| Backgroud and objectiveSubarachnoid hemorrhage (SAH)is a common encountered cerebro-vascular disease with high morbidity and mortality. It is a big threaten to human health ,and predoiminantly attacks young and middle-aged group. In recent years, researchers put up the concept of early brain injury (EBI), and regards it 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 a few years before. It includes the general cerebral injuries from the time aneurysm rapture to vasospasm occurring, of about 72 hours. EBI comprises a series of pathological damages involved multi factors and pathways, but its mechasim is not completely clear. A number of recent studies indicate that inflammation plays an important role in EBI after SAH. Successful intervention to alleviate inflammatory reaction degree may bring up optimistic results of patients with SAH.Adenosine (ADO) is a metabolic product of endogenous adenosine triphosphate (ATP), and participates in multiple diseases through activation of four G-protein-coupled menbraine receptors, the A1, A2A, A2A, and A3 receptor (A3R). In recent years, reseachers pay more and more attention of the anti-inflammatory effect of A3R subunit, and demonstrate that activation of A3R could alleviate the degree of inflammation in rheumatoid arthritis (RA), pneumonia, myocardial infaction, and so on. In vitro studies indicate that A3R involves in the activation of inflammatory cells including microglia, neutrophil, T cells, astroglia etc., regulating the release of various inflammatory cytokines. A3R was observed on microglia cells containing primary cultured microglia, BV2 cell line, and N9 cell line. Thus we are hypothesizeing that activation of A3R could result in suppression of microglia activation, further lead to decreasement of inflammatory mediators including NO?IL-1??IL-6?TNF-?and so on, which generate a protective action after SAH through alleviating toxic effect on neurons and blood-brain barrier (BBB).This study is composed of three parts to testify this hypothesis. Part I consists of experimental SAH rat model establishment through internal carotid perforation using filament, observation of rat mortality, neural function, brain water content, BBB permeability damage, Nissl's staining and TUNEL detection of neurons after application of specific agonist for A3R, to determine if activation of A3R could decrease the extent of EBI after SAH; In part II, we applied fluorescence quantitive RT-PCR and ELISA to detect the transcription and expression level of certain inflammatory factors in rat brain after activation of A3R following SAH; immunofluorescence and histochemistry were used to assess the state of activation of microglia and astroglia in cortex and hippocampus region, in order to determine if A3R activation could suppress inflammatory reaction after SAH; we established PC12 anoxia cell model using cobalt chloride and observed if A3R activation could protect PC12 cells directly. In part III, in vitro experiments including flow cytometry (FCM), ELISA, Laser Scanning Confocal Microscope (LSCM), and Western blot were performed to examine the effect of A3R activation on CD11b expression of microglia, inflammatory cytokines releasement, and cellular signal pathway, so as to verify that A3R activation could effectly suppress microglia activation, and to explore its molecular mechanism.Materials and methodsIn vivo and in vitro experiments were performed in this study. SAH rat model was established and neuroprotective effect and its impact on inflammatory response in brain of A3R activation was examined; PC12 cells substitited for neurons and BV2 microglia cell line were cultured in vitro, and neuroprotective effect and microglia activation suppression effect of A3R activation were evaluated, meanwhile we prelimilarily investigated its molecular mechanism.1. SAH model of Sprague Dawley (SD) Rat was established by internal carotid perforation using filament;2. SD rats were randomly divided into three groups: Sham, SAH, and CL-IB-MECA (A3R specific agonist), and brain water content, BBB permeability, and mortality were detected; 3. Garcina scale system and foreleg palcing test were applied to test neural function of rats in group Sham, SAH, and CL-IB-MECA; Nissl's staining and TUNEL were carried out to determine the level of nerve injury;4. IL-1?, TNF-?, IL-6, and IL-10 in brian tissue were assessed by ELISA at 6, 12, and 24 hours following SAH;5. Fluorescence quantitive RT-PCR was performed to detect mRNA expression of IL-1?, TNF-?, IL-6, and IL-10 at different time points after SAH in all three groups;6. PC12 cell anoxia model via cobalt chloride was established, and cell viability was assessed by MTT method;7. LPS induced BV2 activation cell model was established in vitro, and CL-IB-MECA was applied as an intervention. CD11b expression was detected by FCM; IL-1?, TNF-?, and IL-6 releasement were examined by ELISA; the expression of TRL-4, p38 MAPK, ERK, and Akt/I?Ba were determined by western blot; and state of NF-?B activation was assessed by LSCM.Results1. The mortality of SD rats in group SAH is about 50.00%, which is similar to the death rate of human following SAH;2. CL-IB-MECA as an A3R specific agonist could effectly improve neural function of SD rats with SAH, and significantly decrease its mortality;3. CL-IB-MECA is efficatious in alleviating BBB damage and brain edema following SAH, and also protective to neurons;4. Microglia activation, and expression/releasement of certain cytokines significantly increase in rat brain tissue after SAH, which is considered one of the major mechanism of EBI after SAH. A3R activation could effectively decrease the expression and release of certain inflammatory factors and suppress the activaton of microglia in rats following SAH, consequently play a role in neural protection;5. A3R activation does not directly protect PC12 cells under anoxia with cobalt chloride;6. LPS could activate BV2 cells in vitro and has no impact on proliferation of BV2 cells, however it could promote the release of NO;7. A3R activation could effectively reduce the expression of CD11b on microglia, decrease the release of NO and certain cytokines, but has no influence on microglia proliferation;8. The mechanism of activated A3R suppressing microglia activation may through inhibiting PI3K/AKT/I?Ba pathway and NF-?B translocation to nucleus, while it has no effect on p38/ERK phosphorylation and TLR-4 receptor expression.Conclusions1. A3R activation could effectively reduce brain water content and improve neural function, significantly decrease mortality of rats following SAH, thus bringing about neural protection on EBI;2. Microglia activation, and expression/release of certain cytokines significantly increase in rat brain tissue after SAH, which is considered one of the major mechanism of EBI after SAH;3. A3R activation could effectively decrease the expression and release of certain inflammatory factors and suppress the activaton of microglia in rats following SAH, consequently play a role in neural protection;4. The mechanism of activated A3R suppressing microglia activation may through inhibiting PI3K/AKT/I?Ba pathway and NF-?B translocation to nucleus, while it has no effect on p38/ERK phosphorylation and TLR-4 receptor expression. |
PDF Full Text Request