Involvement of GluR2 up-regulation in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptor in mice

Abstract

We investigated whether glutamate receptor subunit 2 (GluR2) is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors (CB1R) after global cerebral ischemia in mice. Two hours after electric acupuncture (EA) pretreatment, global cerebral ischemia (GCI) was induced by bilateral common carotid artery occlusion (BCCAO) for 20 min. The GluR2 expression was examined in the hippocampus after reperfusion. Cell survival, neuronal apoptosis, the Bax/Bcl-2 ratio and neurological scores were evaluated at 24 h after BCCAO in the presence or absence of the GluR2 inhibitor. Furthermore, the GluR2 was determined in the presence and absence of CB1R inhibitor. Our results showed EA pretreatment enhanced expression of GluR2 in the hippocampus 2 h after reperfusion. Moreover, EA pretreatment improved neurological outcome, promoted cell survival, inhibited neuronal apoptosis, and decreased the Bax/Bcl-2 ratio after reperfusion. GluR2 knockdown by GluR2 siRNA effectively reversed the beneficial effects of EA pretreatment. Furthermore, CB1R siRNA and two CB1R antagonists blocked the elevation of GluR2 expression by EA pretreatment, whereas the two CB1R agonists up-regulated GluR2 expression as EA pretreatment. In conclusion, GluR2 up-regulation is involved in neuroprotection of EA pretreatment against GCI through CB1R, suggesting that GluR2 may be a novel target for stroke intervention.

Figure 1. Expression of GluR2 protein in hippocampus after global cerebral ischemia (n = 5).

(a) Western blot analysis of GluR2 protein expression in sham, global cerebral ischemia 24 h, global cerebral ischemia 48 h, and global cerebral ischemia 72 h groups. The upper part is the photograph of GluR2 and its corresponding β-Actin bands. The GluR2 protein expression level did not significantly change at 24 h, 48 h, and 72 h after reperfusion compared to the Sham group. (b) Western blot analysis of GluR2 protein expression in hippocampus in the sham, global cerebral ischemia 2 h, global cerebral ischemia 6 h. The upper part is the photograph of GluR2 and its corresponding β-Actin bands. (*P < 0.05 vs. Sham).

Figure 2. Effect of EA pretreatment on GluR2 expression in hippocampus after global cerebral ischemia (n = 5).

(a) Western blot analysis of the expression of GluR2 protein in the Sham group, GCI group, EA+GCI group, and EA group. The upper part is the photograph of GluR2 and its corresponding β-Actin bands. (*P < 0.05 vs. GCI). (b) Double immunofluorescent staining for GluR2 (in green) and neurons (in red) in the hippocampus 2 h after reperfusion. EA pretreatment increased cell-specific expression of GluR2 (Upper) immunolabeling in the CA1 pyramidal cell layer. Scale bars = 50 μm.

Figure 3. Neurological scores at 24 h after reperfusion in the mice with 20 min of global cerebral ischemia (n = 8).

(a, c) Western blotting analysis of GluR2 at 72 h after cell transfection and hippocampal injection of GluR2 siRNA Mm-Gria2-2. Mm-Gria2-2 remarkably reduced the expression of GluR2 protein in neuronal cells and hippocampus(*P < 0.05 vs. Sham). The upper part is the photograph of GluR2 and its corresponding β-Actin bands. (b) Analysis of the GluR2 mRNA in the neuronal cells. GluR2 siRNA remarkably decreased the mRNA of GluR2 (*P < 0.05 vs. Sham). (d) Neurological scores 24 h after reperfusion in the mice with 20 min of global cerebral ischemia. Pretreatment with EA significantly improved the neurological scores, whereas the GluR2 siRNA reversed the beneficial effect of EA pretreatment (*P < 0.05 vs. GCI; ^#P < 0.05 vs. EA+GCI).

Figure 4. Neuronal survival, Neuronal apoptosis at 24 h after reperfusion in the mice with 20 min of global cerebral ischemia (n = 5).

(a)Identification of neuronal survival by Nissl-staining in rat hippocampus 24 h after reperfusion. The cell counting showed the number of viable cells in the EA+GCI group is higher compared to the GCI group (*P < 0.05 vs. GCI). GluR2 siRNA decreased the number of viable cell compared to the EA+GCI group (^#P < 0.05 vs. EA+GCI). Scale bars = 20 μm. (b) Neuronal apoptosis was assessed by TUNEL staining 24 h after reperfusion in the mice. The cell counting showed an attenuation in TUNEL-positive neuronal in the hippocampus in the EA+GCI group compared to the GCI group (*P < 0.05 vs. GCI). In siRNA-treated mice, TUNEL- positive cells were significantly higher than in EA-treated mice (^#P < 0.05 vs. EA+GCI). Scale bars = 50 μm. (c) The ratio of Bax/Bcl-2 expression in ischemic mice pretreated with EA or GluR2 siRNA before global cerebral ischemia was analyzed by western blot. The upper part is the photograph of Bax or Bcl-2 and its corresponding β-Actin bands. (*P < 0.05 vs. GCI; ^#P < 0.05 vs. EA+GCI).

Figure 5. Pretreatment with 2-AG up-regulated GluR2 expression via CB1R (n = 5).

(a) Western blot analysis of GluR2 expression in Sham, GCI, 2-AG, AM251+2-AG and vehicle groups (*P < 0.05 vs. GCI; ^#P < 0.05 vs. 2-AG). The upper part is the photograph of GluR2 and its corresponding β-Actin bands. (b) Western blot analysis of GluR2 expression in 2-AG, AM630+2-AG and vehicle groups. The upper part is the photograph of GluR2 and its corresponding β-Actin bands.

Figure 6. Pretreatment with CB1R agonists up-regulated GluR2 expression (n = 5).

(a, b) Western blot analysis of GluR2 expression in GCI, ACEA+GCI, WIN55212-2+GCI, EA+GCI and Vehicle+GCI group. The upper part is the photograph of GluR2 and its corresponding β-Actin bands. (*P < 0.05 vs. GCI; ^#P < 0.05 vs. GCI).

Figure 7. CB1R antagonists and CB1R siRNA dampened the regulatory effect of EA pretreatment on GluR2 expression (n = 5).

(a, b) Western blot analysis of GluR2 expression in GCI, EA+GCI, AM251+EA+GCI, SR141716A+EA+GCI and Vehicle+EA+GCI. The upper part is the photograph of GluR2 and its corresponding β-Actin bands. (*P < 0.05 vs. EA+GCI; ^#P < 0.05 vs. GCI). (c) Western blotting analysis of CB1R expression at 72 h after lateral ventricle injection of CB1R siRNA. CB1R siRNA remarkably reduced the expression of CB1R protein (*P < 0.05 vs. Sham). The upper part is the photograph of CB1R and its corresponding GAPDH bands. (d) Western blot analysis of GluR2 expression in GCI, EA+GCI, CB1 siRNA+EA+GCI, control siRNA+EA+GCI. There was a significant difference between EA+GCI group and CB1 siRNA+EA+GCI group. (*P < 0.05 vs. EA+GCI; ^#P < 0.05 vs. GCI).