The Role Of Silent Information Regulator 1(SIRT 1) In Second Brain Injury After Experimental Intracerebral Hemorrhage

Intracerebral hemorrhage(ICH)accounts for 10-15%of all strokes and is associated with high mortality and morbidity.Up to date,no effective treatment is available to improve clinical outcomes in patients with ICH.Rupture of blood vessels within the brain parenchyma cause primary injury and secondary injury.The primary injury is induced by the hematoma expansion associated with mass effect and the secondary injury contained many parallel pathways including cytoxicity of blood compnents,oxidative stress,inflammation and so on together lead to irreversible injury,eventually cause the disability or even the death.In recently,more evidences suggest that inflammatory play an key role in secondary injury after ICH.Nuclear factor-kappa B(NF-κB)plays a key role in inflammatory response.Without stimulation,NF-κB is retained in the cytoplasm through binding to the inhibitor of NF-κB(IκB)protein.Various stimuli induce the IkB kinase(IKK)to phosphorylate IkB,resulting in their degradation,which permits nuclear translocation of NF-κB subunits and activation of NF-κB target genes.The NF-κB has been confirmed to be actived after ICH and has been suggested to contribute to inflammation and second brain injury after ICH.In recent years?more evidences indicate that the reversible acetylation of RelA/p65 subunit can also modulate NF-κB signaling,which depending on the acetylation specific sites of lysine residues.In particular,acetylation of lysine 310 of p65 is required for full transcriptional activity of p65 and for activation of NF-κB.Interestingly,Silent information regulator 1(SIRT1),a highly conserved NAD+-dependent protein deacetylase which is involved in kinds of cellular processes,may play a role in negative regulation of inflammation by directly deacetylating the p65 protein at lysine 310.What’s more,SIRT1 have nuroprotective effects in kinds of brain injury.However,little is known about the role of SIRT1 in the brain injury after ICH.We supposed that the SIRT1 protects the brain against ICH and suppresses the inflammation though deacetylation of lysine 310 of p65.Part 1Object:we aimed to determine the expression and cell distribution of SIRT1 surrounding the hematoma after ICH.Methods and Results:Male ICR mice were randomly divided into sham group and ICH groups at 8 h and on day 1,day 2,day,3 day,5 day and 7 day(n=6 for each subgroup).ICH model was induced by intracerebral injection of collagenase IV into the striatum.SIRTI expression was measured by Western blot,,immunohistochemistry and immunofluorescence.Acetylated p65(lys 310)protein,the major subunit of NF-κB,was also detected.Our data demonstrated that the expression levels of SIRT1 was markedly decreased,while acetylated p65(lys 310)protein sgnificantly increased after ICH.Moreover,there was a significant negative correlation between the expression of SIRT1 and that of acetylated p65 protein.Double immunofluorescence staining showed that SIRT1 was expressed by neuron rather than astrocyte and microglia after ICH.In addition,a hemoglobin-(Hb-)induced primary neurons ICH model was also used to detect the expression of SIRT1 and found that SIRT1 expression was dramatically decreased in a time-dependent menner.Conclusion:The SIRT1 expression was reduced in neurons after ICH and SITR1 may be involved in the brain injury and inflammatory response after ICH.Part 2Object:The accurate role of SITR1 in ICH has not been studied.So in this part,resveratrol(a potent activator of SIRT1)and EX527(a specific inhibitor of SIRT1)were used to to investigate the role of SIRT1 in the brain injury and inflammatory response after ICHMethods and Results:ICH model was induced by intracerebral injection of collagenase Ⅳ into the striatum.Mice received vehicle or resveratrol at 30 min after ICH.Neurological deficits were assessed on post-operative Days 1-3 and brain water content were evaluated at 72 h after ICH.Western blot and immunofluorescence was performed to quantify acetylation of nuclear factor κB(p65).TNF-α and IL-1β were measured by enzyme-linked immunosorbent assay(ELISA).Neuronal cell death was evaluated with terminal deoxynucleotidyl transferase-mediated uridine 5’-triphosphate-biotin nick end-labeling staining(TUNEL).Additionally,EX527 was administered to manipulate the proposed pathway.Resveratrol(60 mg/kg)improved neurological deficits and reduced brain water content and Neuronal cell death after ICH.Resveratrol decreasing the expression of acetylated p65(lys 310)protein and proinflammation cytokines(IL-1β and TNF-α).EX527 reversed the effects of treatment.Conclusion:These results suggested that SIRT1 contributes to nuroprotective effects by de-acetylation of lysine 310 of p65 and inhibition NF-κB-denpend inflammation after ICH.SIRT1’ activator,resveratrol,could attenuate the brain injury after ICH.Part 3Object:Fisetin is a flavonoid and has multiple biological effects,including neuroprotective and antiinflammatory properties.Evidences also indicate fisetin activate SIRT1 and extend lifespan in lower organisms,which is similar with resveratrol.Howere,if fisetin can modulate the SITR1/NF-κB pathway and protect brain injury after ICH is unknown.Therefore,In this part,we aim to investigat the effects of fisetin on brain injury and SITR1/NF-κB pathway after ICH.Methods and Results:ICH model was induced by intracerebral injection of collagenase Ⅳ into the striatum.Fisetin(25mg/kg or 50mg/kg)were administrated 1 hour after injury.Fistin could alleviate neurological function and decrease brain edema 72h after ICH.The fisetin-induced upregulation of SIRT1 was also associated with an decrease in acetylated p65(lys 310)protein and proinflammation(IL-1β and TNF-a).However,EX527 could blocke the effects of fistin on deacetylation of NF-κB through up-regulation of SIRT1.Conclusion:These results suggest the fisetin could ameliorate ICH-induces brain injury and deacetylation of NF-κB through up-regulation of SIRT1.In conclusion,our word indicated that SIRT1 was down-regulated after ICH and activation of SIRT1 by resveratrol or fisetin can ameliorate ICH-induces brain injury though deacetylation of lysine 310 of p65,which inhibiting NF-κB-denpend inflammation.These reuslts highlight that SIRT1 may be a potential target for treatment ICH and esveratrol and fisetin have therapeutic value for ICH.