| Summary: | 博士 === 國立臺灣大學 === 解剖學暨生物細胞學研究所 === 99 === Neurons in different location require axons and dendrites to communicate with each other. Neurological diseases, e.g. stroke, head trauma, and spinal cord injury, often damage axons and dendrites and result in deficits of motor, sensory and language functions. How to promote growth of neuronal processes (neurites) is a major issue in the field of basic and clinical neuroscience. Our researches utilized in vitro cultured rat neurons to search for potential neuritogenic molecules and its signaling mechanisms. The first part of the research focused on schisandrin, a major ingredient of the Chinese herb Schisandra chinensis, and studied its effect on dorsal root ganglion (DRG) and hippocampal neurons. After treatment with schisandrin for 24 hours, DRG neurons showed increased lengthening and branching of neurites, and maximal effect was seen at a concentration of 3 μg/ml. Both large and small DRG neurons responded to schisandrin. To study the signaling pathway, KT5720 (PKA inhibitor), PD98059 (MEK inhibitor), or LY294002 (PI3K inhibitor) were applied before schisandrin treatment in DRG neurons, and only LY294002 blocked the neuritogenic effect of schisandrin. Western blot analysis showed that schisandrin enhanced phosphorylation of PI3K and Akt, which were blocked by pretreatment of LY294002. Schisandrin also increased phosphorylation of CREB, which was also inhibited by pretreatment of LY294002. Therefore, schisandrin activated PI3K-Akt-CREB pathway to enhance neurite outgrowth. Besides neuritogenic effect on DRG neurons, schisandrin also increased neurite length and branching complexity in hippocampal neurons. This effect was reversed by pretreatment with KN93 (CaMKII inhibitor), εV1-2 (PKCε inhibitor), or PD98059 (MEK inhibitor). Schisandrin also induced calcium inflow into hippocampal neurons in 10 minutes. Western blot showed that schisandrin activated CaMKII, PKCε, MEK and CREB, and the activation of schisandrin-induced PKCε, MEK, and CREB were blocked by pretreatment with KN93, εV1-2, and PD98059, respectively. The result indicated schisandrin caused calcium inflow and activated CaMKII-PKCε-MEK-CREB pathway to increase neuritogenesis. In addition, schisandrin increased expression of post-synaptic protein 95 and uptake of FM1-43, suggesting its role in synaptogenesis of hippocampal neurons.
The second part of this study focused on daidzein, a major component of isoflavone. Following treatment of daidzein for 24 hours, both small and large DRG neurons demonstrated increased lengthening and branching of neurites, and maximal effect occurred at 30 μM. Despite structural similarity of daidzein to estrogen, ICI 182780 (estrogen α/β receptor inhibitor) and G15 (membrane estrogen receptor GPR-30) failed to inhibit the neuritogenic effect of daidzein, indicating that daidzein did not stimulate neurite outgrowth via the estrogen receptors. On the other hand, PP2 (Src inhibitor), staurosporin (pan-PKC inhibitor), rottlerin (PKCδ inhibitor), and U0126 (MEK inhibitor) pretreatment abolished the neuritogenic response of DRG neurons to daidzein. Increased phosphorylation of Src, PKCδ, and MEK occurred after daidzein treatment, and activation of the latter two kinases were blocked by PP2 and rottlerin respectively. Therefore, daidzein activated Src-PKCδ-ERK pathway, which led to neuritogenesis in DRG neurons.
In conclusion, the results of our research demonstrate that both schisandrin and daidzein have neuritogenic effect, and act by different signaling mechanisms. Schisandrin enhanced neurite outgrowth of both DRG and hippocampal neurons, also via different pathways of signal transduction.
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