Estrogen or estrogen receptor agonist inhibits lipopolysaccharide induced microglial activation and death

Abstract

Inflammation is an important pathogenic mechanism in many neurodegenerative disorders. Activated microglia play a pivotal role in releasing pro-inflammatory factors including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) for inducing inflammation. While microglia mediated inflammation is essential in maintaining CNS homeostasis, chronic inflammation results in activation of proteases for cell death. Here, we examined the effect of PPT (estrogen receptor α agonist), DPN (estrogen receptor β agonist), and estrogen on rat primary microglia following exposure to lipopolysaccharide (LPS). Exposure of microglia to LPS (200 ng/ml) for 24 h induced cell death. After LPS toxicity for 15 min, microglia were treated with 25 nM PPT, 25 nM DPN, or 100 nM estrogen that prevented cell death by attenuating the release of IL-1α, IL-1β, TNF-α, and COX-2. Treatment of cells with 100 nM fulvestrant (estrogen receptor antagonist) prior to addition of PPT, DPN, or estrogen significantly decreased their ability to prevent cell death, indicating involvement of estrogen receptor (ER) in providing PPT, DPN, or estrogen mediated cytoprotection. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed alterations in mRNA expression of Bax, Bcl-2, calpain, and calpastatin during apoptosis. We also examined mRNA expression of ERβ and ERα following exposure of microglia to LPS and subsequent treatment with PPT, DPN, or estrogen. We found that estrogen or estrogen receptor agonists upregulated expression of ERs. Overall, results indicate that estrogen receptor agonist or estrogen uses a receptor mediated pathway to protect microglia from LPS toxicity.

Fig. 1

Measurement of microglia viability using the trypan blue dye exclusion test following LPS exposure and estrogen treatment. Cell viability was measured as a percentage of total cell population. LPS = lipopolysaccharide; PPT = ERα agonist; DPN = ERβ agonist. **P ≤ 0.01 compared to control; #P < 0.05 compared to LPS exposure; ##P ≤ 0.01 compared to LPS exposure

Fig. 2

Measurement of relative NO production by primary rat microglia across all treatment groups. Production of NO was measured by quantifying nitrite concentration spectrophotometrically. Relative NO production for each group was determined as a ratio to NO production in the control group. **P ≤ 0.01 compared to control; #P < 0.05 compared to LPS exposure; ##P ≤ 0.01 compared to LPS exposure

Fig. 3

Quantification of ERα and ERβ mRNA levels following LPS toxicity and subsequent treatment with estrogen or estrogen receptor agonist using RT–PCR. #P ≤ 0.01 compared to control ERα expression; **P ≤ 0.01 compared to control ERβ expression

Fig. 4

mRNA levels of pro-inflammatory peptides expressed by activated primary rat microglia calculated using RT–PCR. *P < 0.05 compared to control; #P < 0.05 compared to LPS exposure

Fig. 5

Expression of pro-apoptotic and anti-apoptotic proteins by microglia following exposure to LPS and subsequent treatment with estrogen or ER agonist. *P < 0.05 compared to control; #P < 0.05 compared to LPS exposure