| Objective:To investigate the molecular mechanism of the myocardin-related transcription factor-A promoting autophagy to alleviate neuronal damage in rats induced by cerebral ischemia-reperfusion.Methods:1.To explore the best conditions for lentivirus infection in the ventricle:(1)Thirty-five SD rats were randomly divided into 7 groups:control group,the group with high expression for 5,7,and 9 days,and the group with suppressed expression for 5,7,and 9 days.At the dose of 1.6×10~6 TU,the control group was injected with lentiviral vector,the high expression group was injected with high expression MRTF-A lentivirus,and the suppression expression group was injected with inhibitory expression of MRTF-A lentivirus.After injection,brain tissues were taken on the 5th,7th,and 9th days.Detecting the MRTF-A expression by Western blot used to determine the best time for lentivirus to infect the ventricle.(2)Twenty SD rats were randomly divided into 4 groups:control group,low-dose group,middle-dose group and high-dose group.The control group was injected with lentiviral vector(1.6×10~6TU),and the other groups were respectively injected high expression of MRTF-A lentivirus with different doses(low:0.8×10~6TU,medium:1.6×10~6TU,high:2.4×10~6TU).Seven days later,brain tissue was taken for frozen section,Detecting the fluorescence MRTF-A expression used to determine the optimal dose of lentiviral injection.(3)Twenty SD rats were randomly divided into 4 groups:control group,P1 group(1.72mm posterior bregma,1mm para sagittal suture,depth 2.6mm),P2 group(1.72mm posterior bregma,2mm para sagittal suture,depth 2.6mm),P3 group(0.72mm posterior bregma,2mm para sagittal suture,depth 2.6mm),The injection dose of each group was1.6×10~6TU,brain tissue was taken for frozen section on the 7th day.Detecting the fluorescence MRTF-A expression used to determine the optimal injection site of lentivirus.2.The effect of MRTF-A on ischemic stroke:Eighty SD rats were randomly divided into 4 groups:sham group,cerebral ischemia/reperfusion model group,high expression MRTF-A group and suppression expression MRTF-A group.At a dose of 1.6×10~6 TU,the sham operation group was injected with lentiviral vector,the high expression group was injected with high expression MRTF-A lentivirus,and the suppression expression group was injected with suppression of MRTF-A lentivirus expression.The rat was treated by a middle cerebral artery occlusion method that was cerebral ischemia 2h and reperfusion 24h at 7 days after lentivirus injection.(1)Western blot was used to detect the expression level of MRTF-A;(2)Longa's5-point neurological function score and 2,3,5-chlorotriphenyltetrazolium(TTC)staining to evaluate nerve injury;(3)Wet and dry weight method to measure brain water content;(4)HE staining was used to observe the morphological changes of brain tissue;(5)Transmission electron microscopy was adopted to observe the ultrastructural changes of cells in cerebral cortex and hippocampus CA3 area.These methods were used to determine the effect of MRTF-A on nerve damage.3.The effect of MRTF-A on regulating autophagy followed by ischemic strokeForty SD rats were randomly divided into 4 groups:sham group,cerebral ischemia/reperfusion model group,high expression MRTF-A group and suppression expression MRTF-A group.At a dose of 1.6×10~6 TU,the sham operation group was injected with lentiviral vector,the high expression group was injected with high expression MRTF-A lentivirus,and the suppression expression group was injected with suppression of MRTF-A lentivirus expression.The rat was treated by a middle cerebral artery occlusion method that was cerebral ischemia 2h and reperfusion 24h at 7 days after lentivirus injection.(1)Observing the formation of autophagolysosomes in the cortex and hippocampus CA3 area using transmission electron microscopy;(2)Using Western blot to detect the expression levels of autophagy-related proteins(LC3-?/?,Beclin-1,ATG5,P62),and the autophagy signaling pathway protein(p AKT/AKT,pULK1/ULK1,pmTOR/mTOR).These methods were for exploring the specific molecular mechanism of MRTF-A regulating autophagy.Results:1.The best conditions for lentivirus infection in the ventricle:(1)The lentivirus could effectively upregulate or downregulate the expression of MRTF-A on the 7th-9th days after injection.Compared with the control group,it was statistically significant(P<0.05).