2-arachidonyl glycerol modulates astrocytic glutamine synthetase via p38 and ERK1/2 pathways

Abstract

Background: The glutamine synthetase (GS), an astrocyte-specific enzyme, is involved in lipopolysaccharide (LPS)-induced inflammation which activates the mitogen-activated protein kinase (MAPK) signaling. Endocannabinoid 2-arachidonyl glycerol (2-AG) has been described to serve as an endogenous mediator of analgesia and neuroprotection. However, whether 2-AG can directly influence astrocytic GS and MAPK expressions remains unknown.

Methods: In the present study, the effects of 2-AG on astrocytic GS expression, p38 and ERK1/2 expression, cell viability, and apoptosis following LPS exposure were investigated.

Results: The results revealed that LPS exposure increased GS expression with p38 activation in the early phase and decreased GS expression with activation of ERK1/2, decrease of cell viability, and increase of apoptosis in the late phase. Inhibition of p38 reversed GS increase in the early phase while inhibition of ERK1/2 reversed GS decrease in the late phase induced by LPS exposure. 2-AG protected astrocytes from increase of apoptosis and decrease of cell viability induced by the late phase of LPS exposure. In the early phase of LPS exposure, 2-AG could suppress the increase of GS expression and activation of p38 signaling. In the late phase of LPS exposure, 2-AG could reverse the decrease of GS expression and activation of ERK1/2 induced by LPS.

Conclusion: These findings suggest that 2-AG could maintain the GS expression in astrocytes to a relatively stable level through modulating MAPK signaling and protect astrocytes from LPS exposure.

Keywords: 2-AG; Astrocyte; ERK; Glutamine synthetase; MAPK; p38.

Fig. 1

LPS exposure induced activation of astrocytes (a), changes of GS expression in astrocytes (b), decrease of cell viability in astrocytes (c), and activation of MAPK (d). Astrocytes were treated with LPS at different dosages for 6 h and GFAP expression was analyzed (a). Then astrocytes were treated with 1 μg/ml LPS for different times and GS expression (b), cell ability (c), and activation of MAPK were analyzed (d). Error bars were ± SD. n = 3. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control

Fig. 2

Effects of 2-AG on the change of cell apoptosis and viability of astrocytes induced by LPS. Astrocytes were pretreated with 0.01 μM 2-AG for 2 h and exposed to 1 μg/ml LPS for 12 h. Cell apoptosis was measured by Hoechst 33342 assay (a, b) (the red arrows indicate the apoptotic cells) and MTT was used to measure cell viability (c). The expressions of Bcl-xl and Bax were evaluated through western blotting assay (d, e). Scar bar = 20 μm, error bars were ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

Effects of inhibition of MAPK on the phosphorylation of p38 (a) and ERK1/2 (b), and GS expression (a, b). Astrocytes were pretreated with SB203580 (a) or PD98059 (b) for 1 h and exposed to 1 μg/ml LPS for 2 h (a) or 12 h (b). The protein level of GS, p-p38 and p-ERK1/2 were measured, respectively (a, b). Error bars were ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

Effects of 2-AG on phosphorylation of p38 (a), ERK1/2 (b), and GS expression (a, b) induced by LPS. Astrocytes were pretreated with 0.01 μM 2-AG for 2 h and exposed to 1 μg/ml LPS for 2 h (a) or 12 h (b). The protein level of GS, p-p38 and p-ERK1/2 were measured, respectively (a, b). Error bars were ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 5

Effects of 2-AG on translocation of p38 (a), ERK1/2 (b), and expression of GS (a, b) in astrocytes induced by LPS. Astrocytes were pretreated with 0.01 μM 2-AG for 2 h and exposed to 1 μg/ml LPS for 2 h (a) or 12 h (b). Immunocytochemistry assay was used to analyze the translocation of p38 and ERK1/2 and the change of GS expression. Scar bar = 10 μm

Fig. 6

Effects of inhibitors of CB₁R and CB₂R antagonists on phosphorylation of p38 (a), ERK1/2 (b), and GS expression (a, b) in astrocytes induced by LPS. Astrocytes were pretreated with 1 μM AM281 or AM630 for 1 h before cells were treated with 2-AG and LPS. The protein level of p-p38 (a), p-ERK1/2 (b), and GS were measured by western blotting. Error bars were ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

Schematic drawing of the 2-AG on the GS expression and MAPK activation in astrocytes induced by LPS