WITHDRAWN - Nerve growth factor inhibits TLR3-induced inflammation and oxidative injury in human corneal epithelial cells

Abstract

Purpose : TLR3 activation is considered to play important roles in the pathogenesis of herpes simplex keratitis (HSK). It was found that NGF could effectively treat HSK and prevent the relapse of the disease, whilst the detailed mechanisms remain unclear. The aim of this study is to investigate the effects of NGF on inflammatory responses and oxidative injury triggered by TLR3 activation in human corneal epithelial cells (HCECs) in vitro.

Methods : HCECs were stimulated with TLR3 ligand, poly(I:C), in the absence or presence of NGF. Levels of proinflammatory were measured by ELISA or RT-PCR. NF-κB activation was examined using Western blotting and immunofluorescence staining. The levels of ROS generation were measured by DCF-DA method, and the expression of oxidative marker, mitochondria DNA 8-OHdG was observed.

Results : In HCECs, poly(I:C) stimulation increased the secretion of proinflammatory cytokines, while such efforts could be attenuated by NGF treatment, probably through its inhibition on NF-κB activation. In addition, the increased generation of ROS and the elevated oxidative injury caused by TLR3 activation were ameliorated by NGF.

Conclusions : Nerve growth factor could inhibit inflammation and oxidative injury in HCECs, suggesting that NGF might be a novel therapeutic agent for herpes simplex keratitis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Figure 1. NGF significantly suppressed the expression of proinflammatory cytokines and inhibited NF-κB activation induced by poly(I:C) in HCECs. (A) Increased secretion of IL-6 and IL-8 in HCECs cultured with various conservations of poly(I:C) for 24 hours. (B-D) HCECs were treated with NGF (50ng/ml) and/or poly(I:C) (25.0µg/ml) for 18 hours to determine mRNA level by RT-PCR or for 24 hours to measure proinflammatory cytokines. (E-F) Effect of NGF on TLR3 and TrkA mRNA expressions. (G-H)The levels of p-IκBα and nuclear translocation of NF-κB p65 (I) in HCECs. Data were presented as mean±SD from six separated experiments, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

 

Figure 2. NGF decreased TLR3 induced oxidative injury in HCECs. HCECs were stimulated with NGF (50ng/ml) and/or poly(I:C) (25.0µg/ml) for 10 hours. (A-B)Staining with DCF-DA, the confocal fluorescence microscopy observations and relative fluorescence intensity analysis in HCECs. (C) Expression of oxidative marker, 8-OHdG (green) in HCECs. Data were presented as mean±SD from three separated experiments, ^**p < 0.01. Scale bars: 50µm.