京尼平对α-TN4晶状体上皮细胞系纤维化行为的抑制作用

Genipin suppression of fibrogenic behaviors of the alpha-TN4 lens epithelial cell line.

作者信息

Kitano Ai, Saika Shizuya, Yamanaka Osamu, Reinach Peter S, Ikeda Kazuo, Okada Yuka, Shirai Kumi, Ohnishi Yoshitaka

机构信息

Department of Ophthalmology, Wakayama Medical University, Kimiidera, Wakayama, Japan.

出版信息

J Cataract Refract Surg. 2006 Oct;32(10):1727-35. doi: 10.1016/j.jcrs.2006.05.015.

DOI:10.1016/j.jcrs.2006.05.015

PMID:17010875

Abstract

PURPOSE

To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO).

SETTING

Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan.

METHODS

In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium.

RESULTS

Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic.

CONCLUSION

Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.

摘要

目的

在晶状体上皮细胞系α-TN4中，确定中药茵陈蒿汤的肠道代谢产物京尼平是否能抑制促纤维化肌成纤维细胞的生成以及纤维化细胞因子的上调，并评估该药物在预防后囊膜混浊（PCO）方面的潜在益处。

设置

日本和歌山县和歌山医科大学眼科。

方法

在本研究中，采用比色法、划痕试验、免疫组织化学和蛋白质印迹分析，检测α-TN4细胞的增殖、迁移以及肌成纤维细胞生成的标志物α-平滑肌肌动蛋白（α-SMA）的表达。通过实时逆转录-聚合酶链反应来表征转化生长因子-β1（TGF-β1）和结缔组织生长因子（CTGF）的基因表达。此外，通过蛋白质印迹和免疫组织化学评估p38丝裂原活化蛋白激酶（p38 MAPK）信号通路、细胞外信号调节激酶（ERK）信号通路和Smad信号通路。使用市售的比色法试剂盒检测培养基中核基质蛋白41/7（NMP41/7），评估京尼平的细胞毒性。

结果

京尼平抑制细胞增殖和迁移，同时抑制Smad和p38 MAPK磷酸化，尽管ERK信号通路增强。京尼平抑制TGF-β1和CTGF的mRNA表达。基于α-SMA免疫细胞化学染色强度降低，细胞质纤维形成减少。然而，α-SMA蛋白表达实际上并未改变。这一阴性结果表明京尼平减弱了含α-SMA细胞骨架的形成。用京尼平处理细胞48小时并未增加NMP41/7向培养基中的释放，表明该化合物无细胞毒性。