Endoplasmic reticulum stress regulates autophagy and tumor necrosis factor-α secretion of RAW264.7 cells induced by silica
10.3760/cma.j.issn.1001-9391.2020.02.003
- VernacularTitle: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
- Author:
Huiping CHEN
1
;
Yu ZHOU
;
Xiaofeng QIN
;
Lei WANG
;
Xiaofang LIN
;
Hui CHEN
;
Yongbin HU
Author Information
1. 410000 长沙，中南大学湘雅医院病理科
- Keywords:
Silica;
Macrophages, alveolar;
Tumor necrosis factor-alpha;
Transforming growth factor beta1;
Autophagy;
Endoplasmic reticulum stress
- From:
Chinese Journal of Industrial Hygiene and Occupational Diseases
2020;38(2):91-95
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the role of endoplasmic reticulum stress (ERS) in the autophagy of RAW264.7 cells induced by SiO 2 and its effect on the secretion of tumor necrosis factor-α. Methods:RAW264.7 cells stimulated by 200 μg/ml SiO 2 were used as an vitro cell model, and different treatment times of SiO 2 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, LC3Ⅱ and 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, SiO 2, 1 μmol/L 4-PBA+SiO 2, 10 μmol/L 4-PBA+SiO 2, 20 μmol/L 4-PBA+SiO 2 treatment group, Western blotting testing LC3Ⅰ, LC3Ⅱ and expression of Beclin1 changes. Results:Compared with the control group, SiO 2-induced fluorescence intensity in RAW264.7 cells was significantly increased, with statistically significant differences ( P<0.05) . Compared with control group, with SiO 2 processing time prolonged, LC3Ⅰ, LC3Ⅱ Beclin1 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 SiO 2 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 SiO 2 stimulation group, the TNF-α secretion level of RAW264.7 cells significantly decreased of 1, 10, 20 μmol/L 4-PBA+SiO 2 treatment group, and the difference was statistically significant ( P<0.05) . Conclusion:ERS induced by SiO 2 is involved in the secretion of autophagy and TNF-α in RAW264.7 cells.
