Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion

Chao Fang1, Jia-wei Guo1, Ya-jun Wang1, Xiao-qun Li1, Hao Zhang1, Jin Cui1, Yan Hu1, Ying-ying Jing2, Xiao Chen1,3, Jia-can Su1,2,4
1 Department of Orthopedics Trauma, Shanghai Changhai Hospital, Naval Medical University, Shanghai 200433, China
2 Institute of Translational Medicine, Shanghai University, Shanghai 201901, China
3 Department of Chemistry, Fudan University, Shanghai 200433, China
4 Shanghai Clinical Research Center for Aging and Medicine, Shanghai 200040, China
Correspondence to: Ying-ying Jing:, Xiao Chen:, Jia-can Su:,
DOI: 10.1038/s41401-021-00747-9
Received: 26 January 2021
Accepted: 17 July 2021
Advance online: 11 August 2021


Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg–1·d–1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 μM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.
Keywords: osteoarthritis; Panax notoginseng; diterbutyl phthalate; osteoclastogenesis; subchondral bone; ERK/c-fos/NFATc1 pathway; two-dimensional cell membrane chromatography (2D/CMC); ACLT mice

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