Endocannabinoid 2-AG Is Involved In Modulating LPS-induced Astrocytic Inflammatory Response

Objective: Previous studies have shown that astrocytes are involved in the generation and maintenance of neuroinflammation and chronic pain.Modulating the expression of glutamine synthetase(GS),an astrocyte-specific enzyme,could significantly suppress the neuroinflammation and chronic pain.Endocannabinoids have been described to serve as endogenous mediators of anti-inflammation,analgesia and neuroprotection.In this study,primary cultured astrocytes were used to investigate the effects of endocannabinoid 2-arachidonylglycerol(2-AG)in modulating LPS-induced astrocytic inflammatory response and explore the mechanism of anti-inflammation and neuroprotection of 2-AG in astrocytic inflammatory response.Methods : In this study,lipopolysaccharide(LPS)was used to induce the astrocytic inflammation response and effects of 2-AG on modulation of GS expression and MAPK signaling pathways were observed.Experimental groups: control group,LPS alone group,2-AG alone group,2-AG + LPS group,MAPK inhibitor group(JNK,p38 or ERK1/2 inhibitor + LPS-exposure)and cannabinoid receptors antagonist group(CB1 or CB2 antagonist + 2-AG + LPS).Results: The does-dependent experiment showed that 0.01-10 ?g/ml LPS could activate astrocytes and increase the expression of GFAP,and 1 ?g/ml was the optimal concentration of LPS-activated astrocytes.Subsequently,astrocytes were stimulated with 1 ?g/ml LPS and the expression of GS was observed.The results showed that GS expression increased within 0~3 h and peaked at 1 h,and then decreased after 3 h,at 12 h,GS expression got the minimum when compared with control group.Meanwhile,the cell viability decreased and apoptosis occurs with the prolongation of the stimulation time.The results of activating MAPK signaling pathway showed that the JNK signaling pathway began to be activated at 15 min,peaked at 30 min,and then gradually decreased with LPS-exposure,was close to the level of control group at 9 h.The p38 signaling pathway began to be activated at 30 min,and peaked at 2 h,then gradually decreased with LPS stimulation but was still at a high level at 9 h;ERK1/2 signaling pathway was activated at 1 h and the activation of ERK1/2 get the peak at 3 h,then gradually decreased with LPS stimulation but was still at a high level at 9 h.According to the characteristic of GS expression changes induced by LPS,the LPS stimulation phase was divided into early phase(2 h)and late phase(12 h).In early phase,activation of p38 and JNK signaling pathways increased GS expression,while 2-AG inhibited the activation of p38 signaling pathway,and inhibited the upregulation of GS expression induced of LPS stimulation.In the late phase,activation of ERK1/2 induced the down-regulation of GS expression,while 2-AG inhibited the activation of ERK1/2 and reversed the down-regulation of GS expression induced by of LPS stimulation.Further study found that 2-AG could activate CB2 and inhibit activation of the p38 in the early phase of LPS-exposure,thereby inhibit LPS-induced up-regulation of GS expression.Additionally,2-AG activated CB1 and inhibits LPSinduced up-regulation of GS expression,but the process was not through the p38 signaling pathway.In late phase of LPS-exposure,2-AG could activate CB1 and CB2,inhibit activation of ERK1/2 signaling pathway,and invert decrease of GS expression induced by LPS.Conclusion: LPS can induce astrocytic inflammation response and GS expression increases within 0~3 h and decreases after 6 h.In the early phase of LPSexposure,endocannabinoid 2-AG could suppress the LPS-induced GS expression increase through binding to CB2 and inhibiting the activation of p38.In the late phase of LPS-exposure,endocannabinoid 2-AG could reverse the LPS-induced GS expression decrease through binding to CB1 and CB2 and inhibiting the activation of ERK1/2.