Interleukin 6 induces osteogenic differentiation and calcification of human umbilical artery smooth muscle cells via trans-signaling pathway / 中华风湿病学杂志

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.

LncRNA MIAT downregulates IL-1ß, TNF-ɑ to suppress macrophage inflammation but is suppressed by ATP-induced NLRP3 inflammasome activation.

Cardiovascular disease (CVD) has been identified as the leading cause of premature deaths in rheumatoid arthritis (RA), accounting for about 40 to 50% of all deaths. Macrophage inflammation is regarded as a key point to link to the two diseases. Recently, long non-coding RNAs (lncRNAs) have acknowledged as a regulator of inflammation significantly. Here, we firstly found that lncRNA myocardial infarction associated transcript (lncRNA MIAT), a crucial lncRNA to regulate CVD, expressed increasingly in synovium and myocardial tissues of collagen-induced arthritis (CIA) mice. Besides, we also verified that the increased infiltration of macrophage occurred in those tissues of the CIA. In vitro, we found that macrophage inflammation induced by LPS could up-regulate lncRNA MIAT expression. LncRNA MIAT seemed to inhibit the expression of IL-1ß, TNF-ɑ and be suppressed by ATP-induced NLRP3 inflammasome activation pathway. Therefore, these data indicated an anti-inflammatory effect of lncRNA MIAT in macrophage and an original research direction for high cardiovascular risk in RA.

The experimental gene therapy with lentiviral-mediated RNA interference targeting tumor necrosis factor-α / 中华风湿病学杂志

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.