| ObjectiveNel-like molecule-1 (Nell-1) is a novel secreted protein that was overexpressed in pre-fused areas of craniosynostosis patients. Nell-1 played crutial role in inducing osteoblast differentiation and bone regeneration. Previous studies have suggested an anti-inflammation effect of Nell-1 in inhibiting bone inflammation induced by BMP-2. Dental tissue and craniofacial bone are all neural crest-derived tissues and share several similarities in genetic structure. So we hypothesized that Nell-1 may be involved in the procedure of pulpitis. In this study the goal was to explore the effect of human recombinant Nell-1 (hrNell-1) on lipopolysaccharide (LPS)-induced inflammation in human dental pulp cells (hDPCs) and the related intracellular signaling pathways.Methods1. The human primary dental pulp cells were separated and cultured by tissue block enzyme digestion method. The cells with passages 4-6 at sub-confluence were used in all experimental procedures.2. To explore the effect of Nell-1 on the expression of inflammatory cytokines in human dental pulp cells and to determine the lowest effective concentration of Nell-1, tackled the pulp cells with lOng/ml of Escherichia coli lipopolysaccharide (LPS) and hrNell-1 protein at concentrations of 0, 100ng/ml,200ng/ml,400ng/ml, after co-cultured the cells for one day, removed the cell culture medium and collected the total RNA. The qRT-PCR was carried out to confirm the gene synthesis degree of IL-6 and IL-8.3. The hDPCs were planted into 6-well cell-plates at 1.5×106 cells/well, and then co-treated the cells with lOng/ml of LPS and the minimum effective concentration of hrNell-1 (200ng/ml) for 3 hour,6 hour,12 hour,1 day,3 day,7 day. Then the translation degree of IL-6, IL-8 in various time points were detected by qRT-PCR as well as ELISA.4.To investigate the mechanism, the cells were co-treated for one day with ten ng/ml LPS,200ng/ml, 10μmol/L MAPK inhibitor. Then total RNA was collected and the qRT-PCR was carried out to quantify the gene translation of IL-8 and IL-6.Results1. The primary hDPCs were obtained with tissue-block-enzyme-digestion method successfully, and the cultured cells were obtained after subculture.2. The hDPCs were co-cultured with different concentration of hrNell-1 and lOng/ml of LPS for one day. The data showed that 10ng/ml of LPS could increase the synthesis of IL-6 and IL-8 obviously (p<0.01) and 200ng/ml Nell-1 protein could significantly inhibit the expression of IL-6 (p<0.05) and IL-8 (p<0.01) in human dental pulp cells.3. The hDPCs were co-tackled with ten ng/ml LPS and 200ng/ml hrNell-1 for 3 hour, 6 hour,12 hour,1 day,3 day,7 day, the results showed that the inflammatory effect induced by LPS has a time dependence. The inflammation induced by LPS reached crest at 3 days but decreased in 7 days group. Meanwhile hrNell-1 significantly inhibited LPS-induced inflammation when stimulated for 24 h (p<0.01) and reached crest at 3 days. But in the 3 h,6 h,12 h,7 days groups, the fuction of restrain was not distinct.4. Eventually, to further explore whether the MAPK signalings mediated the anti-inflammatory effect of hrNell-1 we treated the hDPCs with specific MAPKs inhibitors. As pre-treated hDPCs with ERK and p38 MAPK inhibitors, the gene translation of IL-8 and IL-6 couldn’t be suppressed by hrNell-1. While in the JNK MAPK inhibitor group, hrNell-1 still exerted obvious effect on attenuating inflammation.Conclusions1. The enzyme digestion tissue block cell cultrue meithod is easy to operate. The cell morphology was changeful in primary passage. After subcultured, the cell proliferation was stable and grew well, and the cell morphology was uniform, which was suitable for the experimental study.2. The pulpitis model was established successfully by using lOng/ml LPS. The hrNell-1 protein could inhibit the LPS induced inflammation in hDPCs which had dosage and time-dependent manner.3. hrNell-1 may inhibit the inflammation in hDPCs by means of ERK and p38 MAPK signaling channels, rather than JNK singaling channel. |
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