Effect Of Buyang Huanwu Decoction On Hippocampal Nerver Regeneration In Rats With Post-stroke Depression From MAPK/ERK Signal Pathway

Objectives:To observe the effect of Buyang Huanwu Decoction on the ethology, the morphology, cell proliferation and Neural stem cells regeneration of hippocampus, as well as ERK1/2, P-ERK1/2, CREB, P-CREB and BDNF genes and proteins expressions of in model rats with post-stroke depression (PSD), and to explore the mechanisms of Buyang Huanwu Decoction regeneration hippocampus nerve of the PSD rats from the MAPK/ERK signal pathway, which providing experimental evidences for traditional Chinese medicine in the treatment of clinical post-stroke depression drug.Methods:The PSD model rats were established with separately breeding and chronic unpredictable moderate stress (CUMS) after middle cerebral artery occlusion,90Male SD rats were randomly divided into five groups (18rats in each group):sham operation group, cerebral ischemic stroke group, post-stroke depression (PSD) group, fluoxetine group, and Buyang Huanwu Decoction group. From the first day after stress, fluoxetine group and Buyang Huanwu Decoction group were intragastric administrated with fluoxetine hydrochloride soup (1.8mg·kg-1·d-1) and Buyang Huanwu Decoction (contented with12.8g·kg-1·d-1), respectively, for21days, the remaining groups with distilled water. Six rats were selected randomly from each group to intraperitoneal inject BrdU(100mg kg·-1·d-1) for proliferation cell markers. The rats were examined dynamically at the stress before and7th,14th,21th days after stress by body weight, neurological score, sugar consumption test and open-field test. Six cerebral tissues were selected randomly to make paraffin sections from each group, and then morphology changes of hippocampal cell were observed using HE staining, Nestin expressions were tested using immunohistochemical staning. And6cerebral tissues were selected to make frozen sections for hippocampal cell proliferation determination via BrdU immunohistochemistry staining. Hippocampus were separated from6cerebral tissues, Nestin, B DNF, CREB mRNA expression were detected by Realtime-PCR, ERK1/2、P-ERK1/2、P-CREB protein expressions were detected by Western-blotting, respectively.Result:(1) From the results of the ethology, Compared with sham operation group and cerebral ischemic stroke group, weight, sugar consumption and open-field spontaneous activity in PSD model group decreased, and higher neurological function deficits (P<0.01or P<0.05). Compared with PSD group, Buyang Huanwu Decoction group got significantly higher scores of weight,sugar consumption and spontaneous activity, improved neurological function deficits(P<0.01or P<0.05).(2) From the results of HE staining, the obvious injury of the neurons in hippocampal CA1, CA3and DG areas of the PSD group rats were observed, which degeneration, necrosis and lost more, scattered cell arrangement, changed nuclear membrane structure, inconspicuous nuclei, karyopyknosis and deeper color. However, in the Buyang Huanwu Decoction group, the neuron morphology is improved significantly with integrity cell structure, neatly arranged, big and round nuclei, clear nucleolus and only a little of cellls degeneration.(3) The results of BrdU immunohistochemistry showed that there were a certain numbers of proliferating cells in hippocampal CA3 and DG region of all groups. The numbers of proliferating cells in hippocampal CA3and DG region of the Buyang Huanwu Decoction group increased(P<0.01or P<0.05) significantly compared with the PSD group and fluoxetine group.(4) From the results of Nestin expression, Compared with cerebral ischemic stroke group, the expression of Nestin in hippocampal decrease in the PSD group (P<0.01), protmoted chronic stress suppresses that can cause the inhibition of hippocampal neurogenesis. Compared with PSD group, fluoxetine group and Buyang Huanwu Decoction group appears immune response positive products and gene level increased, the difference had statistically significant (P<0.01).(5) From the results of genetic testing, Compared with sham operation group, cerebral ischemic stroke group, the expression of BDNF, CREB mRNA in hippocampal were significantly lower (P<0.01). After Buyang Huanwu Decoction interference treatment which were significantly increased (P<0.01).(6) From the results of protein detection, the expression of P-ERK1/2、p-CREB protein in the hippocampus of PSD group rats were significantly lower (P<0.01) than that of sham operation group. P-ERK1/2、p-CREB protein expression levels in the hippocampal of Buyang Huanwu Decoction group rats were significantly increased (P<0.01) compared with that of PSD group, however the total ERK1/2protein change was no significant difference.Conclusion:(1) Through MCAO combine CUMS method and solitary compound model method was successfully preparation PSD model, PSD rats exists abnormal behavior, the core symptoms shows weight loss, spontaneous activity decreases, lack of pleasure(2) Buyang Huanwu Decoction can significantly improve the behavioral manifestations of PSD rats, get significantly higher scores of weight, sugar consumption and spontaneous activity, improve neurological function deficits, achieve the antidepressant effection.(3) Buyang Huanwu Decoction have neuroprotective effects, which reducing chronic stress-induced injury of rat hippocampal neurons and recovering hippocampal nerve function.(4) Buyang Huanwu Decoction can enlarge the damaged brain tissue self-repair mechanisms, promote post-stroke depression in rat hippocampus nerve cell regeneration.(5) Buyang Huanwu Decoction can increase the BDNF expression, activate MAPK/ERK signaling pathway, neuronal regeneration through the signal transduction cascade induced a few key molecule, which may play one of its antidepressant mechanism.