摘要
目的:观察淫羊藿苷对成熟破骨细胞铁代谢的影响,探讨其可能作用机制。方法:建立破骨细胞模型,随机分为对照组及低浓度淫羊藿苷组(0.1μmol/L)、中浓度淫羊藿苷组(1μmol/L)、高浓度淫羊藿苷组(10μmol/L),分组干预后,采用CCK-8测定各组细胞的增殖能力后选择合适的药物浓度进行后续的实验,加入合适浓度淫羊藿苷-NAC(氧化剂)组,采用抗酒石酸酸性磷酸酶(TRAP)特异性染色测定破骨细胞分化水平,采用ROS试剂盒及亚铁离子荧光探针检测破骨细胞中ROS、亚铁离子水平,采用谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-PX)试剂盒检测破骨细胞内GSH、GSH-PX水平,采用逆转录聚合酶链反应(RT-PCR)及蛋白免疫印迹法检测各组破骨细胞Nrf2/HO-1/GPX4信号通路相关蛋白表达。结果:淫羊藿苷可抑制破骨细胞形成,且抑制作用呈剂量依赖性,高浓度淫羊藿苷对破骨细胞活性抑制作用最强(P<0.05);与对照组比较,淫羊藿苷可促进破骨细胞内ROS含量、亚铁离子含量升高,GSH及GSH-Px含量下降;淫羊藿苷可上调氧化应激相关Nrf2/HO-1信号传导通路,促进Nrf2、HO-1基因表达,抑制破骨细胞铁代谢相关的GPX4基因及转录蛋白表达。结论:淫羊藿苷可能通过上调Nrf2/HO-1信号传导通路,促进破骨细胞氧化应激改变,增加破骨细胞内ROS水平,提高破骨细胞内亚铁离子水平,下调铁代谢相关的GPX4基因表达,进而调控破骨细胞内铁代谢水平,增加其氧化应激损伤,从而抑制破骨细胞形成和骨吸收。
Objective:To observe the effect of icariin on iron metabolism in mature osteoclasts and explore its possible mechanism.Methods:Osteoclast model was established and randomly divided into control group,low concentration icariin group(0.1μmol/L),medium concentration icariin group(1μmol/L)and high concentration icariin group(10μmol/L).After the intervention,CCK-8 was used to determine the proliferation ability of cells in each group,and appropriate drug concentration was selected for subsequent experiments.The experimental groups were added with appropriate concentration of icariin-NAC(oxidant).The differentiation level of osteoclasts was measured by tartrate resistant acid phosphatase(TRAP)specific staining.The levels of ROS and ferrous ions in osteoclasts were detected by reactive oxygen species(ROS)kit and ferrous ion fluorescent probe.The levels of GSH and GSH-Px in osteoclasts were detected by glutathione(GSH)and glutathione peroxidase(GSH-Px)kits.And the related protein expression of Nrf2/HO-1/GPX4 signal pathway in osteoclasts were measured by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.Results:Icariin inhibited the formation of osteoclasts in a dose-dependent manner.High concentration icariin had the strongest inhibitory effect on the activity of osteoclasts(P<0.05).Compared with the control group,icariin increased the levels of ROS and ferrous ions in osteoclasts,and decreased the levels of GSH and GSH-Px.Icariin up-regulated oxidative stress-related Nrf2/HO-1 signal transduction pathway and promoted the gene expression of Nrf2,HO-1,inhibited the expression of GPX4 gene and transcriptional protein related to iron metabolism in osteoclasts.Conclusion:Icariin may promote the changes of oxidative stress in osteoclasts,increase the level of ROS in osteoclasts,increase the level of ferrous ions in osteoclasts,down-regulate GPX4 gene expression related to iron metabolism by up-regulating Nrf2/HO-1 signal transduction pathway,in order to regulate the level of iron metabolism in osteoclasts and increase its oxidative stress injury,which inhibits osteoclast formation and bone resorption.
作者
李倩楠
陈大琴
吕春明
何翔
贺旭峰
杨莉莉
陈文君
丁佩军
张慧敏
LI Qiannan;CHEN Daqin;LV Chunming;HE Xiang;HE Xufeng;YANG lili;CHEN Wenjun;DING Peijun;ZHANG Huimin(Department of Dermatology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Science and Technology Experiment Center,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《上海中医药大学学报》
CAS
2022年第S01期232-239,共8页
Academic Journal of Shanghai University of Traditional Chinese Medicine
基金
上海中医药大学附属曙光医院四明科研基金项目(SGKJ-202014)
作者简介
李倩楠,女,硕士,主治医师,主要从事中医药骨损伤修复的机制及病理性瘢痕修复研究;通信作者:张慧敏,主任医师,教授,E-mail:lion1962@qq.com
