CN113559165A - 甜橙黄酮在制备破骨细胞活性抑制剂中的应用 - Google Patents
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Abstract
本发明公开了甜橙黄酮在制备破骨细胞活性抑制剂中的应用，本发明明确了甜橙黄酮对破骨细胞分化和激活的抑制作用，这些结果提示，甜橙黄酮极有可能通过抑制破骨细胞过度生成和激活从而在溶骨性相关疾病(如骨质疏松症、植入体无菌性松动、骨髓炎、牙周炎、多发性骨髓瘤、Paget’s病、恶性肿瘤的高钙血症、牙槽骨缺失、免疫抑制治疗或长期使用糖皮质激素的引起的骨质丢失等)中具备潜在制药作用。
Description
技术领域
本发明涉及医药应用技术，特别涉及甜橙黄酮在制备破骨细胞活性抑制剂中的应用。
背景技术
破骨细胞在骨重建过程中发挥启动和先锋的作用。作为体内唯一具有骨吸收功能的细胞，破骨细胞有极强的溶骨能力，其功能过度活跃时，骨吸收亢进，将会导致一系列骨吸收过度活跃所致疾病，如骨质疏松症、植入体无菌性松动、骨髓炎、牙周炎、多发性骨髓瘤、Paget’s病、恶性肿瘤的高钙血症、牙槽骨缺失、免疫抑制治疗或长期使用糖皮质激素引起的骨质丢失。靶向抑制破骨细胞的活性和功能是预防和治疗此类疾病最有效的策略之一。目前针对破骨细胞的药物并不多，临床上常用的抑制破骨细胞活性的药物包括双膦酸盐(Bisphosphonates，BPs)、雌激素(Estrogen)及其受体调节剂(SERMs)、降钙素(Calcitonin)等，其中双膦酸盐类药物应用最广泛。这些药虽然有一定效果，但仍有不同程度的局限，比如唑来膦酸(双膦酸盐类)可能会引发肾毒性和心脏毒性等副作用；迪诺塞麦(Denosumab)长期使用可能对免疫系统功能产生潜在影响，且价格昂贵，并不适用于大多数家庭。因此研发更为经济且毒副作用小的药物是非常有必要的。
目前尚未见甜橙黄酮对破骨细胞影响的相关报道。
发明内容
发明目的：针对上述现有技术，本发明提供了甜橙黄酮在制备破骨细胞活性抑制剂中的应用。
技术方案：本发明公开了甜橙黄酮在制备破骨细胞活性抑制剂中的应用。
本发明还公开了甜橙黄酮在制备治疗溶骨性相关疾病的药物中的应用。
进一步的，所述药物是通过抑制破骨细胞过度生成和激活达到治疗效果。
其中，所述溶骨性相关疾病包括骨质疏松症、植入体无菌性松动、骨髓炎、牙周炎、多发性骨髓瘤、Paget’s病、恶性肿瘤的高钙血症、牙槽骨缺失、免疫抑制治疗或长期使用糖皮质激素的引起的骨质丢失。
本发明还公开了一种药物组合物，包括甜橙黄酮和药学上可接受的载体。
本发明还公开了所述药物组合物在制备治疗溶骨性相关疾病的药物中的应用。
进一步的，所述药物组合物通过抑制破骨细胞过度生成和激活达到治疗效果。
本发明通过体外细胞培养，首先检验甜橙黄酮(SIN)对小鼠单核巨噬细胞(BMM细胞)的毒性，发现在320μM浓度及以下时，对BMM细胞无毒性作用。在后续的实验中，所用SIN的浓度低于或等于20μM。随后通过TRAP染色检测了SIN对RANKL诱导的破骨细胞分化的抑制作用。结果发现，SIN对体外培养的破骨细胞形成有抑制作用，且随着SIN的药物浓度的增加其抑制作用越明显。进一步检测SIN对破骨细胞分化抑制的阶段时发现，SIN在破骨细胞分化早期抑制作用明显，长期加药效果最好，晚期给药抑制效果最不理想。NFATc1是破骨细胞分化的重要转录调节因子，使用SIN(20μM)检测对BMMs分化过程中NFATc1表达的影响，发现SIN明显抑制了NFATc1的表达量。
本发明还通过建立LPS诱导钙化骨溶解动物模型，通过比较假手术组、模型组、SIN给药高剂量组、SIN给药低剂量组的小鼠颅骨骨溶解情况，发现SIN给药后使小鼠造模后的颅骨损伤情况得到改善。
有益效果：本发明明确了SIN对破骨细胞分化和激活的抑制作用，这些结果提示，SIN极有可能通过抑制破骨细胞过度生成和激活从而在溶骨性相关疾病(如骨质疏松症、植入体无菌性松动、骨髓炎、牙周炎、多发性骨髓瘤、Paget’s病、恶性肿瘤的高钙血症、牙槽骨缺失、免疫抑制治疗或长期使用糖皮质激素的引起的骨质丢失)中具备潜在制药作用。
