Association of fatty acid binding protein 4 downregulation with the development and treatment of osteoarthritis

Abstract

Osteoarthritis (OA) was a degenerative disease widely infecting millions of individuals. Pain and joint disability were its vital clinical symptoms and challenged the routine activities of patients. It was deserved to be studied on its pathogenesis and new therapy due to the high cost and heavy burden of current treatments. Considering the pathophysiology of osteoarthritis, marrow lesions and bone resorption in subchondral bone due to macrophage related inflammation were important in development of osteoarthritis. There were two main subtypes of macrophages involved in osteoarthritis: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. The dysregulated balance between M1/M2 macrophages might be critical in the pathophysiology of osteoarthritis. FABP4 was a lipid transport protein and expressed in macrophages and adipocytes. In our previous studies, FABP4 expression was increased in plasma and synovial fluid in the patients with osteoarthritis. Osteoarthritis was relieved in FABP4-knockout mice. However, the role of FABP4 in subchondral bone marrow inflammation and resorption in OA progression is still unclear.

Based on previous studies, there are three hypotheses in this study. 1. FABP4 inhibition could relieve subchondral bone marrow resorption and cartilage erosion in vivo. 2. M1 macrophage polarization, osteoclastogenesis, and bone resorption function of osteoclasts could be inhibited by FABP4 suppression. 3. Levofloxacin was proved to be a FABP4 inhibitor and could relieve OA. DMM-induced OA models could be suppressed by Levofloxacin. Two osteoarthritis models were established. One was FABP4 globally knockout (KO) mice with 6-month high-fat diet (HFD) model. And the other was destabilization of the medial meniscus (DMM) induced OA model transfected by si-FABP4 (KD) in bone marrow or synovium. Histological assessment and micro-CT analysis in both two models were performed. Further, bone marrow-derived macrophages (BMDMs) or RAW 264.7 were used to establish M1 polarization and osteoclastogenesis model with/without si-FABP4 transfected. Flow cytometry, TRAP staining, pit assay, and qPCR were used to evaluate the modulation of FABP4 on M1 polarization and the effectiveness of Fabkin on osteoclastogenesis. Western blot was used to clarify the signaling pathways involved in it. For the third hypothesis, DMM-induced models were established to confirm whether Levofloxacin could suppress the development of OA.

Results showed that cartilage degradation and subchondral bone resorption were alleviated in FABP4-KD DMM model in bone marrow and FABP4-KO HFD mice with significantly lower modified OA scores instead of FABP4-KD DMM model in synovium joint. According to in vivo and in vitro tests, M1 polarization was inhibited after the suppression of FABP4 treatment by JNK/p65/IRF5 pathways. FABP4 modulated RANKL-induced osteoclastogenesis and calcium resorption by Fabkin. Western blot showed that the PKA/CREB/p65 pathway was involved in the modulation of Fabkin in osteoclastogenesis and calcium resorption. Further, Levofloxacin relieved DMM-induced subchondral bone marrow resorption, cartilage erosion, and inhibited M1 macrophage polarization.