Role Of HSP27 In Brain Injury After SAH And TAT-HSP27 Improving Neurologic Deficits After Experimental SAH

Subarachnoid hemorrhage(SAH)is a subtype of stroke with high mortality and morbidity rate,which is mainly caused by the rupture of intracranial aneurysm.Although ruptured aneurysms are treated by clipping or coiling surgically,67%of SAH patients still have neurological sequelae due to early brain injury(EBI)and delayed brain injury.Neuronal apoptosis is the most important pathophysiological change underlying EBI.Therefore,neuroprotective agents targeting neuronal apoptosis have become an important strategy to against EBI after SAH.HSP27 is a member of the small HSPs family,has molecular chaperone activity,decreases protein aggregation and helps degradation by the proteasome,and also suppresses cell apoptosis.Previous studies demonstrated that overexpression of HSP27 provides neuroprotection in multiple neurological disease models,include cerebral ischemia,kainateinduced neuronal death and Alzheimer’s disease,which were mainly credited to its antiapoptotic effect.Under SAH pathology,the change of HSP27 expression and its phosphorylation in brainstem and cerebral vessels had been observed in SAH model,suggesting that HSP27 is associated with brain injury after SAH.However,is HSP27 related to the clinical process of SAH patients?Does HSP27 also play a neuroprotective role in SAH model?Does HSP27 protect EBI by regulating cell apoptosis?Is the targeted regulation of HSP27 a potential protective strategy to improve the prognosis of SAH?These problems remain to be answered and need systematic research.In this study,we measured the concentration of HSP27 in CSF from aSAH patients,analyzed the correlation between HSP27 expression and disease severity,investigated the effect of knockdown or overexpression of HSP27 on brain injury in rat SAH model.We screened the effective peptide from HSP27 on cell apoptosis in an in vitro hemolysatedamaged cortical neuron model,and explored the effect of screened short peptide on neurological deficit and cell apoptosis in rat SAH model.The research contents include:1.Research on HSP27 expression change in CSF of aSAH patientsAfter approval by the ethical committee of Shandong Provincial Hospital and patients,73 aSAH patients and 20 NPH patients were included.CSF was collected by lumbar puncture or extraventricular drainage,and the level of HSP27 in CSF was detected by ELISA.Statistical analysis showed that compared with NPH patients,the level of HSP27 in CSF of aSAH patients was significantly higher on day 1 following aSAH.HSP27 level of day 2 to 4 was gradually decreased in comparison to day 1 and then increased on day 5 to 7.Furthermore,there was a significant difference in HSP27 level(day 1)between aSAH patients with HH grade of Ⅰ-Ⅲ vs.Ⅳ-Ⅴ,WFNS grades of Ⅰ-Ⅲ vs.Ⅳ-Ⅴ,and Fisher scores of 1-2 vs.3-4.It suggests that HSP27 plays a role in the EBI of SAH.Therefore,the followup experiments of this study will explore the role of HSP27 within 72h after SAH.2.Research on HSP27 expression change in CSF and basal cortical neurons of SAH model ratsIn order to further explore the specific role of HSP27 within 72 h after SAH,we established the SAH rat model by single injection of autologous blood into the occipital cistern.Rats were randomly divided into 5 groups:sham group,6h after SAH,12h after SAH,24h after SAH and 72h after SAH groups.At the corresponding time points,CSF was collected from the occipital cisterna.The concentration of HSP27 in rat CSF after SAH was detected by ELISA.After perfusion,the rat brain was taken to make coronal sections,and the expression of HSP27 in the neurons of rat basal cortex after SAH was detected by immunofluorescence.ELISA showed that HSP27 level of CSF was obviously increased at 12 h in comparison to that in sham group and then declined significantly.HSP27 can be colocated with NeuN(a marker for neuron),whereas rarely co-located with Iba-1(a marker for macrophages/microglia).Moreover,HSP27 staining was significantly increased at 12 h compared with that of the sham group,whereas obviously declined at 72 h.It is suggested that the main site of where HSP27 exerts its protective effect in EBI of SAH may be in neurons.The expression of HSP27 was significantly increased in both aSAH patients and SAH model rats at the early stage of the disease,suggesting that HSP27 plays an important role in the early stage after SAH.3.Research on knockdown of endogenous HSP27 expression affecting neurological deficit after SAHIn order to further explore the specific role of endogenous HSP27,we constructed pAKD-CMV-EGFP-H1-shRNA-HSP27 vector and designed animal experiments.Rats were randomly divided into 4 groups:sham、SAH+Vehicle(injection of normal saline)、SAH+NC(injection of pAKD-CMV-EGFP-H1-shRNA-NC virus)、SAH+shRNA(injection of pAKDCMV-EGFP-H1-shRNA-HSP27 virus).The packaged virus was injected into the lateral ventricle of rats 19 days before SAH modeling.Neurobehavioral score,Immunofluorescence(coronal section),Western blot(basal cortex tissue)and TUNEL were performed 48h after SAH.Results showed