Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (2): 245-253.doi: 10.12307/2025.205

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Carnosic acid inhibits osteoclast differentiation by inhibiting mitochondrial activity

Li Haishan1, 2, 3, Wu Yuheng3, 4, Liang Zixuan1, 2, 3, Zhang Shiyin1, 2, 3, Zhang Zhen1, 2, 3, Mai Bin1, 2, 3, Deng Wei1, 2, 3, Li Yongxian1, 2, Tang Yongchao1, 2, Zhang Shuncong1, 2, Yuan Kai1, 2   

  1. 1First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 2Guangdong Clinical Research Academy of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 3Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 4Seventh Clinical College of Medicine of Guangzhou University of Chinese Medicine, Shenzhen 518100, Guangdong Province, China
  • Received:2023-12-15 Accepted:2024-01-25 Online:2025-01-18 Published:2024-05-24
  • Contact: Yuan Kai, MD, Associate chief physician, 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 Co-corresponding author: Zhang Shuncong, MD, Chief physician, 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
  • About author:Li Haishan, Master candidate, Physician, 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
  • Supported by:
    Guangzhou Science and Technology Project, No. 202201020295 (to YK); The Natural Science Foundation of Guangdong Province, No. 2021A1515012168 (to ZSC); Guangzhou Science and Technology Project, No. 202201020500 (to TYC); Guangzhou Science and Technology Project, No. 202201020533 (to ZSC); Guangzhou Science and Technology Project, No. 202102021040 (to LYX); The Young and Middle aged Key Talent Cultivation Project of The First Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 2023.10 (to LYX); Youth Talent Lifting Project of Chinese Society of Traditional Chinese Medicine, No. 2022-QNRC2-B11 (to LYX)

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Abstract: 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 30 μmol/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 
(30 μmol/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 function. The results of western blot and RT-PCR revealed that carnosic acid could suppress the expression of NFATc1, CTSK, MMP9, and C-fos proteins associated with osteoclast differentiation, and downregulate the expression of NFATc1, Atp6vod2, ACP5, CTSK, and C-fos genes related to osteoclast differentiation. Furthermore, carnosic acid enhanced the expression of antioxidant enzyme proteins and reduced the generation of ROS during the process of osteoclast differentiation. Overall, carnosic acid exerts its inhibitory effects on osteoclast differentiation by inhibiting the phosphorylation modification of the P38/ERK/JNK protein and activating the MAPK signaling pathway in bone marrow-derived macrophages.
中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: carnosic acid, reactive oxygen species, mitochondrial activity, RANKL, osteoclasts

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