Anti-inflammatory and antioxidative effects of gallic acid on experimental dry eye: in vitro and in vivo studies

Abstract

Abstract Background To investigate the anti-inflammatory and antioxidative effects of gallic acid (GA) on human corneal epithelial cells (HCECs) and RAW264.7 macrophages as well as its therapeutic effects in an experimental dry eye (EDE) mouse model. Methods A cell counting kit-8 (CCK-8) assay was used to test the cytotoxicity of GA. The effect of GA on cell migration was evaluated using a scratch wound healing assay. The anti-inflammatory and antioxidative effects of GA in vitro were tested using a hypertonic model (HCECs) and an inflammatory model (RAW264.7 cells). The in vivo biocompatibility of GA was detected by irritation tests in rabbits, whereas the preventive and therapeutic effect of GA in vivo was evaluated using a mouse model of EDE. Results In the range of 0–100 μM, GA showed no cytotoxicity in RAW264.7 cells or HCECs and did not delay the HCECs monolayer wound healing within 24 h. Ocular tolerance to GA in the in vivo irritation test was good after seven days. In terms of antioxidative activity, GA significantly reduced the intracellular reactive oxygen species (ROS) in lipopolysaccharide (LPS) activated RAW264.7 macrophages and HCECs exposed to hyperosmotic stress. Furthermore, after pre-treatment with GA, the expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase-1 (NQO-1) were significantly upregulated in RAW264.7 macrophages. GA also exhibits excellent anti-inflammatory properties. This is mainly demonstrated by the ability of GA to effectively downregulate the nuclear transcription factor-κB (NF-κB) pathway in LPS-activated RAW264.7 macrophages and to reduce inflammatory factors, such as nitric oxide (NO), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α). In vivo efficacy testing results in a mouse model of EDE showed that GA can effectively prevent and inhibit the apoptosis of corneal epithelial cells (CECs), reduce inflammatory factors in the cornea and conjunctiva as well as protect goblet cells. Conclusion In vitro and in vivo results indicate that GA possesses potent anti-inflammatory and antioxidative properties with no apparent cytotoxicity within the range of 0–100 μM. It is a promising eye drop formulation for the effective prevention and treatment of dry eye disease (DED).