(2)The lentivirus spread to the whole brain on the 7th day after injection,and the optimal injection dose was 1.6×10~6TU,which was statistically significant compared with the control group(P<0.05).(3)The best injection site is 1.72 mm behind bregma,2 mm beside sagittal suture,and injection depth is 2.6 mm.Compared with the control group,it has statistical significance(P<0.01).2.MRTF-A can effectively inhibit nerve injury mediated by ischemic stroke:(1)By detecting brain injury,cerebral infarction volume and cerebral edema,we found that compared with the control group,the ischemic side of the model group increased significantly infarction volume and cerebral edema,and severe neurological impairment(P<0.01).Compared with the model group,high expression of MRTF-A significantly decreased neurological function score,cerebral infarction volume and cerebral water content(P<0.05),while the group of inhibition MRTF-A significantly increase neurological function score,cerebral infarction volume and cerebral water content(P<0.05).(2)By observing the ultrastructural changes of nerve cells,the major damage area that mediated by the cerebral ischemia-reperfusion was in the cortical and the hippocampal CA3 area.It can be observed that the brain tissue structure was disordered,the nerve cell nuclear shrinkage was densely stained,and a large number of necrotic cells were generated.Nerve cell nuclear membrane,perinuclear organelles and synapses were abnormal in shape and structure.Organelles were swollen and deformed,accompanied by the formation of autophagolysosomes.With MRTF-A highly expressed,it could significantly alleviate abnormal changes in the structure of nerve cells,and inhibition of MRTF-A further deepens the damage.3.MRTF-A induces autophagy of nerve cells to inhibit ischemic stroke injury:(1)There was no significant difference in the number of autolysosomes around the cell nucleus between the model group and the control group(P>0.05).Compared with the model group,a large number of autolysosomes were found in the ultrastructure of the highly expressing MRTF-A group(P<0.05),but the MRTF-A inhibited group had the small amount of autolysosomes and the results were not statistically significant(P>0.05).(2)Compared with the control group,the change in expression levels of Beclin-1,ATG5,LC3/I and P62 after establish model was too small to be statistically significant(P>0.05).When the brain tissue was over-expressed MRTF-A,the expression levels of Beclin-1,ATG5 and LC3?/I were significantly increased compared with the model group(P<0.05 or P<0.01),and the expression level of P62 was significantly reduced(P<0.05).The results were reversed when MRTF-A was inhibited compared with the model group(P<0.01).(3)The phosphorylation level of m TOR,AKT,ULK1 in the model group was not significantly different from the sham group(P>0.05);when the brain tissue was over-expressed MRTF-A,the phosphorylation of mTOR,AKT protein was significantly decreased(P<0.05),and the phosphorylation level of ULK1 was significantly increased(P<0.05).When MRTF-A was inhibited,the opposite phenomenon was observed(P<0.05or P<0.01).Conclusions:1.The whole brain was effectively infect by lentivirus that once reach effective dose and duration of action in this studies.2.During the ischemic stroke reperfusion,MRTF-A can effectively inhibit injury by reducing infarct volume,relieving nerve cell damage,and enhancing autophagy level,while inhibiting the expression of MRTF-A aggravate the brain damage.This result suggest that MRTF-A can effectively inhibit nerve injury mediated by ischemic stroke.3.Overexpression of MRTF-A in the ischemic area can significantly upregulate the expression of Beclin-1,ATG5,LC3II/I,and downregulate the expression of P62,which to induce the occurrence of autophagy;Meanwhile,the autophagy level is promoted by regulating the PI3K/AKT-m TOR-ULK1 signaling pathway.These findings indicated that the neuroprotective effect of MRTF-A is related to the induced autophagy. |
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