ZBP1/RIPK1信号通路在LPS-ATP致小鼠巨噬细胞焦亡中的作用  

Role of ZBP1/RIPK1 signaling pathway in lipopolysaccharide-adenosine triphosphate-induced pyroptosis in mouse macrophages

作　　者：熊瑞驿 于春锐 王宜博 王蓓莹 张晓[1] 马福国[1] 孙立新[1] Xiong Ruiyi;Yu Chunrui;Wang Yibo;Wang Beiying;Zhang Xiao;Ma Fuguo;Sun Lixin(Department of Anesthesiology,Qingdao Municipal Hospital,Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学附属青岛市市立医院麻醉科,青岛266071

出　　处：《中华麻醉学杂志》2024年第6期733-737,共5页Chinese Journal of Anesthesiology

摘　　要：目的:评价Z-DNA结合蛋白1(ZBP1)/受体相互作用蛋白激酶1(RIPK1)信号通路在脂多糖(LPS)-三磷酸腺苷(ATP)致小鼠巨噬细胞焦亡中的作用。方法:常规培养小鼠RAW264.7巨噬细胞,采用随机数字表法分为6组( n=9):空白对照组(C组)、LPS-ATP组、LPS-ATP+转染阴性对照scRNA组(LPS-ATP+scRNA组)、LPS-ATP+ZBP1小干扰RNA组(LPS-ATP+siRNA组)、LPS-ATP+二甲基亚砜组(LPS-ATP+DMSO组)和LPS-ATP+RIPK1抑制剂nec-1组(LPS-ATP+nec-1组)。LPS-ATP+siRNA组使用siRNA技术抑制ZBP1的表达,LPS-ATP+nec-1组给予nec-1抑制RIPK1的表达;C组常规培养,余5组给予10 μg/ml LPS孵育24 h,然后加入5 mmol/L ATP孵育30 min构建细胞焦亡模型。采用CCK-8法检测细胞存活情况;ELISA法检测细胞上清液IL-1β、IL-6、IL-18和TNF-α浓度;碘化丙啶荧光染色法测定细胞焦亡情况;Western blot法检测ZBP1、RIPK1、半胱氨酸天冬氨酸蛋白酶1(caspase-1)和消皮素D(GSDMD)表达。 结果:与C组比较,LPS-ATP组细胞存活率降低,细胞焦亡率升高,上清液IL-1β、IL-6、IL-18及TNF-α浓度升高,细胞ZBP1、RIPK1、caspase-1和GSDMD表达上调( P<0.05);与LPS-ATP组比较,LPS-ATP+scRNA组和LPS-ATP+DSMO组上述各指标差异无统计学意义( P>0.05);与LPS-ATP+scRNA组比较,LPS-ATP+siRNA组细胞存活率升高,细胞焦亡率降低,上清液IL-1β、IL-6、IL-18及TNF-α浓度降低,细胞ZBP1、RIPK1、caspase-1和GSDMD表达下调( P<0.05);与LPS-ATP+DSMO组比较,LPS-ATP+nec-1组细胞存活率升高,细胞焦亡率降低,上清液IL-1β、IL-6、IL-18及TNF-α浓度降低,RIPK1、caspase-1和GSDMD表达下调( P<0.05),ZBP1表达差异无统计学意义( P>0.05)。 结论:ZBP1/RIPK1信号通路激活参与了LPS-ATP致小鼠巨噬细胞焦亡的过程。Objective To evaluate the role of Z-DNA-binding protein 1(ZBP1)/receptor-interacting protein kinase 1(RIPK1)signaling pathway in lipopolysaccharide(LPS)-adenosine triphosphate(ATP)-induced pyroptosis in macrophages of mice.Methods The RAW264.7 macrophages from mice were routinely cultured and divided into 6 groups(n=9 each)using a random number table method:control group(group C),LPS-ATP group,LPS-ATP+transfection negative control scRNA group(group LPS-ATP+scRNA),LPS-ATP+ZBP1 small interference RNA group(group LPS-ATP+siRNA),LPS-ATP+dimethyl sulfoxide group(group LPS-ATP+DSMO),and LPS-ATP+RIPK1 inhibitor nec-1 group(group LPS-ATP+nec-1).The siRNA technique was used to inhibit the expression of ZBP1 in group LPS-ATP+siRNA.The RIPK1 inhibitor nec-1 was given to inhibit the expression of RIPK1 protein in group LPS-ATP+nec-1.Group C was routinely cultured.Cells were incubated with 10μg/ml LPS for 24 h,then 5 mmol/L ATP was added,and the cells were incubated for 30 min to develop the cell pyroptosis model in the remaining 5 groups.The cell survival was detected by the CCK-8 assay.The concentrations of interleukin-1beta(IL-1β),IL-6,IL-18 and tumor necrosis factor-alpha(TNF-α)in cell supernatant were determined by enzyme-linked immunosorbent assay.The pyroptosis was determined by propidium iodide fluorescence staining.Western blot was used to detect the expression of ZBP1,RIPK1,caspase-1 and GSDMD.Results Compared with group C,the cell survival rate was significantly decreased,the cell pyroptosis rate and concentrations of IL-1β,IL-6,IL-18 and TNF-αin the supernatant were increased,and the expression of ZBP1,RIPK1,caspase-1 and GSDMD was up-regulated in group LPS-ATP(P<0.05).Compared with group LPS-ATP,no significant change was found in the parameters mentioned above in group LPS-ATP+scRNA and group LPS-ATP+DSMO(P>0.05).Compared with group LPS-ATP+scRNA,the cell survival rate was significantly increased,the cell pyroptosis rate and concentrations of IL-1β,IL-6,IL-18 and TNF-αin the supernatant were decreased,and

关 键 词：脂多糖类 三磷酸腺苷 细胞焦亡 巨噬细胞 DNA结合蛋白质类 受体作用蛋白丝氨酸苏氨酸激酶类 

分 类 号：R614[医药卫生—麻醉学]