丙泊酚通过NLRP3/ASC/Caspase-1通路对肺癌A549细胞增殖及焦亡的影响

Effects of Propofol on Proliferation and Pyroptosis of Lung CancerA549 Cells Through NLRP3/ASC/Caspase-1 Pathway

目的探讨丙泊酚通过核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/凋亡相关斑点样蛋白(ASC)/半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)通路对肺癌A549细胞增殖及焦亡的影响。方法将肺癌A549细胞随机分为空白对照组、低浓度丙泊酚组(30μmol/L)、中浓度丙泊酚组(60μmol/L)、高浓度丙泊酚组(120μmol/L)、高浓度丙泊酚+NLRP3抑制剂组(120μmol/L+100 nmol/L);各组进行相应处理后。四甲基偶氮唑蓝法检测肺癌A549细胞增殖;酶联免疫吸附法检测肺癌A549细胞上清白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)水平;Hoechst/碘化丙啶(PI)双重染色检测肺癌A549细胞焦亡;荧光定量PCR和蛋白免疫印迹法分别检测肺癌A549细胞增殖[原癌基因(c-Myc)、细胞周期蛋白D1(CyclinD1)]、焦亡[消皮素D-N端(GSDM D-N)、IL-1β]及NLRP3、ASC、Caspase-1信使核糖核酸(mRNA)和蛋白表达水平。结果空白对照组细胞呈蓝色荧光;低、中、高浓度丙泊酚组红色荧光逐渐增加;与高浓度丙泊酚组相比,高浓度丙泊酚+NLRP3抑制剂组红色荧光较少;表明丙泊酚可促进肺癌A549细胞焦亡,NLRP3抑制剂可反转高浓度丙泊酚的作用效果。与空白对照组相比,低、中、高浓度丙泊酚组肺癌A549细胞存活率、c-Myc、CyclinD1 mRNA和蛋白表达水平依次降低,上清IL-6、IL-1β、IL-18水平、GSDM D-N、IL-1β、NLRP3、ASC、Caspase-1 mRNA和蛋白表达水平依次升高(P<0.05);与高浓度丙泊酚组相比,高浓度丙泊酚+NLRP3抑制剂组肺癌A549细胞存活率、c-Myc、CyclinD1 mRNA和蛋白表达水平升高,上清IL-6、IL-1β、IL-18水平、GSDM D-N、IL-1β、NLRP3、ASC、Caspase-1 mRNA和蛋白表达水平降低(P<0.05)。结论丙泊酚可能通过激活NLRP3/ASC/Caspase-1通路来抑制肺癌A549细胞增殖,促进细胞焦亡。

Objective To investigate effects of propofol on the proliferation and pyroptosis of lung cancer A549 cells through nucleotide binding oligomerization domain like receptor protein 3(NLRP3)/apoptosis-associated speck-like protein containing a CARD(ASC)/Caspase-1 pathway.Methods Lung cancer A549 cells were randomly divided into groups as following:blank control group,low concentration propofol group(30μmol/L),medium concentration propofol group(60μmol/L),high concentration propofol group(120μmol/L),and high concentration propofol+NLRP3 inhibitor group(120μmol/L+100 nmol/L);Each group was then treated accordingly.Methyl thiazolyl tetrazolium assay was used to detect the proliferation;Enzyme-linked immunosorbent assay was used to detect levels of interleukin-6(IL-6),interleukin-1β(IL-1β)and interleukin-18(IL-18);Hoechst/propidium iodide(PI)double staining was used to detect pyroptosis;fluorescence quantitative PCR and western blotting were used to detect the proliferation(c-Myc,CyclinD1),pyroptosis[gasdermin D-N(GSDMD-N),IL-1β],NLRP3,ASC,Caspase-1 messenger RNA(mRNA)and protein expression levels,respectively.Results The cells in the blank control group showed blue fluorescence;Red fluorescence increased gradually in low,medium and high concentration propofol groups;Compared with the high concentration propofol group,the high concentration propofol+NLRP3 inhibitor group had less red fluorescence.The results showed that propofol could promote lung cancer A549 cells pyroptosis,and NLRP3 inhibitor could reverse the effect of high concentration propofol.Compared with the blank control group,the survival rate,c-Myc,CyclinD1 mRNA and protein expression levels of lung cancer A549 cells in low,medium and high concentration propofol groups were decreased in turn,while the supernatant IL-6,IL-1β,IL-18 levels,GSDMD-N,IL-1β,NLRP3,ASC,Caspase-1 mRNA and protein expression levels were increased in turn(P<0.05);Compared with the high concentration propofol group,the survival rate,c-Myc,CyclinD1 mRNA and protein expression levels of lung cancer A549 cells in the high concentration propofol+NLRP3 inhibitor group were increased,while the supernatant IL-6,IL-1β,IL-18 levels,GSDMD-N,IL-1β,NLRP3,ASC,Caspase-1 mRNA and protein expression levels were decreased(P<0.05).Conclusion Propofol may inhibit lung cancer A549 cells proliferation and promote pyroptosis by activating NLRP3/ASC/Caspase-1 pathway.