Erythrocyte Lysis And Clearance After Intracerebral Hemorrhage And The Role Of RNF146 In Glutamate Excitotoxicity

Intracerebral hemorrhage(ICH)is a common neurologic event,which has a high morbidity or mortality in the aged population.The incidence of ICH is almost 10%-15% every year and increased along with the grown population.In the past several decades,there is no significant declined trend in the morbidity or mortality,which the 30 days' mortality maintained in 30%-50%,and only 20% patients could regain their ability after 6 months.Plenty of erythrocytes enter the brain parenchyma and release the hemoglobin after lysed by an unclear mechanism.The metabolism products released from erythrocytes,including iron,biliverdin and carbon monoxide,which induced cascade molecular events such as glutamate release or oxidative stress after ICH.These events contribute to the brain edema,blood-brain barrier damage and neurologic degeneration.The series of reactions after ICH are the key pathogenic factors leading to further hemisphere injury,especially the elevation of calcium or oxidative stress response due to glutamate releasing.RNF146,a ubiquitination E3 ligase,is believed as the neuroprotection factor after brain ischemic diseases.However,the underlying protective mechanism of erythrocytes lysis and the role of RNF146 in glutamate-induced injury is still not fully understood.Efficient decreasing the metabolism products after ICH may largely attenuate the damage of central nervous system and induced excess glutamate releasing.Besides,RNF146 treatment may reduce the following injury caused by downstream cascade events.Our study showed that(1)Erythrocytes lysis occurred at 4 hours in ICH.(2)Hemoglobin degeneration products,especially iron could increase the level of CD163 and haptoglobin.(3)glutamate excitotoxicity mediated the autophagy in neuronal cells.(4)RNF146 involved in the protection mechanism of glutamate-induced autophagy.This study is divide into three parts:Part1 : Role of acute erythrocytes lysis and its mechanism in intracerebral hemorrhageBackground.Blood is leaked from rupture vascular,then enter the brain tissue,which caused severe damage.Numerous evidence indicated blood cells are the major factors in neurologic damage.It is generally believed that erythrocytes lysis occurred at 1days after ICH,which released many toxic products mediated brain edema and neuron death.Thus,there is a connection between erythrocytes lysis and brain damage.Heparin is an anticoagulant that prevents the formation of blood clots.However,the methods of lysis degree and the role of heparin in erythrocytes lysis are still not fully understood.Methods.There are three parts in our study.Firstly,we separated SD rats into two groups,100?l saline or 100?l autologous blood injection.After 4 hours,the rats received MRI scanning with the sequences of T2,T2*.We measured the brain edema percentage and erythrocytes lysis percentage depend on the MRI images and HE-staining.Secondly,we applied lysed blood cells and packed blood cells injection model to detect the erythrocyte lysis degree or iron content in T2,T2* and R2* mapping.In addition,we also detected blood-brain barrier by western-blot or immunohistochemistry.HE and Fluoro-jade C staining were also applied in this part.At last,we added 5-unit heparin in autologous blood to detect the role of heparin in erythrocytes lysis.The same MRI sequences were used in this part.We also proceeded the HE and Fluoro-Jade C staining.Results.We proved that brain midline shift occurred in 4 hours after ICH.Erythrocytes already lysed in 4 hours after ICH.Besides,we found that the high signal area in lesion indicates the erythrocytes lysis area.In lysed blood injection group,the brain edema was more severe than packed blood injection group as well as the blood-brain barrier damage.Finally,our results showed that heparin reduced the early stage erythrocytes lysis,and brain edema and neuronal cell degeneration.Conclusion.Erythrocytes lysed in the early stage after ICH,which induced severe blood-brain barrier damage and brain edema.Heparin could reduce the toxic effects from early erythrocyte lysis and attenuate the neuronal cell degeneration.Part2:Role of CD163 and Haptoglobin in hemoglobin clearance after intracerebral hemorrhageBackground.Intracerebral hemorrhage(ICH)is principally a lethal disease of the elderly population.Plenty of erythrocytes are released into the extracellular milieu in the brain after ICH occurred.Hemoglobin(Hb)derived from Erythrocyte lysis is believed as the original factor leading to brain injury after ICH.The degradation products of Hb,heme and iron,trigger severe oxidative and excitotoxicity effects eventually leading to brain damage.The toxicity of Hb is associated with the formation of oxygen radicals and the scavenging of nitric oxide(NO).Iron overload also contributes to brain edema and brain atrophy following ICH.Previous study illustrated that Hb also appeared in neurons and glia cells after ICH.However,the protection mechanism by which Hb degenerated is still not fully comprehended.CD163 is identified as a member of scavenger receptor