Ye J, Xu Y-F, Lou L-X, Jin K, Miao Q, Ye X, Xi Y
Department of Ophthalmology, The Second Affiliated Hospital of Zhejiang University, College of Medicine, Hangzhou, Zhejiang, China.
Eye (Lond). 2015 Jul;29(7):964-71. doi: 10.1038/eye.2015.62. Epub 2015 May 8.
This study assessed the anti-inflammatory effect and mechanism of action of hinokitiol in human corneal epithelial (HCE) cells.
HCE cells were incubated with different concentrations of hinokitiol or dimethylsulfoxide (DMSO), which served as a vehicle control. Cell viability was evaluated using Cell Counting Kit-8 (CCK-8) assay. After polyriboinosinic:polyribocytidylic acid (poly(I:C)) stimulus, cells with or without hinokitiol were evaluated for the mRNA and protein levels of interleukin-8 (IL-8), interleukin-6 (IL-6), and interleukin-1β (IL-1β) using real-time PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. Nuclear and cytoplasmic levels of nuclear factor kappa B (NF-κB) p65 protein and an inhibitor of NF-κB α (IκBα) were evaluated using western blotting.
There were no significant differences among the treatment concentrations of hinokitiol compared with cells incubated in medium only. Incubating with 100 μM hinokitiol significantly decreased the mRNA levels of IL-8 to 58.77±10.41% (P<0.01), IL-6 to 64.64±12.71% (P<0.01), and IL-1β to 54.19±8.10% (P<0.01) compared with cells stimulated with poly(I:C) alone. The protein levels of IL-8, IL-6, and IL-1β had similar trend. Further analysis revealed that hinokitiol maintained the levels of IκBα and significantly reduced NF-κB p65 subunit translocation to the nucleus which significantly inhibiting the activation of the NF-κB signal pathway.
Hinokitiol showed a significant protective effect against ocular surface inflammation through inhibiting the NF-κB pathway, which may indicate the possibility to relieve the ocular surface inflammation of dry eye syndrome (DES).
本研究评估了扁柏酚对人角膜上皮（HCE）细胞的抗炎作用及其作用机制。
将HCE细胞与不同浓度的扁柏酚或作为溶剂对照的二甲基亚砜（DMSO）孵育。使用细胞计数试剂盒-8（CCK-8）测定法评估细胞活力。在用聚肌苷酸：聚胞苷酸（poly（I：C））刺激后，分别使用实时PCR分析和酶联免疫吸附测定（ELISA）评估有无扁柏酚处理的细胞中白细胞介素-8（IL-8）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）的mRNA和蛋白质水平。使用蛋白质印迹法评估核因子κB（NF-κB）p65蛋白和NF-κBα抑制剂（IκBα）的核水平和细胞质水平。
与仅在培养基中孵育的细胞相比，扁柏酚各处理浓度之间无显著差异。与仅用poly（I：C）刺激的细胞相比，用100μM扁柏酚孵育可使IL-8的mRNA水平显著降低至58.77±10.41%（P<0.01），IL-6降低至64.64±12.71%（P<0.01），IL-1β降低至54.19±8.10%（P<0.01）。IL-8、IL-6和IL-1β的蛋白质水平呈现相似趋势。进一步分析表明，扁柏酚维持IκBα水平，并显著减少NF-κB p65亚基向细胞核的转位，从而显著抑制NF-κB信号通路的激活。
扁柏酚通过抑制NF-κB途径对眼表炎症显示出显著的保护作用，这可能提示其具有缓解干眼综合征（DES）眼表炎症的可能性。
