Transcriptome Study Of Protective Effect And Mechanism Of Gastrodia Elata On Neurons In Parkinson Mice

Objective:To study the protective effect of Gastrodia elata on A53T alpha synuclein transgenic Parkinson mice,and to explore the molecular mechanism of the neuroprotective effect of Gastrodia elata and the improvement of the symptoms of Parkinson's disease?PD?from the gene transcriptome level..Methods:1.A53T transgenic PD mice were used as experimental animals,and the mice were amplified and propagated.The transgenic PD positive mice were screened through the genotyping test of the rat tail,which was prepared for the follow-up experiment.2.The 4 month old A53T transgenic PD mice were used to set up blank group,model group,positive group,Gastrodia elata high(14.4 g·kg^-1),middle(7.2 g·kg^-1),low dose group(0.72 g·kg^-1),continuous perfusion of stomach for 4 weeks.The behavior of climbing rods and the hematoxylin-eosin?HE?staining and neuron count of substantia nigra in mice were detected.3.The transcriptional library of the blank group,model group,model group and Gastrodia elata group was constructed by high throughput transcriptome sequencing technology,and differentially expressed genes were screened out.The differential gene was annotated and enriched by GO,COG and KEGG database,the key genes and metabolic pathway of transgenic PD mice protected by Gastrodia elata were analyzed,and qRT-PCR?Real-time fluorescent quantitative PCR?technique was used to verify the expression abundance of the candidate gene c-Fos,p38MAPK,MAPKAPK3,NPY,RASGRP1,DRD3.Results:1.A total of 140 weaning mice,80 males and 60 females,were obtained from breeding.74 of the transgenic PD positive mice were identified by genotype,including 42 males and 32 females.2.The results of climbing pole behavior showed that the total number of scores in the model group,positive group,Gastrodia elata high,middle and low dose group were statistically significant compared with that of the blank group?P<0.05?.The total score of high,middle and low dose groups of Gastrodia elata was higher than that in the model group and lower than that in the blank group,in which the model dose fraction of the Gastrodia elata was close to the positive group,and the effect is most significant,but it's not statistical significance compared with the model group?P>0.05?.3.The results of HE staining and neuron count showed that the number of neurons in the model group decreased significantly compared with the blank group and the difference was statistically significant?P<0.05?,in addition,the morphologic model group had a part of the nerve cell body deep staining,glial cell proliferation,and the appearance of Parkinson's disease.The difference between the middle dose group and the model group had significantly different?P<0.05?,thenumberofneuronswasclosetothepositive group.Morphological observation showed that there were more neurons in the remaining substantia nigra,and the number of neurons in slender and deep staining was less.While the number of substantia nigra neurons in the high and low dose groups of Gastrodia elata was less.4.The results of climbing rod behavior test,HE staining and neuron counting showed that the median dose of Gastrodia elata(7.2 g·kg^-1)was the optimal dosage of transgenic PD mice.5.A total of 74.34 Gb Clean Data was obtained by transcriptional sequencing.The Clean Data of each sample reached 7.04 Gb,and the Q30 base percentage was above 85.92%.The comparison efficiency of samples and reference genes ranged from79.70%to 86.35%,and the comparison efficiency was higher.6.The results of transcriptional analysis showed that 788 different genes were screened in the blank group than the model group,of which 369 genes were up-regulated,419 genes were down regulated,and the difference of gene function annotation was found in 215 pathways.466 different genes were screened in the model group compared with the Gastrodia elata group,of which 292 genes were up-regulated,174 genes were downregulated,and the differential gene functional annotations were distributed in 177 pathways.GO and KEGG enrichment analysis showed that the neuroactive ligand receptor interaction pathway,phosphatidylinositol-3 kinase/protein kinase B pathway,Ca²⁺signaling pathway,Ras?proto oncogene of rat sarcoma?pathway in model group were clearly activated.The key genes c-Fos,p38MAPK,and MAPKAPK3 were down regulated in the Gastrodia elata group compared with the model group,while NPY,RASGRP1 and DRD3 were up-regulated in the Gastrodia elata group.Finally,it was verified by qRT-PCR test.Conclusions:1.A53T transgenic PD mice can better simulate the progress of Parkinson's disease.As a PD model animals,it's relatively stable and reliable.2.Gastrodia elata decoction can improve the activity of A53T transgenic PD mice and substantia nigra neuron lesion,especially in the middle dose group of Gastrodia elata(7.2 g·kg^-1).3.The transcriptional sequencing was carried out on the mesencephalic nigra tissue of transgenic PD mice and blank normal mice,which were not given Gastrodia elata,Gastrodia elata and blank normal mice,and the results showed that the pathogenesis of PD is related to the activation of neuroactive ligand receptor interaction pathway,phosphatidylinositol-3 kinase/protein kinase B pathway,Ca²⁺signaling pathway,Ras pathway and so on.The effect of Gastrodia elata on PD protection may be related to down regulation of c-Fos,p38MAPK,MAPKAPK3 and other apoptosis related genes,and up regulation of NPY,RASGRP1,DRD3 and other neurotrophic factor and neurotransmitter related genes.The 6 candidate genes were enriched in the neuroactive ligand receptor interaction pathway,phosphatidyl inositol-3 kinase/protein kinase B pathway and Ras pathway.It may be the neuroprotective effect of Gastrodia elata to the target gene of PD.