Phorbol myristate acetate induces cellular senescence in rat microglia in vitro

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 beta-galactosidase (beta-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 alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) 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 beta-Gal staining showed that microglial beta-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-alpha and IL-1 beta. 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.