鼠尾草酸影响线粒体功能抑制破骨细胞分化  

Carnosic acid inhibits osteoclast differentiation by inhibiting mitochondrial activity

作　　者：李海山 吴宇桁 梁梓炫 张诗茵 张朕 麦彬 邓威 李永贤[1,2] 唐永超 张顺聪[1,2] 袁凯 Li Haishan;Wu Yuheng;Liang Zixuan;Zhang Shiyin;Zhang Zhen;Mai Bin;Deng Wei;Li Yongxian;Tang Yongchao;Zhang Shuncong;Yuan Kai(First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;Guangdong Clinical Research Academy of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;Lingnan Medical Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;Seventh Clinical College of Medicine of Guangzhou University of Chinese Medicine,Shenzhen 518100,Guangdong Province,China)

机构地区：[1]广州中医药大学第一附属医院,广东省广州市510405 [2]广东省中医临床研究院,广东省广州市510405 [3]广州中医药大学岭南医学研究中心,广东省广州市510405 [4]广州中医药大学第七临床医学院,广东省深圳市518100

出　　处：《中国组织工程研究》2025年第2期245-253,共9页Chinese Journal of Tissue Engineering Research

基　　金：广州市科技计划项目(202201020295),项目负责人:袁凯;广东省基础与应用基础研究基金项目(2021A1515012168),项目负责人:张顺聪;广州市科技计划项目(202201020500),项目负责人:唐永超;广州市科技计划项目(202201020533),项目负责人:张顺聪;广州市科技计划项目(202102021040),项目负责人:李永贤;广州中医药大学第一附属医院中青年重点人才培养项目(2023.10),项目负责人:李永贤;中华中医药学会青年人才托举工程项目(2022-QNRC2-B11),项目负责人:李永贤。

摘　　要：背景:鼠尾草酸是在迷迭香中发现的一种生物活性化合物,已被证明可减少炎症和活性氧,但在破骨细胞分化进程中的作用机制尚不明确。目的:探讨鼠尾草酸对破骨细胞活化、活性氧产生及线粒体功能的影响。方法:体外提取培养小鼠来源的原代骨髓源巨噬细胞,使用CCK-8细胞活力实验检测不同浓度(0,10,15,20,25和30μmol/L)鼠尾草酸对骨髓源巨噬细胞的增殖和毒性作用,筛选出安全作用浓度。将骨髓源巨噬细胞按照浓度梯度分组培养,核因子κB受体活化因子配体诱导破骨细胞5-7 d后,分别进行抗酒石酸酸性磷酸酶染色、F-actin染色、H2DCFDA探针和线粒体活性氧、Mito-Tracker荧光检测,观察鼠尾草酸对破骨细胞分化和功能的影响;通过Western Blot及RT-PCR实验检测鼠尾草酸对核因子κB受体活化因子配体诱导的丝裂原激活蛋白激酶信号通路上下游基因和蛋白的影响。结果与结论:①抗酒石酸酸性磷酸酶和F-actin染色显示:鼠尾草酸以浓度依赖性地抑制体外破骨细胞分化及细胞骨架肌动蛋白环形成,其中鼠尾草酸30μmol/L组的抑制作用最显著;与其他干预时期相比,鼠尾草酸在破骨分化的早期(第1-3天)抑制作用最显著;②H2DCFDA探针和线粒体活性氧、Mito-Tracker荧光显示:鼠尾草酸抑制细胞活性氧和线粒体内活性氧的产生,同时减少线粒体膜电位,影响线粒体的功能;③Western Blot及RT-PCR结果显示:鼠尾草酸可抑制与破骨分化相关的NFATc1、CTSK、MMP9、C-fos蛋白的表达,下调与破骨分化相关的NFATc1、Atp6vod2、ACP5、CTSK和C-fos基因的表达;鼠尾草酸还可以增强抗氧化酶蛋白的表达,减少破骨分化过程中活性氧的产生;④鼠尾草酸抑制P38/ERK/JNK蛋白的磷酸化修饰,通过激活MAPK信号通路抑制骨髓源巨噬细胞破骨细胞分化。BACKGROUND:Carnosic acid,a bioactive compound found in rosemary,has been shown to reduce inflammation and reactive oxygen species(ROS).However,its mechanism of action in osteoclast differentiation remains unclear.OBJECTIVE:To investigate the effects of carnosic acid on osteoclast activation,ROS production,and mitochondrial function.METHODS:Primary bone marrow-derived macrophages from mice were extracted and cultured in vitro.Different concentrations of carnosic acid(0,10,15,20,25 and 30μmol/L)were tested for their effects on bone marrow-derived macrophage proliferation and toxicity using the cell counting kit-8 cell viability assay to determine a safe concentration.Bone marrow-derived macrophages were cultured in graded concentrations and induced by receptor activator of nuclear factor-κB ligand for osteoclast differentiation for 5-7 days.The effects of carnosic acid on osteoclast differentiation and function were then observed through tartrate-resistant acid phosphatase staining,F-actin staining,H2DCFDA probe and mitochondrial ROS,and Mito-Tracker fluorescence detection.Western blot and RT-PCR assays were subsequently conducted to examine the effects of carnosic acid on the upstream and downstream proteins of the receptor activator of nuclear factor-κB ligand-induced MAPK signaling pathway.RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining and F-actin staining showed that carnosic acid dose-dependently inhibited in vitro osteoclast differentiation and actin ring formation in the cell cytoskeleton,with the highest inhibitory effect observed in the high concentration group(30μmol/L).Carnosic acid exhibited the most significant inhibitory effect during the early stages(days 1-3)of osteoclast differentiation compared to other intervention periods.Fluorescence imaging using the H2DCFDA probe,mitochondrial ROS,and Mito-Tracker demonstrated that carnosic acid inhibited cellular and mitochondrial ROS production while reducing mitochondrial membrane potential,thereby influencing mitochondrial functio

关 键 词：鼠尾草酸 活性氧 线粒体活性 RANKL 破骨细胞 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R285