Phorbol myristate acetate induces cellular senescence in rat microglia in vitro

Int J Mol Med. 2020 Jul;46(1):415-426. doi: 10.3892/ijmm.2020.4587. Epub 2020 Apr 23.

Abstract

The present study aimed to establish a cellular model to test the hypothesis that oncogene-induced senescence (OIS) is triggered by aging-related activation of microglia. Primary microglia were incubated with phorbol 12-myristate 13-acetate (PMA), and β-galactosidase (β-Gal) staining was applied to subsequent assessment of cellular senescence. Moreover, flow cytometry was employed for examinations of cell cycle arrest and senescence-associated proteins, p53 and p21 were measured by western blotting. Furthermore, examination of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were carried out with microglia supernatants undergoing age-related degenerative diseases in the nervous system, using ELISA. PC12 cells were co-cultured with microglia activated by aging-related alteration(s) to evaluate whether apoptosis was increased in PC12 cells. Cellular senescence-associated β-Gal staining showed that microglial β-Gal expression gradually increased with prolonged PMA stimulation. Microglia in the group receiving 72 h of PMA stimulation displayed the highest percentage of cells arrested in G0/G1, the highest amount of senescence-associated expression of p53 and p21, and the most prominent secretion of TNF-α and IL-1β. In comparison with controls, an increase of apoptotic PC12 cells was detected, which were co-cultured with aging microglia. Taken together, microglia tend to undergo senescence after PMA treatment, suggesting that microglial senescence is associated with inactivation of certain oncogenes.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / genetics
  • Cell Cycle Checkpoints / drug effects
  • Cellular Senescence / drug effects
  • Cellular Senescence / genetics
  • Enzyme-Linked Immunosorbent Assay
  • Interleukin-1beta / metabolism
  • Microglia / metabolism
  • Microglia / pathology*
  • PC12 Cells
  • Rats
  • Tetradecanoylphorbol Acetate / pharmacology*
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Interleukin-1beta
  • Tumor Necrosis Factor-alpha
  • Tetradecanoylphorbol Acetate