Department of Biological Sciences, College of Optometry, State University of New York, New York, New York 10065, USA.
Invest Ophthalmol Vis Sci. 2011 Jan 21;52(1):485-93. doi: 10.1167/iovs.10-5801. Print 2011 Jan.
To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs).
Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8.
A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC.
Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.
通过瞬时受体电位香草酸通道 1（TRPV1）信号通路的激活，确定高渗应激是否会促进人角膜上皮细胞（HCEC）中炎症细胞因子的释放增加。
通过在等渗 Ringers 溶液中添加蔗糖来制备高渗培养基。使用单细胞荧光成像系统测量 HCEC 中 fura2-AM 负载的 Ca2+信号。Western blot 分析评估 EGFR、ERK、p38 MAPK 和核因子（NF）-κB 的磷酸化状态。ELISA 评估 TRPV1 激活对白细胞介素（IL）-6 和 IL-8 释放的影响。
450 mOsm 的高渗应激引起 2 倍的 Ca2+瞬变，该瞬变被 TRPV1 选择性拮抗剂辣椒素和 JYL 1421 抑制。这种瞬变被 PGE2 增强。用辣椒素、基质金属蛋白酶 1（MMP1）抑制剂 TIMP-1、广谱 MMP 抑制剂 GM 6001、肝素结合（HB）-EGF 抑制剂 CRM 197 或 EGFR 抑制剂 AG 1478 预先孵育 HCEC 可抑制高渗诱导的表皮生长因子受体（EGFR）转激活。在高渗和时间依赖性方式下，EGFR 转激活后 ERK 和 p38 MAPK 和 NF-κB 的激活。用辣椒素、AG 1478、ERK 抑制剂 PD 98059、p38 抑制剂 SB 203580 或 NF-κB 抑制剂 PDTC 处理可抑制高渗诱导的白细胞介素（IL）-6 和趋化因子 IL-8 的释放增加。
高渗应激诱导的 TRPV1 通道刺激通过引发 EGFR 转激活、MAPK 和 NF-κB 激活，介导促炎细胞因子 IL-6 和趋化因子 IL-8 的增加。TRPV1 活性或其信号转导介质的选择性药物调节可能为抑制干燥综合征中发生的炎症反应提供一种新方法。
