MicroRNA-149 Suppresses Inflammation in Nucleus Pulposus Cells of Intervertebral Discs by Regulating MyD88.

BACKGROUND Intervertebral disc degeneration (IDD) is associated with low back and neck pain, but the mechanisms underlying its pathogenesis are unclear. In this study, we explored the function of microRNA-149 (miR-149) in inflammatory response mediated by lipopolysaccharide (LPS) in nucleus pulposus (NP) cells. MATERIAL AND METHODS Quantitative real-time PCR was used to detect miRNA and mRNA levels, while Western blotting was utilized to determine protein levels. ELISA was used to examine chemokine production. The correlation between miR-149 and MyD88 was assessed by reporter assay. Apoptosis was examined by flow cytometry. RESULTS miR-149 expression was significantly decreased after LPS exposure in NP cells. Overexpression of miR-149 reversed LPS-induced inhibition in aggrecan and collagen II expression and attenuated LPS-mediated promotion in the levels of MMP3, ADAMTS4, and inflammatory cytokines. Moreover, we found that miR-149 exerted its function by targeting MyD88 in NP cells. CONCLUSIONS miR-149 can inhibit the inflammatory response mediated by LPS in NP cells, and might be a potential target for the treatment of IDD.

Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.

BACKGROUND: Aseptic loosening is a significant impediment to joint implant longevity. Prosthetic wear particles are postulated to play a central role in the onset and progression of periprosthetic osteolysis, leading to aseptic loosening of the prosthesis. METHODS: We investigated the inhibitory effects of a lentivirus-mediated short hairpin RNA that targets the TNF-alpha gene on the particle-induced inflammatory and osteolytic changes via macrophages both in vitro and in vivo. An siRNA sequence targeting the mouse TNF-alpha gene from four candidates, transcribed in vitro, was screened and identified. A lentivirus vector expressing short hairpin RNA (shRNA) was then constructed in order to facilitate efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into cells of the mouse macrophage line RAW 264.7. Ceramic and titanium particles were introduced 24 h after lentivirus transduction to stimulate cells. TNF-alpha expression, represented by both mRNA and protein levels, was quantified with real-time PCR and ELISA at all time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model, followed by local injection of particles. A multi-slice spiral CT scan was used to evaluate the osteolysis of the calvaria by detecting the width of the cranial sutures. RESULTS: Macrophages developed pseudopods when co-cultured with particles. Lentivirus-mediated shRNA was shown to effectively inhibit the expression of TNF-alpha at both the mRNA and protein levels in RAW 264.7. The multi-slice spiral CT scan showed that the lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria. CONCLUSIONS: Our investigation highlighted the results that lentivirus-mediated shRNA targeting the TNF-alpha gene successfully inhibited particle-induced inflammatory and osteolytic changes both in vitro and in vivo. Therefore, lentivirus-mediated gene therapy may provide a novel therapeutic approach to aseptic joint loosening.

MyD88-dependent Toll-like receptor 4 signal pathway in intervertebral disc degeneration.

Lower back pain (LBP) is a common and remitting problem. One of the primary causes of LBP is thought to be degeneration of the intervertebral disc (IVD). The aim of the present study was to investigate the role of the myeloid differentiation primary-response protein 88 (MyD88)-dependent Toll-like receptor 4 (TLR4) signal pathway in the mechanism of IVD degeneration. IVD nucleus pulposus cells isolated and cultured from the lumbar vertebrae of Wistar rats were stimulated by various doses of lipopolysaccharide (LPS; 0.1, 1, 10 and 100 µg/ml) to simulate IVD degeneration. Cells were rinsed and cultured in serum-free Dulbecco's modified Eagle's medium/F12. Reverse transcription-quantitative polymerase chain reaction was used to determine the levels of TLR4, MyD88, tumor necrosis factor α (TNFα), and interleukin-1ß (IL-1ß) mRNA expression after 1, 3, 6, 9 and 12 h of incubation. Additionally, western blot and enzyme-linked immunosorbent assay analyses were used to determine the levels of TLR4, MyD88, TNFα, and IL-1ß protein expression after 24, 48 and 72 h of incubation. The levels of TLR4, MyD88, TNFα and IL-1ß mRNA all increased in the cells stimulated by 10 µg/ml LPS at 3, 6 and 9 h (all P<0.001). Furthermore, the levels of TLR4, MyD88, TNFα and IL-1ß protein all increased at 24, 48 and 72 h (all P<0.001). Additionally, the mRNA and protein levels of TLR4, MyD88, TNFα and IL-1ß increased significantly in the cells stimulated by 1, 10 and 100 µg/ml LPS compared with the control group, and reached a peak in the 10 µg/ml LPS group (all P<0.001). These results suggest that the MyD88-dependent TLR4 signal pathway is a target pathway in IVD degeneration. This pathway is time phase- and dose-dependent, and when activated can lead to the release of inflammatory factors that participate in IVD degeneration.

