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

Objective: 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. Results: 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-. 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-. 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-. 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.