The Effect And Molecular Mechanism Of SIRT3 For Early Brain Injury Following Subarachnoid Hemorrhage In Rat Model

Background and objectiveAneurysmal subarachnoid hemorrhage(SAH) is a common and frequently devastating disease which accounting for 5% of all types of stroke. It is reported that approximately 1/10000 people around the world suffer from an aneurysmal SAH per year which resulting in a greater than 50% combined morbidity with mortality rate. Despite advances in medical equipment and surgical treatment for SAH, therapeutic interventions are still poor and clinical outcomes remain disappointing. Traditionally, delayed cerebral vasospasm(CVS) has been taken into account the most important factor for poor outcomes; however anti-vasospasm agent has not brought tremendous improvement in the human patient outcome after SAH.Recently early brain injury(EBI) was emphasized as a critical etiology for poor outcome in SAH patients; it is generally defined as the global cerebral injury occurred within 72 hours after aneurysm rupture. It is considered that many important pathological mechanisms are closely involved with the poor prognosis, such as oxidative stress, inflammation apoptosis and so on. Since the integrated mechanisms of action have yet to be elucidated, breakthrough in this field is expected to provide an important clinical strategy for patients suffered from SAH to improve survival and prognosis.SIRT3, belonging to the highly conserved family of Sirtuin proteins, is a deacetylase depended on Nicotinamide adenine dinucleotide and expressed abundantly in brain tissue. As a pivotal regulator for energy homeostasis, SIRT3 has been shown to regulate the energy metabolism of mitochondria, substrate oxidation, inflammation chemotaxis and cellular apoptosis by posttranslational modification for mitochondrial proteins deacetylation, and play the important role in the pathological processes of a lot of diseases, especially in oxidative damage, inflammation and ischemic damage. Although recently, remarkable strides concerning SIRT3 have been made in the fields of cardiac, hepatic and renal diseases, few studies has been mentioned in the field of hemorrhagic stroke. Since it has been confirmed that the mechanisms of free radical injury and inflammation played the crucial role in the pathological process of EBI, we speculate reasonably that SIRT3 is associated with development of EBI after SAH, show neuroprotection by regulating the function of mitochondria against inflammation and free radical injury.Research methodsThis dissertation was divided into three parts. In the first part, experimental SAH rat model was established by internal carotid perforation using filament, pretreated with Resveratrol, a activator of SIRT3, then the quality control of animal model and neuroprotection of SIRT3 activation against EBI were evaluated by neurological score, brain water content, blood brain barrier and neuron apoptosis of cortex. Further studies about transcriptional and translational expression of SIRT3 were evaluated by RT-PCR, Western blot and immunohistochemistry in the second part, for purpose of specific cellular distribution detection, double labeled immunofluorescence was adopted. In the last part, we screened the optimal condition for PC12 cell injury induced by Co Cl2 to simulate the environment of neuron injury in vitro, regulated the SIRT3 expression by use of specific activator and inhibitor, and then evaluated impact of SIRT3 by western blot method on cellular signaling pathway, such as TLR4, PI3k/AKT and Nrf2/ARE. It was supposed to define the molecular mechanism of SIRT3 neuroprotection in EBI following with SAH.1、Experiment in vivo:1)Experimental SAH rat model was established by internal carotid perforation;2)All the experimental animals were divided into four groups: control group, sham group, SAH group and Resveratrol pretreatment group;3)Quality control of animal model and neuroprotection of SIRT3 activation against EBI were evaluated by neurological score, brain water content, blood brain barrier and neuron apoptosis of cortex;4)Transcriptional and translational expression of SIRT3 were evaluated by RT-PCR, Western blot and immunohistochemistry, for purpose of specific cellular distribution detection, double labeled immunofluorescence was adopted.2、Experiment in vitro:1)The optimal condition for PC12 cell injury induced by Co Cl2 was screened to simulate the environment of neuron injury in vitro;2)Under the condition mentioned above, the impact of SIRT3 on cellular signaling pathway, such as TLR4, PI3k/AKT and Nrf2/ARE was evaluated by western blot.Experimental results1、 Experimental SAH rat model was established successfully by internal carotid perforation, the model quality was satisfying;2、 Pretreatment with resveratrol, a specific activator of SIRT3, was shown the explicit neuroprotection for experimental SAH animals against early brain injury.3、The expression of SIRT3 in cortex of experimental rat decreased significantly, further detection about SIRT3 expression in both transcription and translation process by RT-PCR and Western blot showed reduced obviously;4、By technology of double labeled immunofluorescence, it was confirmed that SIRT3 was mainly located in cytoplasm of neurons rather than astrocytes, and dropped remarkably at the early stage after experimental SAH;5、The optimal condition for PC12 cell injury induced by Co Cl2 was screened successfully to simulate the environment of neuron injury in vitro;6、Up-regulated SIRT3 might play the pivotal neuroprotective role via activating the Nrf2/ARE signaling pathway, and show uncorrelated with TLR4 and PI3k/AKT signaling pathway.Conclusions1、SIRT3 activation play the neuroprotective role against EBI in experimental SAH rats, which contributing to alleviate brain edema and damage of blood brain barrier, improve the neurological score;2、The expression of SIRT3 in cortex of experimental rat decreased significantly both in transcription and translation process;3、SIRT3 mainly locates in the cytoplasm of neurons rather than in that of astrocytes, in the early stage after SAH, the expression of SIRT3 in neurons dropped dramatically.4、SIRT3 might play the pivotal neuroprotective role via upregulating the Nrf2/ARE signaling pathway, and show uncorrelated with TLR4 and PI3k/AKT signaling pathway.