Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland.
Pharmacol Res. 2011 Nov;64(5):501-8. doi: 10.1016/j.phrs.2011.05.027. Epub 2011 Jun 7.
Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.
核因子 kappa B（NF-κB）活性和白细胞介素 6（IL-6）分泌升高参与了几种年龄相关和炎症相关疾病的病理学，包括年龄相关性黄斑变性（AMD），其中视网膜色素上皮细胞是关键靶点。最近的研究发现，热休克蛋白 70（Hsp70）可能影响 NF-κB 的调节。在目前的研究中，使用塞拉托林（一种新型抗炎化合物）评估 Hsp70 表达对人视网膜色素上皮细胞（ARPE-19）中 NF-κB RelA/p65 活性的影响。通过酶联免疫吸附试验（ELISA）测量 LPS 暴露期间 IL-6 和内源性 NF-κB 水平的表达来确定塞拉托林的抗炎特性。通过 MTT 和 LDH 测定法测量细胞活力，并通过 Western blot 分析 Hsp70 表达水平。为了减弱 Hsp70 表达，用 hsp70 小干扰 RNA（siRNA）转染 ARPE-19 细胞，并使用 NF-κB 共有结合 ELISA 测量 NF-κB RelA/p65 的活性。与单独 LPS 暴露相比，LPS 和塞拉托林同时暴露可降低 ARPE-19 细胞中 IL-6 表达水平以及磷酸化 NF-κB 在丝氨酸 536（Ser536）的活性。此外，当 Hsp70 反应被 siRNA 沉默时，塞拉托林抑制 NF-κB RelA/p65 活性的作用减弱。塞拉托林的有利抗炎浓度没有显示出细胞毒性反应的迹象。我们的研究结果表明，塞拉托林是一种新型植物化合物，通过 NF-κB 和 Hsp70 调节抑制人视网膜色素上皮细胞的固有免疫反应，Hsp70 是 NF-κB 的关键调节因子。
