Potential Role And Mechanisms Of HnRNPA1 In Secondary Brain Injury Induced Intracerebral Hemorrhage In Rats

Part Ⅰ:Changes in hnRNPA1 protein levels and distribution after intracerebral hemorrhage.Objective:To study the expression and distribution of hnRNPA1 protein in brain tissue after intracerebral hemorrhage(ICH)in rats.Methods:(1)48 rats were randomly selected and divided into the following 8 groups:sham operation group,3 h,6 h,12 h,24 h,48 h,72 h and 7 d after intracerebral hemorrhage(ICH),6 rats in each group.All rats were killed at corresponding time points after intracerebral hemorrhage.The brain tissues surrounding the hematomas of rats were collected,and the protein level and distribution of hnRNPA1 were detected by Western blot analysis and immunofluorescence technology.(2)According to the results of the first step,12 rats were randomly selected and divided into:sham operation group and ICH 48 h group,6 rats in each group.All rats were killed at 48 h after modeling.The brain tissues surrounding the hematomas of rats were collected,and nuclear and cytoplasmic proteins were separated.The protein levels of hnRNPA1 in the nucleus and cytoplasm were detected by Western blot analysis.Results:1.The total protein level of hnRNPA1 increased within 3 hours and decreased in 3 to 6 hours after intracerebral hemorrhage in rats.The hnRNPA1 protein level showed a upward trend from 6 hours to 48 days,and a downward trend from 48 hours to 7 days after intracerebral hemorrhage in rats.The hnRNPA1 protein expression peaked at 48 hours after intracerebral hemorrhage in rats.(P<0.01).2.the level of protein hnRNPA1 in the nucleus of rats showed a downward trend after intracerebral hemorrhage(ICH),while the level of protein hnRNPA1 in cytoplasm showed an upward trend.(P<0.01).3.In vivo immunofluorescence results showed that hnRNPA1 was mainly located in the nucleus,but the localization of cytoplasm increased significantly 48 hours after intracerebral hemorrhage.Conclusion:After intracerebral hemorrhage(ICH),the expression level of hnRNPA1 increased significantly,and hnRNPA1 transfered from the nucleus of neurons to the cytoplasm.Part Ⅱ:The role of hnRNPA1 in secondary brain injury induced by intracerebral hemorrhage.Objective:To explore the expression and distribution of hnRNPA1 in cells after intracerebral hemorrhage through the intervention of hnRNPA1 plasmid and small interfering RNA,and the role of its changes in secondary brain injury induced by intracerebral hemorrhage in rats.Methods:108 rats were randomly divided into 6 groups:sham group,ICH group,ICH+vector group,ICH+Over-hnRNPA1 group,ICH+CtrsiRNA group,ICH+hnRNPA1 siRNA group,18 rats in each group.ICH model was established 48 hours after transfection of hnRNPA1 plasmid and small interfering RNA.Six rats selected from each group were used for Morris water maze test,and were performed at the time points of 1 day,3 days,and 5 days after modeling.The remaining rats were scored for behavioral scores before killed at 48 hours after modeling.Then,the rats were anesthetized and killed and brain tissues surrounding the hematoma were taken for Western blot analysis,TUNELand FJB staining.Results:1.Overexpression of hnRNPA1 significantly increased the expression level and distribution of hnRNPA1 in brain tissues,and the level and distribution of hnRNPA1 decreased significantly after treatment with small interfering RNA.2.According to in vivo experiments,TUNEL staining and FJB staining showed that over-expressing hnRNPA1 could reduce the apoptosis and necrosis of neurons in secondary brain injury induced by intracerebral hemorrhage,and inhibiting the expression of hnRNPA1 could increase the apoptosis and necrosis of neurons.(P<0.05)3.Animal neurobehavioral score and Morris water maze test results show that over-expressing hnRNPA1 could improve neurobehavioral function,learning and memory abilities after intracerebral hemorrhage in rats,and down-regulating the expression of hnRNPA1 would weaken neurobehavioral function,learning and memory abilities.(P<0.05)Conclusion:HnRNPA1 plays a protective role in secondary brain injury induced by intracerebral hemorrhage in rats.Increasing the level of the level of hnRNPA1 could alleviate the secondary brain injury induced by intracerebral hemorrhage,while decreasing the level of hnRNPA1 could aggravate secondary brain injury induced by intracerebral hemorrhage.Part Ⅲ The mechanism of HnRNPA1 plays a protective role in secondary brain injury induced by intracerebral hemorrhage.Objective:To infer the potential mechanism of hnRNPA1 plays a protective role in secondary brain injury induced by intracerebral hemorrhage by analyzing the changes of protein levels related to pathways after intervention of HnRNPA1 plasmid and small interfering RNA.Methods:(1)36 SD rats were randomly assigned to the following 6 groups:sham operation group,ICH group,ICH vector group,ICH Over-hnRNPA1 group,ICH Ctr siRNA group,ICH hnRNPA1 siRNA group,6 rats in each group.The model of intracerebral hemorrhage was established 48 hours after transfection of hnRNPA1 plasmid and small interfering RNA.Rats in the corresponding group were killed 48 hours after intracerebral hemorrhage,and brain tissues surrounding the hematoma were taken and analyzed by qPCR to analyze the trend of Bcl-xl mRNA.(2)Using the second part of the experimental samples,Western blot was performed on the sham operation group,ICH group,ICH vector group,ICH Over-hnRNPA1 group,ICH CtrsiRNA group,and ICH hnRNPA1 siRNA group to analyze the change trend of Bcl-xl.Results:1.Overexpression of hnRNPA1 could increase the expression of Bcl-xl mRNA after intracerebral hemorrhage,and inhibition of hnRNPA1 expression could reduce the expression of Bcl-xl mRNA.(P<0.05)2.Overexpression of hnRNPA1 could increase the expression of Bcl-xl after intracerebral hemorrhage,and inhibition of hnRNPA1 expression could reduce the expression of Bcl-xl.(P<0.05)Conclusion:HnRNPA1 could improve the secondary brain injury after intracerebral hemorrhage in rats by regulating Bcl-xl,and it could be used as a potential target and research direction for secondary injury after ICH.