Figure 1 Spinosin protected N2a/APP695 cells from H2O2-induced damage. Cells were treated with spinosin (25 μM) for 22 h alone or after H2O2 model construction (6.25 μM for 2 h). A: Aβ1-42 in conditioned medium was measured by ELISA kits; B: the deposition of Aβ1-42 is detected by ThT staining experiment; C: representative Western blot of p-Tau (Ser199), p-Tau (Ser202) and p-Tau (Ser396); D: quantitative analysis of p-Tau (Ser199) expression normalized to β-actin expression; E: quantitative analysis of p-Tau (Ser202) expression normalized to β-actin expression; F: quantitative analysis of p-Tau (Ser396) expression normalized to β-actin expression; G: representative Western blot of SYP and PSD95; H: quantitative analysis of SYP and PSD95 expression normalized to β-actin expression. N2a: Neuro-2a; ELISA: enzyme linked immunosorbent assay; SPI: spinosin; SYP: synaptophysin; PSD95: postsynaptic density protein 95. The statistical analysis was performed using one-way analysis of variance followed by Tukey’s multiple comparisons test. Values are expressed as mean ± standard error of mean (n = 3). aP < 0.05 vs Ctrl group; bP < 0.05 vs H2O2 group.

Figure 2 Effect of spinosin on the expression changes of MAPK family proteins caused by H2O2 Cells were treated with spinosin (25 μM) for 22 h alone or after H2O2 model construction (6.25 μM for 2 h). A: representative Western blot of JNK, p-JNK, ERK, p-ERK, p38 and p-p38; B: quantitative analysis of p-JNK expression normalized to JNK expression; C: quantitative analysis of p-ERK expression normalized to ERK expression; D: quantitative analysis of p-p38 expression normalized to p38 expression. MAPK: mitogen-activated protein kinase; SPI: spinosin; JNK: c-Jun N-terminal kinase; ERK: extracellular regulated protein kinases. The statistical analysis was performed using one-way analysis of variance followed by Tukey’s multiple comparisons test. Data are mean ± standard error of mean of three independent experiments (n = 3). aP < 0.05 vs Ctrl group; bP < 0.05 vs H2O2 group.

Figure 3 Effect of BIRB796 on oxidative stress, Aβ1-42 secretion, Tau phosphorylation and synaptic plasticity in N2a/APP695 cells Cells were treated with spinosin (25 μM) for 22 h alone or after H2O2 model construction (6.25 μM for 2 h). For the p38 inhibitor group, cells were treated with H2O2 and BIRB796 (2 μM) for 2 h, which were then replaced with fresh medium for the following 22 h. A: intracellular MDA and LDH levels were measured by ELISA kits; B: Aβ1-42 in conditioned medium was measured by ELISA kits; C: representative Western blot of p-Tau (Ser396); D: quantitative analysis of p-Tau (Ser396) expression normalized to β-actin expression; E: cytoskeleton was stained with FITC-phalloidin, and then photographed with a confocal fluorescence microscope, ×200. E1: Ctrl group cells were labeled by FITC-phalloidin; E2: Ctrl group cells were labeled by DAPI; E3: FITC-phalloidin and DAPI stain labeled overlapping image of Ctrl cells; E4: H2O2 group cells were labeled by FITC-phalloidin; E5: H2O2 group cells were labeled by DAPI. E6: FITC-phalloidin and DAPI stain labeled overlapping image of H2O2 cells; E7: H2O2 + SPI group cells were labeled by FITC-phalloidin; E8: H2O2 + SPI group cells were labeled by DAPI. E9: FITC-phalloidin and DAPI stain labeled overlapping image of H2O2 + SPI cells; E10: H2O2 + BIRB796 group cells were labeled by FITC-phalloidin; E11: H2O2 + BIRB796 group cells were labeled by DAPI. E12: FITC-phalloidin and DAPI stain labeled overlapping image of H2O2 + BIRB796 cells. The statistical analysis was performed using one-way analysis of variance followed by Tukey’s multiple comparisons test. N2a: Neuro-2a; ELISA: enzyme linked immunosorbent assay; SPI: spinosin; MDA: malondialdehyde; LDH: lactate dehydrogenase; DAPI: 4′,6-diamidino-2-phenylindole. Data are mean ± standard error of mean of three independent experiments (n = 3). aP < 0.05 vs Ctrl group; bP < 0.05 vs H2O2 group.