| Part one:Influence of HMGB-1 inhibitor glycyrrhizin acts on blood brain barrier and neurons of Li-pilo status epilepticus model ratsObjective:To investigate the influence of HMGB-1 inhibitor glycyrrhizin acts on blood brain barrier and neurons of Li-pilo status epilepticus model rats.Methods:1.Adult male SD rats were used for these trials which were divided into control group, epileptic group, glycyrrhizin low dose group, glycyrrhizin high dose group randomly, the epileptic group and glycyrrhizin group were both injected with lithium chloride-pilocarpine to induce the status epilepticus(SE),2h after SE, glycyrrhizin low dose group and glycyrrhizin high dose group were respectively given 10mg/kg and 30mg/kg glycyrrhizinn. 2. Seventy-two hours after SE,2% evans blue 4ml/kg were given to the rats by femoral vein injection, circulating 1 hours after cardiac perfusion, quickly decapitating brain on the ice plate, detecting the evans blue content in brain tissue.3.Seventy-two hours after SE, taking out the rat brain tissue quickly and placing in a sealed test tube after anesthesia, measuring the water content in brain by dry-wet-weight-method.4.To study the degeneration of neurons on SE rats,we used the the FJB staining.5.Recording the mortality of rats at one week after SE. Results:1.Evans blue:Seventy-two hours, the test of the content of evans blue in four groups showed that epilepsy model group compared with blank control group, the content of evans blue was significantly elevated (p<0.05); compared the low dose glycyrrhizinn group with epilepsy model group, evans blue leakage was not obvious difference (p>0.05); compared high dose glycyrrhizinn groups with epilepsy model group, evans blue leakage was significantly decreased (p<0.05).2.The water content of brain tissue:Seventy-two hours after SE, according to date, epilepsy model group compared with blank control group, water content of brain tissue was significantly elevated (p<0.05); compared the low dose glycyrrhizinn group with epilepsy model group, water content of brain tissue was not obvious difference (p>0.05); compared high dose glycyrrhizinn groups with epilepsy model group, water content of brain tissue was significantly decreased (p<0.05). 3.FJB staining:Seventy-two hours after SE, normal control group:rat hippocampal CA1, CA3, DH area had no FJB positive cells; epilepsy model group:large continuous distribution FJB positive neurons were observed; low dose glycyrrhizinn group:FJB positive neurons were also observed at large parts of CA1, CA3, DH area, but slightly less than the epilepsy group, especially the DH area; in high dose glycyrrhizinn group:FJB positive neurons were significantly reduced by comparing to the epilepsy group at CA1, CA3, DH area.4.Mortality:Recording the mortality of rats at one week after SE, the result showed that blank control group was 0.0%(0/8); epilepsy group was 42.1%(8/19); low dose glycyrrhizinn group was 38.9%(7/18); high dose glycyrrhizinn group was 18.75%(3/16). Compared with blank control group, mortality of epileptic model group was significantly increased (p<0.05); low dose glycyrrhizinn group compared with epileptic model group, mortality was declined, but it was not statistically (p>0.05); high dose glycyrrhizinn group compared with the epilepsy model group, mortality was decreased significantly (p<0.05). Conclusion:SE can cause blood brain barrier damage, brain edema, neuron degeneration and increase the mortality, but by giving the treatment of HMGB-1 inhibitor glycyrrhizinn which can reduce the blood brain barrier damage, acute brain edema, hippocampal neuron degeneration and mortality. It indicates that glycyrrhizinn acts as HMGB-1 inhibitors may have the potential to reduce the injury after SE, there is a need to have further researchs on it.Part two:The mechanism of HMGB-1 inhibitor glycyrrhizin protect the blood-brain barrier integrityObjective:To investigate the mechanism of HMGB-1 inhibitor glycy rrhizin protect the blood-brain barrier integrity. Methods:1.Adult male S D rats which were used for these experiments were divided into control group and epileptic group randomly, pilocarpine were injected to the epil eptic group in order to induce the sustatus epilepticus and epileptic group rates were killed at 3h,24h,72h, 1w following the success of model m aking. The rats in control group were treated identically but received sali ne (NS) instead of pilocarpine. Then using the western blot (Western) to measure the expression of HMGB-1 in hippocampus of rats.2.Adult ma le SD rats were were divided into control group, epileptic group, glycyrr hizin high dose group randomly, the epileptic group and glycyrrhizin gro up were both injected with lithium chloride-pilocarpine to induce the stat us epilepticus(SE),2h after SE, glycyrrhizin high dose group was given 30mg/kg glycyrrhizinn, the rats in control group were treated identically but received saline (NS) instead of pilocarpine,72h after SE, detected ex pression of TNF-a in rats cerebral cortex and hippocampus by western b lot. Results:1.Western blot showed that small amount of HMGB-1 protei n expressed in the normal control group,3h after SE, the HMGB-1 prote in had began to upregulate,24 h after SE,the expression quantity of HM GB-1 reached the peak,72h after SE,it slightly decreased, after 1W, the expression level was still higher than that of the control group (p< 0.05). 2, Seventy-two hours after SE, the level of protein TNF-a in cotex and hi ppocampus were increased in the epileptic group compared with control group(p< 0.05), and were decreased in glycyrrhizin treatment group com pared with the epileptic group (p< 0.05). Conclusion:HMGB-1 and TNF-a were significantly upregulated after SE. The treatment of HMGB-1 inh ibitor glycyrrhizin decreased the level of TNF-a in cotex and hippocampu s of SE model rats, TNF-a play a important role in maintaining the inte grity of BBB (blood brain barrier), so, glycyrrhizin may be though inhibi t the expression of TNF-a to protect the blood-brain barrier from injury. |
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