Erbin在小鼠脓毒症相关性脑病中的作用及其与NLRP3炎症小体的关系

Role of Erbin in sepsis-associated encephalopathy in mice and the relationship with NLRP3 inflammasomes

目的评价ErbB2相互作用蛋白(Erbin)在小鼠脓毒症相关性脑病(SAE)中的作用及其与NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体在其中的作用。方法SPF级健康雄性野生型C57BL/6小鼠和Erbin(-/-)C57BL/6小鼠各60只,8~10周龄,体重20~25g,采用随机数字表法分为4组(n=30):野生型假手术组(WT+Sham组)、野生型SAE组(WT+SAE)、Erbin(-/-)假手术组(EKO+Sham组)和Erbin(-/-)+SAE组(EKO+SAE组)。采用盲肠结扎穿孔法制备小鼠SAE模型。于造模后7 d时行旷场实验(移动总距离),造模后8 d时行新物体识别实验(识别指数),造模后10 d时行Morris水迷宫实验(目标象限停留时间)。于造模后24 h时处死小鼠取海马组织,采用HE染色观察海马组织病理学结果,并计数神经元,计算神经元存活率;采用Western blot法检测NLRP3、caspase-1和凋亡相关斑点样蛋白(ASC)表达;采用免疫组化法计数NLRP3阳性细胞;采用ELISA法检测IL-1β、TNF-α和IL-18含量。结果与WT+Sham组比较,WT+SAE组目标象限停留时间缩短,识别指数和神经元存活率降低,海马组织IL-1β、IL-18、TNF-α含量和NLRP3阳性细胞计数升高,NLRP3、caspase-1和ASC表达上调(P<0.05);与EKO+Sham组比较,EKO+SAE组目标象限停留时间缩短,识别指数和神经元存活率降低,海马组织IL-1β、IL-18、TNF-α含量和NLRP3阳性细胞计数升高,NLRP3、caspase-1和ASC表达上调(P<0.05);与WT+SAE组比较,EKO+SAE组目标象限停留时间缩短,识别指数和神经元存活率降低,海马组织IL-1β、IL-18、TNF-α含量和NLRP3阳性细胞计数升高,NLRP3、caspase-1和ASC表达上调(P<0.05);4组移动总距离比较差异无统计学意义(P>0.05)。结论Erbin可通过抑制NLRP3炎症小体的活化,对SAE小鼠产生内源性保护作用。

Objective To evaluate the role of ErbB2 interacting protein(Erbin)in sepsis-associated encephalopathy(SAE)in mice and the relationship with nod-like receptor thermoprotein domain associated protein 3(NLRP3)inflammasomes.Methods Sixty SPF-grade healthy male wild-type C57BL/6 mice and 60 Erbin(-/-)C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups(n=30 each)by a random number table method:wild-type sham operation group(WT+Sham group),wild-type SAE group(WT+SAE group),Erbin(-/-)sham operation group(EKO+Sham group)and Erbin(-/-)plus SAE group(EKO+SAE group).The model of SAE was established by cecal ligation and perforation in anesthetized mice.Open field test(total distance moved)was performed at 7 days after establishing the model,new object recognition test(recognition index)was performed at 8 days after establishing the model,and Morris water maze test(time of staying at target quadrant)was performed at 10 days after establishing the model.The mice were sacrificed,and hippocampal tissues were removed for microscopic examination of pathologic changes(by HE staining)and for determination of neuron count,expression of NLRP3,caspase-1 and apoptosis-associated speck-like protein containing a CARD(ASC)(by Western blot),the number of NLRP3 positive cells(by immunohistochemistry),and contents of interleukin-1beta(IL-1β),tumor necrosis factor-alpha(TNF-α)and IL-18(by enzyme-linked immunosorbent assay).The cell survival rate was calculated.Results Compared with group WT+Sham,the time of staying at target quadrant was significantly shortened,the recognition index and cell survival rate were decreased,the contents of IL-1β,IL-18 and TNF-αand the number of NLRP3 positive cells were increased,and the expression of NLRP3,caspase-1 and ASC was up-regulated in group WT+SAE(P<0.05).Compared with group EKO+Sham,the time of staying at target quadrant was significantly shortened,the recognition index and cell survival rate were decreased,the contents of IL-1β,IL-18 and TNF-αand the number of NLRP3 positive cells were increased,and the expression of NLRP3,caspase-1 and ASC was up-regulated in group EKO+SAE(P<0.05).Compared with group WT+SAE,the time of staying at target quadrant was significantly shortened,the recognition index and cell survival rate were decreased,the contents of IL-1β,IL-18 and TNF-αand the number of NLRP3 positive cells were increased,and the expression of NLRP3,caspase-1 and ASC was up-regulated in group EKO+SAE(P<0.05).There was no significant difference in total distance moved between the four groups(P>0.05).Conclusion Erbin can exert endogenous protection by inhibiting the activation of NLRP3 inflammasomes in mice with SAE.