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1.
FIGURE 4.

FIGURE 4. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

Cannabinoids WIN55,212-2 and CP55,940 fail to prevent LPS-induced elevation of hippocampal COX-2. a1, immunoblot analysis of COX-2 expression in hippocampi from mice treated with vehicle, LPS, LPS + WIN, or LPS + CP. WIN and CP were injected 30 min before LPS injection. COX-2 protein was detected 4 h after the injections. a2, quantifications of COX-2 expressions under different treatments (n = 3). **, p < 0.01 compared with vehicle controls. b1, immunoblot analysis of COX-2 expression in hippocampi from mice treated with vehicle, LPS, LPS + 2-arachidonyl glycerol ether (2-AGE), or LPS + AA. 2-Arachidonyl glycerol ether and AA were injected 30 min before LPS injection. COX-2 protein was detected 4 h after injections. b2, quantifications of COX-2 expressions under different treatments (n = 3). **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
2.
FIGURE 7.

FIGURE 7. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

Gi/o protein mediates 2-AG suppression of COX-2. a, immunoblot analysis of COX-2 expression and ERK1/2 and NF-κB phosphorylation in mixed culture of hippocampal neurons and astroglial cells treated with LPS, 2-AG, and PTX. Neurons in culture were treated with PTX (50 mg/ml) for 30 min before application of 2-AG and 60 min before application of LPS. Proteins were detected 6 h after treatments. b–d, quantifications of COX-2 expression, p38 MAPK, and NF-κB phosphorylation. *, p < 0.05, and **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS (n = 3).

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
3.
FIGURE 6.

FIGURE 6. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

ERK, MAPK, phosphoinositide 3-kinase, and NF-κB signaling pathways are involved in the 2-AG-induced suppression of COX-2 elevation. a1, Western immunoblot analysis of phosphorylation of p38 MAPK in hippocampal tissue from mice that received vehicle, LPS, LPS + 2-AG, and LPS + 2-AG + SR-1. a2, quantifications of p38 MAPK phosphorylation. b1, immunoblot analysis of NF-κB phosphorylation in the mouse hippocampus. b2, quantifications of NF-κB phosphorylation (n = 3). **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS. c1, Western blot analysis of ERK1/2 and Akt phosphorylation in hippocampal tissue from mice injected with vehicle, LPS, and LPS + 2-AG. c2 and c3, quantifications of ERK1/2 and Akt phosphorylation. **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS (n = 3). d1, Western blot analysis of ERK1/2 and Akt phosphorylation in hippocampal tissue from mice injected with vehicle, KA, KA + 2-AG, and KA + 2-AG + SR-1. c2 and c3, quantifications of ERK1/2 and Akt phosphorylation. **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with KA (n = 3).

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
4.
FIGURE 1.

FIGURE 1. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

2-AG suppresses LPS-induced elevation of COX-2 expression. a1, immunoblot analysis of COX-1 and COX-2 expression in mouse hippocampus. 2-AG was injected intraperitoneally 30 min before intraperitoneal injection of LPS, and COX proteins were analyzed 4 h after LPS injection. a2, quantifications of COX-1 and COX-2 expression. 2-AG induces a dose-dependent reduction of COX-2 elevation in response to LPS injection (n = 6). b, real-time PCR analysis of COX-2 expression in mixed culture of hippocampal neurons and astroglial cells (n = 3). The culture was treated with LPS, and mRNA was assayed 12 h after LPS treatment. Results are from three independent cultures with duplicate wells. *, p < 0.05, and **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
5.
FIGURE 9.

FIGURE 9. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

2-AG inhibits COX-2 elevation-induced enhancement of hippocampal excitatory synaptic transmission. a1, representative sweeps of mEPSCs recorded in control, IL-1β (10 ng/ml)-, IL-1β + 2-AG (1 μm)-, and IL-1β + 2-AG + SR-1 (1 μm)-treated neurons. The culture was treated with IL-β for 16 h. 2-AG or 2-AG + SR-1 was added 30 min before IL-1β application. a2, cumulative probability of mEPSC frequency recorded in neurons with different treatments. a3, mean percentage of change in the frequency of mEPSCs in neurons with different treatments (n = 10 to 16). a4, cumulative probability of mEPSC amplitude. a5, mean percentage of change in the amplitude of mEPSCs. Scale bar:20 pA/2 s. b1, representative sweeps of mEPSCs recorded in control, LPS (1 μg/ml)-, LPS + 2-AG (1 μm)-, and LPS + 2-AG + SR-1 (1 μm)-treated neurons. The culture was treated with LPS for 24 h. 2-AG or 2-AG + SR-1 was added 30 min before application of LPS. b2, cumulative probability of mEPSC frequency recorded in neurons with different treatments. b3, mean percentage of change in the frequency of mEPSCs (n = 7–12). b4, cumulative probability of mEPSC amplitude. b5, mean percentage of change in the amplitude of mEPSCs. Scale bar: 20 pA/2 s. **, p < 0.01 compared with the control; ##, p < 0.01 compared with IL-1β or LPS.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
6.
FIGURE 8.

