小鼠巨噬细胞焦亡时TIPE2与caspase-11的关系

Relationship between TIPE2 and caspase-11 during pyroptosis in macrophages of mice

目的评价小鼠巨噬细胞焦亡时肿瘤坏死因子α诱导蛋白8样分子2(TIPE2)与caspase-11的关系。方法采用随机数字表法将小鼠J774A.1巨噬细胞分4组(n=6):非特异性siRNA(Scr-siRNA)组(S组)、Scr-siRNA+LPS/ATP组(S+LPS/ATP组)、TIPE2-siRNA组(T组)和TIPE2-siRNA+LPS/ATP组(T+LPS/ATP组)。各组分别加入相应的siRNA和Lipofectamine2000转染试剂,转染24~48 h,然后S+LPS/ATP组和T+LPS/ATP组加入LPS 1.0μg/ml,孵育5 h后再加入ATP 5.0 mmol/L孵育1 h。收集细胞,采用Western blot法检测TIPE2、caspase-11、NOD样受体热蛋白结构域相关蛋白3(NLRP3)和caspase-1表达;收集上清液,测定LDH和MPO活性,采用ELISA法检测IL-1β和IL-18浓度。结果与S组比较,S+LPS/ATP组细胞TIPE2表达下调,caspase-11、NLRP3和caspase-1的表达上调,上清液LDH和MPO活性、IL-1β和IL-18浓度升高(P<0.05)。与T组比较,T+LPS/ATP组细胞TIPE2表达下调,caspase-11、NLRP3和caspase-1表达上调,上清液中LDH和MPO活性、IL-1β和IL-18浓度升高(P<0.05)。与S+LPS/ATP组比较,T+LPS/ATP组细胞TIPE2表达下调,caspase-11、NLRP3和caspase-1表达上调,上清液LDH和MPO活性、IL-1β和IL-18浓度升高(P<0.05)。结论TIPE2可通过下调caspase-11表达,抑制小鼠巨噬细胞焦亡。

Objective To evaluate the relationship between tumor necrosis factor-alpha-induced protein 8-like 2(TIPE2)and caspase-11 during pyroptosis in macrophages of mice.Methods J774A.1 macrophages of mice were divided into 4 groups(n=6 each)using a random number table method:non-specific siRNA(Scr-siRNA)group(S group),Scr-siRNA plus LPS/ATP group(S+LPS/ATP group),TIPE2-siRNA group(T group)and TIPE2-siRNA plus LPS/ATP group(T+LPS/ATP group).The corresponding siRNA and Lipofectamine2000 transfection reagents were added to each group,and transfection was performed for 24-48 h,and in addition LPS 1.0μg/ml was then added,cells were incubated for 5 h,then ATP 5.0 mmol/L was added,and cells were incubated for 1 h in S+LPS/ATP and T+LPS/ATP groups.Cells were collected to detect the expression of TIPE2,caspase-11,NOD-like receptor familypyrin domain containing 3(NLRP3)and caspase-1(by Western blot).The supernatant was collected for determination of lactic dehydrogenase(LDH)and myeloperoxidase(MPO)activities and concentrations of interleukin-1beta(IL-1β)and IL-18(by enzyme-linked immunosorbent assay).Results Compared with group S,the expression of TIPE2 was significantly down-regulated,the expression of caspase-11,NLRP3 and caspase-1 was up-regulated,and the activities of LDH and MPO and concentrations of IL-1βand IL-18 in supernatant were increased in group S+LPS/ATP(P<0.05).Compared with group T,the expression of TIPE2 was significantly down-regulated,the expression of caspase-11,NLRP3 and caspase-1 was up-regulated,and the activities of LDH and MPO and concentrations of IL-1βand IL-18 in supernatant were increased in group T+LPS/ATP(P<0.05).Compared with group S+LPS/ATP,the expression of TIPE2 was significantly down-regulated,the expression of caspase-11,NLRP3 and caspase-1 was up-regulated,and and the activities of LDH and MPO and concentrations of IL-1βand IL-18 in supernatant were increased in group T+LPS/ATP(P<0.05).Conclusion TIPE2 inhibits pyroptosis in macrophages through down-regulating the expression of caspase-11 in mice.