CN114931571A - 没食子酸甲酯在制备骨关节炎治疗药物中的应用 - Google Patents
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Abstract
本发明涉及医药技术领域，具体是没食子酸甲酯在制备骨关节炎治疗药物中的应用。没食子酸甲酯可减轻软骨细胞的损伤及改善骨关节炎，直接保护软骨细胞；还可靶向抑制软骨细胞激活的Pi3k/Akt信号通路，从而减少炎症因子释放。此外，没食子酸甲酯还可通过减轻骨关节炎的炎性微环境引起的软骨细胞活力抑制和凋亡，起到间接保护软骨的作用，从而治疗和减缓骨关节炎。本发明为更好地理解骨关节炎的病理过程，以及今后对治疗骨关节炎药物的开发提供了新的思路与借鉴。
Description
技术领域
本发明涉及医药技术领域，具体地说，是没食子酸甲酯在制备骨关节炎治疗药物中的应用。
背景技术
骨关节炎是目前全球范围内致残率、致畸率较高的进行性慢性关节疾病之一，主要以软骨细胞减少、关节基质破坏降解为特征。目前治疗上主要是以缓解临床症状为主，缺乏有效改善软骨退变的药物。在治疗骨关节炎方面，中医药具有疗效确切、副作用小等优点，已经被应用于临床，并且在联合中药治疗后患者康复情况明显好于单纯性常规治疗。说明中医药在治疗骨关节炎中具有良好的疗效。但如何利用中医药防治骨关节炎是我们研究的方向。
软骨细胞凋亡与膝骨关节炎严重程度之间存在相关性。软骨细胞凋亡可能会导致细胞外基质功能的丧失和软骨破坏。尽管尚不清楚软骨细胞凋亡是软骨退化的诱导剂还是软骨受损后的产物，但软骨细胞凋亡是骨关节炎发病机理的重要方面。因此，关节软骨细胞凋亡及异常炎症反应是导致关节组织发生退行性变化的起始病症之一，如何缓解关节内局部炎症压力及其诱发的软骨细胞凋亡等成为治疗该病症的关键。
没食子酸甲酯(Methyl gallate，结构式如式I所示)存在与多种中药中，一种具有抗氧化，抗癌和抗炎活性的植物酚类。也有报道没食子酸甲酯可以调节免疫反应、抑制炎症、保护血管。
中国专利文献CN114028376A公开了MG在制备高尿酸血症肾病和/或痛风性关节炎的NLRP3通路抑制剂中的应用。中国专利文献CN108530314A公开了一种没食子酸甲酯类似物及其用于制备抗炎药物的应用。但是关于没食子酸甲酯在制备型骨关节炎治疗药物中的应用目前还未见报道。
发明内容
本发明的目的在于提供没食子酸甲酯在制备骨关节炎治疗药物中的应用。本发明首次发现，没食子酸甲酯通过抑制Pi3k/Akt信号通路激活，抑制软骨细胞凋亡而保护软骨细胞，从而减缓和治疗骨关节炎。
为了实现上述目的，本发明基于以下技术方案：
(1)提取大鼠的原代软骨细胞，检测没食子酸甲酯是否直接减轻炎症下的软骨细胞退变；
(2)炎症诱导后同时给予没食子酸甲酯，用PCR、Western Blot、免疫荧光检测凋亡相关的Pi3k/Akt信号通路，探究Pi3k/Akt下游相关凋亡表达改变情况，评估没食子酸甲酯保护软骨细胞的作用；
具体涉及以下实验过程：
1.原代软骨细胞的提取与鉴定；
2.CCK-8法筛选没食子酸甲酯干预的最佳浓度；
3.没食子酸甲酯抑制骨关节炎模型软骨细胞的分解代谢能力；
4.没食子酸甲酯缓解骨关节炎模型软骨细胞的合成能力；
5.没食子酸甲酯下调Pi3k/Akt通路影响软骨细胞功能；
6.没食子酸甲酯影响软骨细胞凋亡基因的影响。
基于上述技术方案，本发明的第一方面，提供没食子酸甲酯在制备骨关节炎治疗药物中的应用。
进一步的，所述的骨关节炎包括但不限于白介素-1β型骨关节炎或风寒湿痹型骨关节炎。
进一步的，所述的应用中，没食子酸甲酯用于制备减缓和治疗因炎症对软骨细胞的损伤而导致的骨关节炎的药物。
进一步的，所述的应用中，没食子酸甲酯通过减轻炎症对软骨细胞的损伤，直接保护软骨细胞，从而减缓和治疗骨关节炎。
