| Glycogen synthase kinase3(Glycogen synthase kinase-3, referred to GSK-3), a multi-Ser/Thr protein kinases, widely expressed in eukaryotic cells, belongs to the glycogen synthase kinase family. GSK-3includes two subtypes, GSK-3a and GSK-3β, GSK-3β is expressed in all tissues, and participates in a variety of signal transduction pathways. Studies have showed that activation of GSK-3β can reduce the expression of cAMP response element binding protein (CREB) and inhibit its transcriptional activity, but the mechanism is not clear.In the current study, we choose the mouse as the experimental object to systematically explore the effects of PP2-induced alternation of GSK-3β activity on CREB activity and related mechanisms. Eight-week-old male Kunming (Km) mice were randomly divided into four groups, control group, wort group, PP2group, and wort+PP2group. After six-day navigation test, the intracerebroventricular administration was carried out on mice, after24h, the impact of the drugs on memory capacity was detected by Morris water maze. Subsequently, the hippocampus tissues of mice were prepared to detect the levels of related proteins by Western blot. The results showed that:(1) Morris Water Maze showed:after six days of navigation test, mice can find hidden platform in a very short period, the memory capacity of mice was relatively stable and there was no significant difference among these groups. After24h of injection, the latency to find the hidden platform was tested. Compared with the control group, the latency of wort group mice to find the hidden platform was significantly longer (p<0.001), the numbers of crossing the hidden platform quadrant and the time in the quadrant where hidden platform were both reduced (p<0.01); the latency of PP2group was shorter, the numbers of crossing the hidden platform quadrant and the time in the quadrant were both reversed slightly (p<0.01).(2) Western blot analysis showed:compared with the control group, the total level of GSK-3P did not change significantly in wort group, but the level of phosphorylation of GSK-3β Ser9site decreased significantly (p<0.05), it indicated the GSK-3β activity increased. The level of phosphorylation of GSK-3β-Ser9site did not change significantly in PP2group; while the phosphorylation of GSK-3β Ser9site in wort+PP2group had no obviously change than wort group.(3) Compared with the control group, Fyn phosphorylation was significantly increased in wort group (p<0.01), but PP2group was significantly lower than the control group (p<0.01); compared with the wort group, the level of Fyn phosphorylation was significantly lower in wort+PP2group (p<0.01).(4) Compared with the control group, the level of Tyr1472phosphorylation of NR2B was increased significantly (p<0.01) in wort group and PP2can decrease the level of Tyr1472phosphorylation of NR2B (p<0.001), but the total NR2B had no obvious changes among the groups.(5) Compared with the control group, the level of phosphorylation of CaMKII at Thr286site was significantly increased after wort injection (p<0.01), and the level of phosphorylated CaMKII at Thr286site minimized in wort+PP2group (p<0.05). The total level of CaMKII didn’t change among different groups.(6) The total level of CREB didn’t change in each group; compared with the control group, the level of phosphorylation of CREB at Ser133was lower in wort group (p<0.01); compared with the wort group, the phosphorylation of CREB-Ser133increased in wort+PP2group (p<0.05). Conclusion:as an activator of GSK-3β, wotrmannin increased the expression of GSK-3β. Since GSK-3β is an upstream kinase of Fyn, the activation of GSK-3β would affect the expression of Fyn. NR2B subunit is an important composition of NMDA receptor and its phosphorylation can be regulated by the Fyn which consequently affected the opening of ion channel and Ca2+influx. Ca2+can bind to CaM and form the complex to further regulate the CaMKII activity. The changed CaMKII activity has an influence on the CREB activity which subsequently affects the learning and memory. PP2(non-specific inhibitor of Fyn) inhibited the activity of Fyn to reverse a series of downstream reaction changed by wort, ultimately protected against the impairment of learning and memory induced by wort.This study proposes a new signaling pathway (GSK-3βâ†'Fynâ†'NR2Bâ†'CaMKIIâ†'CREB) to illstruate that PP2may be involved in the mechanism of regulation of CREB activity by GSK-3β and proposed theoretical basis for the treatment of a variety of neurological disorders (such as AD, PD). |
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