Inhibitory effects of rosmarinic acid on pterygium epithelial cells through redox imbalance and induction of extrinsic and intrinsic apoptosis

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Abstract

Pterygium is a common tumor-like ocular disease, which may be related to exposure to chronic ultraviolet (UV) radiation. Although the standard treatment for pterygium is surgical intervention, the recurrence rate of pterygium is high when no effective inhibitory drug is used after surgery. Rosmarinic acid (RA) is a polyphenol antioxidant with many biological activities, including anti-UV and anti-tumor properties. This study aimed to examine the inhibitory effects of RA on pterygium epithelial cells (PECs). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to examine the cell cytotoxicity of PECs after RA treatment. A fluorescent probe, DCFH-DA (2′,7′-dichlorofluorescin diacetate), was stained with PECs to measure intracellular reactive oxygen species (ROS) levels. Antioxidant activity assays were used to measure the levels of superoxide dismutase (SOD) and catalase (CAT) in PECs. Western blot analysis was used to determine the protein expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), quinone acceptor oxidoreductase 1 (NQO1), and apoptosis-associated proteins. RA significantly reduced the cell viability of the PECs. Treatment with RA remarkably increased the Nrf2 protein expression levels in the nucleus, HO-1 and NQO1 protein expression levels, and the activities of SOD and CAT. As a result, intracellular ROS levels in PECs were decreased. Additionally, the induction of extrinsic apoptosis on PECs by RA was associated with increasing expressions levels of Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor-alpha (TNF-α), and caspase 8 protein. Moreover, the induction of intrinsic apoptotic cell death in PECs was confirmed through upregulation of cytochrome c, Bax, caspase 9, and caspase 3 and downregulation of Bcl-2 and pro-caspase 3. Our study demonstrated that RA could inhibit the viability of PECs through regulation of extrinsic and intrinsic apoptosis pathways. Therefore, RA may have potential as a therapeutic medication for pterygium.

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