进一步的，所述的应用中，没食子酸甲酯通过抑制Pi3k/Akt信号通路激活，抑制软骨细胞凋亡而保护软骨细胞，从而减缓和治疗骨关节炎。
本发明的第二方面，提供没食子酸甲酯在制备减轻炎症对软骨细胞损伤的药物中的应用。
本发明的第三方面，提供没食子酸甲酯在制备抑制Pi3k/Akt信号通路激活的药物中的应用。
本发明的第四方面，提供一种骨关节炎治疗药物，所述的药物的活性成分为没食子酸甲酯。
进一步的，所述的药物还包括药学上可接受的载体或辅料。
本发明优点在于：
1、本发明首次公开了没食子酸甲酯的保护体外软骨细胞作用及其在治疗骨性关节炎中的应用。
2、本发明实验结果证明，没食子酸甲酯可减轻软骨细胞的损伤，直接保护软骨细胞；还可靶向抑制控制软骨细胞激活的Pi3k/Akt信号通路，从而减少炎症因子释放。此外，没食子酸甲酯还可通过减轻骨关节炎的炎性微环境引起的软骨细胞活力抑制和凋亡，起到间接保护软骨的作用，从而治疗和减缓骨关节炎。
3、本发明得到了大量实验数据的支持，表明没食子酸甲酯具有显著的软骨细胞保护作用，并可在未来治疗骨关节炎中发挥重要作用。本发明为更好地理解骨关节炎的病理过程，以及今后对治疗骨关节炎药物的开发提供了新的思路与借鉴。
附图说明
图1为没食子酸甲酯对大鼠软骨细胞的细胞毒性检测图；其中，A为24小时CCK-8检测评估细胞的生存能力，B为48小时CCK-8检测评估细胞的生存能力。
图2为没食子酸甲酯抑制骨关节炎模型中软骨细胞的分解代谢能力图；其中，A为RT-PCR检测SD新生大鼠软骨细胞经处理后的促炎因子(IL-6、IL-1β)和分解代谢能力(ADAMTS5、MMP13)的mRNA表达。B为Western Blot检测SD新生大鼠软骨细胞经处理后的iNos、Cox-2和MMP9的蛋白表达。
图3为没食子酸甲酯缓解骨关节炎模型软骨细胞的合成能力图；其中，A为Col2α1与DAPI(细胞核)的各组免疫荧光图像。B为各组的Col-2α1的荧光强度情况。C为WesternBlot检测各组的Col2α1、Acan和Sox-9的蛋白表达。
图4为没食子酸甲酯对软骨细胞Pi3k/Akt通路情况图。
图5为没食子酸甲酯对软骨细胞凋亡基因的影响图。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1：没食子酸甲酯对大鼠软骨细胞的细胞毒性
取出生24h的SD大鼠四肢关节处软骨，提取软骨细胞。P1-2代细胞进行研究。CCK8法检测没食子酸甲酯对大鼠软骨细胞的细胞毒性。
不同浓度没食子酸甲酯在不同时间点对软骨细胞活力的影响(图1)。不同浓度没食子酸甲酯单体干预P1代软骨细胞后，分别于24小时和48小时两个时间点检测450nm处的吸光度值。如图1显示，100μM的没食子酸甲酯干预软骨细胞24、48h时，细胞出现明显生长抑制现象。因此，50μM的没食子酸甲酯对软骨细胞没有毒性。
实施例2：没食子酸甲酯在骨关节炎中的保护作用
取出生24h的SD大鼠四肢关节处软骨，提取软骨细胞。P1-2代细胞进行研究。
模拟体外软骨细胞炎症状态：白介素-1β(IL-1β)20ng/ml+风寒湿血清10％共同干预软骨细胞。风寒湿血清制备方法：首先运用改良Hulth法+风寒湿人工气候箱干预建立风寒湿骨关节炎模型(条件设置+为湿度95±2％、温度11±1℃、风力3级模拟风寒湿环境因素)，分离大鼠血清，灭活备用。
设置分组，共计4组：分别是对照组、模型组(IL-1β20ng/ml)、风寒湿血清组(IL-1β20ng/ml+风寒湿血清10％)、没食子酸甲酯(MG)组(IL-1β20ng/ml+风寒湿血清10％+MG 50μM)。
