探討丹參素鈉對於紫外線B輻射所造成眼球角膜損傷之保護作用

丹參(Salvia miltiorrhiza)具有生物活性且為中國傳統醫學植物性用藥。其中丹參素(Danshensu)為其主要的水溶性活性成分。丹參素分子在自然界中不穩定，丹參素鈉(Sodium Danshensu, Salvianic acid A sodium; SAS) 為其衍生的藥理分子構形。丹參素鈉分子相對穩定，傳統上多用於活血化瘀、緩解胸憋悶及心絞痛。傳統中醫藥學提及丹參可用於緩解眼球疾病的問題，然而丹參素鈉對於眼角膜的藥理作用與生物功效至今尚未清楚。在小鼠模式，紫外線B (UVB)會造成眼角膜的發炎性損傷，並可作為藥效篩選與驗證的生物平台。本研究以此模式探討丹參素鈉是否能預防紫外線B所誘導的角膜表面發炎損傷，並就其藥理功效進行驗證。小鼠分為空白對照組和三個紫外線曝曬組，包含(1) UVB曝曬但無丹參素鈉投藥組 (UVB-no SAS) (2) UVB曝曬並口服使用丹參素鈉1 mg/kg組 (UVB-SAS 1 mg/kg, 每天2次) 和 (3) UVB曝曬並口服使用丹參素鈉10 mg/kg組 (UVB-SAS 10 mg/kg, 每天2次)。實驗方法採取角膜平滑度(Corneal Smoothness)、角膜地形圖(Corneal Topography)，角膜不透明度(Corneal Opacity)及角膜損傷染色(Corneal lissamine green staining)分析策略。組織病理分析採取蘇木紫-伊紅染色(Hematoxylin-Eosin)及免疫化學染色方法來觀察角膜組織之病理改變的程度。實驗結果指出經紫外線B曝曬後，UVB-no SAS組的小鼠角膜表面呈現不規則變化，角膜上皮層剝落及組織細胞結構排列混亂，並且異常活化與增加角膜組織上皮層Fas及COX-2蛋白的表現。在口服丹參素鈉的預防組別，發現角膜損傷和組織病理的改變有減緩的趨勢，並且角膜組織結構的完整性較高。口服丹參素鈉可減少紫外線B所誘導基質層發炎細胞(polymorphonuclear cells, PMNs)的浸潤。統計分析，在口服丹參素鈉的預防組別，於進行紫外線B曝曬後，角膜表面的損傷及基質層發炎細胞浸潤的發生率有顯著的下降，同時亦減少Fas及COX-2在角膜組織的表現。實驗結果說明丹參素鈉10 mg/kg在預防角膜損傷比1 mg/kg更有保護效果。由以上結果得知，丹參素鈉可用於預防紫外線B所誘導發炎性角膜損傷與病理的退化，並在小鼠模式提供了在角膜的藥理功效證據。

Salvia miltiorrhiza (SM) has biological activity and used in traditional Chinese medicine. Danshensu is the main water-soluble active ingredient of SM. Danshensu molecules are unstable in nature and sodium danshensu (Salvianic acid A sodium, SAS), which derived from SM, is more stable and with pharmacokinetic constituents. SAS molecules are relatively stable, traditionally used for blood circulation, relieve chest tightness and angina pectoris. Traditional Chinese medicine mentioned that SM can be used to alleviate the problem of eye diseases, but SAS for corneal pharmacological or biological effects has not been described clearly. Ultraviolet B (UVB) radiation may induced corneal inflammatation in the mouse model as a biological platform for screening and validating the pharmalogical efficacy. The purpose of this study explored whether SAS could prevent inflammation of corneal surface induced by UVB and verify its pharmacological efficacy. Mice were divided into one blank control group, and three UVB radiation groups, i.e. (1) SAS-untreated group, and groups orally administered (2) 1 mg/kg SAS and (3) 10 mg/kg SAS. Pathologic changes to corneal surface were assessed based on smoothness, topographic distortion, opacity, as well as staining with lissamine green. Hematoxylin-Eosin and immunochemical staining were used to observe the morphological and pathological changes of corneal tissue. After exposure to UVB, the corneal surface and pathologic changes of mice in the non-treatment group showed irregular distortion, epithelial exfoliation, disorganized epithelium of cornea and activation of corneal epithelium Fas and COX-2 performance. Prophylactic oral administration of SAS can significantly reduce corneal damage and inflammatory changes, prevent the integrity of the organizational structure from degeneration against UVB radiation damage, and reduced polymorphonuclear cells infiltration in the cornea. Statistical analysis showed a significant decrease in the incidence of corneal surface damage and inflammatory cell infiltration in the prevention group of oral SAS, while reducing the performance of Fas and COX-2. SAS 10 mg/kg in the prevention of corneal injury is more protective than 1 mg/kg. From the above results, SAS can be used to prevent the degeneration of inflammatory corneal pathways induced by UVB, and provide evidence of pharmacology in mouse patterns.