Objective: To investigate the effects of Scutellaria barbata flavonoids (SBF) on abnormal changes of N2a cell protein Phosphatase-1 (PP-1) and related Tau protein phosphorylation induced by β-amyloid25-35 (Aβ25-35).

Methods: N2a cells were cultured and randomly divided into 7 groups, including control group, model group, PP-1 inhibitor OA group, Aβ25-35+OA group and SBF three dose treatment groups. The control group was not treated. Aβ25-35 of 40 µmol/L was added to the model group, and work for 12 h. OA with a final concentration of 15 nmol /L was added to the PP1 inhibitor OA group, and fully act for 12 h. SBF of 1.125 mg/L, 2.25 mg/L and 4.5 mg/L were added to SBF dose treatment groups respectively, and the inhibitor OA with a final concentration of 15 nmol /L was added at the same time, after acting cells for 12 h, Aβ25-35 with a final concentration of 40 µmol/L was added to continue the action for 12 h. The morphology and state of cells were observed under 100X microscope. The injury degree and survival rate of N2a cells were measured by Lactic dehydrogenase release and MTT assay. Western blot was used to detect the phosphorylation expression levels of PP-1 and Tau protein at Thr205, Thr231, Ser404 and Ser199 of N2a cells in each group.

Results: After overnight cultured, the cells in the control group were observed to be intact, the cell membrane was transparent and the morphology of the cells was shuttle. In the model group, after the cells were treated with a final concentration of 40 µmol/L Aβ25-35 for 12 h, the cell membrane was observed to be incomplete and damaged under the microscope, the cells appeared agglomeration, the cell bulges was not obvious and the growth was slow. After treated with three doses of SBF for 24 hours, the three groups of cells was observed that they had different degrees of improvement under the microscope, the growth rate of the cells was faster and the cell membrane was intact. Compared with the control group, the LDH release of N2a cells increased by 8% in the model group (p< 0.01), and significantly decreased by 1.82% in the OA group (p < 0.01). Compared with the model group, the LDH release of N2a cells decreased by 2.64% in Aβ25-35+OA group (p < 0.01). Compared with Aβ25-35+OA group, LDH release of N2a cells decreased by 2.89% in SBF 1.125 mg/mL group (p < 0.01), and decreased by 3.41% in SBF 2.25 mg/mL group (p< 0.01), and decreased by 4.34% in SBF 4.5 mg/mL group (p< 0.01). Compared with the control group, the survival rate of N2a cells decreased by 17.89% in the model group (p< 0.01), and decreased by 16.82% in the OA group (p< 0.01). Compared with the model group, the survival rate of N2a cells increased by 23.03% in Aβ25-35+OA group (p< 0.01). Compared with Aβ25-35+OA group, the survival rate of N2a cells increased by 0.61% in SBF 1.125 mg/mL group (p< 0.01), and increased by 12.89% in SBF 2.25 mg/mL group (p< 0.01), and increased by 25.90% in SBF 4.5 mg/mL group (p< 0.01). Compared with the control group, expect for the decreased protein expression levels of PP-1 and p-Tau (Thr205), the protein expression levels of p-Tau (Ser199), p-Tau (Ser404) and p-Tau (Thr231) were increased in the model group. In the OA group, expect for the decreased protein expression levels of PP-1, the phosphorylated Tau protein expression levels at Ser199, Thr205, Ser404 and Thr231 these four sites were increased (p< 0.01). Compared with the model group, protein expression levels of PP-1 and p-Tau (Thr231) were decreased, the phosphorylated Tau protein expression levels at Ser199, Thr205 and Ser404 sites were increased. Compared with the Aβ25-35+OA group, expect for the decreased protein expression levels of p-Tau (Ser404), the protein expression levels of PP-1, p-Tau (Ser199), p-Tau (Thr205) and p-Tau (Thr231) were increased in the SBF low dose treatment group. In the SBF medium dose treatment group, the protein expression levels of PP-1, p-Tau (Thr205) and p-Tau (Thr231) were increased, while the phosphorylated Tau protein expression levels at Ser199 and Ser404 sites were both decreased. In the SBF high dose group, expect for the increased protein expression levels of PP-1, the protein expression levels of p-Tau (Ser199), pTau (Thr205), p-Tau (Ser404) and p-Tau (Thr231) were decreased.

Conclusion: SBF can up-regulate the activity of PP-1 to inhibit the hyperphosphorylation of Tau protein expression levels at Thr205, Thr231, Ser404 and Ser199 sites.