| Kai Xin San (KXS) is a famous traditional Chinese medicine formula for the treatment of mental disorders, recorded in many medicine books. It was composed of four herbs:Radix and Rhizome Ginseng, Radix Polygalae, Rhizoma Acori Tatarinowii and Poria. Our previous studies indicated that KXS had notable antidepressant effects in numerous experiments. In this study, we investigated the antidepressant effect of KXS on despair model and focused on test its correlation between5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF).3,6’-disinapoyl sucrose (DISS) is an active oligosaccharide ester component of KXS. Our previous studies showed that DISS has notable antidepressant effects. In pharmacological depression models, it has a potentiation effect on central5-HT and hypothalamic-pituitary-adrenal cortex axis (HPA) systems and oxidative systems in chronic stress animals. Our recent study also found that DISS increased expression of noradrenergic-regulated plasticity gene (CREB) and one neurotrophic factor (BDNF) in the hippocampus. Based on these results, this study continued to investagate the neuroprotective effect of DISS and its mechanism based on CREB phosphorylation related upstream and downstream signal pathway. Using singal pathway inhibitors and many molecular biology technologies, the specfic targets and gene activation by DISS were studied, which may be useful to new antidepressant drug development.1Effect of KXS on depression behavior and hippocampal brain-derived neurotrophic factor in miceThe effect of KXS on the immobility time of mice in the TST was observed. The levels of norepinephrine (NE), dopamine (DA), and5-HT in the brain were determined by high-performance liquid chromatography (HPLC). Western blotting analysis was used to examine the expression of CREB, p-CREB, and BDNF in the hippocampus. BDNF mRNA expression was also examined by RT-PCR. KXS could reduce the immobility time of mice in the TST, increase the brain levels of monoamine transmitters and hippocampal levels of CREB, p-CREB and BDNF compared with the control group. Moreover, the anti-depression effect of KXS was correlated with BDNF level.2The effect of DISS on regulating CREB phosphorylation and its antiapoptosis activityBased on the characteristics of SH-SY5Y cells in our laboratory, the cell growth curve was measured by CCK-8assay. SH-SY5Y neuronal cells were pretreated with blank DMEM/F-12for24h followed by co-treatment with DISS for different time and concentrations. Cell viability was determined by CCK-8assay, and apoptosis was confirmed by flow cytometry assay, evaluated with propidium iodide dye. Next, we examined the concentration-dependency of DISS on the SH-SY5Y cells proliferation. Compared with the control, treatment with DISS (6,10,30and60μmol·L-1) increased cell viability dose-dependently and aslo inhibited apoptosis percentage.Western blotting analysis was used to examine the expression of CREB, p-CREB and BDNF after being treated with DISS (6,10,30,60μmol·L-1) with different time. The result showed that, compared with the control, being treated with DISS (60μmol·L-1) for10-30min can increase the expression of p-CREB and also p-CREB/CREB (p<0.05), and reached a maximum at15min.The expression of BDNF also increased after DISS treated for10min. This result indicated that the neuroprotective effect of DISS may be involved in activating CREB, which can induce the downstream BDNF expression.SH-SY5Y neuronal cells were pretreated with H89, KN93, U0126, K252a, LY294002, and subsequently treated with DISS for15min, and then tested the expression of CREB, BDNF and phosphorylation of CREB by Western blotting. Compared with the control, the expression of CREB, BDNF and p-CREB in cells with inhibitors were decreased significantly; Compared with vihicle, the DISS-induced phosphorylation of CREB and expression of BDNF incresing was specifically inhibited by inhibitors of tropomyosin-related kinase B (TrkB), Calcium and calmodulin-dependent protein kinase (CaMKII) and Extracellular signal-regulated kinase (ERK). These results suggested that TrkB/ERK and CaMKII-pathway induced the CREB phosphorylation which may be involved in DISS neuroprotective effect on SH-SY5Y cells.3The effect of DISS on the CREB upstream passway and its detail targetsWe examined the time course of DISS-induced expression of TrkB, CaMKII, p-CaMKII, ERK, PKA, p-PKA and PI3K by Western blotting analysis and the rusults showed that the phosphorylation of CaMKII induced by DISS was increased between10min-1h, and reached a maximum at10min. The expression of CaMKII and ERK also increased after being treated with DISS for1h and4h. Acute DISS treatment did not affect PKA, p-PKA, TrkB and PI3K in SH-SY5Y cells.The Path Detectcis-reporting system was applied to study the luciferase reporter gene activity reflects the CRE-mediated gene expression. SH-SY5Y cells were pretreated with H89, KN93and U0126, and then treated with DISS or forskolin. DISS or forskolin treatment significantly increased CRE activity. Furthermore, the results were consist with the provious results. The DISS-induced CRE activity as well as phosphorylation of CREB was inhibited by U0126and KN93. The relation between TrkB/ERK and CaMK signal pathway was investigated in our research too. SH-SY5Y cells were pretreated with KN93, U0126, K252a, and subsequently treated with DISS for15min, the expression of CaMKII, p-CaMKII, ERK and TrkB were examined by Western blotting analysis. Compared with the control, KN93, U0126and K252a alone did not have any significant effect on the level of these protein expression; Compared with vihicle, the enhanced ERK level was significantly blocked by KN93. These data suggested that DISS could adjust both of the pathway which is the downstream of Trk B, and showed synergism effect.4The effect of DISS on intracellular calcium activityFlow cytometry analytical technique and Fluo-4AM were used to observe the changes of intracellular Ca2+concentration in SH-SY5Y cells treated with DISS. Intracellular Ca2+showed an acute increase and then revive to the basal level. This result showed that DISS can increase intracellular Ca2+concentration with acute treatment, and the effect will be weaken and lost for a long time treatment.In summary, our in vitro data represented DISS-induced BDNF activation may provide neuroprotection and synaptic plasticity via mechanism, at least in part, involving CREB transcription related upstream signaling systems in neuronal culure. The key finding of the present study in the up-regulation of DISS-induced BDNF gene expression and transcription is linked with enhancement of CREB-mediated transcription via ERK and CaMK signaling pathways. |
