1.The Neuron Protective Effects Of Eysteamine On The Early Brain Injury In A Rat Subarachnoid Hemorrhage Model 2.the Inhibition Potential Of Bruceine A And Ketoconazole Towards Cytochrome P450 3A4

Part ONE The neuron protective effects of cysteamine on the early brain injury in a rat subarachnoid hemorrhage model[Objective] Aneurysmal subarachnoid hemorrhage is a disastrous stroke subtype with significant morbidity and mortality, and often results in lasting neurological deficits for survivors. Previous studies have indicated that early brain injury, which refers to the acute injuries to the whole brain within the first 72 h after SAH, including increased intracranial pressure, decreased cerebral perfusion pressure, disturbed microcirculation, brain edema formation, oxidative stress, and delayed cerebral vasospasm and damage to the microvascular system. In particular, oxidative stress and neuron apoptosis plays an important role in the pathogenesis of early brain injury following subarachnoid hemorrhage. The aim of this study was to assess whether cysteamine prevents post-SAH oxidative stress injury via its antioxidative and anti-apoptotic effects.[Methods] Experimental subarachnoid hemorrhage was induced in Sprague-Dawley rats using double blood injection into cisterna magna method. The experimental groups consisted of sham-operated group (n=24), vehicle-treated SAH group (n= 24), and cysteamine-treated SAH group (n=24). In cysteamine-treated SAH group, dose (0.1 ml,20 mg/kg/day) of cysteamine was administered after first blood injection. Rats of vehicle-treated SAH group received equal volumes of 0.9% saline intraperitoneal administration (0.1 ml) at corresponding time points. After the neurological assessment, all the rats were killed at 48h after SAH. Six rats in each group were decollated, then the brain sample was removed for molecular biological and biochemical experiments. Six rats in each group were for detecting blood-brain barrier impairment. Six rats in each group were for detecting brain water content. Six rats in each group were for immunohistological staining study. The following indicators were observed:1. Indicators for early brain injury (including neurobehavioral defects, assessment of brain edema according to the wet/dry method, blood-brain barrier permeability assessed by Evans blue dye extravasation, TUNEL and immunohistological staining for the evaluation of cortical neuron apoptosis).2. The reactive oxygen species level, the concentration of malondialdehyde and glutathione were detected; and the activity of lactate dehydrogenase and glutathione peroxidase determination was measured using commercial assay kit.3. The concentration of 3-nitrotyrosine,8-hydroxydeoxyguanosine and brain-derived neurotrophic factor was measured with commercial enzyme-linked immunosorbent assay kit for rats.[Results] It was observed that intraperitoneal administration of cysteamine (20 mg/kg/day) could significantly alleviate EBI (including neurobehavioral deficits, brain edema, blood-brain barrier permeability, and cortical neuron apoptosis) after SAH in rats. Meanwhile, cysteamine treatment reduced post-SAH elevated the reactive oxygen species level, the concentration of malondialdehyde,3-nitrotyrosine, and 8-hydroxydeoxyguanosine and increased the glutathione peroxidase enzymatic activity, the concentration of glutathione and brain-derived neurotrophic factor in brain cortex at 48 h after SAH.[Conclusion] These results indicated that intraperitoneal administration of cysteamine (20 mg/kg/day) confers protection against SAH-induced EBI in rats through the inhibition of oxidative stress and the promotion of BDNF production in vivo. Neurobehavioral deficit was improved; brain edema and blood-brain barrier permeability were attenuated; oxidative stress and cortical apoptosis were reduced after cysteamine treatment. These finding suggest that cysteamine may be a candidate treatment for EBI after SAH.Part TWO The inhibition potential of bruceine a and ketoconazole towards cytochrome P450 3A4[Objective] Cerebral haemorrhage, a kind of intracranial hemorrhage occurring in the brain tissue, severely damages the health of human body. It can be induced by brain trauma, and sometimes occurs spontaneously with hemorrhagic stroke. Searching efficient medicines for cerebral haemorrhage is an important and necessary task. The following therapeutic regimens are needed:1) antihypertensive therapy is employed to lowerthe blood pressure; 2) drugs to reduce elevated intracranial pressure.3) inhibition of secondary inflammatory response injury and apoptosis after cerebral hemorrhage. Bruceine a, with the potential of antitumor cytotoxic and inhibiting the nuclear factor-kappa B p65, is a potential therapeutic drug for cerebral haemorrhage. The present study aims to determine the inhibition potential of bruceine a towards cytochrome P450 (CYP) 3A4 which catalyzed most of clinical drugs.[Methods] Protein data bank is the source to get the crystal structure of CYP3A4. Among the list of CYP3A4 crystal structures, we select the crystal structure for which the activity center was bound with the inhibitor ketoconazole. Chemdraw software in the ChemOffice package was used to draw the two-dimensional structure of bruceine a, and the structure was drawn using standard bond lengths and angles. Ranked by Chemscroe values, we just selected the most suitable binging conformation among the top five poses. The carbonyl group located in the benzene ring was the metabolic site which has 1.98 A distance towards the catalytic center of CYP3A4. Strong hydrogen bond was formed between the structure of bruceine a and amino acid Ile301 in the protein sequence of CYP3A4. The docking process of bruceine a into the activity cavity of CYP3A4 was performed using Surflex-Dock. Co-docking method was used to compare the binding potential between bruceine a and ketoconazole.[Results] Bruceine a exerted stronger binding potential than ketoconazole towards the activity center, indicating the importance to monitor the drug-drug interaction between bruceine a and substrates mainly undergoing CYP3A4-catalyzed metabolism.[Conclusion] Different from the in vitro screening method, bioinformatics-guided molecular docking method has more rapid screening capability. This method has also been employed to screen the inhibitors of CYP3A4. In this study, bruceine a was demonstrated to be a strong inhibitor of CYP3A4. The present study also demonstrates the power of molecular docking prediction in the metabolic behavior prediction.