摘要
目的探讨甜菊素在体外对破骨细胞活性的影响及其作用机制。方法原代单核细胞提取自成年C57BL/6小鼠肱骨和股骨中,采用CCK-8法检测甜菊素对正常细胞存活率的影响,TRAP染色研究甜菊素对RANKL诱导的破骨细胞生成的作用;运用靶点预测和分子对接技术寻找甜菊素的潜在作用靶点,并采用Western blot法验证甜菊素对预测靶点表达的影响;采用RT-qPCR、Western blot等方法研究甜菊素对NF-κB通路中关键蛋白IκBα、破骨细胞溶骨相关基因(Acp5,V-ATpase-d2,CTSK)和调节分化相关转录因子(NFATc-1,c-Fos)表达的影响。结果10μmol/L甜菊素能够抑制破骨细胞生成(P<0.01),而且几乎无细胞毒性(P>0.05);甜菊素与AKR1B1结合较稳定,而且能抑制AKR1B1下游通路相关蛋白IκBα的表达及破骨细胞溶骨相关基因、调节分化相关转录因子的表达(P<0.01)。结论甜菊素在体外对破骨细胞活性有较好的抑制作用,具有被开发为治疗破骨细胞溶骨功能亢进导致的骨质疏松药物的前景。
AIM To investigate the effects and mechanism of steviosin on osteoclast activity in vitro.METHODS Primary monocytes were extracted from the humerus and femur of adult C57 BL/6 mice,and had their cell viability determined under the influence of steviosin by CCK-8 method.Steviosin had its effects on RANKL-induced osteoclastogenesis investigated by TRAP staining;its potential action targets observed by target prediction and molecular docking technology;its effects on the predicted target expression verified by Western blot;and its effects on the expressions of the key protein IκBαin the NF-κB pathway,osteoclast osteolysis-related genes(Acp5,V-ATpase-d2,CTSK)and regulation of differentiation-related transcription factors(NFATc-1,c-Fos)investigated by RT-qPCR and Western blot.RESULTS 10μmol/L steviosin inhibited osteoclastogenesis(P<0.01)with almost no cytotoxicity(P>0.05).With stable binding to AKR1 B1,steviosin inhibited the expression of AKR1 B1 downstream pathway-related protein IκBα,the expressions of osteoclast osteolysis-related genes,and regulation of differentiation-related transcription factors as well(P<0.01).CONCLUSION With its good inhibitory effects on the activity of osteoclasts in vitro,steviosin highlights the pharmaceutical development for the treatment of osteoporosis caused by hyperosteolysis of osteoclasts.
作者
陈莎
周佳瑛
储非凡
林芝
张伟琪
朱爱芳
CHEN Sha;ZHOU Jia-ying;CHU Fei-fan;LIN Zhi;ZHANG Wei-qi;ZHU Ai-fang(Shaoxing Keqiao Women and Children’s Hospital,Shaoxing 312030,China;The Second School of Clinical Medicine,Wenzhou Medical University,Wenzhou 325035,China;The First School of Clinical Medicine,Wenzhou Medical University,Wenzhou 325035,China)
出处
《中成药》
CAS
CSCD
北大核心
2022年第6期1804-1809,共6页
Chinese Traditional Patent Medicine
基金
国家级大学生创新创业训练计划项目(201910343018,202010343030,202010343041)
浙江省大学生科技创新活动计划暨新苗人才计划项目(2020R413031,2020R413053,2020R413054)。
作者简介
陈莎(1991—),女,住院医师,从事绝经后骨质疏松研究。Tel:15906873898,E-mail:1529129949@qq.com;共同第一作者:周佳瑛(1999—),女,本科生,研究方向为脊髓损伤。Tel:15258033161,E-mail:zhoujiaying51@sina.com;通信作者:朱爱芳(1978—),女,副主任医师。E-mail:778295185@qq.com。
