Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Aug 20.
Published in final edited form as: Biochem Biophys Res Commun. 2022 May 30;617(Pt 1):30–35. doi: 10.1016/j.bbrc.2022.05.090

BAY 11-7082 inhibits the secretion of interleukin-6 by senescent human microglia

Maxwell Cook a,#, Houmin Lin a,#, Sandeep K Mishra a, Gavin Y Wang a,b,*
PMCID: PMC9540971  NIHMSID: NIHMS1839873  PMID: 35671608

Abstract

The accumulation of senescent cells in aged tissues has been implicated in a variety of age-related diseases, including cancer and neurodegenerative disorders. Recent studies have demonstrated a link between age-associated increase of senescent glial cells in the brain and the pathogenesis of Alzheimer’s disease (AD). However, there is a lack of in vitro cellular models of senescent human microglia, which significantly limits our approaches to study AD pathogenesis. Here, we show for the first time that ionizing radiation (IR) dose-dependently induces premature senescence in HMC3 human microglial cells. Senescence-associated β-galactosidase activity, a well-characterized marker of cellular senescence, was substantially increased in irradiated HMC3 cells compared with control cells. Furthermore, we found that phosphorylated p53 levels and p21 expression levels were markedly higher in IR-induced senescent microglia than in control cells. Senescent human microglia exhibited the senescence-associated secretory phenotype (SASP), as evidenced by the increased secretion of pro-inflammatory cytokine interleukin-6 (IL-6). Treatment with an NF-κB inhibitor, BAY 11–7082, inhibits the secretion of IL-6 by senescent HMC3 cells. Collectively, our studies have established an in vitro cellular model of human microglial senescence and suggest that the NF-κB pathway may play a critical role in regulating the SASP of senescent HMC3 cells.

1. Introduction

Senescent cells accumulate in aged tissues and are thought to play a critical role in age-related diseases, including cancer and neurodegenerative disorders [1, 2]. Replicative senescence was originally observed in cultured normal human fibroblasts after they reached the maximum number of cell doublings or replication cycles, which is referred to as the Hayflick limit [3]. Apart from replicative cellular senescence, cells can undergo premature senescence, also known as stress-induced senescence, in response to a variety of cell-intrinsic and extrinsic stimuli, such as DNA damage, oxidative stress, oncogenic activation, and exposure to radiation [4–7]. Senescent cells can survive for a long period of time and produce large amounts of pro-inflammatory cytokines, such as interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha (TNFα), termed as the senescence-associated secretory phenotype (SASP) [2]. Microglia, the main resident macrophages of the central nervous system (CNS), have been implicated in the pathogenesis of several neurological disorders [8, 9], including Alzheimer’s disease (AD). A recent study reported that microglia could undergo replicative senescence in a mouse model of AD and that the prevention of senescence induction led to reduced accumulation of amyloid beta and diminished synaptic damage [10]. We and others have shown that ionizing radiation (IR) is a potent inducer of premature senescence in various types of mammalian cells [11–14]. However, whether IR can induce premature senescence in human microglial cells has yet to be determined, which was a major goal of this study.

Cellular senescence is a hallmark of aging [15], and aging is the main risk factor for neurodegenerative disorders [16]. Thus, it is conceivable that senescent cells may play a significant role in AD pathogenesis and progression. Indeed, increased number of senescent glial cells has been observed in mouse models of AD as well as in the brains of AD patients [10, 17]. Notably, depletion of senescent cells has the potential to improve tau pathology and alleviate cognitive impairments in preclinical models [17, 18]. These results suggest that targeted clearance of senescent cells in the brain may represent a new therapeutic approach for AD treatment. Nevertheless, in vitro cellular models of human microglial senescence have yet to be established and characterized. In this study, we demonstrate that IR is highly effective at inducing premature senescence in human HMC3 microglial cells. We also show that senescent microglia secrete high levels of neuroinflammatory cytokine IL-6 and that blocking of NF-κB by BAY 11–7082 inhibits the release of IL-6 by senescent HMC3 cells. These findings point to a role of the NF-κB signaling pathway in modulating the SASP of senescent human microglia.

