Effect of N-acetylcysteine on the transforming growth factor β1-induced transdifferentiation of retinal pigment epithelial cell to myofibroblast / 中华实验眼科杂志

ABSTRACT

Background The transdifferentiation of retinal pigment epithelial (RPE) cell to myofibroblast is a central event in the development and progression of proliferative vitreoretinopathy (PVR).N-acetylcysteine (NAC) can inhibit the transdifferentiation of multiple cells to myofibroblasts via suppressing the production of reactive oxygen species (ROS) and the phosphorylation of MAPK induced by transforming growth factor-β1 (TGF-β1),but the effect of NAC on the TGF-β1-induced transdifferentiation of human RPE cell line ARPE-19 cell to myofibroblast and its underlying molecular mechanism is still unclear.Objective This study was to investigate the effect of NAC on the TGF-β1-induced transdifferentiation of ARPE-19 cell to myofibroblast and its underlying mechanisms.Methods Human ARPE-19 cells were divided into control group,TGF-β1 treatment group,NAC intervention group and simple NAC group.Cells in the control group were not disposed,10 ng/ml TGF-β1,10 ng/ml TGF-β1 +5 mmol/L NAC and 5 mmol/L NAC were used to interfere the ARPE-19 cells for 48 hours in the other 3 groups.The morphological changes of the cells were observed by phase contrast microscopy.Real-time quantitative PCR,Western blot,immunofluorescence and ELISA were used to detect the expression of transdifferentiation marker genes-α-smoot h muscle actin (α-SMA),fibronectin and type Ⅰ collagen.The expression level of intracellular ROS induced by TGF-β1 in the presence or absence of NAC was detected using the non-fluorescent probe DCFH-DA performed by flow cytometry.The phosphorylation level of p38MAPK,ERK1/2 and SAPK/JNK were detected by Western blot.The cell counting kit-8 (CCK-8) assay was used to investigate the effect of NAC on the viability of ARPE-19 cells.Results The expressions of α-SMA,fibronectin and type Ⅰ collagen mRNA in the TGF-β1 treatment group were significantly increased,and were (2.15±0.29),(9.54 ± 1.08),(25.78 ±0.66) times higher than those of the control group,with significant differences between the two groups (all at P＜0.05).The expressions of α-SMA,fibronectin and type Ⅰ collagen protein were significantly increased,and were (8.49± 0.32),(2.53 ± 0.69),(4.45 ± 1.05) times higher than those of the control group,respectively,with significant differences between the two groups (all at P＜0.05).The expressions of α-SMA,fibronectin and type Ⅰ collagen mRNA in the NAC intervention group were (66.70± 12.57)％,(66.11±8.35)％ and (33.19±6.90)％ lower than those of the TGF-β1 treatment group,and the expressions of protein were (52.30±4.83)％,(55.03 ±2.58)％ and (56.08 ±3.65)％ lower than those of the TGF-β1 treatment group,respectively,with significant differences between the two groups (all at P＜0.05).Flow cytometry results showed that the expression level of intracellular ROS in the TGF-β1 treatment group was (2.12±0.20) times higher than that of the control group,and the intracellular ROS in the NAC intervention group was (57.41 ±9.45) ％ lower than that of the TGF-β1 treatment group,with significant differences between the groups (both at P＜0.01).Western blot results showed that the phosphorylation levels of p38MAPK,SAPK/JNK and ERK1/2 in the TGF-β1 treatment group were (9.18±1.00),(4.87±0.81) and (4.20±0.69) times higher than those of the control group,respectively,showing significant differences between the two groups (all at P＜0.05).The phosphorylation level of p38MAPK,SAPK/JNK and ERK1/2 in the NAC intervention group were (48.16± 14.82) ％,(67.90±2.90) ％ and (74.52±4.00)％ lower than those of the TGF-β1 treatment group,respectively,showing significant differences between the two groups (all at P＜0.05).Conclusions NAC inhibits the TGF-β1-induced transdifferentiation of ARPE-19 cells possibly via suppressing the production of intraeellular ROS and the phosphorylation of p38MAPK,SAPK/JNK and ERK1/2.