Mechanism Of Stachydrine In Improving Neurodegeneration Induced By Traumatic Brain Injury

With the rapid development of modern machinery,large machinery,high-rise buildings and various large vehicles can be seen everywhere.Therefore,the cases of traumatic brain injury(TBI)caused by accidental fall,falling objects and traffic accidents are increasing.Because of TBI has high disability rate,high mortality rate,complex and diverse mechanism of occurrence and development,TBI has no particularly good treatment drugs and means.This study explored the effect of stachydrine and molecular mechanism on TBI,and revealed the potential application value of stachydrine as a therapeutic drug for TBI.Stachydrine is of great significance for the treatment of TBI.Construction and neurobehavioral evaluation of TBI model ratsObjective: To explore the successful construction of TBI model rats and the effect of stachydrine on the neurobehavior of TBI rats.Methods: The rats were divided into four groups: shame operated group,TBI group,30mg/kg stachydrine treatment group and 60mg/kg stachydrine treatment group.We established TBI model with Pinpoint PCI3000 system,and then give stachydrine treatment for 2 weeks after 2 hours of TBI model induction.The effect of stachydrine was determined by evaluating the modified nervous system severity score(mNSS),brain water content percentage and Morris water maze test in rats with TBI.Results:(1)The TBI model rats was established by using Pinpoint PCI3000 system.It was found that the mNSS and brain water content percentage of the TBI group were higher than those of the shame operated group,and the Morris water maze test proved that the TBI group rats had cognitive dysfunction.(2)mNSS and brain water content percentage in stachydrine treated group were lower than those in TBI group,and cognitive dysfunction was reduced.Conclusion: TBI model rats are successfully constructed.Stachydrine can reduce mNSS score,brain water content percentage and reduce cognitive impairment and memory impairment of TBI rats.Transcriptome analysis of injured tissues in four groups of ratsObjective: To explore the mechanism of stachydrine affecting TBI,and to predict the pathways and biological processes regulated by stachydrine.Methods: The four groups of rats were collectively killed after two weeks of drug treatment,then the injured hippocampal tissue was taken out by decapitation,and then all m RNA in the cell tissue was affinity enriched by polythymine.Finally,the eluted enriched m RNA was reverse transcribed by RNA polymerase II,and then the mature m RNA and c DNA were sequenced by mass spectrometer,and the sequencing results were analyzed by bioinformatics,Then the genetic performance of each combination is evaluated to obtain the relative differential expression genomes between each group.At the same time,GO and KEGG functional annotation and enrichment analysis are provided for these different genomes,so as to predict the occurrence conditions and development rules of the disease.Results:(1)Compared with the shame operated group,the TBI group had the largest number of differential genes,and the number of differential genes decreased significantly after stachydrine treatment.(2)The differential genes in the shame operated group and the TBI group were clustered in biological processes such as apoptosis,inflammatory reaction,autophagy,cancer formation and synaptic dysfunction,and the degree of differential gene aggregation in these biological processes decreased in the stachydrine treatment group.