2. Materials and Methods

2.1. Cell line, reagents, and antibodies

The HMC3 human microglial cell line (ATCC CRL-3304) and EMEM medium (ATCC 30–2003) were purchased from American Type Culture Collection (ATCC, Manassas, VA). BrdU (59–14-3) and mouse anti-BrdU (B2531) monoclonal antibody (mAb) were purchased from Sigma (St. Louise, MO). BAY 11–7082 was obtained from Cayman Chemical (Ann Arbor, MI, cat# 10010266). Human IL-6 DuoSet ELISA (DY206) kits were purchased from R&D Systems (Minneapolis, MN). Phospho-p53 (Ser 15) antibody (#9284), p53 mouse mAb (#2524), NF-κB p65 rabbit mAb (#8242), p21 Waf1/Cip1 rabbit mAb (#2947), and β-Actin mouse mAb (#3700) were purchased from Cell Signaling (Danvers, MA). Alexa Fluor-594 labeled goat anti-mouse IgG (#A10680) and Alexa Fluor-647 labeled goat anti-rabbit IgG (#A21244) were purchased from Thermo Fisher (Waltham, MA). Mouse anti-human p16 mAb (cat# 51–1325GR) was obtained from BD Biosciences. Mouse anti-human CD68 mAb (Cat# 14–0688-82, clone: KP1) was obtained from Thermo Fisher. Pro-Long Gold Anti-Fade Reagent (#9071) was obtained from Cell Signaling Technology (Danvers, MA).

2.2. Irradiation and cell growth curve assay

HMC3 cells were cultured using EMEM medium containing 10% FBS, 2 mM L-glutamine and 100 μg/mL of penicillin-streptomycin (Thermo Fisher). Cells were plated in 35 mm culture dishes and exposed to 10 Gray (Gy) of ionizing radiation (IR) using a 137Cs γ-irradiator (JL Shepherd, Glendale, CA). Irradiated cells and the non-irradiated control cells were cultured at 37 °C with 5% CO2 for 1 to 5 days. Changes in the number of live cells were determined using trypan blue exclusion method after 1, 3, and 5 days of culture.

2.3. Senescence assay

Senescence-associated β-galactosidase (SA-β-gal) assays were performed using a senescence β-galactosidase staining kit (Cell Signaling, #9860) as we previously reported [6].

2.4. BrdU incorporation and confocal microscopy

BrdU incorporation assays were performed as we previously reported [13], with some minor modifications. Briefly, HMC3 cells were cultured in 4-well CELLview™ cell culture dishes (Greiner Bio-One North America) and incubated with BrdU (10 μM) for 12 h. Cells were blocked with 5% normal goat serum for 30 min prior to incubation with mouse anti-BrdU mAb (1:200, Sigma) overnight at 4 °C. After extensive washes, cells were incubated with goat anti-mouse IgG-Alexa Fluor 647 (1:500) for 2 h at room temperature (RT). Microscopic imaging data were acquired using an LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany).

2.5. Western blotting

Western blot analyses were performed as previously described [19]. Briefly, protein samples were prepared using cell lysis buffer (Cell Signaling) supplemented with a cocktail of proteinase inhibitors (Sigma). Protein concentrations were quantified using a Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, Hercules, CA). Proteins were resolved on 4 – 20% Mini-Protean TGX gels (Bio-Rad) and transferred onto 0.2 μm PVDF membrane (Millipore). Blots were blocked with 5% non-fat milk at RT for 1 h before incubating with primary antibodies at 4°C overnight. After extensive washing with TBS-T, blots were incubated with appropriate HRP-conjugated secondary antibodies at RT for 1.5 h. Protein bands were detected using an ECL Plus Western Blotting Detection System and an Odyssey Fc Imaging System (LI-COR Biosciences).

