Chen Huiyu, Zhang Jing, Dai Yiqin, Xu Jianjiang
Department of Ophthalmology and Visual Science, Eye & ENT Hospital, NHC Key Laboratory of myopia (Fudan University); Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai Medical College of Fudan University, Shanghai, 200031 China.
J Inflamm (Lond). 2019 Dec 26;16:27. doi: 10.1186/s12950-019-0232-0. eCollection 2019.
In herpes simplex epithelial keratitis, excessive TLR3-induced cellular responses after virus infection evoke inflammatory cascades that might be destructive to the host cornea. Nerve growth factor (NGF), a pluripotent neurotrophic factor with immune regulatory effect, was proved to be effective in Herpes simplex keratitis (HSK) treatment, although the detailed mechanisms remain unclear. This study aims to investigate the effects of NGF on modulating inflammatory responses triggered by TLR3 activation in human corneal epithelial cells (HCECs) in vitro.
HCECs were stimulated with TLR3 agonist, poly(I:C), in the absence or presence of NGF. Cell viability and cytotoxicity were measured by a CCK-8 assay and LDH release assay, respectively. The activation of NF-κB signaling pathway was examined using immunofluorescence staining and western blotting. Levels of proinflammatory cytokines were determined by ELISA or RT-qPCR. ROS generation and 8-OHdG positive cells were examined by a fluorometric analysis.
It was shown that NGF significantly inhibited the generation of proinflammatory cytokines in HCECs triggered by TLR3 activation ( < 0.05), probably via suppressing NF-κB activation. NGF also impeded the upstream signal to initiate NF-κB activation by scavenging ROS by approximately 50% ( < 0.05). In addition, 8-OHdG positive cells were substantially attenuated by NGF treatment ( < 0.01).
Taken together, this study indicates that NGF could inhibit TLR3-induced inflammatory cascades in HCECs, suggesting NGF as a potential therapeutic agent for HSK.
在单纯疱疹性上皮性角膜炎中，病毒感染后Toll样受体3（TLR3）诱导的过度细胞反应引发炎症级联反应，这可能对宿主角膜具有破坏性。神经生长因子（NGF）是一种具有免疫调节作用的多能神经营养因子，已被证明在单纯疱疹性角膜炎（HSK）治疗中有效，但其详细机制仍不清楚。本研究旨在探讨NGF对体外人角膜上皮细胞（HCECs）中TLR3激活引发的炎症反应的调节作用。
在存在或不存在NGF的情况下，用TLR3激动剂聚肌苷酸-聚胞苷酸（poly(I:C)）刺激HCECs。分别通过CCK-8法和乳酸脱氢酶（LDH）释放法测定细胞活力和细胞毒性。采用免疫荧光染色和蛋白质印迹法检测核因子κB（NF-κB）信号通路的激活情况。通过酶联免疫吸附测定（ELISA）或逆转录定量聚合酶链反应（RT-qPCR）测定促炎细胞因子水平。通过荧光分析检测活性氧（ROS）的产生和8-羟基脱氧鸟苷（8-OHdG）阳性细胞。
结果表明，NGF显著抑制了TLR3激活引发的HCECs中促炎细胞因子的产生（<0.05），可能是通过抑制NF-κB激活。NGF还通过清除约50%的ROS（<0.05）来阻碍启动NF-κB激活的上游信号。此外，NGF处理可显著减少8-OHdG阳性细胞（<0.01）。
综上所述，本研究表明NGF可抑制HCECs中TLR3诱导的炎症级联反应，提示NGF作为HSK的一种潜在治疗药物。
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