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.
Abstract
Objective To evaluate the role of Z-DNA-binding protein 1(ZBP1)/receptor-interac-ting 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+dim-ethyl 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 10 μg/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-1 beta(IL-1β),IL-6,IL-18 and tumor necrosis factor-alpha(TNF-α)in cell supernatant were determined by en-zyme-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 Com-pared with group C,the cell survival rate was significantly decreased,the cell pyroptosis rate and concentra-tions of IL-1β,IL-6,IL-18 and TNF-α in 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 sig-nificantly increased,the cell pyroptosis rate and concentrations of IL-1β,IL-6,IL-18 and TNF-α in the su-pernatant were decreased,and the expression of ZBP1,RIPK1,caspase-1 and GSDMD was down-regulated in group LPS-ATP+siRNA(P＜0.05).Compared with group LPS-ATP+DMSO,the cell survival rate was significantly increased,the cell pyroptosis rate and concentrations of IL-1β,IL-6,IL-18 and TNF-α in the supernatant were decreased,the expression of ZBP1,caspase-1 and GSDMD was down-regulated(P＜0.05),and no significant change was found in the expression of ZBP1 in group LPS-ATP+nec-1(P＞0.05).Conclusions Activation of ZBP1/RIPK1 signaling pathway is involved in LPS-ATP-induced pyroptosis in macrophages of mice.
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