Anandamide Protects HT22 Cells Exposed to Hydrogen Peroxide by Inhibiting CB1 Receptor-Mediated Type 2 NADPH Oxidase
Figure 1
Experimental protocol diagram. (a) The HT22 cells were assigned into seven groups. The control cells were cultured in drug-free medium, and the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. MTT assay was taken to determine the injury degree. (b) The cells were divided into six groups; except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA for 3 h. MTT assay was taken to evaluate the injury degree. (c) The cells were assigned into six groups, including control, AEA, H2O2, AEA + H2O2, AM251 + AEA + H2O2, and AM251 + H2O2 groups. After an incubation of 3 h, MTT assay, LDH release, and western blotting were taken to determine the roles of CB1 and Nox2 in AEA-induced protection. (d) The cells were divided into three groups, including control, CB1-siRNA, and SC-siRNA groups; after an incubation of 5 h, western blotting was used to evaluate the silencing rate of CB1 protein expression. (e) Then, the cells were divided into five groups, including control, H2O2, AEA + H2O2, CB1-siRNA + AEA + H2O2, and SC-siRNA + AEA + H2O2; the cell injury was evaluated by MTT and LDH release at 3 h after incubation, and ROS generation was evaluated by measuring fluorescence intensity. (f) The cells were divided into five groups, including control, H2O2, AEA + H2O2, apocynin + AEA + H2O2, and apocynin + AEA + H2O2; western blotting, MTT assay, and LDH release were taken to measure Nox2 expression and cell injury.
