内质网应激调控二氧化硅诱导的RAW264.7细胞自噬及肿瘤坏死因子-α分泌

Endoplasmic reticulum stress regulates autophagy and tumor necrosis factor-αsecretion of RAW264.7 cells induced by silica

目的探讨内质网应激(ERS)在二氧化硅(SiO2)诱导的RAW264.7细胞自噬中的作用及对肿瘤坏死因子-α(TNF-α)分泌的影响.方法以200μg/ml SiO2刺激的RAW264.7细胞为体外细胞模型,以SiO2不同处理时间分组,包括0h组(对照组)、6、12、24和48 h组,通过吖啶橙及单丹(磺)酰戊二胺荧光(MDC)染色检测细胞自噬小体的形成.应用实时聚合酶链反应(实时PCR)检测自噬相关分子Beclin1 mRNA表达,蛋白免疫印迹(western blotting)检测自噬相关蛋白LC3Ⅰ、LC3Ⅱ与Beclin1的表达;应用实时PCR和western blotting检测ERS特异标记分子BiP的表达;酶联免疫吸附法(ELISA)检测RAW 264.7细胞转化生长因子-β1(TGF-β1)和TNF-α的分泌.以ERS抑制剂4-PBA进行干预试验,包括空白对照组、SiO2组、1μmol/L 4-PBA+SiO2组、10μmol/L 4-PBA+SiO2组、20μmol/L 4-PBA+SiO2组,检测各组LC3Ⅰ、LC3Ⅱ与Beclin1蛋白的表达及TGF-β1、TNF-α的分泌变化.结果与对照组比较,SiO2诱导RAW264.7细胞各时间组荧光强度均明显增强,差异均有统计学意义(P<0.05);与对照组比较,SiO2诱导RAW264.7细胞各时间组的Beclin1 mRNA表达均明显增加,LC3Ⅰ、LC3Ⅱ和Beclin1蛋白表达均明显增加,差异均有统计学意义(P<0.05);与对照组比较,SiO2诱导RAW264.7细胞6、12、24 h组BiPmRNA和蛋白表达均明显增加,6h达高峰,差异均有统计学意义(P<0.05);与SiO2组比较,随着4-PBA浓度增加,RAW264.7细胞的LC3-II和Beclin 1蛋白水平逐渐下调,差异均有统计学意义(P<0.05);与SiO2组比较,1、10、20μmol/L 4-PBA+SiO2组RAW264.7细胞TNF-α分泌水平明显下降,差异均有统计学意义(P<0.05).结论SiO2诱导的ERS可能参与了RAW264.7细胞自噬以及TNF-α的分泌过程.

Objective To investigate the role of endoplasmic reticulum stress(ERS)in the autophagy of RAW264.7 cells induced by SiO2 and its effect on the secretion of tumor necrosis factor-α.Methods RAW264.7 cells stimulated by 200μg/ml SiO2 were used as an vitro cell model,and different treatment times of SiO2 were used as variables.They were divided into 0 h treatment group(blank control group),6 h,12 h,24 h,and 48 h treatment group.The formation of autophagospores was detected by acridine orange and mondane-sulfonate(MDC)staining.Application of real-time quantitative PCR(Real-time PCR)to detect autophagy related molecular Beclin1 mRNA expression and protein immunoblot(Western Blotting)detecting autophagy related proteins LC3Ⅰ,LC3Ⅱand expression of Beclin1.Real-time PCR and Western blotting were used to detect the expression of ERS specific marker BiP.Secretion of RAW 264.7 cell transforming growth factor-β1(TGF-β1)and tumor necrosis factor-α(TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA).ERS inhibitors 4-PBA intervention experiment,including blank control group,SiO2,1μmol/L 4-PBA+SiO2,10μmol/L 4-PBA+SiO2,20μmol/L 4-PBA+SiO2 treatment group,Western blotting testing LC3Ⅰ,LC3Ⅱand expression of Beclin1 changes.Results Compared with the control group,SiO2-induced fluorescence intensity in RAW264.7 cells was significantly increased,with statistically significant differences(P<0.05).Compared with control group,with SiO2 processing time prolonged,LC3Ⅰ,LC3ⅡBeclin1 mRNA and protein expression and protein expression increased,6 h,24 h,the height of the differences were statistically significant(P<0.05);Compared with the control group,the mRNA and protein expression level of BiP reached the peak for 6 h,and the expression level in 6 h,12 h and 24 h groups increased significantly,and the difference was statistically significant(P<0.05).Compared with the SiO2 stimulation group,the LC3Ⅱand Beclin 1 protein levels of RAW264.7 cells were gradually down-regulated by increasing the dose of 4-PBA.With the increase of 4-PBA concentration,the down-regulated levels were more significant,and the difference was statistically significant(P<0.05).Compared with the SiO2 stimulation group,the TNF-αsecretion level of RAW264.7 cells significantly decreased of 1,10,20μmol/L 4-PBA+SiO2 treatment group,and the difference was statistically significant(P<0.05).Conclusion ERS induced by SiO2 is involved in the secretion of autophagy and TNF-αin RAW264.7 cells.