title: Down-regulation of TNF-alpha by small interfering RNA inhibits particle-induced inflammation in vitro.
abstract: Aseptic joint loosening is a key factor that reduces the life span of arthroplasty. There are currently few effective treatments for joint loosening except surgical revision. We explored the inhibitory effects of tumor necrosis factor (TNF)-alpha-targeted small interfering RNA (siRNA) on particle-induced inflammatory cytokine expression in the murine macrophage cell line, RAW264.7. siRNA targeting TNF-alpha was chemically synthesized and transfected into RAW264.7 cells by cationic liposomes. Ceramic, titanium, and polyethylene particles of different sizes (0.2-1.2 µm and 1.2-10 µm) were prepared to stimulate cells 24 h after siRNA transfection. The down-regulation of TNF-alpha mRNA and protein levels was quantitatively determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively, at different time intervals. Fluorescence microscopy showed that the siRNA transfection efficiency was 85.2% ± 3.5%. Real-time PCR showed that the levels of TNF-alpha mRNA in the particle stimulation plus RNA interference (RNAi) groups were significantly lower compared with the particle stimulation-only groups 3 h after stimulation (P < 0.05). Similarly, ELISA essays showed that the protein levels of TNF-alpha in the RNAi-treated groups were significantly decreased 24 h after transfection (P < 0.05). There were no significant differences in the inhibitory effect among the groups stimulated by different particle types or particle sizes (P > 0.05). Our results indicated that siRNA targeting TNF-alpha can inhibit TNF-alpha release from RAW264.7 cells when stimulated by ceramic, titanium, and polyethylene particles of different sizes and that the inhibitory effects were not related to the type or size of particle.
authors.affiliation: Department of Orthopaedic Surgery, The Second Affiliated Hospital of SUN YAT-SEN University, Guangzhou, China.
authors.forename: Chu-qiang
authors.initials: CQ
authors.lastname: Qin
authors.forename: Yue
authors.initials: Y
authors.lastname: Ding
authors.forename: Dong-sheng
authors.initials: DS
authors.lastname: Huang
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.forename: Ruo-fan
authors.initials: RF
authors.lastname: Ma
authors.forename: Jian-bin
authors.initials: JB
authors.lastname: Huang
mesh_terms: D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D007249:Inflammation / Q000235:genetics* / Q000276:immunology; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000627:therapeutic use*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000276:immunology
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha