Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (6): 1144-1151.doi: 10.12307/2025.300

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Cistanoside A mediates p38/MAPK pathway to inhibit osteoclast activity 

Li Yueyao1, Zhang Min2, Yang Jiaju2   

  1. 1Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; 2Second Hospital of Shanxi University, Taiyuan 030001, Shanxi Province, China
  • Received:2024-01-29 Accepted:2024-03-06 Online:2025-02-28 Published:2024-06-20
  • Contact: Zhang Min, MD, Chief physician, Second Hospital of Shanxi University, Taiyuan 030001, Shanxi Province, China
  • About author:Li Yueyao, Master candidate, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China
  • Supported by:
    Chinese Medicine Scientific Research Project of Shanxi Provincial Administration of Traditional Chinese Medicine (to ZM) 

Abstract:
BACKGROUND:
Cistanoside A has the effects of anti-inflammation, antioxidation, antioxidation, reducing renal damage and anti-osteoporosis, but its effect on osteoclast differentiation, function and its underlying molecular mechanisms remain unclear.
OBJECTIVE: To investigate the effect of Cistanoside A on osteoclast differentiation and bone resorption induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in vitro and its mechanism.
METHODS: Bone marrow macrophages were obtained from the femur and tibia of 4-6-week-old C57BL/6 mice. The cytotoxic effect of Cistanoside A (5, 10, 20, 40, 80, and 160 μmol/L) on bone marrow macrophage viability was examined using the cell counting kit-8 assay kit. Tartrate-resistant acid phosphatase staining was performed to observe the effect of different concentrations of Cistanoside A on osteoblast differentiation and its effective intervention concentration was determined. There was positive control group, Cistanoside A low, medium, and high dose groups (40, 80, and 160 μmol/L). After cell attachment, 50 ng/mL RANKL was added to induce osteoblast differentiation, and the corresponding dose of Cistanoside A was added to the Cistanoside A low, medium, and high dose groups, respectively. F-actin ring and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride staining were performed to detect the effects of Cistanoside A on the formation of osteoclasts. Toluidine blue staining of bone abrasion slices was used to observe the effects of Cistanoside A on bone resorption function of osteoclasts. The expression of upstream and downstream proteins of the JNK/MAPK pathway was detected by Western blot. The expression of genes related to osteoclast differentiation and bone resorption function such as tartrate-resistant acid phosphatase, DC-STAMP, Nfatc-1, Ctsk and c-Fos was detected by RT-qPCR. 
RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay showed that Cistanoside A significantly inhibited RANKL-induced osteoclast differentiation and bone resorption in a dose-dependent manner compared with the positive control group. The results of RT-qPCR showed that compared with the positive control group, both high and low dose groups of Cistanoside A could significantly downregulate the mRNA expression of tartrate-resistant acid phosphatase, DC-STAMP, Nfatc-1, Ctsk and c-Fos in a dose‐dependent manner. The results of western blot assay showed that the high dose group of Cistanoside A significantly inhibited the expression of p-JNK protein at 10, 20, 30 and 60 minutes of intervention; compared with the positive control group, Cistanoside A significantly inhibited the expression of Nfatc1 and c-Fos proteins in a dose-dependent manner. To conclude, Cistanoside A could inhibit the formation and bone resorption of osteoclasts by reducing the level of p-JNK protein, inhibiting the activation of MAPK pathway and the expression of key genes in osteoclasts.

Key words: Cistanoside A, osteoclast, MAPK, JNK, RANKL, RANK, osteoporosis, phenylethanoid glycosides

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