2.6. CD68 Immunofluorescence

CD68 expression levels were assessed using immunofluorescence and confocal microscopy. Briefly, irradiated and control HMC3 cells were cultured in 4-well CELLview™ dishes (Greiner Bio-One North America). Cells were fixed with 4% paraformaldehyde (PFA) for 15 min, and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min. Cells were incubated with mouse anti-human CD68 (1:100) overnight at 4°C. Then, cells were incubated with Alexa Fluor-647-labeled goat anti-mouse IgG (1:500) at RT for 2 h. Images were captured using an LSM 880 Airyscan super-resolution confocal microscope (Carl Zeiss, Oberkochen, Germany). Mean fluorescence intensity (MFI) was quantified using ImageJ (NIH).

2.7. Enzyme-linked immunosorbent assay (ELISA)

ELISA was employed to measure IL-6 levels. Briefly, IR-induced senescent or control HMC3 cells were plated in 12-well plates. Three days after IR, cell culture media were replaced with serum-free EMEM and cells were treated with BAY 11–7082 or DMSO as a vehicle control. Supernatants were collected at 24 h after drug treatment and cells debris were removed by centrifuging at 4,000 rpm for 5 min. IL-6 levels were measured using a human IL-6 DuoSet ELISA kit (R&D Systems, cat# DY206) according to the manufacturer’s instructions. Plates were read using a SYNERGY H1 microplate reader (BioTek).

2.8. Real-time reverse transcriptase-PCR (RT-PCR)

Total RNA was extracted using TRIzol reagent (Thermo Fisher). First-strand cDNA was synthesized from 1 μg of total RNA using an iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. The PCR primers used for this study are IL-1β (Forward: 5’-AACCTCTTCGAGGCACAAGG-3’, reverse: 5′-AGCCATCATTTCACTGGCGA-3’), IL-6 (forward: 5′-GAAGGCAGCAGGCAACAC-3′, reverse: 5’-TGAACTCCTTCTCCACAAGCG-3’), TNF-α (forward: 5’-CTTCTGCCTGCTGCACTTTG-3’, reverse: 5’-GTCACTCGGGGTTCGAGAAG-3’), and GAPDH (forward: 5’-GACAGTCAGCCGCATCTTCT-3’, reverse: 5’-GCGCCCAATACGACCAAATC-3’). Expression levels of human IL-6, IL-1β and TNF-α mRNA were measured using iTaq Universal SYBR® Green Supermix (Bio-Rad) on a LightCycler® 480 System (Roche). GAPDH expression levels were used as an internal reference to calculate the changes in target mRNA expression using the 2−ΔΔCT method as described previously [14].

2.9. NF-κB p65 nuclear translocation assay

HMC3 cells were plated in 4-well CELLview™ dishes (Greiner Bio-One North America) and cultured at 37°C with 5% CO2. Immunofluorescence was performed at 1 h after IR to determine NF-κB p65 nuclear translocation. Briefly, cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100. After blocking with 5% normal serum, cells were incubated with p65 antibody (1:100) over night at 4°C. After washing with PBS, cells were incubated with goat anti-rabbit IgG Alexa Fluor 647 (1:500) at RT for 2 h. Images were acquired using an LSM 880 Airyscan super-resolution confocal microscope (Carl Zeiss, Oberkochen, Germany). Mean fluorescence intensity (MFI) was measured using ImageJ (NIH).

2.10. Statistical analysis

Data are presented as mean ± SEM. Comparisons between two groups were carried out using Student’s t-test. Multiple comparisons were performed using one-way analysis of variance (ANOVA). Differences were considered statistically significant at p < 0.05. All analyses were carried out using GraphPad Prism software (GraphPad Software, Inc. San Diego, CA).

