| The inflammatory response of temporal lobe epilepsy in the brain is mainly characterized by the activation of glial cells.In particular,the activation of microglia plays an important role in the process of epilepsy,and may be partially regulated by high mobility group protein 1(HMGB1).However,the role of Toll like receptors(TLRs)in the activation of microglia by HMGB1 needs to be further explored.On the other hand,the antiepileptic effect of boxa,a selective inhibitor of HMGB1 receptor,on the signal regulation of HMGB1,and the optimal time and dose of action are not clear.Therefore,the purpose of this study is to investigate the regulatory effect of Hmgb1-Tlrs(especially TLR2 and TLR4)signal axis on the activation of microglia in hippocampus of acute epilepsy model.Part One: the optimization of the antiepileptic effects of HMGB1 receptor antagonist BoxA in rat model of SE Objective: The anticonvulsant effect of BoxA,a selective inhibitor of HMGB1 receptors,has been demonstrated in several experimental models of epilepsy.Therefore,the purpose of this study is to determine the antiepileptic efficacy of BoxA and the optimal injection time and dosage in a rat model of Pilocarpine-induced SE.Methods: 72 male Sprague-Dawley rats were divided into 8 groups.Rats were given lithium chloride and Pilucapine to induce seizures,and experiments were performed in two steps.In the first step,BoxA injection(10 μg)(BoxA group)was performed in the experimental group at 30 minutes,4 hours or 12 hours before the seizure was induced,and the control group(Control group)received an equal amount of saline injection to determine the optimal time of BoxA injection.The second step is to inject different doses of BoxA(250 ng,1 μg,10 μg)at the optimal time before seizure is induced,and the Control group received an equal amount of saline injection.The antiepileptic effect was evaluated according to time latency and frequency of seizures.Results: Compared with the Control group,BoxA had the smallest effect on the overall state of seizures in the 30 min group and the frequency of seizures did not change significantly compared with the Control group(P> 0.05).However,earlier time points including the 4 hours and 12 hours groups,the seizure latency and frequency were improved significantly compared with the Control group(P <0.05),and the 4 hours group Prolonged the seizure latency better than the 12 hours group(P<0.05).Using fixed intervention time(4 hours),further studies on doses found that compared with the Control group,the 250 ng group had no significant difference in seizure suppression(latency and frequency)(P> 0.05).Compared with the Control group,10 μg group significantly prolonged the seizure latency and decreased the frequency of seizures(P <0.05)and even better compared with the 250 ng and 1 μg groups(P <0.05).The above results suggest that 10 μg BoxA using stereotactic injection may be the optimal dose.Conclusion: This study shows that BoxA,a specific inhibitor of HMGB1 receptors,exerts anticonvulsant effects in rat models of seizures,and achieves the best effect at a dose of 10 μg per rat(about 200 mg)injected 4 hours before the seizures are induced.Part Two: the regulatory effect of HMGB1 receptor antagonist BoxA on neuroinflammatory response and microglial activation in rat model of SE Objective: To observe the regulatory effect of BoxA,a selective inhibitor of HMGB1 receptor,on neuroinflammatory response and microglial activation in a rat model of SE.Methods: A rat model of Status epilepticus(SE)model was established using Pilocarpine.Rat model,using HMGB1 antagonist BoxA(can selectively block the binding of HMGB1 and TLRs)to observe the study.Rats were divided into the following 4 groups: sham operation grou P(6 rats),sham operation + BoxA group(6rats),the SE model group(8 rats)and the SE model + BoxA group(8 rats).Except for the sham operation group and the sham operation + BoxA group,the model group and the model + BoxA group all used Pilocarpine to induce the rat SE model.24 hours after SE induction,the blood brain barrier(BBB)permeability and brain water content were evaluated in the brain of epileptic rats;TUNEL staining was used to observe the apoptosis of hippocampal neurons,and immunohistochemical staining was used to evaluate the morphological and functional changes of microglia.Then cleaved caspase-3,cas Pase-3 proteins were determined by Western blotting.And the expression of proinflammatory genes of IL-1beta,IL-6 and TNF-alpha were evaluated by polymerase chain reaction(q PCR)in vivo.Results: Com Pared with the mice without BoxA treatment,BoxA treatment group can significantly increase the seizure latency and reduce the seizure frequency,but no seizures were found in the sham operation + BoxA group;2.After the induction of SE model,the permeability of BBB in the hippocampus of rats increased,and the content of brain water increased.3.TUNEL staining was used to evaluate the apoptosis of nerve cells.The apoptotic index of SE model group+BoxA group was significantly lower than the index of SE model group,and the protein expression level of cleaved caspase 3 in hippocampal CA1 area was significantly lower than that of SE model group.4.Compared with SE model group,activation of microglia was found in SE model + BoxA group,and the expression of IL-1beta,IL-6 and TNF-alpha related pro-inflammatory cytokines was significantly decreased by polymerase chain reaction(q PCR);Conclusion: HMGB1 antagonist BoxA can prevent proinflammatory changes and microglial activation in acute seizure models,increase seizure latency and reduce seizure frequency,inhibit BBB breakdown and neuronal apoptosis in hippocampus.Part Three: BoxA Regulates Microglial Inflammatory Factors and HMGB1 Signaling in LPS Stimulated Isolated Microglia Model Objective: To observe the regulatory effect of BoxA,a selective inhibitor of HMGB1 receptor,on the secretion of inflammatory factors and the expression of HMGB1-TLR2/4 in isolated microglial cells after SE ex vitro.Methods: After BoxA-pretreatment and the induction of SE,microglia were isolated from hippocampal CA1 region 24 h-Post SE ex vivo.Lipopolysaccharide(LPS)was used to activate and q PCR was used to detect the expression levels of inflammatory cytokines IL-1beta,IL-6 and TNF-alpha.HMGB1,TLR2 and TLR4 proteins were determined by Western blotting.Results: Compared with the SE model group,the SE model + BoxA group found that the activation of microglial cells was significantly inhibited,and q PCR results showed that the expression of related pro-inflammatory cytokines IL-1beta,IL-6 and TNF-alpha was significant reduced;Western blot results showed that the expression levels of HMGB1,TLR2 and TLR4 in SE model group were significantly increased.However,the expression of TLR2 and TLR4 was only inhibited in SE + BoxA group,but the ex Pression of HMGB1 was not inhibited.Conclusion: HMGB1 antagonist BoxA can down-regulate the expression of TLR2 and TLR4.The results of this study suggest that the HMGB1-TLR2/TLR4 signal axis plays an important role in the regulation of microglia activation during SE.This signal axis has important value as a potential target for SE clinical treatment. |
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