Morphine reduced glutamate-induced astrocytes apoptosis. (A) Following 14 days’ culture, astrocytes were stained by green fluorescence marked GFAP antibody, nuclei were stained with blue fluorescence by Hoechst 33342 dye. Scale bar = 50 μm; (B) following cells were treated with glutamate in the presence/absence of different doses of morphine for 24 h, MTT assay was performed to detect cell viability, * p < 0.05, *** p < 0.001 compared to glutamate only treated group, ###
p < 0.001 compared to morphine only treated group, n = 6; (C) following 24 h treatment, the cells were lysed by lysis buffer and the protein was extracted, then the expression of cleaved caspase-8, caspase-9, and caspase-3 were detected by western blot, β-actin was internal control, blots were representative of three independent experiments; (D) following 24 h treatment, the cells were fixed and stained with Hoechst 33342 dye, bright colored condensed nuclei indicated apoptotic cells. Scale bar = 50 μm and referred to all panels; (E) statistical analysis of Hoechst apoptotic staining, *** p < 0.001 compared to glutamate only treated group, ##
p < 0.01, ###
p < 0.001 compared to morphine only treated group, n = 3.