title: Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs).
abstract: Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC) characterized by multi-lineage differentiation making it an attractive choice for tissue regeneration. However, before DPSCs can be used for cell-based therapy, we have to understand their biological properties in response to intrinsic and extrinsic stimuli such as lipopolysaccharide (LPS). DPSCs were therefore stimulated with LPS and senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, with cell number and cell-cycle arrest being examined by BrdU assay and flow cytometry, respectively. The morphology of DPSCs was characterized by their flat shape, increased size and increased SA-β-gal activity after repeated stimulation (3 or 6 times) with LPS. Reactive oxygen species (ROS) staining showed that the number of ROS-stained cells and the DCFH fluorescent level were higher in the LPS-treated DPSCs compared with those in the untreated DPSCs. Protein and mRNA expression levels of γ-H2A.X and p16(INK4A) were also increased in DPSCs with repeated LPS stimulation. We found that the LPS bound with Toll-like receptor 4 (TLR4) and that TLR4 signaling accounted for p16(INK4A) expression. Further results indicated that the senescence of DPSCs stimulated repeatedly with LPS was reversed by p16(INK4A) short interfering RNA. The DNA damage response and p16(INK4A) pathways might be the main mediators of DPSC senescence induced by repeated LPS stimulation. Thus, DPSCs tend to undergo senescence after repeated activation, implying that DPSC senescence starts after many inflammatory challenges. Ultimately, these findings should lead to a better understanding of DPSC-based clinical therapy.
authors.affiliation: Department of Stomatology, Affiliated Hospital of Nantong University, Nantong, China.
authors.forename: Xingmei
authors.initials: X
authors.lastname: Feng
authors.forename: Guijuan
authors.initials: G
authors.lastname: Feng
authors.forename: Jing
authors.initials: J
authors.lastname: Xing
authors.forename: Biyu
authors.initials: B
authors.lastname: Shen
authors.forename: Wei
authors.initials: W
authors.lastname: Tan
authors.forename: Dan
authors.initials: D
authors.lastname: Huang
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Lu
authors.forename: Tao
authors.initials: T
authors.lastname: Tao
authors.forename: Jinlong
authors.initials: J
authors.lastname: Zhang
authors.forename: Liren
authors.initials: L
authors.lastname: Li
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Gu
mesh_terms: D000293:Adolescent; D000328:Adult; D017209:Apoptosis; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D003782:Dental Pulp / Q000166:cytology*; D006657:Histones / Q000096:biosynthesis; D006801:Humans; D008070:Lipopolysaccharides; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008964:Molar, Third / Q000166:cytology*; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D055815:Young Adult; D001616:beta-Galactosidase
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; C468783:H2AX protein, human; D006657:Histones; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D001616:beta-Galactosidase