3. Results

3.1. IR induces premature senescence in human HMC3 microglia

We and others have demonstrated that IR is a potent inducer of premature senescence in several types of mammalian cells, including hematopoietic stem cells [11–14]. However, it has yet to be determined if IR induces senescence in human microglia. To fill this knowledge gap, we exposed HMC3 cells to a range of IR doses. Then, senescent cells were detected using SA-β-gal assay because increased SA-β-gal activity is a well-characterized biomarker of senescence [20]. Our data demonstrates that the number of SA-β-gal positive senescent cells increases with IR doses in irradiated HMC3 cells (Fig. 1A & B). These results indicate that IR dose-dependently induces premature senescence in human microglia. Moreover, the senescent HMC3 cells exhibited a morphological change, showing enlarged cell size, one of the typical characteristics of senescent cells.

Fig. 1. IR dose-dependently induces premature senescence in HMC3 cells.

Fig. 1.

Open in a new tab

(A) SA-β-gal assays were performed 5 days after irradiation to identify senescent cells. Shown are representative microscopic images of SA-β-gal positive (blue stained) senescent and control HMC3 cells. (B) The percentage of SA-β-gal positive senescent cells was quantified and graphed. (C) Cell growth curve assays were performed to determine the proliferating potential of HMC3 cells. (D) BrdU incorporation assays were performed 4 days after IR using a published protocol [13]. (E) The percentage of BrdU positive cells was quantified and graphed. Data are presented as a mean ± SEM of three independent assays. Scale bar = 25 μm, and it applies to all microscopic images. **p < 0.01, ***p < 0.001 versus control (Ctrl or 0 Gy irradiation).

Subsequent cell growth curve analyses show that the irradiated HMC3 cells exhibit a persistent growth arrest. In contrast, the control cells continuously proliferate and increase their numbers during the entire period of culture (Fig. 1C). Furthermore, BrdU incorporation assays revealed that irradiated HMC3 cells almost completely lost their ability to incorporate BrdU for DNA synthesis (Fig. 1D & E), indicating that they were in a state of irreversible cell cycle arrest, a hallmark of cellular senescence.

3.2. IR-induced senescence of HMC3 cells was associated with activation of the p53/p21 pathway

Our previous studies have demonstrated that activation of the p53/p21signaling pathway plays a critical role in IR-induced premature senescence [12]. Moreover, DNA damage induces phosphorylation of p53 at Ser15, leading to p53 activation [21]. To gain insight into the mechanism by which IR induces senescence in human microglia, we investigated the effects of IR on the p53/p21signaling pathway. Western blot analyses reveal that IR substantially increases expression levels of phosphorylated p53 (p-p53) in a dose-dependent manner, indicating that IR activates the p53 pathway in HMC3 cells (Fig. 2A & B). The activation of p53 by IR was further confirmed by the finding that expression levels of p21, a key downstream target of p53, were markedly increased in irradiated HMC3 cells (Fig. 2A & C). However, there was no clear correlation between p16 expression levels and IR doses (Fig. 2A)

Fig. 2. IR-induced senescence of HMC3 cells was associated with activation of the p53/p21 pathway.

Fig. 2.

Open in a new tab

(A) Expression levels of phosphorylated p53 (p-p53, Ser15), total p53, p21, and p16 in HMC3 cells were assessed at 48 h after IR using Western blotting. Representative immunoblotting images are shown. (B, C) Relative expression levels of p-p53 (B) and p21 (C) were quantified using ImageJ and graphed. (D) Expression levels of p-p53 (Ser15), p53, p21, and p16 were analyzed by immunoblotting at different time points after IR. (E, F) Relative expression levels of p-p53 (E) and p21 (F) were quantified using ImageJ. All data are presented as a mean ± SEM of three independent assays. *p < 0.05, **p < 0.01, ***p < 0.001.

