Research Of The Role And Potential Mechanism Of TRAF6 On Early Brain Injury After Subarachnoid Hemorrhage In Rats

Part I: The expression level changes of TRAF6 at different time point after SAH in ratsObjective:to investigate expression level changes of TRAF6 at different time point after SAH in rats.Methods:60 Sprague-Dawley(SD)rats were randomly divided into 5 groups of 12 rats each: sham group,SAH-12 h group,SAH-24 h group,SAH-48 h group,SAH-72 h group.A rat SAH model was induced by injection of 0.3ml fresh,non-heparinized autologous arterial blood into prechiasmatic cistern in about 20 s.All Sprague-Dawley(SD)rats were killed at matching time point.The brain cortex of six rats in each group was taken for western blot analysis and immunofluorescence analysis respectively,to investigate the expression of TRAF6.Results:western blot analysis indicate that the expression level of TRAF6 increased gradually first after SAH,then decreased again,and peaked at 24 h(P<0.05),immunofluorescence analysisget the same result.Conclusion:The expression level of TRAF6 increased gradually after SAH,and peaked at 24 h,indicating that TRAF6 may have some link to EBI after SAH,and 24 h after SAH could serve as a time point for further experiments.Part II: The role and potential mechanism of TRAF6 on early brain injury after SAH in ratsObjective:through TRAF6 overexpression plasmid,small RNA interference and site-directed mutagenesis studies,we nextsearch the role and potential mechanism of TRAF6 on early brain injury after SAH in rats.Methods:Experiment 1: 108 Sprague-Dawley(SD)rats were randomly divided into 6 groups of 18 rats each: sham group,SAH group,SAH + vehicle group,SAH + TRAF6 overexpression group,SAH + control si RNA group,SAH + si RNA group.The brain cortex of six rats in each group wastaken for western blot analysis to test the expression level of TRAF6,LC3 II/LC3 I and albumin,and for assay of ROS indicators.Another six rats were killed for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining and FIB staining,to evaluate the neuro apoptosis and necrosis.The last six rats were used for brain edema and assess.Experiment 2: 108 Sprague-Dawley(SD)rats were randomly divided into 6 groups of 18 rats each: SAH + vehicle group,SAH + GFP-TRAF6-wt group,SAH + GFP-TRAF6-C70 A group,SAH + TRAF6 overexpression group,SAH + TRAF6 overexpression + MG132 group,SAH + CLI-095 group.The brain cortex of six rats in each group wastaken for western blot analysis to test the expression level of GFP-TRAF6-ub?ULK1-ub/ULK1-total?ECSIT-ub/mitochondria-pro?LC3 II/LC3 I and albumin,and for assay of ROS indicators.Another six rats were killed for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining and FIB staining,to evaluate the neuro apoptosis and necrosis.The last six rats were used for brain edema and assess.Results:experiment 1:TRAF6 overexpression plasmid and si RNA can separately increase and decrease the expression level of TRAF6 after SAH(P<0.05,P<0.01);separately decrease and increase autophagy activity after SAH(P<0.05,P<0.05);separately increase and decrease the number of neuronal apoptosis(P<0.05,P<0.05)and cell necrosis(P<0.01,P<0.01);separately increase and decreasethelevel of ROS(P<0.05,P<0.05),brain water content(P<0.01,P<0.05)and albuminlevel(P<0.01,P<0.05).Experiment 2:Compared with GFP-TRAF6-wt group,the ubiquitin level of TRAF6,ULK1 and ECSIT in GFP-TRAF6-C70 A group not increased(P<0.05);autophagy activity increased clearly(P<0.05);the number of neuronal apoptosis and cell necrosis not increased(P<0.05);the level of ROS,brain water content and albumin level not increased as well(P<0.05).In addition,TRAF6 overexpression lead to the rise of ULK1 expression level(P<0.01),the use of MG132 can increase the expression level of ULK1(P<0.05).TLR4 inhibitor can decrease the ubiquitin level of TRAF6(P<0.01).Conclusion:TRAF6 can exacerbate EBI after SAH by inhibiting autophagy and promoting production of ROS,including the increase of neuro apoptosis/necrosis number,the aggravation of brain edema and blood brain barrier(BBB)damage.The role of TRAF6 in EBI after SAH is determined mainly by its E3 ubiquitin ligase activity.TLR4 is the upstream molecule of TRAF6,and its activity can directly affect expression and ubiquitination level of TRAF6,thus affecting the degree of EBI.