Fig. 5
Ex-4 increases the Ins2-encoding insulin to reduce the tau hyperphosphorylation in the high-glucose-damaged HT22 cells. A–D HT22 cells under the control (CON) or high glucose (50 mM, HG) environment for 48 h, then part of cells in HG group treated with exendin-4 (10 nM, HG + Ex-4) for 48 h. A mRNA levels of Ins2 in groups were measured by RT-qPCR assay. β-actin was the internal control. n = 4. B The insulin levels of the culture supernatant in groups were detected by an ELISA kit. n = 4. C The insulin production and insulin signalling activation as P-AKTS473 to total AKT and P-GSK-3βS9 to total GSK-3β were examined through Western blot analysis and their gray density. n = 4. D Represented immunofluorescence staining of HT22 cells was performed for insulin including proinsulin (green) and DAPI (blue). Scale bar = 100 μm. E–G HT22 cells were cultured in high glucose (50 mM, HG) environment for 48 h after infection with Ins2-knockdown and its negative control lentivirus. Then, they were cultured in high glucose (50 mM, HG) environment with or without Ex-4 (10 nM) for another 48 h. E Immunoblot and gray density demonstrated the levels of phosphorylated tau at specific sites and total tau. n = 4. F Insulin levels in culture supernatant in groups measured by ELISA kit. n = 4. G Immunoblot and the gray density for insulin with insulin signalling factors, including total AKT, P-AKTS473, total GSK-3β, and P-GSK-3βS9. n = 4. Results are representative of three independent experiments. Values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; N.S. no significant difference