Inhibition of the PI3K/AKT pathway reduces tumor necrosis factor-alpha production in the cellular response to wear particles in vitro.

Joint replacement is the most effective treatment for end-stage osteoarticular disease. However, macrophage-mediated aseptic loosening of joint prosthesis severely hampers the clinical effects of joint replacement. Until now, the mechanism by which macrophages regulate the secretion of inflammatory cytokines after particle stimulation is not clear. It is well known that the PI3K/AKT pathway participates in multiple cellular processes, including cell growth, survival, and inflammation. However, whether the PI3K/AKT pathway participates in the proinflammatory response of macrophages after particle stimulation and secondary aseptic loosening is still unknown. In this study, ceramic and titanium particles of different sizes were prepared to stimulate macrophages. LY294002, a specific inhibitor of PI3K, was pretreated prior to particle stimulation. The expression of tumor necrosis factor-alpha (TNF-α) and all the subunits of PI3K and AKT were detected by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot. The result showed that LY294002 could suppress the RNA and protein expression of TNF-α in RAW264.7 cells after stimulation of different particles. The subunits of PI3K (p110ß and p85ß), followed by activation of phosphor-AKT (Ser473), participated in the regulation of activating macrophages by wear particles, ultimately resulting in the secretion of TNF-α.

In vitro comparison of the biological activity of alumina ceramic and titanium particles associated with aseptic loosening.

Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aimed to compare the biological activity of ceramic and titanium particles that are associated with particle-induced, aseptic joint loosening. Different sizes of alumina-ceramic particles and titanium particles were prepared to stimulate murine macrophage cells RAW 264.7, of which the expressions of tumor necrosis factor alpha (TNF-alpha) and receptor activator of nuclear factor-κB ligand (RANKL) were measured by qPCR and ELISA at various time points. In the presence of all particles, the expression of TNF-alpha increased in a time-dependent manner, whereas the expression of RANKL showed no regular expression patterns. Notably, particles of smaller sizes provoked significantly higher levels of TNF-alpha and RANKL than those of larger sizes. Compared to the titanium particles, the ceramic particles provoked a significantly lower production of TNF-alpha. Thus, the bioactivities of titanium and alumina ceramic particles were inversely proportional to the sizes of the particles, and the expression of RANKL was not parallel to that of TNF-alpha. The successful outcome of ceramic-on-ceramic artificial joint prostheses may be attributed to the low biological activity of ceramic particles, as evidenced here.

Down-regulation of TNF-alpha by small interfering RNA inhibits particle-induced inflammation in vitro.

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.

[Application of allograft and autologous bone graft in posterolateral lumbar fusion].

OBJECTIVE: To evaluate the clinical effect of local autogenous bone chips extended with allogeneic bone grafts in the posterolateral lumbar fusion. METHODS: From March 2005 to April 2007, 22 cases which underwent posterolateral lumbar fusion with allograft bone mixed with local autograft bone were analyzed retrospectively. The postoperative temperature, drainage flow and healing time of the incision were analyzed; postoperative lumbar pain was evaluated by visual analog scale (VAS) and JOA score; the postoperative efficacy was assessed by MacNab criteria and Oswestry disability index (ODI); the fusion rate was defined by Jorgenson fusion criteria. RESULTS: All cases were followed up for 17-35 months with an average of 21 months, the wound all healed by first intention; no red swelling, exudation and infection occurred. The excellent and good rate was 81.8% for JOA score (excellent in 4 cases, good in 14 cases, fair in 4 cases), 77.3% for MacNab criteria (excellent in 4 cases, good in 13 cases, fair in 5 cases) and 90.9% for ODI index (excellent in 3 cases, good in 17 cases, fair in 2 cases). The postoperative X-ray fusion rate within 1 year was 90.9%. CONCLUSION: Allograft bone mixed with local autograft bone can achieve good efficacy and fusion rate in posterolateral lumbar fusion.