Research On The Neuroprotective Effect And The Involved Mechanisms Of Gastrodin

【Background】That the Tian ma(Gastrodia eleta blume) is a useful traditional Chinese herb medicine in China ancient time is considered to have several beneficial properties in treating headaches, dizziness, tetanus, epilepsy, infantile convulsions, and numbness of the limbs.The compound 4-Hydroxybenzyl alcohol 4-O-beta-D-glucopyranoside, which is also known as gastrodin(GAS), is the main active constituent isolated from Tian ma. Recent studies indicate that GAS has a neuroprotective action against hypoxia in the cultured cortical neuron, protect primary cultured rat hippocampal neurons against amyloid-beta peptide-induced neurotoxicity and ameliorate cerebral damage after transient focal cerebral ischemia. It also reported that gastrodin could inhibit expression of inducible NO synthase(NOS), cyclooxygenase-2(COX2) and pro-inflammatory cytokines in cultured LPS-stimulated microglia, and exhibits anxiolytic-like effects via the GABAergic nervous system. In addition, gastrodin may improve learning and facilitate memory consolidation and retrieval, and protect lead-induced synaptic plasticity deficits in rat hippocampus. Moreover, recent studies showed that the beneficial effects of gastrodin might result from its antioxidant properties. GAS could protect primary cultured rat hippocampal neurons against amyloid-beta peptide-induced neurotoxicity and attenuate the reduction of catalase(CAT) and superoxide dismutase(SOD) expression via the activation of ERK1/2-Nrf2 pathway. These observations lead to the hypothesis that gastrodin may also be effective in improving psychiatric symptoms and provide neuroprotection against ischemic brain injury. 【Aims】1.To investigate the antidepressant-like effect and the protective effect on astrocyte of gastrodin.2.To test the possibility that GAS may suppress specific signaling pathways associated with the immune response, thereby maintaining hippocampal function and preventing behavioral signs of depression.3.To investigate the neuroprotection of GAS against ischemic brain injury. 【Methods】1. To investigate the therapeutic effects of the gastrodin(GAS) in a chronic unpredictable stress(CUS) rat model, SD rats were randomly assigned to the following 8 groups(n = 10 for each groups): sham, sham + GAS(L), sham + GAS(M), sham + GAS(H), CUS, CUS + GAS(L), CUS + GAS(M) and CUS + GAS(H). The sham group received only saline, and the sham + GAS(L, M or H) group received GAS(50, 100 or 200 mg/kg daily). The rats in the CUS group were subjected to CUS, and then received saline as a vehicle for 7 days(1 ml/kg daily). The CUS + GAS(L, M or H) group also experienced CUS, but they received GAS(50, 100 or 200 mg/kg daily) for 7 days. GAS was the chemical control reagent produced by biotransformation(purity was more than 99.2%) and supplied by the Kunming Pharmaceutical Corporation(Kunming, China). The solubility of GAS was more than 300 mg/m L, and it was stable for more than 2 years at room temperature when dissolved in sterile water. In the present study, GAS was dissolved in saline, and all the treatments were administered intraperitoneally for all groups at the same time. The body weight for each rats were measured weekly. After 7 days of treatment with GAS or vehicle, rats were experienced sucrose preference test and forced swim test, then sacrificed and the expressions of GFAP and BDNF in the hippocampus were determined by using immunohistochemistry or Western blot. To assess the effect of GAS on the hippocampal-derived astrocytes and the expression of BDNF and pERK1/2, the astrocytes were obtained by a culture assay and exposed to different concentrations of GAS(sham, 5, 10, 20, 50 and 100 μg/mL) for 48 and 72 h, then the cell viability and the expression of pERK1/2 and BDNF were tested by WST-1 and Western blot assay. To further assess the protective effect of GAS on astrocytes, the cells were exposed to serum-free condition(DMEM only) and received different concentrations of GAS(sham, 5, 10, 20, 50 and 100 μg/mL) for 48 and 72 h, then the cell viability were tested by WST-1 assay. All experiments were carried out using the