Phorbol myristate acetate induces cellular senescence in rat microglia in vitro

Cao,Dan; Li,Xiao-Hong; Luo,Xiao-Guang; Yu,Hong-Mei; Wan,Li-Shu; Wei,Ling; Ren,Yan

doi:10.3892/ijmm.2020.4587

Phorbol myristate acetate induces cellular senescence in rat microglia in vitro

Authors:
- Dan Cao
- Xiao-Hong Li
- Xiao-Guang Luo
- Hong-Mei Yu
- Li-Shu Wan
- Ling Wei
- Yan Ren
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Affiliations: Department of Geriatrics, The Fourth People's Hospital of Shenyang, Shenyang, Liaoning 110031, P.R. China, Department of Neurology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China, Department of Neurology, The First Hospital of Dandong, Dandong, Liaoning 118000, P.R. China
Published online on: April 23, 2020 https://doi.org/10.3892/ijmm.2020.4587
Pages: 415-426

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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.

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