| Kai-xin-san(KXS), first being recorded by Sun Simiao in Beiji Qianjin Yaofang<Thousand Formulae for Emergency> in Tang Dynasty, is a classical traditional Chinese medicine prescription to treat mental disorders, which is composed of Radix Ginseng, Radix polygala, Poria, and Rhizoma acoru tatarinowii. The herbal recipe can cure symptoms including restlessness, melancholy, forgetfulness etc, which are similar to depression, anxiety and difficulty in study and memorization in the West medicine. Our early pharmacological studies show that oligose esters, main active components of Polygala tenuifolia willd, are essential to antidepression. Tenuifoliside A (TFSA), an active oligose ester component from Polygala tenuifolia willd, can exert neuroprotective effects on SH-SY5Y thus relieve Cort-induced injury by promoting DNA synthesis of new-born cells, and increase the expression of phospho-cAMP response element binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus of depression rats model caused by chronic mild stress(CMS), and regulate HPA axis function in CMS rats. Evidences suggest BDNF has been implicated in the pathophysiology of depression and plays an important role in the mechanism of antidepression action.The Neurotrophic Hypothesis of Antidepressant Actions states that BDNF secretion reduction or the signal transduction pathway impairment related to BDNF, as one of key links leads to depression and also is the hot spot in antidepression research around the world. BDNF is known to mediate its action through activating various signaling transduction pathways after binding with tyrosine kinase receptor B (TrkB), which are important therapeutic targets for mood disorders.This study contains two parts. First, the classical depression animal model (chronic mild stress model) is established to detect the change of key link proteins on the BDNF/TrkB pathway of depressant animals, including TrkB, ERK, CREB, PI3K, Akt, GSK3β and BDNF. In comparison, to unfold the antidepressant-like action of Kai-Xin-San, a classical prescription on the same pathway to elucidate the molecular mechanism(s) underlying the antidepressant-like action of KXS in depth. Second, the neuroprotective effects of TFSA, extracted from Polygala tenuifolia willd in KXS, are explained by models of the rat glioma cell (C6cell) and the rat primary cultured hippocampal neurons. Moreover, according to the new theory of depression pathogenesis, the molecular and cellular mechanism in antidepression of TFSA is clarified by specific antagonists from the BDNF-TrkB signal transduction pathways perspective.To explore the molecular mechanism(s) underlying the antidepressant-like action of KXS, the depression model in rat was established by breeding separately and chronic mild stress procedure. The changes of critical protein (TrkB, p-ERK, p-CREB, PI3K, Akt, p-GSK3β, BDNF) expression were observed through TrkB/BDNF/ERK and TrkB/BDNF/PI3K signaling transduction pathways in hippocampus and prefrontal cortex within rats brains. In comparison with unstressed control rats, a5-week CMS exposure significantly reduced the level of TrkB receptor and BDNF expression in hippocampus and prefrontal cortex, the level of PI3K, Akt and p-ERK were decreased too. We found that treating the CMS rats with370mgkg-1of KXS for2weeks could normalize low TrkB receptor levels caused by CMS in hippocampus and prefrontal cortex, and it promoted the protein levels of p-CREB, p-ERK, PI3K and Akt as well as BDNF in hippocampus, however, the level of p-GSK3β protein was not changed markedly. Additionally, KXS increased the level of p-ERK, p-CREB and BDNF in prefrontal cortex of model group. These results implicate KXS produced an antidepressant-like effect. Especially, we founded that the mechanism of KXS is at least partly medicated by increasing expression of BDNF and TrkB receptor and reinforcing neuron nutrition via BDNF/TrkB/ERK and BDNF/TrkB/PI3K signaling pathways in hippocampus and via BDNF/TrkB/ERK signaling pathway in prefrontal cortex mostly.In vitro experiments, the injury model was established; C6cells were pretreated with CORT for30min simulating the high CORT concentration, followed by co-treated with30,10and3μmol·L-1of TFSA respectively and5μmol·L-1of