The Role And Mechanism Of Nuclear Receptor Nur77 During Early Brain Injury After Experimental Subarachnoid Hemorrhage In Rats

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disorder with very high morbidity and mortality rates. It predominantly attacks young and middle-aged group. SAH accounts for approximately 5-7% of all strokes despite many therapeutic advances in recent years. Early brain injury (EBI) and cerebral vasospasm are two major complications that often present in patients suffering from aneurysmal SAH. Previous research has primarily focused on cerebral vasospasm;however, poor patient prognosis, even when vasospasm is properly managed, has prompted researchers to investigate modulating EBI as an ameliorative therapy for SAH. In recent years, researchers regard EBI as one of the major causes of poor outcomes after SAH. So studies focused on how to alleviate EBI after SAH may bring optimistic consequences.EBI is a new concept put forth over these years. Significant pathophysiological changes during the EBI period post-SAH (within the first 72 hours of seizure)include: increased intracranial pressure (ICP), attenuation of cerebral blood flow(CBF),blood-brain barrier (BBB) disruption, changes in cerebral perfusion pressure(CPP), brain edema, brain swelling, and acute vasospasm. Complicated molecular mechanisms describing post-SAH EBI have been studied, including multiple apoptotic and inflammatory pathways and protein interactions, among which apoptosis of neurons, astrocytes and microglia cells have been widely demonstrated.Many studies have recently indicated that apoptosis may be essential to the pathogenesis of secondary brain injury after SAH. As a result, the apoptotic cascades present a number of potential therapeutic targets that may be manipulated to ameliorate secondary brain injury after SAH.Nur77 (also known as TR3 or NGFI-B) was originally isolated as an immediate-early response gene induced by a nerve growth factor in PC 12, a microglia cell line. It is a potent pro-apoptotic member of the orphan nuclear receptor superfamily that acts in response to extracellular stimuli. When stimulated by certain apoptotic signals, Nur77 is phosphorylated and migrates from the nucleus to the cytoplasm. It has been found that nuclear export of Nur77 requires dimerization with RXR (retinoid X receptor) to respond to apoptotic stimuli. RXR acts as a carrier to assist in the translocation of Nur77. Through interactions with BCL-2, the anti-apoptotic BCL-2 can be converted to a pro-apoptotic form through conformational changes which trigger cytochrome C (cyto C) release and subsequently induce apoptosis. Nur77 was observed on primary cultured glia and neuron cell line. Thus we hypothesized that activation of Nur77 could increase brain edema, aggravate toxic effect on blood-brain barrier and neurological function through induction of cerebral cell apoptosis.This study is composed of three parts to testify this hypothesis. Part I consists of experimental SAH rat model establishment through prechiasmatic cistern blood injection, we applied fluorescence quantitative RT-PCR and Western blot to detect whether the expression levels of Nur77 (phosphorylated-Nur77,p-Nur77)?Bcl-2?cyto C are correspond to the time point of peak brain water content and neurological scores after activation of Nur77 following SAH; observation whether Cytosporone B(Csn-B; an agonist for Nur77) can exacerbate such effects. Immunohistochemistry and TUNEL were also used to determine if activation of Nur77 could increase the extent of apoptosis and EBI after SAH; In part II, We utilized a rat model of induced SAH in order to investigate whether CsA can reduce brain edema formation and subsequent apoptosis, with the goal of promoting and expediting neurological recovery. Western blot and immunohistochemistry are also been used to detect the expression of Nur77 dependent apoptosis pathway proteins. TUNEL detection of neurons after application of CsA to determine if inhibition of Nur77 could decrease the extent of EBI after SAH, providing unique insights into future therapeutic development. In part III, the role of JNK, Akt and RXRa in Nur77-dependent apoptotic pathway was observed. Hemoglobin (Hb) Incubated Neuron Injury Model was used to simulate the SAH in vitro model. Fluorescence quantitative RT-PCR,western blot and Tunel were used.Materials and methodsSAH rat model was established and early brain injury effect and its impact on cerebral apoptosis of Nur77 activation was examined?1. Prechiasmatic