(3)The differential genes in shame operated group and TBI group were enriched in mTOR,cAMP,PI3K-Akt and MAPK pathways,and the enrichment was weakened after stachydrine treatment..Conclusion: Sachydrine may regulate the inflammatory response,autophagy and apoptosis of neurons by regulating one or more of mTOR,cAMP,PI3K-AKT and MAPK pathways,so as to reduce the harm of TBI.Effects of stachydrine on TBI ratsObjective: To explore the mechanism of stachydrine on TBI rats and verify the conjecture of transcriptome data analysis.The four groups of rats were collectively killed after two weeks of drug treatment,and their injured brain tissues(hippocampus)were sampled for Nissl staining and HE staining to judge the effect of stachydrine on cell apoptosis and TBI damage.The proteins were extracted from the injured brain tissues of four groups of rats,and the expression of p-mTOR / t-mTOR,p-AKT / t-AKT,p-PI3K / t-PI3K,LC-3,TLR-4 and NF-κB were detected by Western blot.For the inflammatory factor in the blood of four groups of rats,the content of INF-γ,IL-1β and TNF-α was determined by ELISA kit.Results:(1)The apoptosis of the TBI group was the most serious,and the apoptosis was relieved after stachydrine treatment.(2)In the TBI group,there was necrosis and pathological changes in the injured brain tissue,and the situation was improved after stachydrine treatment.(3)Compared with the shame operated group,the expression of p-mTOR / t-mTOR,p-AKT / t-AKT and p-PI3K / t-PI3K decreased significantly,and the expression of LC-3,TLR-4 and NF-κB significant increased in the TBI group.After stachydrine treatment,the expression of p-mTOR / t-mTOR,p-AKT / t-AKT and p-PI3K / t-PI3K increased;the expression of LC-3,TLR-4 and NF-κB decreased.(4)Compared with the shame operated group,the contents of inflammatory factors INF-γ,IL-1β and TNF-α in the TBI group increased,and the contents of INF-γ,IL-1β and TNF-α decreased significantly after stachydrine treatment.Conclusion: In animal models,stachydrine can activate PI3K / mTOR / AKT signaling pathway and inhibit neuronal apoptosis.The effect of stachydrine on TLR-4 / NF-κB pathway can inhibit inflammation and autophagy..Effect of stachydrine on injured HT22 cellsObjective: To verify the mechanism of stachydrine in the treatment of TBI at the cellular level.Methods: Take HT22 cells as the representative to carry out the experiment,and carry out the traction experiment + strike on HT22 cells to imitate TBI.Different concentrations of stachydrine were used to treat the injured HT22 cells.Taking the treatment time as the node,the HT22 cells in each group were measured by flow cytometry to obtain their apoptosis rate(early apoptosis + late apoptosis).The most effective concentration and treatment time of stachydrine to inhibit apoptosis were selected for subsequent experiments.The HT22 cells in the 6h injury group were used for tunel staining to verify the successful construction of the injury model.The cells in the 5μM stachydrine + 6h injury group were tunel stained and compared with the 6h injury group to observe the effect of stachydrine on the apoptosis of HT22 cells.The proteins were extracted from the cells of blank group,5μM stachydrine + 6h injury group and 6h injury group,and the expression of p-mTOR / t-mTOR,p-AKT / t-AKT,p-PI3K/ t-PI3K,LC-3,TLR-4 and NF-κB were detected by Western blot.The total RNA was extracted from the cells of blank group,5μM stachydrine + 6h injury group and 6h injury group.Then the gene expression of p-mTOR / t-mTOR,p-AKT / t-AKT,p-PI3K/ t-PI3K,LC-3,TLR-4 and NF-κB were detected by q-PCR.Results:(1)Stachydrine can reduce the apoptosis rate of HT22 cells,and 5μM stachydrine treatment had the best inhibitory effect on apoptosis.(2)Tunel staining showed that the injury model of HT22 cells was successfully established,and the number of apoptosis cell in 5μM stachydrine + 6h injury group was much less than that in 6h injury group.(3)Compared with the blank group,the expressions of p-mTOR / t-mTOR,p-AKT / t-AKT,p-PI3K/ t-PI3K in the 6h injury group decreased significantly,and the expressions of LC-3,TLR-4 and NF-κB increased.After 5μM stachydrine treatment,the expression of p-mTOR / t-mTOR,p-AKT / t-AKT,p-PI3K/ t-PI3K increased,and the expression of LC-3,TLR-4 and NF-κB reduced.(4)The results of q PCR showed that compared with the 6h injury group,The m RNA expression of mTOR,AKT and PI3K in 5μM stachydrine + 6h injury group was increased;and the m RNA expression of LC-3,TLR-4 and NF-κB decreased.Conclusion: In the cell experiment of HT22,stachydrine can activate PI3K / mTOR / AKT pathway,inhibit TLR-4 / NF-κB pathway pathway,inhibit apoptosis,inflammatory response and autophagy,and weaken the damage of TBI to HT22 cells.