Ascorbic acid 6-palmitate (L-AP) is a lipid-soluble derivative of ascorbic acid (Vitamin C, VitC), which has been indicated as an antioxidant [
10,
11]. To confirm the roles of L-AP on lipopolysaccharide (LPS)-induced microglia activation in BV-2 cells independent of cell growth inhibition, we first examined the potential cytotoxicity of L-AP on BV-2 cells in the absence or presence of LPS stimulation for 24 h by CCK8 assay. After 24 h of exposure to the various concentrations of L-AP, the viability of BV-2 cells did not suffer from significant inhibition at the concentrations below 62.5 µM (Fig.
2A). IC
50 value of L-AP was 84.56 µM (Fig.
2B). Therefore, concentrations of 2.5, 12.5 and 62.5 µM L-AP were chosen for subsequent experiments. According to our preliminary study, LPS (1 µg/mL) exposure for 24 h did not significantly change cell growth. And as we expected, after 4 h of pre-treatment with different concentrations of L-AP (2.5,12.5 and 62.5 µM), the viability of BV-2 cells was not significantly inhibited by L-AP at all concentrations in the presence of LPS (1 µg/mL) (
P > 0.05, Fig.
2C). Then, the cellular morphology was observed as an indicator of the microglia activation level in the presence of LPS (1 µg/mL) for 24 h. We found that in the presence of LPS (1 µg/mL), BV-2 cells become spindle-shaped, while in the absence of LPS (1 µg/mL), BV-2 cells are round-shaped (Fig.
2D). Moreover, the ELISA data showed that the concentrations of IL-6 and TNF-α were significantly increased in the presence of LPS (1 µg/mL) for 24 h, while pre-treated with L-AP at 62.5 µM notably prevented LPS-induced IL-6 and TNF-α secretion (
P < 0.05, Fig.
2E). Subsequently, whether L-AP treatment modulates LPS induced microglia M1/M2 polarization, mRNA levels of M1 marker (
inos) and M2 marker (
il-10 and
arg-1) were measured by qRT-PCR. Our data showed that in the presence of LPS (1 µg/mL) for 24 h, the mRNA level of
inos was significantly increased, and the mRNA levels of
il-10 and
arg-1 were significantly decreased, whereas the M2 marker (
il-10 and
arg-1) levels were significantly increased by L-AP pre-treatment at 62.5 µM (
P < 0.05, Fig.
2F). However, L-AP treatment at 62.5 µM did not demonstrate significant inhibition on M1 marker (
inos) transcription (
P > 0.05, Fig.
2F). At the same time, although VitC pre-treatment tended to reduce
inos level and increase the level of M2 markers (
il-10 and
arg-1) in the presence of LPS (1 µg/mL), there was no significant difference (
P > 0.05, Fig.
2F). Taken together, microglia were activated and polarized to the M1 phenotype after LPS exposure and the treatment with L-AP at 62.5µM promoted the polarization of microglia to the alternative M2 phenotype, whereas VitC did not switch microglia M1/M2 polarization in this condition.