H P Chen1, Y Zhou2, X F Qin2, L Wang1, X F Lin2, H Chen2, Y B Hu3. 1. Department of Pathology, Xiangya Hospital, Central South University, Changsha 410000, China. 2. Xiangya Medical College, Central South University, Changsha 410013, China. 3. Department of Pathology, Xiangya Hospital, Central South University, Changsha 410000, China; Xiangya Medical College, Central South University, Changsha 410013, China.
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.
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.