Acrolein Aggravated The Secondary Brain Injury Following Intracerebral Hemorrhage Through Drp1 Mediated Mitochondrial Fission

BackgroundCerebral stroke is divided into two categories,namely ischemic stroke and hemorrhagic stroke.Among them,intracerebral hemorrhagic stroke has a higher mortality and disability rate.Clinical advances in intracerebral hemorrhage(ICH)treatment are restricted by the incomplete understanding of the molecular mechanisms contributing to the secondary brain injury(SBI).Acrolein(CH2=CH–CHO)is a highly active unsaturated aldehyde which is reported to be capable of inducing cellular oxidative injury,impairing the redox balance,and leading to cell apoptosis.Previous studies indicate that acrolein induced neurotoxicity plays an important role in many nervous system diseases,like spinal cord injury,Parkinson’s disease,ischemic stroke and so on.Objective(1)To study the change pattern of acrolein after ICH in mice.(2)To investigate the possible mechanism of acrolein induced brain injury(3)To verify the protective effect of acrolein scavenger on brain injury after ICH in mice.Methods(1)Healthy male C57 mice were randomly divided into sham group and 5 ICH subgroups according to different time points(12h,24 h,48h,72 h,7d post ICH).As for ICH group,0.075 u collagenase VII was injected into mouse striatum with stereotactic injection needle(coordinates: 0.20 mm anterior,2.30 mm right lateral,3.50 mm deep).The operations of sham group were the same except that it is the equal amount of normal saline was injected after skull drilling.At the corresponding time points after surgery,mice were sacrificed and randomly divided into western blot group and immunofluorescence group to detect the expression changes and spatial location of acrolein in the tissues around hematoma.(2)In in vitro experiments,mice primary neurons were cultured.The changes of mitochondrial morphology(via mitotracker staining)and function(ROS content,mitochondrial membrane potential level and cytochrome c release)were observed under the treatment of acrolein with different time gradients and concentration gradients.In terms of the mechanism,we explored the potential regulation of acrolein on mitochondrial fusion proteins Mfn1,Mfn2,OPA1 and mitochondrial fission protein Drp1.(3)Acrolein scavenger hydralazine(5mg/kg)was injected intraperitoneally after ICH surgery.And western blot,electron microscopy and immunofluorescence were used to detect the level of acrolein,the changes of mitochondrial morphology and function,the extent of neural apoptosis.And brain edema,hemorrhage volume and neurobehavioral function were also measured to assess the protective effects of acrolein scavenging on ICH induced brain injuries.Results(1)Western blot showed that the level of acrolein in the brain tissue around the hematoma increased significantly at 12 hours after ICH,reached the peak at 72 hours,and maintained a high level until to the 7th day.Double immunofluorescence staining of the brain slices of 72 h post-ICH mice predominantly revealed an abundant co-location of acrolein with neuron,while little co-labelled with astrocytes and microglia.(2)In in vitro experiments,the results indicated that compared with the vehicle group,acrolein treatment evidently increased mitochondrial fragmentation.At the same time,mitochondrial function was also damaged,which was evidenced by the increased ROS content,facilitated release of cytochrome C,and the decreased MMP.Moreover,The damage degree of acrolein to mitochondrial morphology and function increased in concentration(25 μ m;50 μ m;100 μ m)and time(6h;12h;24h)dependent manners.(3)In vitro experiments,we found that acrolein can promote the mitochondrial fission protein Drp1 to be recruited from the cytoplasm to the outer membrane of mitochondria,triggering excessive mitochondrial fission,while showing no remarkable regulation on mitochondrial fusion proteins OPA1,mfn1 and Mfn2.Additionally,the experimental results showed that mdivi-1(10 μ m,24 h),a selective inhibitor of Drp1,could significantly reduce the mitochondrial fragmentation caused by acrolein,reduce the production of ROS and the release of cytochrome c,and promote the recovery of mitochondrial membrane potential.(4)The results showed that acrolein stimulation significantly reduced the level of pAMPK and inhibited the AMPK pathway,while the level of p-ERK increased and the ERK pathway was activated.However,AICAR,the activator of AMPK pathway,significantly reversed the decrease of phosphorylation level at Ser637 site of Drp1 induced by acrolein.SCH772984,the inhibitor of ERK pathway,significantly inhibited the increase of Ser616 phosphorylation level induced by acrolein.These results indicated that acrolein can regulate the activity of Drp1 through AMPK and ERK pathways.(5)Hydralazine,an acrolein scavenger,inhibited the mitochondrial translocation of Drp1 and attenuated the morphological damage of mitochondria after ICH in mice.Western blot analysis showed that compared with sham group,the mitochondrial level of Drp1 in ICH group was significantly higher,while hydrazine could significantly inhibit the recruitment of Drp1 to mitochondria.Moreover,we observed that hydralazine could significantly reduce mitochondrial fragments,swelling,crista collapse and membrane rupture after ICH.(6)The results showed that both hydralazine and mdivi-1,an inhibitor of Drp1,could significantly reduce neuron apoptosis,brain edema and improve neurological function after ICH,although there was no significant effect on hematoma volume.ConclusionIt is the first time to find that acrolein may be a key pathogenic factor in secondary brain injury after cerebral hemorrhage,and in mechanism,reveal the critical role of Drp1 mediated mitochondrial oxidative damage in the neurotoxicity of acrolein.Moreover,we also verify the protective effect of acrolein scavenger,hydralazine,on mitochondrial damage and brain injuries after ICH in mice.Acrolein scavenging treatment may be promising to be clinically applied and to extensively benefit ICH suffers.