Effects Of Phenolic Component Of Gastrodia Elata Blume On Astrocytes Facilitate Neuronal Repair In Cerebral Ischemia/Reperfusion

Objectives Ischemic Cerebrovascular Disease (ICVD) has a high prevalence in old people and the pathological research has been focused on the neuro-protection and repair. The brain is very susceptible to damage during ischemia induced by oxidative stress due to a high rate of oxidative metabolic activity and inadequate neuronal cell repair activity. Astrocyte, which is rich in certain antioxidants and cytokines, play an important neuroprotective role in brain resistance to the deleterious effects of superoxides, and its role in the Nrf2-mediated cellular defense system has been proposed as an avenue for ischemic stroke treatment. Gastrodia elata Blume., a traditional Chinese medicine which has been proved has the neuroprotective effect on ischemic cerebrovascular diseases, will be tested for its therapeutic action on cerebral ischemia by using its active compounds, the phenolic component (PEEG70), and its Nrf2related mechanism will be also determined.Methods The experiment conducted in two parts.In vivo:Middle cerebral artery occlusion (MCAO) was performed in adult male Sprague-Dawley rats for2h ischemia, and then underwent reperfusion (I/R). Animals were administered with vehicle, Nimodipine,7.29g/kg or0.81g/kg of PEEG70respectively. The neurofunction deficit was scored by Longa5and the neurorepair was scored by beam-walking test at day3,7and14after I/R, then the rats were sacrificed and the brain were moved for immunohistochemistry staining of GFAP, MAP2and the transcription factor Neurogenin2(Ngn2).In vitro:Astrocytes and SH-SY5Y (a human neuroblastoma cell line) were employed in this study. The first section was design to detect the protective effects of PEEG70at the dose of15,25,50ug/ml on the damage of astrocyte or SH-SY5Y cells induced by0.5mM H2O2for4h; The second section was to detect the time course of PEEG70pretreatment of astrocyte or SH-SY5Y cells up to1-48h at the dose of25ug/ml on the damage induced by0.5mM H2O2for4h; The third section was design to determine the effect of PEEG70on the neuroprotection role of astrocytes on neurons. Briefly, the culture medium of atrocytes non/with PEEG70(NACM/PACM) was used for SH-S Y5Y culture at a ratio of1:10,1:5or1:1to normal medium for24h and then exposured to0.5mM H2O2for4h. Cell injury was assessed by measuring extracellular lactate dehydrogenase (LDH) release; the fourth section detected the effect of PACM on oxygen and glucose deprivation/reoxygenation (OGD)-induced SH-SY5Y damage by immunofluorescence.The neuroprotective mechanism of PEEG70via astrocyte-mediated cellular defense system was studied by detecting the Nuclear factor erythroid2-related factor2(Nrf2) pathway including its downstream protein heme oxygenase1(HO-1), NADPH-quinone oxidoreductase (NOQ1) and brain derived neurotrophic factor (BDNF) by Western blotting.Results In vivo:compared to the model group, PEEG70can markedly improve the neurofunction and the neurorepair of rat at day7and14after I/R. It attenuated the activation of astrocytes and improved the morphology both of astrocytes and neuron cells, increased the number of positive reaction cells of GFAP and MAP2(P<0.05, respectively) at the dose of7.29g/kg, as well as the expression of Ngn2(P<0.05).In vitro:LDH assay indicated that PEEG70can protect the astrocytes and neurons in a dose and time dependent manner. Compare to control, significant difference was observed from a dose of25μg/ml PEEG70pretreatment that reduced H2O2-induced cell death in astrocytes (76±11%vs. control, P<0.05), and in SH-SY5Y cells (85±4%vs. control, P<0.05). Both of the NACM and the PACM can protect the SH-SY5Y from oxidative stress induced by OGD, and the effect of PACM better than NACM (P<0.05). The protective effects of PEEG70(25μg/ml) on astrocytes were mediated via activation of the Nrf2pathway by increasing the accumulation of Nrf2in nuclei to1.63±0.16(P<0.01) fold higher than control and its downstream protein including HO-1(1.67±0.17fold vs. control), NQO-1(1.95±0.31fold vs. control), and BDNF (1.83±0.29fold vs. control)(P<0.01) which may contribute to the recovery of neurofunction.Conclusions These results demonstrated that PEEG70exerts neuro-protection and-repair effects by upregulating the transcription factor Ngn2, neurotrophic factor BDNF and improving Nrf2-mediated cellular defense system in astrocytes, which indicated that PEEG70may be act as a potential candidate for neurological disorders treatment.