烟草烟雾提取物通过ROS/NLRP3炎性小体信号通路介导人子宫内膜上皮细胞焦亡

Cigarette smoke extract induces ROS/NLRP3 inflammasome pathway-mediated pyroptosis in human endometrial epithelial cells

目的探讨烟草烟雾提取物(cigarette smoke extract,CSE)诱发人子宫内膜上皮细胞(HEEC)焦亡的机制。方法将永生化HEEC细胞,与不同浓度的(0%、0.5%、1.0%和2.0%)CSE共培养24 h。使用荧光探针(DCFH-DA)试剂盒检测细胞活性氧(reactive oxygen species,ROS)的含量;CCK-8试剂盒检测细胞活性及Western blot检测NOD-like receptor protein 3(NLRP3);Apoptosis-associated speck-like protein with CARD domain(ASC)、Caspase-1、Gasdermin D(GSDMD)和interleukin(IL)-1β蛋白表达。随后,分别给予ROS清除剂(Tempol)与NLRP3选择性抑制剂(MCC950)对CSE暴露的HEEC细胞进行干预,检测细胞内ROS的含量;观察细胞的形态变化,检测NLRP3、ASC、Caspase-1、GSDMD和IL-1β蛋白表达情况。结果与对照组相比,CSE促进HEEC细胞内ROS的蓄积(F=298.60;P<0.001),抑制细胞活性(F=295.80;P<0.01;P<0.001),诱导NLRP3蛋白表达(F=191.70;P<0.001),差异有统计学意义。此外,CSE破坏HEEC细胞膜完整性,导致细胞膜形成肿胀的囊泡(即焦亡小体);而超氧化物歧化酶(SOD)类似物Tempol能改善CSE导致的ROS蓄积(F=1029.00;P<0.001)以及抑制CSE诱导的HEEC细胞焦亡小体的形成和NLRP3炎性小体相关蛋白NLRP3(F=135.00;P<0.01);ASC(F=563.40;P<0.01);Caspase-1(F=88.09;P<0.01);GSDMD(F=48.21;P<0.01);IL-1β(F=74.43;P<0.01)的表达,差异有统计学意义;然而使用MCC950抑制NLRP3的活性(F=174.70;P<0.05),能够改善CSE诱导的HEEC细胞焦亡小体的形成,但并不能改变CSE对细胞内ROS蓄积的影响,差异无统计学意义(F=863.00;P>0.05)。结论CSE通过促进ROS蓄积,激活NLRP3炎性小体信号通路来介导HEEC细胞发生焦亡。

Objective To investigate the mechanism of pyroptosis of human endometrial epithelial cells(HEEC)induced by cigarette smoke extract(CSE).Methods HEEC cells were co-cultured with different concentrations of CSE(0%,0.5%,1.0%,2.0%)for 24 hours.Fluorescent probe(DCFH-DA)kit was used to detect the content of reactive oxygen species(ROS),CCK-8 kit was used to detect cell viability,and Western blot was used to detect the protein expression of NOD-like receptor protein 3(NLRP3).Apoptosis-associated speck-like protein with CARD domain(ASC)NLRP3,ASC,Caspase-1,Gasdermin D(GSDMD)and interleukin(IL)-1β.Subsequently,ROS scavenger(Tempol)and NLRP3 selective inhibitor(MCC950)were given respectively to intervene HEEC cells exposed to CSE,the intracellular ROS content was detected,the morphological changes of cells were observed,and the expressions of NLRP3,ACS,Caspase-1,GSDMD and IL-1βwere detected.Results Compared with control group,the accumulation of ROS in HEEC cells was significantly increased(F=298.60;P<0.001),and the cell viability was inhibited(F=295.80;P<0.01;P<0.001),as well as the expression of NLRP3 protein was promoted(F=191.70;P<0.001).In addition,the integrity of HEEC cell membrane was disrupted,the formation of swollen vesicles(pyroptosis body)on the cell membrane.CSE-induced ROS accumulation was significantly decreased by TempOL(F=1029.00;P<0.001),and the formation of pyroptosis body was inhibition and the expressions of the NLRP3(F=135.60;P<0.01),ASC(F=563.40;P<0.01),Caspase-1(F=88.09;P<0.01),GSDMD(F=48.21;P<0.01),IL-1β(F=74.43;P<0.01)were significantly decreased.However,inhibition of NLRP3 expression(F=174.70;P<0.05)by MCC950 with no effect on CSE-induced ROS accumulation(F=863.00;P>0.05).Conclusion CSE may induce pyroptosis in HEEC cells by promoting ROS accumulation and activating theNLRP3 inflammasome signaling pathway.