| Objective：At the whole animal level to research the effect of MRTF-A on neuronal apoptosisinduced by cerebral ischemia and reperfusion (MCAO/R) and its mechanism inrats.Methods:1. To investigate the distribution and time relationship between the expression oflentivirus-mediated MRTF-A (LV-MRTF-A) in the cerebral cortex on rats.①30SPF level SD rats were randomly divided into5groups, the experimental groupwere injected with with different doses LV-MRTF-A（1μl,2μl,4μl,5μl）in cerebralcortex, while the control group just injected with negative LV(NEG-LV),lentivirus were injection on rats through intraventricular, make the whole braintissues into paraffin sections in the5days, use a confocal fluorescence microscopyto detect the expression and distribution of MRTF-A;②Intracerebroventricularinjected with4μl LV-MRTF-A for5days, and then observe the expression ofMRTF-A in different parts of the cortex by the laser confocal microscope.③36SPF level SD rats were randomly divided into6groups, make the whole braintissues into paraffin sections in1d,3d,5d,7d,9d respectively after injected withLV-MRTF-A. Use a confocal fluorescence microscopy to detect the expression ofMRTF-A.2. To investigate the effect of LV-MRTF-A on neuronal apoptosis induced by cerebralischemia reperfusion on rats:84SPF level SD rats were randomly divided into3groups: control group, model group and experimental group. Control group andmodel group were intracerebroventricular injected with4μl NEG-LV; experimentalgroup were injected with4μl LV-MRTF-A. The experimental and model groupwere constructed the model of ischemia/reperfusion (MCAO2h/R24, R48, R72h) 4days after the injection. Neurological defect scores were examined by Longa’smethod; cerebral infarction volume was evaluated by TTC staining; apoptosisindex was examined by TUNEL assay.3. To investigate the mechanism of MRTF-A against neuronal apoptosis induced bycerebral ischemia reperfusion:54SPF level SD rats were randomly divided into3groups: control group, model group and experimental group. Control group andmodel group were intracerebroventricular injected with4μl NEG-LV; experimentalgroup were injected with4μl LV-MRTF-A. The experimental and model groupwere constructed model of ischemia/reperfusion (MCAO2h/R24h)4days afterthe injection. Use the Real-time PCR method to detect the mRNA expression ofMRTF-A, caspase3and Mcl-1; Western blot method to to detect the proteinexpression of MRTF-A, CC3and Mcl-1; Immunofluorescence assay to detect theexpression of cytC, Apaf-1, CC3and Mcl-1.Results:1. After injected with different doses of LV-MRTF-A for5days, fluorescencemicroscopy detection results showed that1μl and2μl groups had a low levelexpression of MRTF-A, compared to1μl and2μl groups the expression of MRTF-Ain4μl and5μl groups increased significantly, and the expression of two groups hadno significant difference; MRTF-A was widely expressed in cerebral cortex and hada highest level in the injection point and the surrounding, distal end of the injectionpoint MRTF-A’s expression gradually decreased. After injected with4μlLV-MRTF-A, in1d and3d days, there was a letter express of MRTF-A, while from5d to7d MRTF-A’s expression was relatively high and then declined slightly in9dwhen compared to7d.2. After injected with4μl LV-MRTF-A for4days and constructed the cerebral ischemiareperfusion model,2h ischemia and reperfusion24h,48h,72h. Compared with thecontrol group, neurological function scores and infarct volume were significantlydifferent (P <0.01); the neurological function score and cerebral infarction volume of the experimental group corresponding to the same point of time in the modelgroup also there was a decline, resulting in a significant difference (P <0.05or P<0.01); the apoptosis index detected by TUNEL assay showed that compared withthe control group, in the model group it was increased significantly(P <0.05P <0.01), and with the prolongation of reperfusion the apoptosis index decreasedgradually. However, when injected with LV-MRTF-A before MCAO/R, it found thatthe apoptosis index decreased significantly (P <0.05or P <0.01).3. After injected with4μl LV-MRTF-A for4days and then constructed the cerebralischemia reperfusion model,2h ischemia and reperfusion24h. The results showedthat in the rat cerebral cortex MRTF-A mRNA and protein expression were increasedsignificantly. Compared with the control group, the mRNA expression of Caspase-3was significantly increased in model group, Mcl-1mRNA expression wassignificantly decreased (P <0.01). Compared with the model group, in experimentalgroup caspase-3mRNA expression was significantly decreased, expression of Mcl-1mRNA was significantly increased (P <0.01). Western blot results showed thatcompared with the control group, CC3protein expression increased and Mcl-1protein expression were significantly decreased in model group,(P <0.01). Whilecompared with the model group, in experimental group CC3protein expression wasdecreased obviously, Mcl-1protein expression was increased significantly (P <0.01);immunofluorescence results showed that ischemia2h and reperfusion24h caninduced cyt c and Apaf-1a high expression, the expression regions of them overlapin a wide range. What’s more, CC3expression increased significantly but Mcl-1expression decreased significantly. After in jected with LV-MRTF-A, the resultswere just on the contrary.Conclusion:1. The optimal injection dose of LV-MRTF-A was4μl,5d-7d was the highest expressiontime of LV-MRTF-A. And it had a highest expression in the injection point and adiffusion expression in cortex. It proved that LV-MRTF-A can express safely and effectively in rat brain cortex, which provide experimental basis to explore the effectof MRTF-A against neuronal apoptosis induced by cerebral ischemia reperfusion.2. At the whole animal level, MRTF-A can inhibit neuronal apoptosis induced bycerebral ischemia reperfusion and improve neurological function, reduce the volumeof cerebral infarction.3. At the whole MCAO/R rats model, it was found that intracerebroventricularinjection of LV-MRTF-A can inhibit the neuronal apoptosis induced by cerebralischemia/reperfusion and improve neurological function, reduce the volume ofcerebral infarction. The mechanism may related with MRTF-A reduced cytccombined with Apaf-1and down-regulated their expression, down-regulatedpro-apoptotic target genes caspase3, CC3and up-regulated anti-apoptotic target geneMcl-1expression. |
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