The Role And Mechanism Of SIRT1 On Early Brain Iniury In A Rat Experimental Subarachnoid Hemorrhage Model

BackgroundSubarachnoid hemorrhage (SAH) is a lethal disease,30% patients die within the first few days,10% die in the following days according to various complications and the overall mortality rate is more than 50%. SAH consists of 5-7% of total strokes and affects 10 per 100,000 adults each year. Most deaths occur in the early phase of initial bleeding resulting from the increased ICP or acute hydrocephalus.One main issue contributes to the high mortality and morbidity, which is early brain injury (EBI). EBI is defined as brain injury after SAH attack and before 72 hours, and EBI is a very complicated pathophysiological process and mainly related with increased intracranial pressure (ICP), decreased cerebral blood flow (CBF), decreased cerebral perfusion pressure, disruption of the blood-brain barrier and brain edema. Many evidences show that apoptosis is a main factor in the pathogenesis of EBI in experimental SAH. Apoptosis of neurons and endothelial cells are reported in SAH models, both of them may be related to brain edema.Generally tumor suppressor p53 participates in apoptosis regulation, and p53 is the first non-histone protein found to be acetylated. When C-terminal lysine of p53 are acetylated, p53 can transactivate its transcriptional targets, in many pathological conditions p53 up-regulates proapoptotic molecules such as Bax, Puma, Noxa, Bid. In addition acetylation can compete to modify the same lysine residues at which other modification can occur, such as ubiquitination and methylation. Previous experiments show that p53 acetylation is partly dependent on deacetylation, while deacetylation is controlled by deacetylases called HDAC family.Sirtuins belong to class Ⅲ HDAC family and are capable of deacetylating histone or non-histone proteins. SIRT1 is an important member of this family and plays important roles in many physiological or pathological processes. Beside the ability of deacetylating H1, H3 and H4, SIRT1 is also discovered to deacetylate p53. Theoretically SIRT1 is endowed with the ability to regulate cell cycle arrest, apoptosis, and tumor suppression through the modification of p53. In previous studies SIRT1 protected the brain or heart in ischemic models through p53 deacetylation.Resveratrol(RES) is a natural compound and exists in many plants such as skin of grape, blueberries etc, nowadays it is found to extend lifespan. And Sirtinol (SIR) is SIRT1 inhibitor and used to inhibit SIRT1 in many researches. In our study we focus on the effect of resveratrol after S AH, and we hope to find an underlying target which can reverse apoptosis after SAH.MethodsPart 1SAH model was performed using endovascular perforation technique. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH 6h group, SAH 24h group, and SAH 48h group. Neurological deficit scores and brain edema were evaluated. The levels of Proteins SIRT1, apoptotic proteins (p53 and caspase3) were evaluated by western blot analysis. Immunofluorescence staining is performed for neuronal apoptosis related cells.Part 21. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group, SAH+RES group. Neurological deficit scores and brain edema were evaluated.2. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH group, SAH+SOL group, SAH+RES, SAH+RES+DMSO and SAH+RES+SIR group. Neurological deficit scores, brain edema and level of Evans blue were evaluated. The levels of Proteins (ZO-1, Occludin, Claudin5, SIRT1, p53, acetylated p53 and cleaved caspase3) were evaluated by western blot analysis. Double labeled immunofluorescence staining with NeuN and TUNEL was performed for neuronal apoptosis related cells detection. Real-time PCR was used to detect mRNA level of downstream targets of p53.ResultPart1After SAH the water content of brain increased and reached the top at 24 hours after SAH, neurological function decreased and changed to be the worst at 24 hours after SAH. Western blot showed that SIRT1 reached the lowest point at 24 hours after SAH, and the level of p53, ratio of acetylated p53 reached the top at 24 hours after SAH. Immunofluorescence staining showed that TUNEL positive cells increased significantly at 24 hours after SAH and TUNEL and NeuN double-staining demonstrated that neuronal apoptosis was located.Part 21. Based on water content and neurological function evaluation, RES pretreatment reversed the climbing of water content at bilateral hemisphere and decline of neurological function after SAH.2.24 hours after SAH RES pretreatment significantly reduced the amount of brain water content and Evans blue and improved neurological defects, but SIR blocked this effect. By western blot analysis RES pretreatment significantly increased the level of tight junction proteins including ZO-1, Occludin, Claudin5, which decreased after SAH and was closely related with BBB disruption, SIR reversed this effects of RES. RES pretreatment increased the level of SIRT1 and reduced the level of p53 and ratio of acetylated p53 and their apoptotic protein cleaved caspase3, SIR reversed the effect of RES again. TUNEL and NeuN double labeled immunofluorescent staining demonstrated that RES obviously reduced the number of apoptotic neuronal cells, and SIR inhibited this protective effect. By real-time PCR evaluation mRNA of Bax, PUMA, Noxa and Bid, all of them increased after SAH at 24 hours, Bax was reduced by RES pretreatment statistically, but SIR reversed this results.ConclusionPart1Brain edema, level of p53, acetylated p53 and cleaved caspase3 reached top at 24 hours after SAH, at the same time SIRT1 reached at lowest point and neuronal apoptosis was most obviously at 24 hours. These results implied that SIRT1 may take part in EBI related neuronal apoptosis regulation after SAH.Part 2RES pretreatment improved neurological defects, brain edema after SAH through tight junction proteins up-regulation, and meanwhile by increasing SIRT1, reducing p53, acetylating p53 and increasing cleaved caspase3 RES pretreatment protected neuronal cells. SIRT1 inhibitor SIR reversed the effect of RES. Based on these results SIRT1 may play an important role of neuronal protection in EBI after SAH.