Effects Of Exogenous Neuron Growth Factor (NGF) On Lipopolysaccharide (LPS) Induced Inflammatory Reaction In Osteoblasts.

Objective: To study the effect of exogenous nerve growth factor (NGF) on lipopolysaccharide (LPS)-induced inflammatory response and related gene expression in osteoblasts.Methods: Osteoblasts were isolated and cultrured from the newborn SD rats. Von Kossa's method was used for the identification of osteoblasts. At the end of the 2nd generation of primary osteoblasts, cells were trypsinized and resuspended withα-MEM culture medium containing 10% FBS, adjusting the concentration of 1×105 cells / cm3 in culture flasks until to 90% confluence. Cells were exposed to various concentrations of LPS for 24 h and vehichle was used in control group, followed by the detection of cell viability with MTT method. Different concentrations of neuron conditioned medium (NCM, 5, 10 and 20 dilution, respectively) or NGF (100ng/ml, 10ng/ml and 1ng/ml) were added in to the medium, followed by 2h, 12h and 24h LPS (1μg/ml) expousure, respectively. Nitric oxide (NO) in the supernatant was measured and cell viability was assayed with MTT method. The total RNA was extracted and reverse-transcripted. TNF-α, COX-2 and NF-κB mRNA expression was detected using SYBR GREEN method.Results: MTT detection showed that 10, 5 and 2.5μg/ml LPS exposure for 24 h resulted in significant increase in cell viability compared with the control group. However, 1.25μg/ml or lower concentration did not affect cell viability. Cells was treated with 1μg/ml LPS for 1 h, 2 h, 6 h, 12 h and 24 h showed a time-dependent increase in NO produrtion, with a significant difference at 6 h, and highly significant differences at 12 h and 24 h. Therefore, 1μg/ml LPS was choosed for the expriment. Cells treated with 1μg/ml LPS showed no remarkable change in NO production for 2 h, while, NO levels elevated after 12 h and 24 h LPS exposure (p<0.05, p<0.01), compared with the control group. Compared with LPS group, NCM of 1/10 dilution could significantly inhibit NO production at 12 h (p<0.05), and NCM of 1/20,1/10 and 1/5 dilutions suppressed NO production at 24 h (p<0.05, p<0.01, p<0.01). Compared with the control group, 1μg/ml LPS and different concentrations of NGF have little effect on NO production in osteoblasts. 12 and 24h exposure induced significant NO release. Compared with LPS group, 100ng/ml NGF could significantly inhibit NO release in osteoblasts at the 12h and 24h, and 10ng/ml NGF can inhibit osteoblast NO release at 24h. In this range of concentration, cell viability was not affected. Compared with the control group, LPS induced significant increase in TNF-αmRNA expression at 2, 12 and 24h. With the corresponding time point compared with LPS group, high dose of NGF significantly inhibited the TNF-αmRNA expression at 2h. Each dose of NGF could significantly inhibit TNF-α, COX-2 and NF-κB mRNA expression at 12h.Conclusion: NGF could inhibit the NO production, reduce the release of inflammatory mediators, and inhibit TNF-α, COX-2 and NF-κB mRNA in osteoblasts, thus reducing the inflammatory cascade and exterting anti-inflammatory action.