Effect of N-acetylcysteine on the transforming growth factor β1-induced transdifferentiation of retinal pigment epithelial cell to myofibroblast

Authors: Liu Shuai,  Jin Haiying

DOI: 10.3760/cma.j.issn.2095-0160.2017.10.008
Published 2017-10-10
Cite as Chin J Exp Ophthalmol, 2017,35(10): 894-901.

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-smooth muscle actin (α-SMA), fibronectin and typeⅠcollagen.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-β1treatment 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-β1treatment 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 intracellular ROS and the phosphorylation of p38MAPK, SAPK/JNK and ERK1/2.

Key words:

N-acetylcysteine; Retinal pigment epithelial cell; Transforming growth factor-β1; Myofibroblast; Transdifferentiation; α-Smooth muscle actin

Contributor Information

Liu Shuai
Department of Ophthalmology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200093, China
Jin Haiying
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Updated: September 4, 2019 — 1:56 pm