收集各组的总RNA和蛋白，进行RT-PCR检测和Western Blot检测软骨细胞的分解代谢指标(图2)。图2为没食子酸甲酯抑制骨关节炎状态下软骨细胞的分解代谢能力图，结果显示：模型组的促炎因子(IL-6、IL-1β)、分解代谢指标(ADAMTS5、MMP13、iNos、Cox-2和MMP9)的表达量较对照组升高，说明IL-1β干预后的软骨细胞出现炎症状态；风寒湿血清组的促炎因子和分解代谢指标的表达较模型组进一步升高，说明IL-1β联合风寒湿血清干预更能模拟骨关节炎的炎症状态；而加入没食子酸甲酯后，软骨细胞中促炎因子和分解代谢指标表达明显下降，说明没食子酸甲酯能够抑制骨关节炎下软骨细胞的分解代谢能力。
免疫荧光和Western Blot检测软骨细胞的合成能力指标(图3)。图3为没食子酸甲酯缓解骨关节炎状态下软骨细胞的合成能力图，结果显示，模型组的Col2α1、Acan和Sox-9的表达量较对照组下降，说明IL-1β干预后的软骨细胞出现软骨合成能力下降；风寒湿血清组的Col2α1、Acan和Sox-9表达量较模型组下降明显，说明IL-1β联合风寒湿血清干预后软骨细胞合成能力进一步降低；而加入没食子酸甲酯后，软骨细胞中合成指标表达明显上升，说明没食子酸甲酯能够改善炎症状态下骨关节炎模型中软骨细胞的合成能力。
Western Blot检测Pi3k/Akt通路蛋白水平(图4)。图4为没食子酸甲酯对骨关节炎状态下软骨细胞Pi3k/Akt通路情况图，结果显示，模型组的p-Pi3k、p-AKT的表达量较对照组升高；风寒湿血清组的p-Pi3k、p-AKT的表达量较模型组上升明显，说明IL-1β联合风寒湿血清干预后的细胞内Pi3k/Akt通路被激活；而加入没食子酸甲酯后，软骨细胞中p-Pi3k、p-AKT的表达量下降，说明没食子酸甲酯调控炎症状态下的软骨细胞中Pi3k/Akt信号通路失活影响骨关节炎。
Western Blot检测软骨细胞凋亡蛋白水平(图5)。图5为没食子酸甲酯对骨关节炎状态下软骨细胞凋亡基因的影响图，结果显示，模型组的Bax、Cleave-caspase 3的表达量较对照组升高，Bcl-2表达量较对照组降低；风寒湿血清组的Bax、Cleave-caspase 3的表达量较对照组升高，Bcl-2表达量较对照组更低，说明IL-1β联合风寒湿血清干预后的细胞内凋亡情况明显加剧；而加入没食子酸甲酯后，软骨细胞中Bax、Cleave-caspase 3的表达量较对照组降低，Bcl-2表达量较对照组升高，说明没食子酸甲酯降低炎症状态下软骨细胞中凋亡基因的表达，从而在骨关节炎起保护作用。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (8)
1.没食子酸甲酯在制备骨关节炎治疗药物中的应用。
2.根据权利要求1所述的没食子酸甲酯在制备骨关节炎治疗药物中的应用，其特征在于，没食子酸甲酯用于制备减缓和治疗因炎症对软骨细胞的损伤而导致的骨关节炎的药物。
3.根据权利要求1所述的没食子酸甲酯在制备骨关节炎治疗药物中的应用，其特征在于，没食子酸甲酯通过减轻炎症对软骨细胞的损伤，直接保护软骨细胞，从而减缓和治疗骨关节炎。
4.根据权利要求1所述的没食子酸甲酯在制备骨关节炎治疗药物中的应用，其特征在于，没食子酸甲酯通过抑制磷脂酰肌醇-3-激酶/蛋白激酶B(Pi3k/Akt)信号通路激活，抑制软骨细胞凋亡而保护软骨细胞，从而减缓和治疗骨关节炎。
5.没食子酸甲酯在制备减轻炎症对软骨细胞损伤的药物中的应用。
6.没食子酸甲酯在制备抑制Pi3k/Akt信号通路激活的药物中的应用。
7.一种骨关节炎治疗药物，其特征在于，所述的药物的活性成分为没食子酸甲酯。
8.根据权利要求7所述的骨关节炎治疗药物，其特征在于，所述的药物还包括药学上可接受的载体或辅料。
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