To investigate whether forskolin, a protein kinase A agonist, regulates toll-like receptor 4 actions on retinal endothelial cell permeability in vitro. We also evaluated whether PKA could regulate TLR4 signaling independent of exchange protein activated by cAMP in REC in culture. REC were grown in normal (5 mM) or high (25 mM) glucose. Cells were treated with forskolin to increase PKA levels, siRNA against TLR4, siRNA against myeloid differentiation primary response 88, siRNA against translocating chain associated membrane protein 1, siRNA against epac1, or scrambled siRNA, or a combination of these treatments. Western blotting was done for zonula occludens 1 and occludin protein levels, as well as TLR4 signaling cascade proteins. Permeability measurements were done for REC in culture following inhibition of TLR4 or its signaling cascades. Forskolin restored high glucose-associated decreases in ZO-1 and occludin, which was associated with improved in vitro permeability levels. Both forskolin and TLR4 inhibition reduced high glucose-induced increases in REC permeability, but the actions were not cooperative. Forskolin regulated both MyD88-dependent and -independent signaling pathways, independent of Epac1. Finally, blockade of MyD88 or TRAM1 reduced permeability in REC grown in high glucose. A PKA agonist regulated TLR4 signaling independent of Epac1. PKA agonism or TLR4 inhibition is effective at reducing high glucose-induced permeability in REC in vitro. These studies offer new avenues for therapeutic development.

中文翻译：

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Forskolin 在体外通过 TLR4 作用调节视网膜内皮细胞通透性。

目的 研究蛋白激酶 A 激动剂毛喉素 (forskolin) 是否在体外调节 Toll 样受体 4 对视网膜内皮细胞通透性的作用。我们还评估了 PKA 是否可以独立于培养物 REC 中由 cAMP 激活的交换蛋白来调节 TLR4 信号传导。REC 在正常 (5 mM) 或高 (25 mM) 葡萄糖中生长。用毛喉素处理细胞以增加 PKA 水平，用针对 TLR4 的 siRNA、针对骨髓分化初级反应 88 的 siRNA、针对易位链相关膜蛋白 1 的 siRNA、针对 epac1 的 siRNA 或乱序 siRNA 或这些处理的组合进行处理。对闭合小带 1 和 Occludin 蛋白水平以及 TLR4 信号级联蛋白进行蛋白质印迹分析。在抑制 TLR4 或其信号级联后，对培养物中的 REC 进行渗透性测量。Forskolin 恢复了 ZO-1 和 occludin 与高葡萄糖相关的降低，这与体外通透性水平的改善有关。毛喉素和 TLR4 抑制均可减少高葡萄糖诱导的 REC 通透性增加，但作用并不协同。Forskolin 调节 MyD88 依赖性和非依赖性信号通路，不依赖于 Epac1。最后，MyD88 或 TRAM1 的阻断降低了高葡萄糖条件下生长的 REC 的通透性。PKA 激动剂独立于 Epac1 调节 TLR4 信号传导。PKA 激动或 TLR4 抑制可有效降低体外 REC 中葡萄糖诱导的高通透性。这些研究为治疗开发提供了新途径。毛喉素和 TLR4 抑制均可减少高葡萄糖诱导的 REC 通透性增加，但作用并不协同。Forskolin 调节 MyD88 依赖性和非依赖性信号通路，不依赖于 Epac1。最后，MyD88 或 TRAM1 的阻断降低了高葡萄糖条件下生长的 REC 的通透性。PKA 激动剂独立于 Epac1 调节 TLR4 信号传导。PKA 激动或 TLR4 抑制可有效降低体外 REC 中葡萄糖诱导的高通透性。这些研究为治疗开发提供了新途径。毛喉素和 TLR4 抑制均可减少高葡萄糖诱导的 REC 通透性增加，但作用并不协同。Forskolin 调节 MyD88 依赖性和非依赖性信号通路，不依赖于 Epac1。最后，MyD88 或 TRAM1 的阻断降低了高葡萄糖条件下生长的 REC 的通透性。PKA 激动剂独立于 Epac1 调节 TLR4 信号传导。PKA 激动或 TLR4 抑制可有效降低体外 REC 中葡萄糖诱导的高通透性。这些研究为治疗开发提供了新途径。PKA 激动剂独立于 Epac1 调节 TLR4 信号传导。PKA 激动或 TLR4 抑制可有效降低体外 REC 中葡萄糖诱导的高通透性。这些研究为治疗开发提供了新途径。PKA 激动剂独立于 Epac1 调节 TLR4 信号传导。PKA 激动或 TLR4 抑制可有效降低体外 REC 中葡萄糖诱导的高通透性。这些研究为治疗开发提供了新途径。