| Background and Objective:Inflammation and neuronal apoptosis are important factors in secondary brain injury after aneurysmal subarachnoid hemorrhage(SAH),which means it's necessary to explore effective anti-in:flammatory and anti-apoptotic regimens.In this study,the aim was to evaluate the biological activity of anti-inflammatory reaction and anti-neuronal apoptosis of PP2A/TTP after EBI and its mechanism of action after early brain injury by detecting the expression of Tristetraptolin 36(TTP)and its upstream protein phosphatase 2A(PP2A)after subarachnoid hemorrhage in rats,which provides much better ideas for drug development of early brain injury after subarachnoid hemorrhage,and also provides theoretical basis for its clinical application in future.Methods:In this study,firstly,a rat SAH intravascular puncture model was established.Then,Western blot was employed to detect the protein expression level of PP2A and TTP at 6 hours,12 hours,24 hours,48 hours and 72 hours after SAH.According to different intervention factors,it was divided into seven groups:Sham group,SAH + Vehicle(normal saline,NS)group,SAH + ScrsiRNA group,SAH +PP2A siRNA group,SAH + TTP siRNA group,SAH + FTY720 group and SAH +FTY720 + TTP siRNA.Also,itu hybridization in fluorescences and immunofluorescence were used for cell localization.These SAH rats were injected with PP2A agonist FTY720,PP2A siRNA and TTP siRNA at the left lateral ventricle site in 24 hours before modeling,whose aim was evaluated the rats situation of hindbrain injury in each experimental group at 24 hours.Meanwhile,neurobehavioral test(modified Garcia score)was used to evaluate the neurobehavioral of SAH rats.The wet and dry weight method was employed to detect water content of brain tissue to evaluate brain edema.After PP2A agonist FTY720,PP2A siRNA,TTP siRNA intervention,Western blot was used to detect the expression changes of PP2A,TTP and its downstream inflammatory factors(TNF-a,IL-6,IL-8)and apoptosis-related factors(Bcl-2,Caspase-3).Also,neuronal apoptosis was detected by terminal deoxynucleotidyltransferasedUTP nick-end labeling(TUNEL)staining.Results:In this study,the expression of PP2A and TTP reached a peak at 24 hours after SAH,especially the expression of TTP,which is much clearer,while the expression of PP2A was relatively stable.The marker of neuron to know from PP2A and TTP,Neuron specific nuclear protein(NeuN),was co-fluorescence localization,which indicated it's really expressed in neuronal cells.Moreover,Western blot showed that the activation of PP2A by PP2A activator FTY720 significantly increased the expression of TTP unphosphorylation,and unphosphorylated TTP could further degrade the expression levels of TNF-a,IL-6 and IL-8.Meanwhile,the intervention of PP2A siRNA and TTP siRNA also enhanced the expression levels of downstream inflammatory factors:TNF-?,EL-6 and IL-8,which resulted in the much higher positive rate of neuronal apoptotic cells,neurological function score decreased,cerebral edemworseninga,and the brain damage extent aggravated.Especially,it's indicated that TTP siRNA could abolish the pro-inflammatory and anti-apoptotic effects of FTY720;moreover,by adding FTY720,decreased protein expression levels of TNF-?,IL-6 and IL-8 in brain tissue and significantly improve the neurological function score after SAH,the degree of brain edema reduced,the expression of Caspase-3 further inhibited,and the expression level of Bcl-2 up-regulated,which all demonstrated the anti-inflammatory and anti-apoptotic effects of PP2A in synergy with TTP.Conclusions:SAH can induce the expression of PP2A and TTP.PP2A/TTP plays a certain neuroprotective role in EBI after SAH.Its possible specific neuroprotective effect was that it reduced brain injury by inhibiting TNF-?/IL-6/IL-8 inflammatory factor pathway,and further inhibited Caspase-3 expression,up-regulating Bcl-2 expression in order to reduce brain damage caused by neuronal apoptosis. |
PDF Full Text Request