Figure 3.

Clearance of oAβ1-42 and the production of HO-1, MMP-9, and neurotoxic molecules by microglia activated with CpG. A: Western blot analysis of oAβ1-42 in neuronal cultures (neu) and neuron-microglia co-cultures (neu + mi). Twenty-four hours after addition of 5 μmol/L oAβ1-42, oAβ1-42 present in the supernatants of these cultures was detected by Western blotting. B: Western blot analysis of oAβ1-42 in neuron-microglia co-cultures with CpG treatment. Neuron-microglia co-cultures (neu + mi) were treated with 5 μmol/L oAβ1-42 for 24 hours following 3 hours of treatment with 1, 10, or 100 nmol/L CpG. Microglia activated with CpG dose-dependently reduced the amount of oAβ1-42 in the supernatants. C: Semiquantification of oAβ1-42 in B by densitometric analysis. The amount of oAβ1-42 in neuron-microglial co-cultures without CpG (black) was normalized to 100%. oAβ1-42 in co-cultures treated with 1 nmol/L CpG (blue), 10 nmol/L CpG (green), or 100 nmol/L CpG (red) was calculated. ^∗P < 0.05 and ^∗∗P < 0.01 as compared with the intensity of oAβ1-42 in neuron-microglia co-cultures without CpG. Each column indicates the mean ± SEM (n = 6). The production of HO-1 (D) and MMP-9 (E) by microglia activated with CpG in the absence or presence of oAβ1-42. After 3 hours of treatment with CpG, microglial cultures were treated with or without oAβ1-42 for 24 hours. ^∗P < 0.05 as compared with untreated controls. Each column indicates the mean ± SEM (n = 3–5). F: The effect of HO-1 and MMP-9 on oAβ1-42 neurotoxicity. Neuron-microgila co-cultures were treated with 100 nmol/L CpG in the presence of 10 μmol/L tin-mesoporphyrin (SnMP) IX, a specific HO-1 inhibitor, or 50 nmol/L MMP-9 inhibitor for 3 hours, and then oAβ1-42 was added to the cultures for 24 hours. Tin-mesoporphyrin IX, but not the MMP-9 inhibitor, decreased neuronal survival rate. ^∗P < 0.05 as compared with CpG-treated cultures without inhibitors. Each column indicates the mean ± SEM (n = 6–9). G: The suppressive effect of CpG on ROS production by oAβ in the neuron microglia co-cultures. After neuron-microglia co-cultures were treated with or without 100 nmol/L CpG for 3 hours, cells were loaded with fresh nerve culture medium containing 5 μmol/L H₂DCFDA-AM for 30 minutes. After washing, culture medium containing 5 μmol/L oAβ1-42 was added and the increment of the fluorescence was calculated at 5 minutes. ^∗P < 0.05 as compared with untreated controls. **P < 0.05 as compared with co-culture cells treated with oAβ1-42. Each column indicates the mean ± SEM (n = 4). H: The measurement of TNF-α (left), nitrite (middle), and glutamate (right) produced by microglia activated with 100 nmol/L CpG with or without oAβ1-42. After 3 hours treatment with CpG, microglial cultures were treated with or without oAβ1-42 for 24 hours. ^∗P < 0.05 as compared with untreated microglia. Each column indicates the mean ± SEM (n = 7).