Subsequent time course studies show that IR induces a rapid phosphorylation of p-p53 and the increased phosphorylation of p53 sustains for at least 48 h, suggesting that IR may induce a persistent activation of p53 in HMC3 cells (Fig. 2D & E). IR-induced increase in p21 expression was detectable at 8 h after irradiation and this increase peaked at 48 h after IR (Fig. 2D & F). These results demonstrate a role of the p53/p21 pathway in IR-induced senescence in HMC3 cells. Consistent with our finding, it has been shown that p21 can act both as a mediator and a biomarker of cellular senescence [11]. In contrast, changes in p16 expression levels were not closely associated with the time points after IR exposure (Fig. 2D).

3.3. Senescence of HMC3 cells produce high levels of IL-6

It has been well-documented that senescent cells produce pro-inflammatory cytokines and chemokines, which are major components of the SASP [2, 22]. Moreover, the SASP is thought to be a hallmark of senescent cells [23]. To determine if IR-induced senescent human microglia show the characteristic of SASP, we measured IL-6 levels in HMC3 cell culture supernatants using ELISA. We found that IL-6 levels were markedly increased in senescent microglia compared with those in control cells (Fig. 3A). In addition, our data show that IL-6 levels correlate with IR doses and the percentage of SA-β-gal positive senescent cells (Fig. 1B, Fig. 3A). These results confirmed that senescent HMC3 cells exhibit the SASP and secrete high levels of IL-6.

Fig. 3. Senescent HMC3 cells produce high levels of IL-6.

Fig. 3.

Open in a new tab

(A) Levels of IL-6 were measured using ELISA, and the data indicates that IR dose-dependent increases IL-6 secretion from senescent HMC3 cells. (B-D) RT-PCR assays were performed to assess levels of IL-6 (B), IL-1β (C) and TNFα (D) mRNA transcription. (E) Immunofluorescence and confocal microscopic assays were performed to analyze CD68 expression in HMC3 cells. (F) CD68 mean florescence intensity (MFI) was quantified using ImageJ. Data are presented as a mean ± SEM of three independent experiments. Scale bar = 25 μm, and it applies to all microscopic images. ***p < 0.001, ****p < 0.0001.

Next, we examined the transcription levels of IL-6, IL-1β and TNFα mRNA in irradiated HMC3 cells. As shown in Fig. 3B, our data demonstrates that IR dose-dependently promotes IL-6 mRNA transcription in HMC3 cells. However, mRNA expression levels of IL-1β and TNFα were not significantly changed in irradiated HMC3 cells as compared to those in control cells (Fig. 3C & D). Consistent with our finding, it was reported that IL-1β was not detectable in HMC3 cells [24]. It also has been shown that HMC3 cells are not efficient at secreting TNFα [25].

Increased expression of pro-inflammatory cytokines such as IL-6 is a marker of activated microglia in AD [26]. CD68 is also considered as a marker of activated microglia [27, 28], which prompted us to examine CD68 expression levels in senescent HMC3 cells. Our data revealed that CD68 expression levels were markedly higher in senescent than in control HMC3 cells (Fig. 3E & F). These results suggest that senescent HMC3 cells may share the characteristics of activated microglia, such as secretion of pro-inflammatory cytokines and expressing high levels of CD68.

3.4. Treatment with BAY 11–7082 inhibits the secretion of IL-6 by senescent HMC3 cells

To explore the mechanisms by which the SASP is regulated in senescent human microglia, we investigated the effects of IR on the NF-κB signaling pathway. We found that the nuclear immunoreactivity for NF-κB/p65 was substantially higher in irradiated HMC3 cells than in control cells (Fig. 4A & B). These results suggest that IR may promote NF-κB nuclear translocation and thus stimulate its activation in HMC3 cells. In agreement with this idea, we show that treatment with BAY 11–7082, a potent NF-κB inhibitor [29], dose-dependently inhibits the secretion of IL-6 by senescent HMC3 cells (Fig. 4C). Together, our findings suggest that NF-κB may play a critical role in modulating the SASP of senescent HMC3 cells.

