Study On The Mechanism Of MAP4K4 Induced Rapid Blood-Brain Barrier Damage After Subarachnoid Hemorrhage In Mice

Objectives1.Clear MAP4K4 early blood-brain barrier damage after subarachnoid hemorrhage;2.Discuss MAP4K4 induced rapid blood brain barrier damage after subarachnoid hemorrhage possible mechanismMethods1.Protein expression differences of MAP4K4 in time course and cellular location of MAP4K4 after SAH was evaluated.48 mice were randomly assigned into 7 groups(n=6): Sham,SAH 3 hours,SAH 6 hours,SAH 12 hours,SAH 24 hours,SAH 48 hours and SAH 72 hours groups.MAP4K4 expression was detected by Western blotting(WB)in cortex isolated from the ipsilateral/left hemisphere.Immunostaining of MAP4K4 and Lectin(endotheliocyte marker)was performed at 24 hours to confirm the expression of MAP4K4 on endothelial cells in the cortex(n=3).2.To evaluated the intrinsic function of MAP4K4 and screen for effective dosage of MAP4K4 recombinant protein,159 adult C57 B6 J mice were randomly assigned into 8 groups: Sham(n=25),SAH(n=12),SAH+NS(2 μL normal saline,n=19),SAH+MAP4K4 10 ng/μL(n=12),SAH+MAP4K4 50ng/μL(n=19),SAH+MAP4K4 200 ng/μL(n=12),SAH+Scr(scramble)si RNA(n=25),SAH+MAP4K4 si RNA groups(n=25).Neurological impairment were evaluated via Modified Garcia Score at 24 and 72 hours as well as Beam balance test(n=6).Furthermore,brain water content was performed at 24 hours and 72 hours(n=6),Evans blue permeability assessment(n=6)was performed 24 hours post SAH to evaluate the blood-brain barrier injury(n=6).Immunostaining was also utilized at 24 h after SAH induction to detect the endothelial continuity in the cortex(n=3).3.Furthermore,to investigate pathways of BBB damage,60 adult C57B6 J mice were assigned into 5 groups(n=12): Sham,SAH+NS,SAH+MAP4K4 50 ng/μL,SAH+Scr si RNA,SAH+MAP4K4 si RNA groups.Western blot was performed to detect the phosphate-NF-κB(p-p65),zonula occludens-1(ZO-1)and claudin 5 expression(n=6)and zymography(n=6)was used to detect MMP9 levelin ipsilateral/left hemisphere of each group.4.To investigate effect of PF-06260933(PF),an inhibitor of MAP4K4,and screen for optimal dose,135 adult C57B6 J mice were used.Sham(n=31),SAH(n=12),SAH+vehicle(double-distilled water,n=31),SAH+PF-06260933 1mg/kg(n=12),SAH+ PF-06260933 10 mg/kg(n=31),SAH+ PF-06260933 100 mg/kg groups(n=12).1 mg/kg,10 mg/kg or 100 mg/kg PF(Merc,Darmstadt,Germany)were orally delivery at 15 min after SAH.Neurological impairment was assessed by Modified Garcia Score and Beam balance at 24 and 72 hours after SAH(n=6).Brain water content and Evans’ blue extravasation was carried out to analyze BBB injury.Tight junction protein degradation was evaluated by immunohistology(n=3)and Western blot(n=6)was performed to detect the p-p65,ZO-1 and claudin 5 expression.Gelatin zymography was to investigate the protein level of MMP9(n=6)..Results1.A significant increasing of MAP4K4 were found in the ipsilateral/left hemisphere at 12,24,48 and 72 hours,and elevated at 24 h 48 h and 72 h after SAH.Furthermore,immunohistochemical staining at sham group showed that MAP4K4 expression was colocalized with Lectin(endothelial marker)in left/ipsilateral cortex.At 24 h post SAH,MAP4K4 was elevated in endothelial cells obviously.2.The MAP4K4 si RNA could effectively knockdown the MAP4K4 expression after SAH.Compared with the SAH + MAP4K4 group and the SAH + MAP4K4 group,the SAH + MAP4K4 group and the SAH + MAP4K4 group,the SAH + MAP4K4 group and the SAH + NS group had more severe neurological function score reduction,significant increase of brain edema and aggravation of Evans blue extravasation.Comp ared with SAH+Scr group,MAP4K4 si RNA group had significantly improved neurological dysfunction at 24 hours,significantly reduced brain edema,and significantly reduced Evans blue leakage.3.24 hours after SAH,via MAP4K4 recombinant protein injection and MAP4K4 si RNA after pretreatment,the endothelial cells of the ipsilateral cortex and tight junction protein 5(5)Claudin marker staining showed endothelial cells and is closely connected to the integrity of the protein damage,at the same time MAP4K4 the use of recombinant protein increased endothelial cells and tight junction protein discontinuity,and MAP4K4 the continuity of si RNA group improved significantly.4.WB and gelatin,according to the results of enzyme spectrum MAP4K4 recombinant protein increased the level of P-p65 and MMP9 and ZO-1 and Claudin 5 levels,at the same time MAP4K4 si RNA group reversed the trend.5.PF-06260933,a highly selective small molecule inhibitor of MAP4K4,was employed after SAH,10 mg/kg and 100 mg/kg PF significantly improved nerve function and decreased brain water content,while 1mg/kg had no significant effect.6.WB and zymography showed that the expression of p-p65 and MMP9 decreased after PF administration,while the expression of zo-1 and Claudin 5 increased.ConclusionThe present study demonstrated that MAP4K4 increased after SAH,increasing at 24 h,48 h and 72 h,elevated in endothelial cells.Exogenous MAP4K4 protein worsen neurological deficits and brain edema at concentration of 50 ng/μL and 200 ng/μL,while MAP4K4 silencing efficiently reserved those trends.Administration of MAP4K4 inducing BBB disruption by broken up the continuous ECs and tight junction.Further mechanism study indicated that MAP4K4 enhancing p-p65 nuclear translocation and activating downstream MMP9,which as a main factor for degradation tight junction proteins.Taken together MAP4K4 may be a potential target to reduce BBB degradation after SAH...