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

Yao Li; Chengjun Sun; Ge Feng; Yao He; Jie Li; Jinlin Song

doi:10.1016/j.archoralbio.2020.104769

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

Arch Oral Biol. 2020 Sep:117:104769. doi: 10.1016/j.archoralbio.2020.104769. Epub 2020 May 20.

Authors

Yao Li¹, Chengjun Sun², Ge Feng³, Yao He¹, Jie Li¹, Jinlin Song⁴

Affiliations

¹ College of Stomatology, Chongqing Medical University, PR China; Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, PR China; Chongqing Municipal Key Laboratory of Oral Biomedical Engisneering of Higher Education, PR China.
² Stomatological Hospital, Southern Medical University, PR China.
³ College of Stomatology, Chongqing Medical University, PR China.
⁴ College of Stomatology, Chongqing Medical University, PR China; Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, PR China; Chongqing Municipal Key Laboratory of Oral Biomedical Engisneering of Higher Education, PR China. Electronic address: songjinlin@hospital.cqmu.edu.cn.

PMID: 32599516
DOI: 10.1016/j.archoralbio.2020.104769

Abstract

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-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.

Keywords: Autophagy; Interleukin-6; Lipopolysaccharide; Low-intensity pulsed ultrasound; Periodontal ligament cells.

MeSH terms

Autophagy*
Beclin-1 / metabolism
Cells, Cultured
Humans
Lipopolysaccharides / pharmacology*
Microtubule-Associated Proteins / metabolism
Periodontal Ligament / cytology*
Ultrasonic Waves*

Substances

BECN1 protein, human
Beclin-1
Lipopolysaccharides
MAP1LC3B protein, human
Microtubule-Associated Proteins