Cistern Blood Injection SAH model of Sprague Dawley (SD) was established2. SD rats were randomly assigned to three groups: (1) untreated group; (2) treatment control group; (3) SAH groups; the experimental SAH group was divided into four subgroups, corresponding to 12h, 24h, 48h, and 72h after inducing SAH, respectively.3. Csn-B was used to demonstrate that Nur77 could be enriched and used to aggravate EBI after SAH. SD rats were randomly assigned to four groups: control group, SAH,SAH treated with the Csn-B solvent, dimethyl sulfoxide and SAH treated with Csn-B4. Part ?: SD rats were randomly assigned to four groups: control group, SAH, SAH treated with DMSO vehicle and SAH treated with CsA5. Brain water content, mortality were detected in each group; Garcia scale system was applied to test neural function of rats6. Fluorescence quantitative RT-PCR and Western blot were performed to detect mRNA and proteins expression levels of Nur77?p-Nur77?Bcl-2 and Cyto C at different time points after SAH in all groups;7. Immunohistochemistry staining was used to detect immune activity of Nur77?p-Nur77?Bcl-2 and Cyto C in cerebral cells8. Detected the expression of apoptotic pathway final protein caspase-3 through western blot and apoptotic level of cerebral cells via TUNEL staining9. Part ?: JNK inhibitor, SP600125 and Akt agonist,insulin were used to evaluate the role of Nur77 phosphorylation, respectively. Neuroprotective effects of those two drugs had also been studiedResults1. Csn-B as a Nur77 specific agonist could exacerbate EBI after SAH. CsA treatment can ameliorate EBI after experimentally induced SAH by inhibiting a Nur77-dependent apoptotic pathway and significantly decrease its mortality;2. Expression of Nur77 and associated death-signaling pathways, including p-Nur77,BCL-2 and cytochrome C caused severe neurological outcomes. These molecular changes directly led to the enhancement of brain edema and the decrease of neurological scores, reflecting Nur77 plays key role in the regulation of EBI after SAH;3. Csn-B injection directly led to the enhancement of brain edema and the decrease of neurological scores, indicating exacerbated EBI after SAH. CsA directly led to the inhibition of apoptosis, and through this, decreases in brain edema and improvement of neurological outcomes. Both of these results demonstrate that Nur77 contributes to apoptosis in cerebral cells during EBI after SAH;4. Csn-B directly led to the increase of Bcl-2 and cyto C expressions, indicating that Nur77 interacts with BCL-2 and translocates from the nucleus to the cytoplasm,where it targets mitochondria. Mitochondrial localization of Nur77 triggers the release of cytochrome C and subsequently induces apoptosis;5. CsA significantly attenuated the Nur77-dependent apoptotic pathway induced by SAH, consequently play a role in neural protection;6. SP600125 significantly alleviated increases in p-JNK, p-Nur77, Bcl-2, cyto C, and caspase-3 associated with SAH induction. These changes directly led to the inhibition of apoptosis, and through this, decreases in brain edema and improvement of neurological outcomes, both of which contribute to amelioration in EBI after SAH.7. Insulin significantly activated Akt, increased the phosphorylation of Nur77 and alleviated increases in Bcl-2 and cyto C associated with SAH induction. These changes directly led to the inhibition of apoptosis, decrease in brain edema and improvement of neurological outcomes.Conclusions1. Nur77 may be one of the major regulators of EBI-associated apoptosis, which is the main source of poor neurological outcomes in our study;2. It has been shown that Nur77 promotes the conversion of BCL-2 from a pro-survival to pro-apoptotic subset. Therefore, we believe that Nur77 may play an important role in the triggering and progression of apoptosis, which is the main cause of post-SAH EBI;3. Csn-B significantly exacerbated brain injury, evident through lower neurological scores, by promoting the apoptosis of central nervous cells. Bcl-2, one of the major mediators of post-SAH apoptosis, was significantly up-regulated after Csn-B administration, leading to an outburst of Cyto C within the cells;4. CsA treatment can ameliorate EBI after experimentally induced SAH by inhibiting a Nur77-dependent apoptotic pathway. CsA may display potent neuroprotective effects on brain injury in the early period after SAH, providing unique insights into future therapeutic development;5. It has been found that Nur77 inducd apoptosis of is linked to JNK activation and Akt inhibition.