减轻类风湿性关节炎小鼠关节炎症，说明蛇床子素是可用于防治类风湿性关节炎的药物。
Claims (4)
1.一种治疗类风湿性关节炎的药物，其特征是，有效成分为蛇床子素，有效浓度为1-100μM。
2.按权利要求1所述的治疗类风湿性关节炎的药物，其特征是，蛇床子素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。
3.蛇床子素在制备防治类风湿性关节炎的药物或保健品中的用途。
4.根据权利要求3的用途，其特征是，蛇床子素的有效浓度为1-100μM。
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| CN115844876A (zh) * | 2022-11-23 | 2023-03-28 | 中国药科大学 | 一种防治类风湿关节炎和/或骨关节炎的药物及应用 |
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| CN115844876A (zh) * | 2022-11-23 | 2023-03-28 | 中国药科大学 | 一种防治类风湿关节炎和/或骨关节炎的药物及应用 |
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