A new approach to transfect NF-\u03baB decoy oligodeoxynucleotides into the periodontal tissue using the ultrasound-microbubble method

Hiroyuki Yamaguchi,Sawa Kaneko,Takashi Ono,Jun-Ichi Suzuki,Risa Usumi-Fujita,Yasuhiro Shimizu,Yuji Ishida,Jun Hosomichi

doi:10.1038/ijos.2017.10

Hiroyuki Yamaguchi, Sawa Kaneko + Show 6 more

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https://doi.org/10.1038/ijos.2017.10

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Abstract

The objective of this study is to investigate the effect of the ultrasound-microbubble technique in nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN) transfection in the gingival tissue in mice. The 6-FAM-labeled scrambled decoy ODN with microbubbles was applied to the periodontal tissue in 8-week-old male C57BL/6J mice by ultrasound radiation at low (LUM-Sc) and high (HUM-Sc) intensities to optimize the transfection condition of the ultrasound-microbubble method. Histological inspections were performed two hours after transfection to compare the expression with that in the sham-operated group without ultrasound radiation (A-Sc). Then, an NF-κB decoy was transfected into the periodontal tissue using the high-intensity ultrasound-microbubble (HUM-NF) technique to examine the anti-inflammatory effects of the decoy ODN. Western blot analysis was performed to investigate the expression of interleukin(IL)-1β, IL-6 and intercellular adhesion molecule-1 (ICAM-1) in the gingival tissues in the HUM-Sc, the HUM-NF and control groups. The fluorescence microscopy results showed that the fluorescent intensity in the periodontal tissues in the LUM-Sc and HUM-Sc groups was significantly higher than that in the A-Sc and the control groups. The fluorescent intensity in the HUM-Sc group, especially in the gingival connective tissue, was the highest of all groups. Western blot analysis indicated that the protein expression levels of IL-1β, IL-6 and ICAM-1 in the HUM-NF group were significantly lower than those in the HUM-Sc and the control groups. These findings suggest that the high-intensity ultrasound-microbubble technique is an effective tool for decoy transfection into the periodontal tissue.

Highlights

Periodontal disease is associated with the infiltration of inflammatory cells into the gingiva, resulting from the interaction between the host’s defence mechanisms and plaque microorganisms[1,2] and can lead to the destruction of the periodontal tissues, including alveolar bone loss.Recently, prevention of periodontitis and regeneration of the periodontal tissues has gained increasing attention from many researchers and dental clinicians
Transfection of decoy ODN into periodontal tissue via an ultrasound-microbubble method First, we examined the feasibility of transfecting a decoy ODN into murine periodontal tissue using a previously established ultrasoundmicrobubble method
A difference in fluorescent intensity was observed between the LUM-Sc and HUM-Sc groups, in the gingival connective tissue, with that of the HUM-Sc group being greater (Figure 4). These data indicate that the following conditions, as used for the HUM-Sc group in these experiments, comprise an optimal method for decoy ODN delivery into gingival tissues: ultrasound irradiation for 60 s at a frequency of 1.0 MHz, an intensity of 2.0 W·cm − 2 and a duty cycle of 50%

Summary

Introduction

Periodontal disease is associated with the infiltration of inflammatory cells into the gingiva, resulting from the interaction between the host’s defence mechanisms and plaque microorganisms[1,2] and can lead to the destruction of the periodontal tissues, including alveolar bone loss.Recently, prevention of periodontitis and regeneration of the periodontal tissues has gained increasing attention from many researchers and dental clinicians. Periodontal disease is associated with the infiltration of inflammatory cells into the gingiva, resulting from the interaction between the host’s defence mechanisms and plaque microorganisms[1,2] and can lead to the destruction of the periodontal tissues, including alveolar bone loss. Nuclear factor kappa B (NF-κB) is a common signalling molecule involved in inflammation and is known to have an important role in the initiation of immune and inflammatory reactions in periodontal tissues.[3] Notably, NF-κB was the first transcription factor shown to bind a DNA element in a kappa immunoglobulin light-chain enhancer.[4] The expression and activation of NF-κB initiate a downstream signalling cascade involving various inflammatory cytokines, including interleukin(IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α), as well as several adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).[5,6] the NF-κB family of transcription factors and their associated signalling pathways have been shown to be involved in both innate and adaptive immune responses.[7]

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