second passage of astrocyte.2.After the adaptive phase, rats were randomly divided into 8 groups of 12: sham, sham + GAS(L)(low-dose, L = 50 mg/kg/day), sham + GAS(M)(medium dose, M =100 mg/kg/day), sham + GAS(H)(high-dose, H = 200 mg/kg/day), CUS, CUS + GAS(L), CUS + GAS(M), and CUS + GAS(H). From week 1 to week 5, the 4 CUS groups were exposed to two stressors per day in random sequence to maximize the unpredictability. At week 6, rats were treated with saline or GAS once daily for 14 days. Following drug treatments, individual treatment groups were subdivided for behavioral testing and Western blot and Elisa analysis or BrdU immunohistochemistry: All the rats in each group were exposed to sucrose preference test, and 8 of them were then exposed to forced swim test and sacrificed for BrdU detection or Western blotting and Elisa subsequently; the remaining(n = 4) were exposed to Morris water maze. To examine the direct effects on NSCs, different doses of GAS were administered to cultured NSCs for 3 or 5 days and viable cell number estimated by WST-1 assay. To investigate the protective effect of GAS against IL-1β, this cytokine(10 ng/m L) was added to the cell medium for 2 h followed by GAS treatment for 24 or 48 h. Cell number was estimated by WST-1 and BrdU assays.3.To detect the neuroprotective effect of GAS, mice were subjected to MCAO followed by GAS or vehicle treatment, at 24 hours after reperfusion, the neurologic deficiency was detected, and then mice were sacrificed for TTC staining or Nissl staining and Western Blot.To detect if the neuroprotective effect of GAS was long lasting but not transient, mice were subjected to MCAO and then received GAS or vehicle once daily for 7 consecutive days. At 7 days after reperfusion, the neurologic deficiency was detected, and then mice were sacrificed for TTC staining or Nissl staining and Western Blot.To evaluate the protective effect of GAS on the oxidative damage and its anti-inflammatory effect in mice brain induced by MCAO, we determined the protein expression of nuclear factor erythroid 2-related factor 2(Nrf2), Heme oxygenase-1(HO-1), SOD1, phospho-Akt, total Akt as well as the content of MDA and total cerebral SOD activity in the ischemic hemisphere at 6 hours after reperfusion, as well as the protein expression of TNF-α and IL-1β. 【Results】1.GAS administration(100 and 200 mg/kg daily) reversed depressive-like behaviors in rats exposed to CUS paradigm and restored the expression of GFAP and BDNF in the hippocampus. Moreover, in vitro experiments revealed that GAS did not increase the cell viability of astrocytes but protected it from 72 h’s serum-free damage at the dosage of 20 μg/m L. Interestingly, the increased ERK1/2 phosphorylation and BDNF levels were observed after GAS(20 μg/m L) treatment for 72 h.2.GAS(200 mg/kg daily) reversed all tested depression-like behaviors in CUS model rats and up-regulated NSCs proliferation in the hippocampus. Enhanced expression of p-iκB, NF-κB, and IL-1β by CUS was also reversed by GAS. Moreover, in vitro experiments revealed that GAS alone did not increase the viability of NSCs but protected them from IL-1β-induced damage.3.GAS, especially at high dose(100 mg/kg) reduced tested neuronal injury and neurobehavioral deficient in MCAO mice. Enhanced expression of cleaved Caspase 3 and Bax and decreased expression of Bcl-2 by MCAO were also reversed by GAS. Moreover, GAS treatment decreased the MDA content and the expression of TNF-α and IL-1β, and increased amount of SOD activity and the expression of HO-1 and SOD1 in GAS-treated ischemic brain. Furthermore, GAS significantly increased Akt phosphorylation and Nrf2 expression. 【Conclusions】1.Gastrodin possesses antidepressant effect and the changes in the astrocyte activation and the level of BDNF may play a critical role in the pharmacological action of GAS.2.GAS possesses antidepressant and neuroprotective effects, and GAS may reduce depression-like behaviors by protecting hippocampal NSCs against the proinflammatory cytokine IL-1β.3.GAS could protect the brain damage induced by MCAO, and the activation of Akt/Nrf2 pathway may play a critical role in the pharmacological action of GAS.