<p><em>N</em>-Acetyl-l-Leucine-Polyethyleneimine-Mediated Delivery of CpG Oligodeoxynucleotides 2006 Inhibits RAW264.7 Cell Osteoclastogenesis</p>

Abstract

Introduction: CpG oligodeoxynucleotides (CpG ODN) play important roles in resisting inflammation and bone resorption. However, the inherent instability and rapid degradation hinder their wider application. This study aimed to evaluate whether N-acetyl-L-leucinemodified polyethyleneimine (N-Ac-L-Leu-PEI) could effectively deliver CpG ODN 2006 to RAW264.7 cells and and if it can regulate osteoclastogenesis in vitro. Materials and Methods: Gel retardation assay was conducted to evaluate whether N-Ac-L-Leu-PEI and CpG ODN could form a stable complex. RAW264.7 cells were divided into four groups of control group, ODN group, phosphorothioate ODN group and N-Ac-L-Leu-PEI/ODN group. Fluorescence assay was conducted to evaluate the transfection rate of ODNs in different groups. Cell viability was determined by MTT assay. Cell apoptosis was determined by live-dead cell staining and flow cytometry assay. Relative expression levels of osteoclastic differentiation factors, including Nfatc, c-fos, receptor activator of nuclear factor κB (RANK), and matrix metalloproteinase 9 (MMP9), were determined by real-time PCR and Western blot. Results: N-Ac-L-Leu-PEI and CpG ODN could form a stable complex at a mass ratio of 1:1 (w:w). MTT assay showed that the cell viability of N-Ac-L-Leu-PEI was relatively high even at a mass ratio of 8 μg/mL. The transfection rate of N-Ac-L-Leu-PEI-ODN complex was higher than 90%. The cell proliferation and apoptosis was significantly enhanced in N-Ac-L-Leu-PEI-CpG ODN group when compared to those in phosphorothioate CpG ODN. The expression levels of Nfatc, c-fos, RANK, and MMP9 were significantly decreased in N-Ac-L-Leu-PEI/ODN complex group. Discussion: N-Ac-L-Leu-PEI could be a potential gene vehicle for the prevention of periodontitis-mediated bone resorption.

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“…This is a crucial component in resisting inflammation, it is involved in the bone resorption process, and enhances cell apoptosis. 27 …”