| Objective: The mechanism of inflammatory injury induced by ICH is complicated,aggravating the condition and affecting the prognosis.Recently,many studies have found that nuclear factor-?B(NF-?B)is a crucial transcription factor that initiates inflammation in the perihematomal region of ICH.NF-?B essential modulator(NEMO)-binding domain(NBD)peptide,a cell-permeable peptide spanning the NBD of IKK? or IKK?,functions as a highly specific inhibitor of NF-?B.This peptide can negatively regulate the NF-?B pathway.In this study,we used the ICH mouse model established by autologous blood injection,to explore the effects and underlying pathomechanisms of NBD peptides after ICH.Methods: The mice were divided into five groups randomly: control group,ICH group,NBD peptide control group,NBD peptide pre-treatment+ICH group,mut-NBD pre-treatment+ICH group.Experimental animals were administered with NBD or control(mutated)peptides 2 hours before ICH by intracerebroventricular injection(icv.).First,the forelimb asymmetry tests were used to evaluate the neurological deficits and the water content in brain were detected by dry-wet weight.Then,the content of NF-?B,p-I?B?,p-IKK?/?,and TNF-? and microglia activation in perihematomal region were assessed by Western blot,ELISA or immunofluorescence.Finally,NBD peptides were treated after p65 siRNA transient knockdown p65.Western Blot was used to detect the expression of p65 and its phosphorylation in the perihematomal tissue.ELISA was used to detect changes in inflammatory factor TNF?,behavioral test to assess neurological impairment.These methods were used to assess whether NBD peptide specifically inhibits NF-?B activation induced by ICH.Results: These results indicate that NBD peptides significantly reduced the forelimb asymmetry score(p<0.001),and brain water content were decreased in ipsilateral basal ganglia and cortex(p<0.01).What's more,NBD peptides treatment markedly reduced I?B? and p65 phosphorylation(p<0.01),blocked nuclear translocation of p65(p<0.001),and upregulated I?B? expression by NF-?B after ICH induction(p<0.05).We also found that NBD peptides suppressed microglia inflammation and lowered the expression of TNF-? and IL-1?(p<0.01).Further experiments were performed when mice were treated with NBD peptides in the presence of p65 siRNA,NBD peptides cannot reduce p65 phosphorylation and expression(p>0.05).However,in the presence of control siRNA,NBD peptides reduced p65 expression and significantly inhibited its phosphorylation(p<0.05),which was obvious higher than that in the presence of p65 siRNA.It confirmed that the specificity of NBD peptides inhibit ICH-induced NF-?B activation.The mut-NBD peptide does not exert any neuroprotective effects after ICH compared with the NBD peptide group(p>0.05).Conclusion: Summary,this study demonstrated that NBD peptides could improve neurological deficits and reduce brain edema to exert neuroprotective role after ICH.NBD peptide may specifically inhibit the activity of NF-?B signaling pathway induced by ICH by reducing the phosphorylations of IKK?/?,I?B?,NF-?B p65,inhibiting the nuclear translocation of p65,increase the expression of I?B?,and does not affect NF-?B basal activity.In addition,NBD polypeptide may also reduce the inflammatory injury after ICH by inhibiting the activation and proliferation of microglia and the release of inflammatory factors.We believed that NBD peptide might be a potential candidate for a novel therapeutic strategy for ICH by inhibiting perihematomal inflammation. |
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