Low-intensity pulsed ultrasound activates autophagy in periodontal ligament cells in the presence or absence of lipopolysaccharide

LIPUS can promote autophagy in PDLCs irrespective of lipopolysaccharide. Autophagy might be involved in LIPUS anti-inflammatory mechanism in PDLCS.

This study aims to determine if low-intensity pulsed ultrasound (LIPUS) activates autophagy in human periodontal ligament cells (PDLCs) irrespective of lipopolysaccharide. Design: Six groups were designed: control, LIPUS, lipopolysaccharide, LIPUS + lipopolysaccharide, LIPUS+3-Methyladenine, LIPUS + lipopolysaccharide+3-Methyl- adenine. LIPUS pretreated PDLCs for 2 h and lipopolysaccharide treated for different times. Real-time PCR and Western-blot were performed to evaluate mRNA and protein expression levels of autophagic genes Beclin-1 and LC3 respectively. A transmission electronic microscope was used to observe the autophagosome. ELISA was used to test interleukin-6 expression.

Compared with the non-treatment, LIPUS pretreatment increased mRNA expression levels of LC3 (P < 0.05) and Beclin-1 (P < 0.05) at 4 h and 8 h, and enhanced the protein expression levels of LC3-Ⅱ at 8 h (P<0.05) and Beclin-1 at 4 h, 8 h and 16 h(P<0.05). After LIPUS pretreatment and lipopolysaccharide treatment for 8 h, LC3-Ⅱ and Beclin-1 protein expression levels were elevated (P < 0.05) compared with the control. Following further treatment by 3-Methyladenine, Beclin-1 protein expression was decreased (P < 0.05) compared with the LIPUS plus lipopolysaccharide group, but LC3-Ⅱ protein expression was not. Autophagosomes were not found in the LIPUS+3-Methyladenine and LIPUS+lipopolysaccharide+3-Methyladenine groups. After LIPUS pretreatment and lipopolysaccharide treatment for 36 h, intreleukin-6 expression was decreased (P<0.05) compared with the lipopolysaccharide group. However, after addition of 3-Methyladenine, intreleukin-6 expression was elevated (P < 0.05) compared with the LIPUS +lipopolysaccharide group. Conclusions: LIPUS can promote autophagy in PDLCs irrespective of lipopolysaccharide. Autophagy might be involved in LIPUS anti-inflammatory mechanism in PDLCS.