Fig. 4. IR promotes NF-κB/p65 nuclear translocation and blocking of NF-κB inhibits IL-6 secretion.

Fig. 4.

Open in a new tab

(A) Shown are representative confocal microscopic images of NF-κB/p65 nuclear translocation assays. (B) MFI of p65 nuclear immunostaining was analyzed using ImageJ and graphed. (C) ELISA assays were performed to measure IL-6 levels. The results show that treatment with BAY 11–7082 (BAY) dose-dependently inhibits IL-6 secretion by senescent HMC3 cells. Data are presented as a mean ± SEM of three independent experiments. Scale bar = 25 μm, and it applies to all microscopic images. *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

Alzheimer’s disease (AD) is the most common type of neurodegenerative disorders and the leading cause of dementia. It has been estimated that 6.5 million Americans aged 65 and older are currently living with AD [30]. Nevertheless, effective therapeutics for AD and Alzheimer related dementia have yet to be developed. Microglial dysfunction and senescence have been implicated in the pathogenesis of AD [10, 31]. However, the mechanisms whereby human microglia undergo senescence in AD are not fully understood. In the present study, we show that IR induces human microglial senescence in a dose-dependent manner. Mechanistically, we found that IR-induced senescence of human microglia is associated with a sustained activation of the p53 pathway and a time-dependent increase of p21 expression. In contrast, p16 expression levels did not show such a pattern in irradiated human HMC3 microglial cells. This finding is consistent with the previous observations showing that not all the cells expressing p16 are senescent [32] and that not all senescent cells express high levels of p16 [23]. Although activation of the p53/p21 and the p16INK4a/Rb pathways have been implicated in the processes of senescence induction and maintenance [7, 33], our studies indicate that activation of the p53/p21 rather than the p16/Rb pathway may play a key role in IR-induced senescence in HMC3 cells.

Elevated IL-6 levels have been found in brain specimens from AD patients [34]. A meta-epidemiological study indicated that human cognitive functions adversely correlated with serum IL-6 levels [35]. It also has been shown that a high level of IL-6 is associated with the severity of cognitive impairments in AD patients [36]. Moreover, it was reported that cerebral overexpression of IL-6 resulted in transgene dose- and animal age-dependent deficits in avoidance learning as well as progressive neurodegenerative pathologies [37]. Collectively, these results suggest that neuroinflammation-associated high levels of IL-6 secretion may play a role in the pathogenesis of neurodegenerative disorders. However, the cellular sources of the elevated IL-6 levels in AD have yet to be determined. Our data demonstrates, for the first time, that IL-6 levels are markedly higher in IR-induced senescent human microglia than in control cells. This new finding indicates that senescent microglial cells may be a major source of high levels of IL-6 in AD.

Recent studies have suggested a potentially important role of senescent microglial cells in AD development and progression [10, 33]. This concept was further reinforced by the findings that pharmacological clearance of senescent cells using senolytic drugs delayed the progression of AD and alleviated tau-dependent pathology in animal models [17, 18]. Nevertheless, the mechanisms of senescence induction and the biological characteristics of senescent human microglial cells have not been well defined. Our work demonstrates, for the first time, that IR exposure induces premature senescence in human HMC3 microglial cells in a dose-dependent fashion. We also show that senescent microglia produce high levels of IL-6 and that inhibition of NF-κB prevents the release of IL-6 by senescent HMC3 cells. These results suggest that the NF-κB signaling pathway may play a crucial role in regulating the SASP of senescent human microglia. More recently, it has been hypothesized that senescent cells may contribute to the onset and progression of AD via boosting senescence-associated neuroinflammation [31], but experimental data that substantiates such idea is still scarce. Using IR-induced senescent human microglial cells as a model system, our studies provide the first in vitro experimental evidence to support this hypothesis.

Highlights.

