Studies On The Neuroprotection Of MC1568on Rats With Cerebral Hemorrhage As Well As Its Mechanism

PART I Expression patterns of histone deacetylases in rats with cerebral hemorrhageObjective To investigate the expression isoforms in brains of rats with intracerebral hemorrhage for determining specific HDAC subtypes involved in the pathological processes of brain hemorrhageMethods Rats were divided into sham group and cerebral-hemorrhage group. Models of cerebral hemorrhage were made by injection of bacterial collagenase type Ⅳ directly into rats’right striatum, and then morphological studies were performed. And mRNA and protein levels of HDACs in rats’brains were detected respectively by Real-Time PCR and Western blotting technology at3h,6h,12h,24h and48h after cerebral hemorrhage. Subsequently, cellular localizations of HDAC proteins were achieved by immunofluorescence technique.Results HE staining showed that there was a formed round hematoma in the right caudate nucleus. In the hematoma, brain tissue was loose with a lot of vacuoles, nerve cells were significantly reduced. And there were a number of red blood cells and inflammatory cells in the area of perihematoma. Compared with the sham group, mRNA levels of HDAC4, HDAC5and HDAC7were increased, but expression levels of HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, HDAC9, HDAC10, and HDAC11mRNA were not basically changed. Accordingly, elevated protein levels of HDAC4and HDAC5rather than HDAC7were detected by immunoblotting. Finally, both HDAC4and HDAC5were located in neurons.Conclusion Stereotactic injection of bacterial collagenase into rats’ the right striatum have established models of rats’ cerebral hemorrhage successfully. HDAC1-11isoforms are all expressed in the brains of rats with cerebral hemorrhage, but only mRNA and protein levels of both HDAC4and HDAC5are significantly increased, which indicate that these two isoforms are probably involved in the pathological process of cerebral hemorrhage. In addition, both HDAC4and HDAC5are mainly expressed in neurons. PART Ⅱ The neuroprotective effects of MC1568on the rats with cerebral hemorrhageObjective To investigate the neuroprotective effects of MC1568on rats with cerebral hemorrhageMethods IV type of bacterial collagenase induced successfully intracerebral hemorrhage. Then, ICH rats were divided randomly into MC1568group and saline group, which were respectively given MC1568and saline (25mg/kg, twice daily intraperitoneally, consecutively for3days). Subsequently, behavioral scores and head MRI were respectively performed on the1st,3rd day,7th day,14th day and28th day after cerebral hemorrhage, brain water content was measured on the3rd day, and the number of apoptotic nerve cells was examined by TUNEL assay.Results Compared with rats in the saline group, modified neurological severity scores (mNSS) of rats were markedly lowered on the7th,14th and28th day in the MC1568group, but not on the1st,3rd day; hematoma volumes of rats in the MC1568group were decreased on the3rd day; but brain water contents were not reduced on the3rd day; brain tissue loss of rats was significantly reduced in the MC1568group on28th day; apoptotic cells were decreased in the area of perihematoma.Conclusion MC1568can achieve neuroprotection against rats with intracerebral hemorrhage through reducing hematoma volume, decreasing the number of apoptotic nerve cells and lowering the degree of brain tissue loss thereby improving neurological function. PART Ⅲ Study on the mechanism of the protective effects of MC1568on rats with intracerebral hemorrhageObjective To investigate the mechanism of the protective effect of MC1568against rats with intracerebral hemorrhageMethods After bacterial collagenase inducing successfully models of cerebral hemorrhage, saline and MC1568were given to the rats. Then detection of mRNA levels of caspase-3, bcl-2, MMP-9, TNF-α, IL-1β was performed by Real-Time PCR, and protein levels of caspase-3, bcl-2, MMP-9, TNF-α, IL-1β were examined by Western blotting. Ultimately, the number of apoptotic nerve cells was examined by TUNEL assay.Results Compared with rats in the saline group, mRNA and protein levels of MMP-9, TNF-α and IL-1β of rats were all decreased in the MC1568group; mRNA and protein levels of pro-apoptotic factor (caspase-3) were also decreased, but expressions of anti-apoptotic factor (bcl-2) were upregulated in mRNA and protein levels. Besides, TUNEL positive cells were less in the MC1568group than those in the saline group. Conclusion neuroprotection of MC1568against cerebral hemorrhage resulted probably from both reduction of inflammation by decreasing expressions of inflammatory factors and inhibition of nerve cells apoptosis by regulating expressions of apoptotic factors.