| Background:Traumatic brain injury(TBI)is one of the most serious public health problems in the world.TBI affects three to four million people in China each year,accounting for 87 percent of trauma-related deaths,and has become the fourth leading cause of death among young people.In addition,the incidence of TBI is increasing among the elderly.Hydrogen(H2),a colorless and odorless gas,has been observed to have preventive and therapeutic effects on brain trauma and other neurological disorders,but its exact mechanism has not been fully clarified.Autophagy refers to a group of evolutionarily old mechanisms whereby eukaryotic cells eliminate cytoplasmic material through lysosomal degradation,and dysregulation of autophagy contributes to neuronal cell death after TBI.Studies have shown that rutin can provide protection effect against?-radiation-induced brain injury via activation of the PI3K/AKT/GSK-3?pathway through its ability to scavenge free radicals generation and its anti-inflammatory effects,indicating that the signal pathway is involved in the regulation of brain injury.Therefore,this study hypothesized that hydrogen plays a protective role in TBI by activating the PI3K/Akt/GSK3?signal pathway to regulate the inhibition of autophagy.This study intends to adopt Bend.3 cell scratch model,through the detection of cell morphology and growth speed,and the expression of signal pathway protein p-Akt and p-GSK3?,and autophagy protein Beclin-1,LC3-II/I and p62,clarify the hydrogen from the perspective of autophagy in the treatment of craniocerebral trauma concrete mechanism,further explore PI3K/Akt/GSK3?signal pathway in the role of this process,clarify that hydrogen improved the prognosis of TBI partly through inhibition of autophagy,and inhibitory effect was exerted by activating PI3K/Akt/GSK3?signal pathway,which will provide a scientific basis for the hydrogen treatment of TBI.Objective:To observe the effects of hydrogen on the growth rate and cell morphology of Bend.3 cells after scratches,and to detect the effects of hydrogen on the expression levels of signal pathway protein p-Akt and p-GSK3?,and autophagy protein Beclin-1,LC3-II/I and p62 after TBI.Methods:Bend.3 cells were cultured and randomly divided into control group,control+hydrogen group,TBI+hydrogen group,TBI+hydrogen group(TBI+H2 group),TBI+hydrogen+PI3K inhibition group(TBI+H2+LY group),TBI+hydrogen+autophagy activation group(TBI+H2+Rap group)and TBI+hydrogen+autophagy inhibition group(TBI+H2+CQ group).After modeling,the cells in control+H2 group,TBI+H2 group,TBI+H2+LY group,TBI+H2+Rap group and TBI+H2+CQ group were incubated with hydrogen-rich medium.During the same period of hydrogen intervention,cells in the TBI+H2+LY group,TBI+Rap group and TBI+H2+CQ group were treated with PI3K inhibitor Ly294002(20u M),autophagy inducer rapamycin(20u M)and autophagy inhibitor chloroquine(10u M)for 24 hours,respectively.After24 hours of treatment,the cell growth status was observed and the cell proliferation activity was determined by MTT assay.LC3 activity was observed by immunofluorescence assay in each group,and the expression levels of p-Akt,p-GSK3?,beclin-1,LC3-II/I and p62 were determined by western blot assay.Results:The b End.3 cells grew to confluency and mature after 4 days of culture.Then,scratching injury was carried out on the b End.3 cells.The wound can be observed at 0hour after scratching,and was significantly reduced 24 hours after scratching.Furthermore,the cells can be almost re-integrated and re-endothelialized 24 hours after scratching under hydrogen treatment.But,PI3K inhibitor Ly294002 and autophagy inducer rapamycin can reverse the protective effect of hydrogen.After Ly294002 or rapamycin treatment,the reintegration rate of the cells had significantly slowed down.While chloroquine treatment can improve the reintegration rate of the cells after TBI.The expression levels of signal pathway protein p-Akt and p-GSK3?,and autophagy protein Beclin-1 and LC3-II/I were equivalent between the sham group and the sham+H2 group(P>0.05).The expression levels of these four proteins were higher in the TBI group than in the sham group(P<0.05).Hydrogen treatment in the TBI+H2group further up-regulated the expressions of p-Akt and p-GSK3?and inhibited the expression of Beclin-1 and LC3-II/I compared with the TBI group.However,the expression level of p62 was similar among all the groups(P>0.05).The viability of the b End.3 cells was significantly lower in the TBI+H2+LY group than in the TBI+H2group(P<0.05).Similarly,the expression levels of p-Akt,p-GSK3?and p62 were decreased in the TBI+H2+LY group than in the TBI+H2 group(P<0.05).In addition,the expression levels of Beclin-1 and LC3-II/I were higher in the TBI+H2+LY group compared with the TBI+H2 group(P<0.05).The cell viability was decreased in the TBI+H2+Rap group and increased in the TBI+H2+CQ group compared with the TBI+H2 group(P<0.05).The expression levels of LC3-II/I and Beclin-1 were elevated in the TBI+H2+Rap group than in the TBI+H2 group(P<0.05).However,the expression level of p62 was decreased in the TBI+H2+Rap group and increased in the TBI+H2+CQ group than in the TBI+H2 group(P<0.05).In addition,there was no difference in the expressions of p-Akt and p-GSK3?between the TBI+H2 group,the TBI+H2+Rap group and the TBI+H2+CQ group(P>0.05).Conclusion:Hydrogen improved the cell viability of TBI partly through inhibition of autophagy,and inhibitory effect of hydrogen on autophagy was exerted by activating PI3K/Akt/GSK3?signal pathway. |
PDF Full Text Request