Desipramine (DIM) for48h. Then observe the neuroprotective effects of TFSA against neurotoxicity induced by CORT on C6cells. Cell viability of the injury cells affected by TFSA was determined; fluorescence microscope was used to observe the Apoptotic phenotype change to nuclei after Hoechst33258staining; the expression of ERK as well as apoptotic related proteins Bcl-2and Bax were detected too. Caspase-3kinase activity in C6cells either exposed to CORT alone or co-treated with TFSA was also assayed. Compared with the normal control group, the results demonstrated that0.8mmol-L-1of CORT decreased the viability of C6cells significantly to69.58%(P<0.01). Cell nuclei shape changed and appeared apoptotic body; Western blot results showed that the levels of ERK and Bcl-2decreased significantly in C6cells (P<0.01); in contrast, the expression of Bax and the activity of Caspase-3increased markedly. However, this neurotoxicity effect of CORT could be reversed by TFSA. These results suggest that TFSA protects C6cells from CORT-induced damage, and the neuroprotective mechanism might be associated with the enhancing the expression of Bcl-2, suppressing Bax and decreasing the activity of Caspase-3via ERK pathway activated by TFSA.To further study the neuroprotective effect of TFSA on C6cells, normal cultured C6cells were treated with25pg·L-1rhβ-NGF or0.6,3,6,10,30and60μmol·L-1of TFSA for24h, cell survival rate was detected and lactate dehydrogenase (LDH) release. The release of BDNF protein was detected. After treatment of cells with10μmol·L-1TFSA, we examined the important protein phosphorylation in TrkB/BDNF/ERK and TrkB/BDNF/PI3K signaling pathways by setting a sequence of time points at2,5,7,10,15,30,45and60min; protein kinase inhibitors were used to investigate the mechanism underlying the neuroprotective effect of TFSA. The results demonstrated that TFSA (6,10and30μmol·L-1) promoted the cell proliferation and no obvious cytotoxicity. Moreover, TFSA increased TrkB, ERK and PI3K activation as well as GSK3β and CREB phosphorylation in different time points, with enhanced release of BDNF consequently; later on, each of which was completely blocked by TrkB antagonist (K252a), MEK antagonist (U0126) and PI3K antagonist (LY294002), BDNF expression reduction appeared correspondingly. Taking these results together, we hypothesize the neuroprotection of TFSA might be mediated via TrkB/BDNF/ERK and TrkB/BDNF/PI3K signaling transduction pathway in C6cells.Moreover, we explored the possible neuroprotective effect mechanism of TFSA in rat primary cultured hippocampal neurons models. While primary (within12h) hippocampal neurons cells after cultured for seven days, were treated with25pg·L-1BDNF or1,3,10and30μmol·L-1of TFSA for48h, the nerve cell survival rate was detected. BDNF Protein expression was detected too. The results demonstrated that TFSA promoted the cell survival and increased TrkB and ERK phosphorylation. However, the TFSA-induced ERK phosphorylation was completely blocked by TrkB antagonist (K252a). These results suggest that TFSA could promote BDNF production in primary hippocampal neurons possibly by inducing ERK phosphorylation via activating TrkB/ERK/BDNF pathway.To sum up, this research paper has proved the antidepressive effects of KXS from the level of animals, and has found out that the mechanism is through upregulating BDNF and TrkB receptor expression and then activates TrkB-BDNF signal system, KXS in hippocampus also mediates ERK and PI3K signal pathways, while in prefrontal cortex mainly mediates neuroprotective effects developed by ERK signal pathway to brain neurons. Further more, the study from the levels of C6cells line and primary culture nerve cells has clarified the active component TFSA in KXS neuroprotective mechanism is related to activating the TrkB/BDNF mediated ERKand PI3K signal pathways, promoting ERK and Akt phosphorylation and further promoting downstream CREB and GSK3β phosphorylation and finally BDNF expression. Accordingly, the author believes that the neuroprotective and neurotrophic effects of TFSA, which is the main active ingredient in KXS, is the main molecular mechanisms of KXS antidepressants. |
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