  • IR dose-dependently induces premature senescence in HMC3 human microglial cells

  • Senescent HMC3 cells show a sustained activation of the p53/p21 pathway

  • Senescent human microglial cells secrete high levels of interleukin-6 (IL-6)

  • Treatment with BAY 11–7082 inhibits the secretion of IL-6 by senescent HMC3 cells

Acknowledgments

This study was supported in part by the National Institutes of Health (NIH) grants AG068286 and CA210962. M.C. was supported by an NIH training grant, T32 GM132055. The authors wanted to thank Steven Grant Dixon for excellent technical assistance

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflicts of interest

The authors declare no conflict of interest.

References

  • 1.Campisi J, Aging, cellular senescence, and cancer, Annu. Rev. Physiol 75 (2013) 685–705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Childs BG, Durik M, Baker DJ, van Deursen JM, Cellular senescence in aging and age-related disease: from mechanisms to therapy, Nat. Med 21 (2015) 1424–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Shay JW, Wright WE, Hayflick, his limit, and cellular ageing, Nat. Rev. Mol. Cell Biol 1 (2000) 72–6. [DOI] [PubMed] [Google Scholar]
  • 4.Toussaint O, Royer V, Salmon M, Remacle J, Stress-induced premature senescence and tissue ageing, Biochem. Pharmacol 64 (2002) 1007–9. [DOI] [PubMed] [Google Scholar]
  • 5.Luo H, Yang A, Schulte BA, Wargovich MJ, Wang GY, Resveratrol Induces Premature Senescence in Lung Cancer Cells via ROS-Mediated DNA Damage, PLoS One 8 (2013) e60065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Zhong G, Qin S, Townsend D, Schulte BA, Tew KD, Wang GY, Oxidative stress induces senescence in breast cancer stem cells, Biochem. Biophys. Res. Commun 514 (2019) 1204–1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Qin S, Schulte BA, Wang GY, Role of senescence induction in cancer treatment, World J. Clin. Oncol 9 (2018) 180–187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Li Q, Barres BA, Microglia and macrophages in brain homeostasis and disease, Nat. Rev. Immunol 18 (2018) 225–242. [DOI] [PubMed] [Google Scholar]
  • 9.Navarro V, Sanchez-Mejias E, Jimenez S, et al. , Microglia in Alzheimer’s Disease: Activated, Dysfunctional or Degenerative. Front. Aging Neurosci 10 (2018) 140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hu Y, Fryatt GL, Ghorbani M, et al. , Replicative senescence dictates the emergence of disease-associated microglia and contributes to Aβ pathology. Cell Rep 35 (2021) 109228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Fletcher-Sananikone E, Kanji S, Tomimatsu N, et al. , Elimination of Radiation-Induced Senescence in the Brain Tumor Microenvironment Attenuates Glioblastoma Recurrence, Cancer Res 81 (2021) 5935–5947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Luo H, Yount C, Lang H, et al. , Activation of p53 with Nutlin-3a radiosensitizes lung cancer cells via enhancing radiation-induced premature senescence, Lung Cancer 81 (2013)167–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.He X, Yang A, McDonald DG et al. , MiR-34a modulates ionizing radiation-induced senescence in lung cancer cells, Oncotarget 8 (2017) 69797–69807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wang Y, Schulte BA, Larue AC, Ogawa M, Zhou D D, Total body irradiation selectively induces murine hematopoietic stem cell senescence, Blood 107 (2006) 358–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G, The hallmarks of aging, Cell 153 (2013) 1194–217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Niccoli T, Partridge L, Ageing as a risk factor for disease, Curr. Biol 22 (2012) R741–52. [DOI] [PubMed] [Google Scholar]