FIGURE 8. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

2-AG protects hippocampal neurons from proinflammatory and excitotoxic insults. a1–a5, TUNEL images of hippocampal neurons in control (a1), IL-1β (50 ng/ml (a2)), IL-1β + 2-AG (1 μm (a3)), IL-1β + 2-AG (5 μm (a4)), and IL-1β + 2-AG (5 μm) + SR-1 (5 μm (a5)) for 24 h. a6, percentages of injured neurons under different treatments (n = 10–12). b1–b5, TUNEL images of hippocampal neurons in control (b1), Glu (50 μm (b2)), Glu + NS398 (20 μm (b3)), Glu + 2-AG (5 μm (b4)), and Glu + 2-AG (5 μm) + SR-1 (5 μm (b5)) for 24 h. b6, percentages of injured neurons under different treatments (n = 10). c1, Hoechst staining of hippocampal neurons in control, Glu (50 μm), Glu + NS398 (20 μm), Glu + 2-AG (3 μm), and Glu + 2-AG + SR-1 (5 μm) for 24 h. c2, percentages of apoptotic neurons under different treatments (n = 5). d, Western blot analysis of cleaved caspase-3 in control, Glu (50 μm), 2-AG (1 and 5 μm), and SR-1 (1 μm). e, glutamate elevates COX-2 expression, and 2-AG attenuates Glu-induced elevation of COX-2. Hippocampal neurons in culture were treated with Glu for 24 h in the absence and presence of 2-AG and 2-AG + SR-1. **, p < 0.01, compared with control; #, p < 0.05, and ##, p < 0.01 compared with IL-1β or glutamate.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
7.
FIGURE 3.

FIGURE 3. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

Endogenous 2-AG suppresses COX-2 elevation. a1, MGL inhibitors URB602 and ATFMK prevent LPS-induced elevation of COX-2 in hippocampal slices. Hippocampal slices were treated with LPS in the absence and presence of URB602 or ATFMK. COX-2 expression was detected 6 h after treatments. a2, quantifications of COX-2 in slices treated with LPS in the absence and presence of MGL inhibitors (n = 4). b1, MGL inhibitors reduce COX-2 expression in response to LPS in mixed culture of hippocampal neurons and astroglial cells. COX-2 protein was analyzed 24 h after treatments. b2, quantifications of COX-2 in the culture treated with LPS in the absence and presence of MGL inhibitors or SR-1 (n = 3). c1, FAAH inhibitor or CB2R antagonist does not prevent 2-AG-produced suppression of COX-2 elevation in hippocampal tissue. Mice were injected intraperitoneally with LPS, LPS + 2-AG, LPS + 2-AG + SR144528 (SR-2), and LPS + URB597. COX-2 was determined 4 h after injections. c2, quantifications of COX-2 in the hippocampal tissue from animals that received different treatments (n = 3). **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
8.
FIGURE 2.

FIGURE 2. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

CB1 mediates 2-AG suppression of COX-2 elevation in response to inflammatory and excitotoxic stimuli. a1, immunoblot analysis of COX-2 expression in hippocampus from mice treated with vehicle, KA, KA + 2-AG, and KA + 2-AG + SR-1. COX-2 protein was detected 4 h after injections. a2, quantifications of COX-2 expressions under different treatments (n = 3). b, quantitative real-time PCR analysis of COX-1, COX-2, and CB1 expression in hippocampal tissue from mice treated with vehicle, LPS, LPS + 2-AG, and LPS + 2-AG + SR-1 (n = 4). c1, immunoblot analysis of COX-1, COX-2, and CB1 in the hippocampus from mice treated with vehicle, LPS, LPS + 2-AG, and LPS + 2-AG + SR-1. c2, quantifications of protein expressions under different treatments (n = 4). d1, 2-AG failed to suppress COX-2 elevation in response to LPS injection in CB1 knock-out (cnr1-/-) mice. Immunoblot analysis is shown of COX-2 expression in the hippocampus from animals that received vehicle, LPS, or LPS + 2-AG. d2, quantifications of COX-2 protein expressions in CB1 knock-out (KO) and wild type (WT) animals (n = 4). e1, CB1, but not CB2, mediates 2-AG suppression of COX-2. Immunoblot analysis is shown of COX-2 expression in a mixed culture of neurons and astroglial cells. e2, quantifications of COX-2 under different conditions (n = 3). **, p < 0.01 compared with vehicle controls; ##, p < 0.01 compared with LPS or KA.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.
9.
FIGURE 5.

FIGURE 5. From: Endocannabinoid 2-Arachidonoylglycerol Protects Neurons by Limiting COX-2 Elevation.

Neurons and astroglial cells are involved in the 2-AG-produced suppression of COX-2. a1–a3, COX-2 is expressed in neurons and astroglial cells. Shown is COX-2 immunostaining with NeuN (neuronal marker), GFAP (astrocytic marker), and OX-42 (microglial marker) in different cultures. Images were taken with a Zeiss deconvolution microscope using Slidebook 4.0 software with magnification ×40. Scales bars = 10 μm. b1–b3, CB1 receptors are expressed in neurons and astroglial cells. Shown is CB1 immunostaining with NeuN, GFAP, and OX-42 in different cultures. c, 2-AG induces a CB1-dependent suppression of COX-2 elevation in response to IL-1β stimulus in relatively pure neuronal culture (astroglial cells ∼1%). Neurons in culture were treated with IL-1β (50 ng/ml), IL-1β + 2-AG (3 μm), and IL-1β + 2-AG + SR-1 (3 μm). COX-2 mRNA was analyzed using real-time PCR 24 h after treatments. d, 2-AG suppresses COX-2 expression in response to LPS (1 μg/ml) stimulus in mixed culture of hippocampal neurons and astroglial cells (astroglial cells 10∼15%). The culture was treated with LPS, LPS + 2-AG, and LPS + 2-AG + SR-1. e, 2-AG prevents excessive expression of COX-2 in astroglial cell-enriched culture (astroglial cells >95%). Astroglial cells in culture were treated with LPS (0.5 μg/ml), LPS + 2-AG (3 μm), and LPS + 2-AG +SR-1 (3 μm). **, p < 0.01 compared with controls; ##, p < 0.01 compared with IL-1β or LPS.

Jian Zhang, et al. J Biol Chem. 2008 August 15;283(33):22601-22611.

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