cysteine-rich(SRCR)superfamily class B.It plays a crucial character in Hb clearance by mediating the endocytic process of Hemoglobin-Haptoglobin complex.CD163 is also recognized as a marker of anti-inflammation macrophages and regulates activation of the heme oxygenase-1(HO-1)in macrophages.It functions as an anti-inflammatory molecule involved with resolution of inflammation since pro-inflammatory factors restrain its expression.Besides,CD163 positive macrophages are commonly discovered in areas of regeneration tissues after ischemic injury.Methods.The present study is divided into four parts.First,autologous injected ICH model was applied to examine CD163 and haptoglobin expression pattern at day 1,day 3 and day 7.Secondly,SD rats were injected with ferrous chloride to examine iron overload in ICH and sacrificed at day 1 for histology analysis.Thirdly,Minocycline was used to antagonist iron toxicity on cerebral and then detected the CD163 and haptoglobin expression.Finally,we performed primary microglia culture to detect the effect of CD163 effects on hemoglobin clearance.Results.Intracerebral injection of autologous blood induced CD163 and haptoglobin upregulation both in cortex and basal ganglia peaked at day 7.CD163 positive cells in ipsilateral cortex area were largely expressed in neuron,partly emerged in microglial and astrocyte.CD163 co-localized with microglia cells in basal ganglia.Injection of iron in SD rats similarly induced CD163 and Haptoglobin expression in ipsilateral hemisphere at day 1.Furthermore,administration with minocycline eventually decreased CD163 and Haptoglobin expression.CD163 blocking antibody decreased the microglia-phagocytic hemoglobin.Conclusion.We detected that CD163 and haptoglobin emerged at day 1 and peaked at day 7 after ICH.Iron increased the CD163 and haptoglobin expression and Minocycline inverted this effect.CD163 antibody blocked the effects of microglia phagocytosed hemoglobin.Part3:Role of RNF146 in the glutamate excitotoxicity induced autophagy and related mechanisms.Background.Glutamate is an endogenous excitatory neurotransmitter in the central nervous system.Glutamate toxicity is a chief factor which contributes to neuronal cell death in stroke,traumatic brain injury(TBI),and neurodegenerative diseases,such as Parkinson's and Alzheimer's Disease.Autophagy is the major catabolic pathway that is involved in the degradation of damaged or impaired organelles,abnormal protein,and other cytosolic components.It is also an important cellular activity following glutamate-induced excitotoxicity.RNF146,which is also called Iduna,is a Poly(ADP-ribose)(PAR)-dependent E3 ligase and a central regulatory molecule in PAR-polymerase-1(PARP-1)-dependent cell death.RNF146 is a positive regulator of Wnt signaling via mediating the tankyrase-dependent degradation of axin.However,the involvement of this pathway in cell death caused by glutamate-induced excitotoxicity has not been reported.The role of RNF146 in glutamate-induced excitotoxicity and autophagy and its crosstalk with downstream signaling are still unclear.Methods.We aimed to investigate the role of RNF146 in modulating autophagy in HT22 cells under glutamate excitotoxicity injury.First,to identify the effect of RNF146 on glutamate-induced excitotoxicity,HT22 cells were transfected with LV-RNF146 or LV-negative control for 4 h and applied to MTT assay or LDH cytotoxicity assay.After media replacement and 48 h further incubation,cells were treated with glutamate for 24 h.Secondly,to determine the role of autophagy in glutamate excitotoxic injury,we assessed the extent of cell injury after HT22 cells undergoing glutamate and pharmacological agents by which regulated autophagy.Moreover,we investigate the relationship between RNF146 and Wnt signaling in excitotoxic injury.HT22 cells were treated with LV-RNF146 for 24 h and then cultured 2 d before other treatments.To further elucidate the targets of RNF146 in Wnt/?-catenin signaling,we used inhibitors to interfere the different processes of Wnt/?-catenin signaling transduction.To identify the mechanism by which RNF146 modulates autophagy,we first applied Foxy5,a Wnt5 a peptide mimetic,and antagonist IWP2 to examine their function in autophagy.Finally,to examine whether the Wnt/?-catenin pathway is involved in the negative regulation of autophagy by RNF146,we used the Wnt/?-catenin signaling inhibitor JW74.Results.Here we found that induction of RNF146 decreased the cellular damage and excitotoxicity induced by glutamate.RNF146 also suppressed the excessive autophagy,which is detrimental to HT22 cells survival,induced by glutamate or rapamycin treatment.In addition,we find that Wnt/?-catenin was a negative regulation factor for autophagy in glutamate excitotoxicity.Over-expression of RNF146 promoted Wnt/?-catenin signaling,which was related to destabilization of ?-catenin destruction complex.Conclusion.These results indicated that RNF146 acted as a neuroprotective agent against glutamate-induced excitatory damage,and this neuroprotection might be at least partly dependent on the inhibition of excessive autophagy by regulating Wnt/?-catenin signaling.