  • 17.Zhang P, Kishimoto Y, Grammatikakis I, et al. , Senolytic therapy alleviates Aβ-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer’s disease model, Nat. Neurosci 22 (2019) 719–728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bussian TJ, Aziz A, Meyer CF, et al. , Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline, Nature 562 (2018) 578–582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Qin S, He X, Lin H, et al. , Nrf2 inhibition sensitizes breast cancer stem cells to ionizing radiation via suppressing DNA repair, Free Radic. Biol. Med 169 (2021) 238–247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Dimri GP, Lee X, Basile G, et al. , A biomarker that identifies senescent human cells in culture and in aging skin in vivo, Proc. Natl. Acad. Sci. U. S. A 92 (1995) 9363–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Shieh SY, Ikeda M, Taya Y, Prives C, DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2, Cell 91 (1997) 325–34. [DOI] [PubMed] [Google Scholar]
  • 22.Birch J, Gil J, Senescence and the SASP: many therapeutic avenues, Genes Dev 34 (2020) 1565–1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Gorgoulis V, Adams PD, Alimonti A, et al. , Cellular Senescence: Defining a Path Forward, Cell 179 (2019) 813–827. [DOI] [PubMed] [Google Scholar]
  • 24.Dello Russo C, Cappoli N, Coletta I, et al. , The human microglial HMC3 cell line: where do we stand? A systematic literature review, J. Neuroinflammation 15 (2018) 259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hankittichai P, Lou HJ, Wikan N, et al. , Oxyresveratrol Inhibits IL-1β-Induced Inflammation via Suppressing AKT and ERK1/2 Activation in Human Microglia, HMC3, Int. J. Mol. Sci 21 (2020) 6054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Leng F, Edison P, Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here? Nat. Rev. Neurol 17 (2021) 157–172. [DOI] [PubMed] [Google Scholar]
  • 27.Hopperton KE, Mohammad D, Trépanier MO, Giuliano V, Bazinet RP, Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review, Mol. Psychiatry 23 (2018) 177–198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Matsumura A, Suzuki S, Iwahara N, et al. , Temporal changes of CD68 and α7 nicotinic acetylcholine receptor expression in microglia in Alzheimer’s disease-like mouse models, J. Alzheimers Dis 44 (2015) 409–23. [DOI] [PubMed] [Google Scholar]
  • 29.Dai Y, Pei XY, Rahmani M, et al. , Interruption of the NF-kappaB pathway by Bay 11–7082 promotes UCN-01-mediated mitochondrial dysfunction and apoptosis in human multiple myeloma cells, Blood 103 (2004) 2761–70. [DOI] [PubMed] [Google Scholar]
  • 30.Editorial, 2022 Alzheimer’s disease facts and figures, Alzheimers Dement 18 (2022) 700–789. [DOI] [PubMed] [Google Scholar]
  • 31.Guerrero A, De Strooper B, Arancibia-Cárcamo IL, Cellular senescence at the crossroads of inflammation and Alzheimer’s disease, Trends Neurosci 44 (2021) 714–727. [DOI] [PubMed] [Google Scholar]
  • 32.Frescas D, Hall BM, Strom E, Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent, Cell Cycle 16 (2017) 1526–1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Saez-Atienzar S, Masliah E, Cellular senescence and Alzheimer disease: the egg and the chicken scenario, Nat. Rev. Neurosci 21 (2020) 433–444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Bauer J, Strauss S, Schreiter-Gasser U, et al. , Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer’s disease cortices, FEBS Lett 285 (1991) 111–4. [DOI] [PubMed] [Google Scholar]
  • 35.Cojocaru IM, Cojocaru M, Miu G, Sapira V, Study of interleukin-6 production in Alzheimer’s disease, Rom. J. Intern. Med 49 (2011) 55–8. [PubMed] [Google Scholar]
  • 36.Lai KSP, Liu CS, Rau A, et al. , Peripheral inflammatory markers in Alzheimer’s disease: a systematic review and meta-analysis of 175 studies, J. Neurol. Neurosurg. Psychiatry 88 (2017) 876–882. [DOI] [PubMed] [Google Scholar]
  • 37.Heyser CJ, Masliah E, Samimi A, Campbell IL, Gold LH, Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain, Proc. Natl. Acad. Sci. U. S. A 94 (1997) 1500–5. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES