抑制Pannexin1可能通过减少NLRP3炎症小体的激活减轻脓毒症小鼠脑损伤

Inhibition of Pannexin 1 may reduce brain injury in septic mice by reducing NLRP3 inflammasome activation

目的:探究缝隙连接蛋白Pannexin 1(Panx1)是否参与脓毒症小鼠脑损伤的发生发展及其可能的作用机制。方法:清洁级健康成年雄性C57BL/6小鼠45只,采用随机数字表法分为以下3组:假手术(Sham)组、盲肠结扎穿孔(CLP)组、甘珀酸(Panx1通道抑制剂,CBX)预处理+盲肠结扎穿孔(CLP+CBX)组。除Sham组外,其余两组采用CLP法制备脓毒症脑损伤模型。CLP+CBX组于造模前30 min经双侧侧脑室各注射1μL CBX,对照组双侧侧脑室注射等剂量生理盐水。造模后24 h对各组小鼠行神经行为评分,后麻醉、处死小鼠,心脏灌流后取脑组织,HE染色切片观察海马组织病理损伤,ELISA试剂盒法测定海马组织IL-1β与TNF-α含量,Western Blot及免疫组化检测海马组织Panx1蛋白表达水平,Western Blot检测NLRP3、Caspase-1蛋白表达水平,RT-PCR检测Panx1、NLRP3、Caspase-1及IL-1β与TNF-αmRNA表达水平。结果:与Sham组相比,CLP组神经行为评分明显降低,海马神经元病理损伤明显,IL-1β与TNF-α表达量增加,Panx1、NLRP3、Caspase-1蛋白及mRNA表达水平升高,IL-1β与TNF-αmRNA表达增加(P<0.05);与CLP组相比,CLP+CBX组神经行为评分升高,海马组织病理损伤减轻,IL-1β与TNF-α表达量减少,Panx1、NLRP3、Caspase-1蛋白及mRNA表达水平降低,IL-1β与TNF-αmRNA表达减少(P<0.05)。结论:抑制Panx1可降低神经炎症反应,从而减轻脓毒症小鼠脑损伤,这可能与NLRP3炎症小体激活减少有关。

Objective:To investigate whether Pannexin 1(Panx1)is involved in the development of brain injury in septic rats and its possible mechanism.Methods:Forty-five healthy adult male C57BL/6 mice were randomly divided into three groups:Sham operation(Sham)group,cecal ligation and perforation(CLP)group,carbenoxolone(CBX;Panx1 channel inhibitor)pretreatment+CLP(CLP+CBX)group.Except for the Sham group,the other two groups were subjected to CLP to prepare the septic brain injury model.The CLP+CBX group was injected with 1μL CBX in each lateral ventricle 30min before modeling,and the control group was injected with the same dose of normal saline.The mice were anesthetized and sacrificed.After cardiac perfusion,the brain tissue was taken for pathological observation of hippocampal tissue.The contents of IL-1βand TNF-αin hippocampal tissue were measured by ELISA kit,the expression of Panx1 protein in hippocampal tissue was detected by Western Blot and immunohistochemistry.The protein expression levels of NLRP3 and caspase-1 were detected by Western Blot,and the mRNA expression levels of Panx1,NLRP3,caspase-1,IL-1β,and TNF-αwere detected by RT-PCR.Results:Compared with that respectively in the Sham group,the neurobehavioral score of the CLP group was significantly decreased,the pathological damage of hippocampal neurons was obvious,the expression levels of IL-1βand TNF-αwere increased,and the protein and mRNA expression levels of Panx1,NLRP3 and caspase-1 were increased.The mRNA expressions of IL-1βand TNF-αwere increased(P<0.05).Compared with that respectively in the CLP group,the neurobehavioral score of the CLP+CBX group was increased,the pathological damage of hippocampal tissue was alleviated,the expression levels of IL-1βand TNF-αwere decreased,the protein and mRNA expression levels of Panx1,NLRP3 and caspase-1 were decreased,and the mRNA expressions of IL-1βand TNF-αwere decreased(P<0.05).Conclusion:Inhibition of Panx1can reduce neuroinflammatory response,thereby reducing brain injury in sepsis mice,which may be related to the decreased activation of NLRP3 inflammasome.