The influence of actin depolymerization induced by Cytochalasin D and mechanical stretch on interleukin-8 expression and JNK phosphorylation levels in human retinal pigment epithelial cells.
The influence of actin depolymerization induced by Cytochalasin D and mechanical stretch on interleukin-8 expression and JNK phosphorylation levels in human retinal pigment epithelial cells.
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DOI：
10.1186/s12886-017-0437-z
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发表时间：
2017-04-07
影响因子：
2
通讯作者：
Liu W
中科院分区：
文献类型：
--
作者：
Gao M;Wu S;Ji J;Zhang J;Liu Q;Yue Y;Liu L;Liu X;Liu W
本研究探讨了细胞松弛素D（Cytochalasin D）诱导的肌动蛋白细胞骨架解聚和机械牵张对人视网膜色素上皮（RPE）细胞白细胞介素8（IL-8）表达和c-jun氨基末端激酶（JNK）磷酸化水平的影响。使用Flexcell FX-5000张力系统以0.33 Hz对培养的人RPE细胞（ARPE-19）施加循环拉伸，拉伸率为20%，持续0 h、6 h或24 h。细胞单独拉伸或用细胞松弛素D预处理。使用鬼笔环肽免疫荧光染色评估肌动蛋白细胞骨架的重新分布。Western blotting法检测IL-8和JNK在RPE细胞中的表达水平。对照组细胞显示丰富而均匀的鬼笔环肽染色。机械牵张24 h后，鬼笔环肽染色显示细胞骨架不清晰，不规则。在所有细胞松弛素D处理的细胞中，无论机械应力如何，都观察到细胞骨架结构的收缩和破坏。仅用周期性牵张刺激RPE细胞并不诱导IL-8表达和JNK磷酸化水平的显著增加，这与对照组的那些相似。单独用细胞松弛素D预处理后，IL-8表达和JNK磷酸化水平在6 h时无显著差异，但在24 h时分别显著增加约1.2倍（1.18 ± 0.05; P<0.01）和3.0倍（3.01 ± 0.02; P<0.01）。RPE细胞与细胞松弛素D预孵育后，暴露于周期性牵张，IL-8表达和JNK磷酸化水平增加约1.3倍（1.31 ± 0.02; P<0.01）和1.3倍24 h分别增加1.7倍（1.69 ± 0.06; P<0.01）和3.2倍（3.21 ± 0.12; P<0.01）。这项研究表明，细胞松弛素D和机械拉伸破坏肌动蛋白聚合上调人RPE细胞中白细胞介素-8的表达和JNK磷酸化水平，这表明肌动蛋白细胞骨架的完整性可能在RPE细胞的促炎过程中发挥重要作用。
This study explores the role of actin cytoskeleton depolymerization induced by Cytochalasin D and mechanical stretch on the interleukin-8 (IL-8) expression and c-jun N-terminal kinase (JNK) phosphorylation levels in human retinal pigment epithelial (RPE) cells. A Flexcell FX-5000 Tension system was used to apply cyclic stretch to cultured human RPE cells (ARPE-19) at 0.33 Hz with 20% elongation for 0 h, 6 h or 24 h. The cells were stretched alone or pre-treated with Cytochalasin D. The redistribution of the actin cytoskeleton was evaluated using phalloidin immunofluorescence staining. The protein expression levels of IL-8 and JNK in the RPE cells were determined via Western blotting. The cells in the control groups displayed abundant and uniform phalloidin staining. After exposure to mechanical stretch for 24 h, phalloidin staining revealed an unclear and irregular actin cytoskeleton. In all Cytochalasin D-treated cells, the shrinkage and disruption of the cytoskeletal structure was observed regardless of mechanical stress. The stimulation of the RPE cells with cyclic stretch alone did not induce a significant increase in IL-8 expression and JNK phosphorylation levels, which were similar to those of the control groups. After pre-treatment with Cytochalasin D alone, IL-8 expression and JNK phosphorylation levels were not significantly different at 6 h but were significantly increased by approximately 1.2-fold (1.18 ± 0.05; P<0.01) and 3.0-fold (3.01 ± 0.02; P<0.01) at 24 h, respectively. After the pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch, IL-8 expression and JNK phosphorylation levels increased by approximately 1.3-fold (1.31 ± 0.02; P<0.01) and 1.3-fold (1.31 ± 0.02; P<0.01) at 6 h, respectively, and by 1.7-fold (1.69 ± 0.06; P<0.01) and 3.2-fold (3.21 ± 0.12; P<0.01) at 24 h, respectively. This study demonstrates that disruption of actin polymerization by cytochalasin D and mechanical stretch upregulates interleukin-8 expression and JNK phosphorylation levels in human RPE cells, which indicates that the integrity of the actin cytoskeleton may play important roles in the pro-inflammatory processes in RPE cells.