Figure 9. GSPs attenuated STZ-induced mPTP opening by enhancing PI3K-Akt-dependent phosphorylation of GSK-3β (Ser9) in primary cortical neurons. (A) Neurons were co-pretreated with of 50 μg/mL GSPs and LY294002 (20 μM) for 2 h and then treated with STZ (0.5 mM) for 24 h. The cell viability was detected by using MTT assay and is represented as the percentage of the CON (non-treated) cells. (B) The representative images of H2DCFDA staining (green) in primary cortical neurons. (C) The quantification analysis of the H2DCFDA staining among different groups. (D) GSPs inhibited STZ-induced mPTP opening, which was blocked by LY294002. (E) GSPs increased phosphorylation of GSK-3β at Ser9 in the presence of STZ, which was blocked by LY294002. (F) GSPs increased the binding of p-GSK-3β and ANT in the presence of STZ, and LY294002 inhibited the effects of GSPs. (G) Immunoprecipitation experiments showed that GSPs reduced STZ-induced binding of ANT to CypD, which was blocked by LY294002. *P < 0.05, **P < 0.01 vs CON; #P < 0.05 vs STZ; $P < 0.05 vs STZ+GSPs; n=4.