摘要
眼翳(Pterygium)為帶有纖維微血管及上皮層之不正常翼狀組織，由結膜往角膜方向生長，除了影響外觀，也會導致角膜產生不規則散光、影響視力，若過度增生至角膜中央甚至會造成失明。眼翳發生的主要因素是由於過多紫外線(Ultraviolet, UV)照射眼球表面組織後，產生的大量自由基導致角膜輪部幹細胞病變、並誘導結膜細胞增生所致。目前臨床上最普遍的治療方式為直接以手術切除眼翳組織，但眼翳術後復發率高，且臨床上仍未有直接抑制眼翳生長之藥物。迷迭香酸(rosmarinic acid, RA)屬於咖啡酸脂類，具有相當強的抗氧化能力，並具有抗病毒、抗發炎、抗癌、抑制血管新生等生物活性，先前研究已指出迷迭香酸可降低紫外線對皮膚細胞造成的傷害，故本研究以RA探討其對眼翳上皮細胞(pterygium epithelial cells, PECs)之作用及相關機制。結果發現以100 μM RA作用後可有效抑制PECs生長 (p < 0.05)，並透過活化Nrf2表現，增加細胞內HO-1、NQO1、SOD與CAT等抗氧化因子的表現、進而降低細胞當中的ROS含量。PECs經RA作用後，Fas、FADD、TNF-α和caspase 8等外在路徑細胞凋亡相關蛋白表現量有顯著增加，同時也透過增加Bax、cytochrome c、caspase 9的表現，與降低Bcl-2和pro-caspase 3的表現，誘導細胞走向內在路徑的細胞凋亡，因此RA除了可以誘導細胞內部之氧化壓力失衡以外，也可透過細胞凋亡來抑制其生長。由於纖維化(fibrosis)為眼翳生長、復發另一重要調控機轉，在實驗中也發現RA可藉由負調控TGF-β/Smad訊息傳遞路徑，使PECs內的TGF-β1、TGF-βRII、p-Smad1、p-Smad2、p-Smad3、Smad4、p-Smad5及type I collagen蛋白表現量下降。由以上結果可知，RA為相當有潛力應用於抑制眼翳生長之天然物，未來可再進一步以動物模式探討其應用於臨床上之可能性。
並列摘要
Pterygium is a common tumor-like ocular disease, which may be related to exposure of chronic ultraviolet (UV) radiation. Though the standard treatment for pterygium is surgical intervention, the recurrence of pterygium is high when no effective inhibitory drug is used after surgery. Rosmarinic acid (RA) is a polyphenol antioxidant, which has many biological activities including anti-UV and anti-tumor. This study was to examine the inhibiting effects of RA on pterygium epithelial cells (PECs). MTT assay was used for examining the cell cytotoxicity of PECs after RA treatment. Acridine orange (AO)/DAPI staining was stained with PECs for examing cell viability. A fluorescent probe, DCFH-DA (2’,7’-dichlorofluorescin diacetate), was stained with PECs for measuring intracellular ROS (reactive oxygen species) levels. Antioxidant activity assays were measured the levels of superoxide dismutase (SOD) and catalase (CAT) in PECs. Western blot analysis was used to determine the protein expression of Nrf2, HO-1, NQO1, apoptosis associated proteins and TGF-β/Smad signaling associated proteins. RA significantly reduced the cell viability of the PECs. Treatment with RA could remarkably increase the Nrf2 protein expression levels in nucleus, HO-1 and NQO1 protein expression levels, and activities of SOD and CAT. As a result, intracellular ROS levels in PECs were decreased. In addition, the induction of extrinsic apoptosis on PECs by RA was also associated with the increasing of Fas, FADD, TNF-α and caspase 8 protein expression levels. Moreover, the induction of PECs intrinsic apoptosis cell death was confirmed through up-regulation of cytochrome c, Bax, caspase 9 and caspase 3, down-regulation of Bcl-2 and pro-caspase 3. Our study demonstrated that RA could inhibit the viability of the PECs via regulation of extrinsic and intrinsic apoptosis pathway. Since fibrosis is an important mechanism beyond pterygium recurrence, our results showed that RA could down-regulating TGF-β/Smad signaling through decreasing TGF-β1, TGF-βRII, p-Smad1, p-Smad2, p-Smad3, Smad4, p-Smad5 and type I collagen protein expression levels. Therefore, RA may have potential for pterygium therapeutic medication.