that AAV-eGFP-shRNA effectively infected the basal cortex,produced considerable expression of eGFP,and significantly decreased expression of HSP27.Numerous TUNEL positive cortical cells,neurobehavioral scores and activation of caspase-3 significantly increased in SAH+shRNA group as compared with that of SAH+NC group on 48h after SAH.These results indicate that knockdown of endogenous HSP27 increases cell apoptosis and deteriorates neurological deficit in rat SAH.4.Research on overexpression of HSP27 affecting brain injury after SAHTo verify the protective effect of HSP27 in the early stage after SAH,the pAAV-CAGHSP27-3FLAG vector was constructed in this study to detect the effect of exogenous HSP27 overexpression on the early stage after SAH in rats.First,the packaged virus was injected into the lateral ventricles of normal rats,IL-1β and IL-6 in the cerebrospinal fluid of rats was detected by ELISA.The results showed that there was no significant change in the levels of two inflammatory factors between the virus injection group and control group.It suggested that the AAV virus did not cause neuroinflammatory reaction in rats.Then,we designed animal experiments.Rats were randomly divided into 4 groups:sham、SAH+Vehicle(Injection of normal saline)、SAH+Con(injection of pAAV-CAG-3FLAG virus)、SAH+shRNA(injection of pAAV-CAG-HSP27-3FLAG virus).The packaged virus was injected into the lateral ventricle of rats 19 days before SAH modeling.Neurobehavioral score,Immunofluorescence(coronal section),Western blot(basal cortex tissue)and TUNEL were performed 48h after SAH.Western blot confirmed the increased HSP27 protein expression by detecting FLAG expression,and immunofluorescence staining showed AAV-HSP27-3FLAG obviously increased HSP27 expression in the basal cortex.Exogenous overexpression of HSP27 decreased the neurobehavioral scores of SAH rats,significantly reduced the number of apoptotic cells in the basal cortex,and decreased the expression of p-MKK4,p-JNK,p-c-jun and active caspase-3.These results indicate that overexpression of HSP27 decreases cell apoptosis and ameliorated neurological deficit in rat SAH.5.Screening of HSP27 effective peptide for inhibiting cortical neuron apoptosis1-120 amino acids derived from N-terminal region of HSP27 was proved to be a necessary domain for neuroprotection.In order to find a more accurate effect peptide,we designed and synthesized peptides from the N-terminal 1-120 amino acids of HSP27,including HSP271-30、HSP2731-60、HSP2761-90、HSP2765-90、HSP2791-120.CCK8 analysis showed that all peptides have no significant toxicity to primary cortical neurons obtained from E18 rat embryos.Hemolysate in medium(1:50)was used to stimulate neuronal death.TUNEL and Western blot were used to detect the anti-apoptotic activity of five peptides.Results showed that HSP2761-90、HSP2765-90 peptides,but not HSP271-30、HSP2731-60 and HSP2791-120 peptides,effectively inhibited hemolysate-increased the activation of caspase-3 and reduced hemolysate-elevated the number of TUNEL positive cells.These results suggested that the N-terminal 65-90 amino acids of HSP27 are the key area for affecting cell apoptosis.6.Study on validation of TAT-HSP2765-90 in vivoAfter screening the effective peptide HSP2765-90,we designed the fusion peptide TATHSP2765-90 and verified its protective effect in vivo animal experiment.Rats were randomly divided into 4 groups:sham、SAH+Vehicle(injection of normal saline)、SAH+TAT(injection of TAT)、SAH+shRNA(injection of TAT-HSP2765-90).The lateral ventricle injection time of rats was 30min after SAH.Neurobehavioral score,Western blot(basal cortex tissue)and TUNEL were performed 48h after SAH.Results showed that TAT-HSP2765-90 significantly decreased the neurobehavioral scores of SAH rats,reduced the number of apoptotic cells in the basal cortex,and decreased the expression of active caspase-3.These results indicated that microinjection of TAT-HSP2765-90 into the lateral ventricle of rat SAH could decrease cell apoptosis and ameliorated neurological deficit in rat SAH.At the same time,TAT-HSP2765-90-FITC was synthesized and injected into the lateral ventricle of rats at 30 minutes after SAH,and then FITC signal was detected to colocalize with NeuN at 48h after SAH.In conclusion,the present study shows that the level of HSP27 in CSF of aSAH patients was significantly higher on day 1 following aSAH,and early CSF level of HSP27 is related to clinical and hematological severity in aSAH patients.HSP27 level in rat CSF and basal cortex neurons were obviously increased at 12 h after SAH and then declined significantly.Overexpression of HSP27,but not knockdown of HSP27,confers neuroprotection after SAH in rat.HSP2765-90 peptide can effectively inhibit neuronal death in an in vitro hemolysatedamaged cortical neuron apoptosis model,and TAT-HSP2765-90 attenuated neurological deficit after SAH in rat.The above findings suggest that HSP27 plays a role in EBI after SAH,which lays an experimental foundation for further study on the mechanism of HSP27.The screened mimic short peptide has potential clinical transformation value.