The pathological mechanism and possible therapeutic strategy in osteoarthritis

• Main Authors: Chih Chang Yeh, 葉致昌
• Other Authors: C. N. Chen
• Format: Others
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/90181803792803297458

博士 === 長庚大學 === 臨床醫學研究所 === 103 === Osteoarthritis (OA) is the most common degenerative joint disease in the world. The clinical symptoms are pain, stiffness and deformation of affected joints which are characterized with progressive destruction of articular cartilage, joint space narrowing, subchondral sclerosis and spur formation. Aging, joint trauma, obesity and excessive repetitive loading are the well-known risk factors of OA, but the exact mechanisms of OA remain unclear. The aim of this thesis was to investigate the mechanisms underlying macrophage-induced urokinase plasminogen activator (uPA) expression in human chondrocytes and its modulation by fluid shear stress; and to evaluate the protective effects of curcuminoid-loaded liposomes against bone turnover in a cell-based model of osteoarthritis. This thesis addresses the preparation of peripheral blood-macrophage-conditioned medium (PB-MCM), modeling of shear stress in a parallel plate flow chamber, liposome formulation techniques, cell uptake and bioactivity assays. Stimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that the JNK/Akt phosphory- lation and NF-κB activation are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK/Akt phosphorylation, NF-κB activation, and uPA expression. The PB- MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression. Therefore, our data support the hypothesis that uPA upregulation stimulated by macrophages may be modurated with low fluid shear stress. AMPK may potentially provide new molecular target or new ‘entry point’ to develop an effective disease-modifying drugs for the treatment of osteoarthritis. In addition, curcumin (Cur) and bisdemethoxycurcumin (BDMC), extracted from Curcuma longa, are poorly-water-soluble polyphenol compounds that have shown antiinflammatory potential for the treatment of osteoarthritis. To increase cellular uptake of Cur and BDMC in 7F2 mouse osteoblastic cells, soybean phosphatidylcholine (SPC) were used for liposome formulation. There are about 70% entrapment efficiency of Cur and BDMC in the liposomes and the particle sizes are stable after liposome formation. Both Curcuminoid-loaded liposomes can inhibit NO production of macrophage and its osteoclast differential activities. In comparison with free drugs (Cur and BDMC), curcuminoid-loaded liposomes were less cytotoxic, and expressed high cellular uptake of the drugs. Of note is that Cur-Lip can prevent liposome-dependent inhibition of osteoblast differentiation and mineralization, but BDMC-Lip could not. With interleukin (IL)-1β stimulation, curcuminoid-loaded liposomes can successfully down- regulate the expression of inflammatory markers on osteoblasts, and show high osteoprotegerin (OPG)/ Receptor activator of nuclear factor κB ligand (RANKL) ratio to prevent osteoclastogenesis. Our data demonstrated that Cur and BDMC can be successfully encapsulated in liposomes and that can reduce osteoclast activity and maintain osteoblast functions. Therefore, Curcuminoid-loaded liposomes may slow osteoarthritis progression.