Transcriptomics Study On The Protective Effect Of Gastrodin On Rat Cortical Neurons After Oxygen-glucose Deprivation And Reperfusion Injured

Ischemic cerebrovascular disease is a series of symptoms or signs caused by degeneration and necrosis of brain tissue due to decreased or complete occlusion of cerebral blood flow,which belongs to"apoplexy"category in traditional Chinese medical science.It has a high mortality rate,and even if the rescue is timely,paralysis,aphasia,dementia and other sequelae will also occur.Gastrodia elata Bl.known as"God medicine for apoplexy",is one of the commonly used traditional Chinese medicines in the treatment of cerebral ischemia/reperfusion injury.In order to further explore the molecular biological mechanism in treating apoplexy of Gastrodia elata Bl.,the primary cortical neurons as experimental objects and the high-throughput RNA-Seq sequencing as analysis technology were used in this paper to research the key active component gastrodin,which has potential anti-cerebral ischemia effect.The aim of this study is to explore the mechanism of G.elata on injured cells at the transcriptional level,and provide theoretical support for the application of G.elata in clinical cerebral ischemic diseases.Methods:1.Primary cortical nerve cells were obtained by trypsin digestion,and sugar-free Earle's solution combined with Na₂S₂O₄ were used to establish oxygen-glucose deprivation and reperfusion?OGD/R?damage model.The cortical nerve cells were randomly divided into control group,model group,and low,middle and high-dose gastrodin groups,and the survival rate of cortical cells was measured by MTT method and the leakage rate of lactate dehydrogenase?LDH?was measured by kit.2.Extraction of total RNA from cortical neurons in control group,model group and gastrodin preadministration experimental groups,then reverse transcription and construct cDNA library.High-throughput sequencing was carried out using HiSeq X-ten platform to construct the transcripts database of rat cortical cell.By functional annotation,differential expression gene analysis,metabolic pathway enrichment analysis,genes and metabolic pathways related to protective effect of gastrodin on OGD/R damaged cortical cells were screened.3.The mRNA transcriptional activity of some candidate genes screened in the previous step was verified by QRT-PCR technique.Results:1.The model was established with 8 mM Na₂S₂O₄ combined with sugar-free Earle's solution,and the survival rate was 50%⁶0%after 1 h of oxygen-glucose deprivation and reperfusion for 2 h,which was the best model condition for OGD/R damage of primary nerve cells.2.After OGD/R damage,the activity of rat cortical cells decreased significantly and the release of LDH increased,while gastrodin pretreatment can increase the survival rate and significantly decreased the leakage rate of LDH.3.Totally 260,098,083 high quality nucleotide sequences and 76,943,018,898 bp base number were obtained by high-throughput sequencing,and the Q30 base percentage was above 90.32%.The mapped Ratio is higher ranged from 81.19%to83.21%.4.Under the screening criteria of FDR...