附图说明
图1是甜橙黄酮对BMMs细胞活性的影响；
图2是甜橙黄酮抑制破骨细胞分化结果；
图3是SIN在破骨细胞形成早期发挥抑制作用结果；
图4是SIN对BMMs细胞凋亡检测结果；
图5是甜橙黄酮能抑制破骨细胞形成过程中标志性基因的表达；
图6是甜橙黄酮能抑制破骨细胞分化过程中的重要的转录调节因子NFATc1；
图7是SIN在体内对骨溶解的保护作用。
具体实施方式
下面结合具体实施例对本发明作出详细说明。
实验耗材：8周C57BL/6雄鼠采购于北京维通利华实验动物技术有限公司；甜橙黄酮(Sinensetin，SIN，结构式如图1A所示，纯度≥98％)，购自成都瑞芬思生物科技有限公司；α-MEM培养基购自美国Gibco公司；胎牛血清购自美国Invitrogen公司；小鼠重组RANKL和M-CSF购自R&D公司；MTS试剂购自Sigma-Aldrich；Trizol试剂、PrimeScript反转录试剂以及SYBR Premix Ex Taq试剂购自南京诺唯赞生物科技股份有限公司；抗酒石酸酸性磷酸酶染色试剂购自Sigma-Aldrich公司。
小鼠骨髓来源巨噬细胞(BMM)的分离和培养：颈椎脱臼法将8周C57BL/6小鼠处死，75％酒精浸泡消毒5min后，在超净工作台中分离小鼠两条股骨和胫骨，剔除多余组织，剪开股骨和胫骨两端，用1ml注射器吸取α-MEM细胞培养基，反复冲洗至骨髓腔发白。收集含有骨髓细胞的冲洗液，1200rpm离心5min后，用10ml含有M-CSF的α-MEM细胞培养基重悬细胞，接种于两个10cm培养皿中培养；待细胞贴壁换液，同时将未贴壁细胞用PBS清洗掉，继续培养2-3天后用于破骨细胞的诱导分化实验。
实施例1：MTT法检测甜橙黄酮对细胞活力的影响
取对数生长期的BMM细胞，加入终浓度为0μM、2.5μM、5μM、10μM、20μM、40μM、80μM、160μM、320μM的SIN，每个浓度设4个复孔，培养48h后用MTT比色法测定细胞活力，检测OD490nm，结果如图1B所示。根据图示可见，SIN在320μM浓度及以下时，对BMM细胞无毒性作用。
实施例2：甜橙黄酮对破骨细胞分化的影响
BMMs细胞计数后，按照8x103个/孔接种于96孔板中，细胞贴壁后加入含有RANKL(100ng/ml)、M-CSF(30ng/ml)和不同浓度SIN(0μM、5μM、10μM、20μM)的培养基，诱导BMMs形成破骨细胞，在5-7天形成巨型泡状多核细胞后进行TRAP染色，统计每个浓度每个孔破骨细胞数量(细胞核数量≥3)和面积。结果如图2所示，其中(A)(B)在96孔板中用含有RANKL、M-CSF和不同浓度的SIN(0μM、5μM、10μM、20μM)诱导BMMs分化形成破骨细胞，并进行TRAP染色；(C)每个破骨细胞平均面积；(D)细胞核数量大于3的每孔破骨细胞数量。(显著性差异：*P<0.05,**P<0.01,***P<0.001vs SIN 0μM处理组)。根据图示可见，SIN对体外培养的破骨细胞形成有抑制作用，且随着SIN的药物浓度的增加其抑制作用越明显。
实施例3：甜橙黄酮抑制破骨细胞分化的作用阶段
用20μM SIN在不同的时间段处理BMMs细胞，探究SIN在破骨细胞形成的哪个阶段产生抑制作用，药物处理时间段分别是：全程药物处理(5天均加RANKL和SIN)；早期药物处理(前2天为RANKL+SIN共孵育，后3天只加RANKL孵育)；后期药物处理(前2天只加RANKL诱导，后3天为RANKL+SIN共孵育)。对处理后的细胞进行TRAP染色，统计药物处理不同阶段破骨细胞数量(细胞核数量≥3)和面积。结果如图3所示，其中，(A)各药物处理组TRAP染色代表性图片；(B)不同药物处理组破骨细胞数目和面积统计。(显著性差异：**P<0.01和***P<0.001vs Ctrl)。根据图示可见，SIN在破骨细胞分化早期抑制作用明显，长期加药效果最好，晚期给药抑制效果最不理想。
实施例4：甜橙黄酮对BMMs凋亡影响
