The Neuroprotective Effects Of Gastrodin On Cerebral Ischemia-hypoxia Injury

Background and Objective:Hypoxic-ischemic brain damage (HIBD) is a common disease of high mutilation and fatal. The mechanisms of HIBD are complicated and have not been completely understood. Biochemical events such as energy failure, overloading of intracellular calcium, increasing of excitatory amino acids (EAAs) release, accumulation of nitric oxide (NO) by hypoxia-ischemia (HI) may lead to brain dysfunction and neuronal death. These events are not independent, but interact with each other. At present, it has been considered that neuroprotective therapy is a basic measure. Its mechanisms of action are to block up the process of hypoxic-ischemic, to ameliorate the cerebral injury and to promote neurofunctional revert. Currently, many chemical drugs have been developed. However, most of them had severe side-effects on Ischemic Encephalopathy clinically. Because naturally occurring drugs had mechanism of action through multitarget and were hypotoxic, they were researched generally as neuroprotective drug. Therefore, it is necessary to search new types of therapeutic drugs for the treatment of cerebral ischemia.Gastrodin (Gas) is a primary component of the functional extracts from the rhizome of Gastrodia elata BLUME (Orchidaceae) and its chemical name is 4-hydroxymethyl phenyl-β-D-glucopyranoside. It has been found that Gastrodin is a safe sedatives and has the effects of mitigate, analgesia, improving the function of heart and cerebral vessels, anti-inflammatory and anti-free radical. Its feature is safe, low poison and no accum, drug dependence and addiction. In addition, some studies found that Gastrodin could enhance haemal compliance and had protective effects against rat cortical neurons. However, the mechanisms: underlying the neuroprotective effects of Gastrodin are currently unclear.In this study, Gastrodin was added to the cultured cerebral neurons of embryonal rats in vitro, which damaged by hypoxic-ischemic. The neuroprotective effects of Gastrodin were observed through biochemical and molecular biological technique. The aim of this study is to investigat the mechanisms of Gastrodin on cerebral hypoxic-ischemic, which will provide a academic basis of Gastrodin to be used in treatment of cerebral hypoxic-ischemic disease.Materials and Methodology:1. Cerebral neurons of Wistar's embryo rats (16-19d) were used for the culture of neurons.2. The injury model of cultured neurons was made by N₂ and glucose-deprived Earle's solution. The Nitrogen gas and glucose-deprived Earle's solution were administrated for 5 hours in this model. The neurons were randomly divided into 3 groups: the group of pseudo-operation (normal control group, NC group), the group of cerebral hypoxic-ischemic injury (CHII group) and the group of cerebral hypoxic-ischemic injury treated with Gastrodin (G group). Gastrodin (13mg/L, 26mg/L, 52mg/L) was added into the G group before and during neurons damage.3. Morphologic change of neurons was observed under invert microscope.4. The survival rate was analyzed using colorimetric Trypan blue assay.5. The levels of nitric oxide (NO) and the activity of lactate dehydrogenase (LDH) in the supernatant of cultured neurons were measured by NO and LDH kits.6. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of NR1 mRNA semi-quantificationally in hypoxic-ischemic injury in neurons cultured for 10d in vitro.7. SPSS 10.0 statistical package was used for statistical analysis. One-way ANOVA and LSD were used to analyze all experimental data. Differences with P< 0.05 were considered statistically significant.Results:1. Effect of Gastrodin on the morphologic change of hypoxic-ischemic injury in cerebral neurons:Neurons were observed under invert microscope. Neurosome of normal control was turgor vitalis, nervous process was long and became reticulodromous. The neurons cultured for 10 days' were put into an environment of simulated hypoxic- ischemic, and after 5 hours, they became swollen, necrosis. After adding Gastrodin, more neurons remained nervous process and less cell debris.2. Effect of Gastrodin on the survival rate of hypoxic-ischemic injury in cerebral neurons:The survival rate decreased markedly after the hypoxic- ischemic injury of cerebral neurons (P<0.01). Gastrodin at the concentration of 13mg/L, 26mg/L, 52mg/L could increase remarkably the survival rate ( All P<0.01), and the concentration of 26mg/L had the best effect, which suggested that Gastrodin could resist the injury caused by hypoxic- ischemic.3. Effect of Gastrodin on the level of NO in the supernatant of cultured cerebral neurons:The level of NO in the supernatant increased obviously (P<0.01) after the hypoxic-ischemic injury of cerebral neurons. By adding Gastrodin into the nutrient solution, release of NO contents decreased (P<0.05).4. Effect of Gastrodin on the activity of lactate dehydrogenase (LDH) in the supernatant of cultured cerebral neurons:The amount of LDH in the supernatant can be markedly increased after the hypoxic-ischemic injury of cerebral neurons(P<0.01). When treating with Gastrodin, the 13mg/L, 26mg/L and 52mg/L dose could diminished the transudation of LDH (P<0.05), which suggested Gastrodin could antagonize the neurotoxicity of hypoxic-ischemic.5. Effect of Gastrodin on the NR1 mRNA expression of hypoxic-ischemic injury in cultured cerebral neurons:The NR1 mRNA expression increased markedly after the hypoxic-ischemic injury of neural cells(P<0.01), each concentration 13mg/L, 26mg/L, 52mg/L)of Gastrodin could inhibit evidently the NR1 mRNA expression (P <0.01).Conclusions:The Gastrodin obviously improved corpuscular morphous, raised survival rate, inhibited the release of LDH, diminished NO contents and inhibited NR1 mRNA expression. It had obviously protective effect on hypoxic-ischemic injury of cerebral neurons, one of the mechanisms might be related with the inhibition of NR1 mRNA expression induced by Gastrodin. Thereby, less protein of N-Methyl-D-aspartate (NMDA) receptor was generated. So it could inhibit the release of LDH and diminish NO contents for cutting off the pathway of Glu-NO-cGMP. All factors improved corpuscular morphous and raised survival rate.