透明质酸钠修饰过氧化钙纳米粒子诱导胃癌细胞焦亡

Sodium-hyaluronate-modified calcium peroxide nanoparticles induce pyroptosis in gastric cancer cells in vitro

目的探讨透明质酸钠修饰过氧化钙纳米粒子(sodium-hyaluronate-modified calcium oxide nanoparticles,SH-CaO_(2)NPs)诱导人胃癌细胞焦亡的作用及其可能机制。方法运用透射电镜(transmission electron microscope,TEM)、X射线衍射(X-ray diffraction,XRD)、红外光谱及Zeta电位观测SH-CaO_(2)NPs的合成情况;细胞划痕实验验证SH-CaO_(2)NPs对人胃癌细胞迁移能力的影响;CCK-8法检测SH-CaO_(2)NPs对胃癌细胞增殖活性的影响以及使用Nod样受体蛋白3(NOD-like receptor protein 3,NLRP3)、半胱氨酸天冬氨酸蛋白酶1(cysteinyl aspartate specific proteinase,Caspase-1)抑制剂预处理后胃癌细胞的增殖活性;DCFH-DA荧光探针及流式细胞仪检测细胞内活性氧的表达量;免疫荧光法及Western blot检测各组细胞NLRP3、Caspase-1、GSDMD蛋白表达水平。结果TEM、XRD、红外光谱及Zeta电位观测结果提示SH-CaO_(2)NPs成功制备。CCK-8法及细胞划痕实验显示,SH-CaO_(2)NPs作用于人胃癌细胞24 h后,胃癌细胞生长明显受到抑制(P<0.001)。ROS荧光及流式细胞仪结果显示,SH-CaO_(2)NPs作用于人胃癌细胞24 h后肿瘤细胞内ROS含量升高(P<0.001),予以ROS抑制剂(NAC)后细胞内NLRP3蛋白含量升高,Caspase-1、GSDMD剪切体即活性片段表达量上升(P<0.001),予以NLRP3、Caspase-1抑制剂可逆转此过程。结论SH-CaO_(2)NPs可抑制人胃癌细胞活力,可能通过激活ROS/NLRP3/Caspase-1/GSDMD信号通路介导炎症反应和细胞焦亡。

Objective To investigate the role of sodium-hyaluronate-modified calcium peroxide nanoparticles(SH-CaO_(2)NPs)in inducing pyroptosis in human gastric cancer cells and its possible mechanisms.Methods Transmission electron microscopy(TEM),X-ray diffraction(XRD),infrared spectroscopy,and zeta potential test were used to confirm the synthesis of SH-CaO_(2)NPs.Cell scratch assay and CCK-8 assay were employed to observe the impacts of SH-CaO_(2)NPs on the migration and proliferation of human gastric cancer cell line HGC-27.The generation of reactive oxygen species(ROS)was observed with confocal laser scanning microscopy(CLSM)and quantified with flow cytometry in the cells after SH-CaO_(2)NPs treatment or with pretreatment with ROS inhibitor NAC.Furthermore,the effects of pretreatment of NLRP3 inhibitor(MCC950)and Caspase-1 inhibitor(VX765)on the proliferative activity and on expression of their own and their downstream GSDMD in HGC-27 cells were also investigated with CCK-8 assay,immunofluorescence assay and Western blotting.Results TEM images,XRD,infrared spectroscopy,and zeta potential test confirmed the successful preparation of SH-CaO_(2)NPs.Cell scratch assay and CCK-8 assay showed that application of SH-CaO_(2)NPs for 24 h significantly inhibited the proliferation of HGC-27 cells(P<0.001),while,CLSM and flow cytometry indicated the treatment also promoted the production of ROS(P<0.001).Pretreatment of ROS inhibitor NAC resulted in up-regulation of NLRP3,and increased expression levels of cleaved Caspase-1 and N-terminal fragment of GSDMD(P<0.001),while pretreatment of both NLRP3 inhibitor and Caspase-1 inhibitor could reverse the process.Conclusion SH-CaO_(2)NPs inhibit cell viability of human gastric cancer,which may mediate the inflammatory response and pyroptosis by activating the ROS/NLRP3/Caspase-1/GSDMD signaling pathway.