不同浓度的SIN(0μM、5μM、10μM、20μM)处理BMMs细胞24h后，收集细胞，根据Annexin V-FITC/PI细胞凋亡检测试剂盒(凯基公司，南京)说明书操作，采用流式细胞术检测SIN对BMMs凋亡的影响。结果如图4所示，根据图示可见，SIN在选择的浓度范围内对于BMMs凋亡没有影响。
实施例5：Q-PCR检测破骨细胞分化标志性基因的表达
用不同浓度的SIN、100ng/ml的RANKL和30ng/ml的M-CSF处理BMM细胞5天后，提取细胞总RNA，按照Vazyme反转录试剂所给说明书进行逆转录。用Vazyme(SYBR Premix ExTaq)试剂将体系所需引物、模板、酶和无RNA酶水加入八连管中，在实时定量PCR仪上进行检测。相关基因主要包括：CTSK、CTR、TRAP和NFATc1。
引物序列：
用Q-PCR检测，结果如图5所示，CTSK(A)、CTR(B)、TRAP(C)、NFATc1(D)表达量，(*P<0.05,**P<0.01和***P<0.001vs SIN 0μM处理组)。根据图示可见，SIN能抑制破骨细胞分化标志性基因的表达。
实施例6：Q-PCR检测破骨细胞分化过程中的重要的转录调节因子NFATc1的表达
用RANKL(100ng/ml)和RANKL(100ng/ml)+SIN(20μM)分别处理BMMs 5天，3天，1天和0天，并提取细胞总蛋白，进行Western Blot检测NFATc1表达情况。结果如图6所示，(**P<0.01)，SIN(20μM)抑制BMMs分化过程中NFATc1的表达。
实施例7：甜橙黄酮对于LPS诱导的颅骨溶解的治疗作用
将动物随机分为4组(n＝4)：PBS对照(Sham)、LPS注射液(5mg/kg体重)(Vehicle)和不同浓度的SIN(25和50mg/kg)。假手术组皮下注射PBS(100μl)，其他组在小鼠颅骨矢状中线缝处注射LPS(100μl)。SIN处理组或Vehicle组均为隔天注射LPS，其中SIN处理组在不注射LPS时给予相应浓度的药物腹腔注射，周期为7天。将4％多聚甲醛固定后的颅骨进行Micro-CT扫描和重构，颅骨脱钙后包埋，切片得颅骨最大面H&E染色以及TRAP染色。结果如图7所示，(A)Micro-CT骨扫描后，小鼠颅骨形态。以小鼠颅骨表面矢状线和冠状线的交汇处为圆心，选取直径为4cm的圆为感兴趣区域(ROI)，对ROI进行骨参数分析，得出(B)BMD、(C)BV/TV的相关数据。(D)颅骨脱钙后包埋，切片得颅骨最大面H&E染色以及TRAP染色。(E)(F)浸润纤维化面积对组织总组织面积(侵蚀面积，％)、每个骨表面破骨细胞表面的百分比(OcS/BS，％)。根据图示可见，SIN给药后使小鼠造模后的颅骨损伤情况得到改善，骨吸收减少，骨密度增加。
Claims (7)
1.甜橙黄酮在制备破骨细胞活性抑制剂中的应用。
2.甜橙黄酮在制备治疗溶骨性相关疾病的药物中的应用。
3.根据权利要求2所述应用，其特征在于，所述药物通过抑制破骨细胞过度生成和激活达到治疗效果。
4.根据权利要求2所述应用，其特征在于，所述溶骨性相关疾病包括骨质疏松症、植入体无菌性松动、骨髓炎、牙周炎、多发性骨髓瘤、Paget’s病、恶性肿瘤的高钙血症、牙槽骨缺失、免疫抑制治疗或长期使用糖皮质激素的引起的骨质丢失。
5.一种药物组合物，其特征在于，包括甜橙黄酮和药学上可接受的载体。
6.权利要求5所述药物组合物在制备治疗溶骨性相关疾病的药物中的应用。
7.根据权利要求6所述应用，其特征在于，所述药物组合物通过抑制破骨细胞过度生成和激活达到治疗效果。
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| CN114732812A (zh) * | 2022-05-06 | 2022-07-12 | 重庆西南果品营养研究院 | 甜橙黄酮在制备高尿酸血症产品中的应用及产品 |
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