BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed
to play a key role in the development and progression of neurodegenerative diseases. The aim of
this investigation was to determine levels of neurosteroids produced by resting and injured BV-2
microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively
reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability
was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly,
rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then,
BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the
overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone,
5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the
culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.
BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and
pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only
synthesize several neurosteroids, but further increase this production following oxidative damage.
Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in
neurodegenerative diseases.