The Effects Of HDAC3 Selective Inhibitor In Brain Injury After Intracerebral Hemorrhage And Its Molecular Mechanism In Rats

Purposes:This experiment hopes to establish a collagenase-induced cerebral hemorrhage model to explore whether HDAC3 plays a toxic role in aggravating secondary brain injury after cerebral hemorrhage,and to further clarify whether the application of the HDAC3 selective inhibitor RGFP966 could improve neuronal injury and explore its potential mechanism,and finally provide a new therapeutic target for the clinical treatment of cerebral hemorrhage.Methods:1.Thirty rats were randomly divided into five groups: SHAM,6h postICH,1d post-ICH,3d post-ICH and 7d post-ICH.After the modeling,RTq PCR,western-blot and immunofluorescence staining were used to detect the expression and distribution of HDAC3 in the brain tissue of rats at different time points after intracerebral hemorrhage,and to explore whether HDAC3 was involved in the process of brain injury in rats after intracerebral hemorrhage.2.Forty rats were randomly divided into four groups: SHAM,ICH,ICH+RGFP966 and ICH+vehicle.RGFP966 was injected intraperitoneally immediately after modeling.Neurobehavioral scoring,Western-Blot,HE staining,TUNEL staining and FJB fluorescence staining were used to observe the behavioral performance of 3d post-ICH rats and to detect the degeneration and apoptosis of neurons in the surrounding tissue of the cerebral hematoma,and to explore whether inhibiting HDAC3 could improve the rats neuronal damage after intracerebral hemorrhage.3.Forty rats were randomly divided into four groups: SHAM,ICH,ICH+RGFP966 and ICH+vehicle.RGFP966 was injected intraperitoneally immediately after the modeling was completed.Western-Blot,ELISA and immunofluorescence staining were used to detect the expression levels of NF-κB,EAAT2 and glutamate in the brain tissue of 3d post-ICH rats,and finally explore its potential mechanism of inhibition of HDAC3 to improve neuronal damage after cerebral hemorrhage in rats.Results:1.RT-qPCR results showed that compared with SHAM group,HDAC3 m RNA increased after ICH in rats and reached the maximum value on the third day(P<0.001).Western-Blot results showed that compared with SHAM group,the expression of HDAC3 increased after ICH in rats and reached the maximum value on the third day(P<0.0001).Immunofluorescence staining showed that HDAC3 was widely expressed in the cytoplasm and nucleus of neurons,astrocytes and microglia in the perihematomal tissue 3 days after intracerebral hemorrhage.2.The behavioral results showed that compared with the ICH group,the neurobehavioral scores of the ICH+RGFP966 group were significantly reduced(P<0.0001).The results of HE staining showed that compared with the ICH group,the ICH+RGFP966 group had less erythrocyte infiltration and less nuclei condensed.Western-Blot results showed that compared with the ICH group,the expression of HDAC3 in the brain tissue of the ICH+RGFP966 group was significantly decreased(P<0.001);compared with the ICH group,the expression of MPO in the brain tissue of the ICH+RGFP966 group was significantly decreased(P<0.001).TUNEL staining showed that compared with the ICH group,the TUNEL(+)cells in the brain tissue of the ICH+RGFP966 group were significantly reduced(P<0.01).FJB staining showed that compared with the ICH group,the FJB(+)cells in the brain tissue of the ICH+RGFP966 group were significantly reduced(P<0.01).3.Western-Blot results showed that the expression levels of total NF-κB p65 were not significantly different among the groups;compared with the ICH group,the nuclear NF-κB p65 expression in the rat ICH+RGFP966group was significantly decreased(P<0.001).Compared with the ICH group,the expression of EAAT2 in the ICH+RGFP966 group was significantly increased(P<0.001).ELISA results showed that compared with the ICH group,the glutamate content in the ICH+RGFP966 group was significantly decreased(P<0.05).Immunofluorescence staining showed that HDAC3 was expressed in the cytoplasm and nucleus of astrocytes,while EAAT2 was widely expressed on the astrocyte membrane.Conclusion:(1)The expression of HDAC3 increases after intracerebral hemorrhage in rats,and inhibition of HDAC3 expression with the HDAC3 selective inhibitor RGFP966 can improve neuronal damage after intracerebral hemorrhage in rats;(2)RGFP966 may upregulate EAAT2 by preventing the translocation of NF-κB to the nucleus expression,increased glutamate reabsorption,reduced cell excitotoxicity,and improved neuronal damage after intracerebral hemorrhage.