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Objective:To explore the trans-membrane signaling mechanism of interleukin-6 (IL-6)-induced osteogenic differentiation and calcification of human umbilical artery smooth muscle cells (HUASMCs).Methods:HUASMCs were primarily cultured in vitro and were stimulated with IL-6, IL-6+solutable IL-6 receptor (sIL-6R), IL-6+sIL-6R+solutable gp130 (sgp130), or vehicle (blank control). Alizarin red and Von Kossa staining were used for detecting cell calcification, Western blot was used to test the protein expression of tissue-nonspecific alkaline phosphatase (TNAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2) and Runt related transcription factor 2 (Runx2), and immunofluorescence was used to examine the mIL-6R expression of HUASMCs. The comparison of measurement date between the two groups was conducted by t-test. The comparison of measurement date between multiple groups was conducted by one-way analysis of variance (ANOVA). Results:The intensity severity of calcification stain was IL-6+sIL-6R group >IL-6+sIL-6R+sgp130 group>IL-6 group=blank control. After stimulated for 12 hours, the TNAP expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.44±0.08), (0.52±0.14), (0.84±0.16) and (0.55±0.10) respectively ( F=290.96, P<0.001). After stimulated for 3 days, the OPN expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.61±0.84), (0.95±0.16), (1.65±0.24) and (0.99±0.10) respectively ( F=507.72, P<0.001). After stimulated for 12 hours, the BMP-2 expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.77±0.05), (1.69±0.16), (2.81±0.26) and (0.57±0.12) respectively ( F=959.09, P<0.001). After stimulated for 3 days, the Runx2 expression in blank control, IL-6 group, IL-6+sIL-6R group,IL-6+sIL-6R+sgp130 group were (0.57±0.03) , (0.92±0.10), (1.31±0.13) and (0.66±0.06) respectively ( F=1141.27, P<0.001). Comparing with Jurkat cells (positive control) and CEM cells (negative control), HUASMCs limited expressed mIL-6R. Conclusion:IL-6 may induce HUASMCs osteogenic differentiation and calcification mainly via the sIL-6R-mediated trans-signaling pathway.
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Objective To investigate the effects of lentiviral-mediated RNA interference (RNAi) targeting tumor necrosis factor-α(TNF)-αgene on the expression of TNF-α, interleukin (IL)-1β, IL-6 of murine macrophages RAW264.7, and the efficiency of RNAi experimental gene therapy for the murine collagen-induced arthritis (CIA). Methods The RAW264.7 macrophages were infected by lentivirus-RNAi particles, then stimulated by Lipopolysaccharides (LPS). The TNF-α, IL-1β, IL-6 expression of RAW264.7 macrophages were measured with real-time polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA). CIA models were esta-blished in DBA/1 mice using bovine type Ⅱ collagen. The treatment effect of lentivirus-RNAi on CIA were observed through arthritis scores, serum TNF-α measurement and hind paw paraffin section hematoxylin/eosin staining after lentivirus-RNAi particles tail vein injection. Results The TNF-αmRNA relative expression level of lentiviral RNAi group was 0.291 ±0.021, significantly lower than that of negative control group 0.925±0.013 (t=25.4, P<0.01). The inhibition rate in mRNA levels was 68.5%. The serum TNF-α level of lentiviral RNAi group was [(249 ±11) ng/ml], significantly lower than that of negative control [(382±6) ng/ml] (t=10.31, P<0.05). The inhibition rate of protein levels was 34.7%. It had no effect on the IL-1β and IL-6 mRNA expression. On the 8th day after systemic administration, the arthritis score of lentivirus-RNAi group was 2.50±0.19, which was significantly lower than that of blank controls (3.63 ±0.18) and negative controls (3.75 ±0.16) (F=42.8, P<0.01). From now on, arthritis score of lentivirus-RNAi group and positive control decreased slowly to at least 2 weeks after treatment induction. The serum TNF-α levels of lentivirus-RNAi group and positive controls were [(35±6) pg/ml] and [(32±7) pg/ml] significantly lower than that of negative controls [(47±3) pg/ml] (t=3.03, 4.11, P<0.01) respectively. Morphological examination showed that the lentivirus-RNAi decreased CIA pathological manifestations. Conclusion Lentiviral-mediated RNAi targeting murine TNF-α gene can effectively inhibit TNF-α expression both in vitro and in vivo, which also effectively improve the CIA arthritis score. Lentiviral-mediated RNAi targeting TNF-αgene provides a potential strategy for rheumatoid arthritis (RA) treatment.