以白內障之體外疾病模式探討藥物之生化機制

Biochemical Mechanisms of Pirenoxine and Carnosine Investigated by in vitro Cataract Models

林怡伶(I-Lin Lin)

指導教授 ： 吳姿樺

臺北醫學大學/藥學院/藥學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00032

摘要

白內障是導致世界人口失明最主要的原因，其中致白內障因素中又以老化或過度暴露於紫外線下為最為常見。因此本研究欲建立白內障之體外 (in vitro) 疾病模式，用以探討臨床常用但仍缺乏學理之Pirenoxine (Px) 以及市場上逐漸推展的CAR (CAR) 之效用及其生化機制。本研究使用豬水晶體蛋白萃取溶液，以高濃度鈣離子、外加calpainⅡ或不同波長段的紫外線誘發水晶體蛋白混濁，繼而以波長405nm偵測混濁度變化或利用蛋白質電泳 (SDS-PAGE) 分析水晶體蛋白之分子變化情形。研究結果顯示豬水晶體蛋白萃取溶液 (50mg/mL) 與10 mM鈣離子連續觀察五日，其溶液混濁度可由0.135± 0.004上升至0.617± 0.002，具統計上顯著差異 (p< 0.05)。豬水晶體蛋白萃取溶液 (5mg/mL) 外加2 mM鈣離子及每毫克蛋白加7.5單位calpainⅡ經60分鐘加熱後，混濁度可由0.069± 0.004上升至0.247± 0.021，具統計上顯著差異 (p< 0.05)；由PAGE顯示β水晶體蛋白表現減少而α水晶體蛋白有降解產生。以UVB連續照射γ水晶體蛋白 (20mg/mL)，於6小時可觀察到混濁度由0.061± 0.008上升至0.800± 0.006，具統計上顯著差異 (p< 0.05)；而由PAGE顯示γ水晶體蛋白有二聚物及降解產生。以UVC連續照射γ水晶體蛋白 (0.4mg/mL)，於4小時可觀察到混濁度由0.055± 0.006上升至0.544± 0.006，具統計上顯著差異 (p< 0.05)；而由PAGE顯示γ水晶體蛋白產生明顯降解。Px (0, 0.03, 0.1, 0.3, 1 μM) 對於10 mM鈣誘發之水晶體蛋白連續觀察五日混濁度值之數值分別上升0.482± 0.002、0.321± 0.042、0.272± 0.091、0.229± 0.001、0.202± 0.004，相較於未加藥組均達統計上顯著差異 (p< 0.05)，且其作用與劑量呈正相關；另UVC (14 J/cm2/hr) 誘導之γ水晶體蛋白混濁現象形成及降解之效果，其4小時混濁度值在Px (0、1 mM)分別增加0.489± 0.007、0.401± 0.015，彼此間具統計上顯著差異 (p< 0.05)，而由PAGE觀察到Px組其在21kDa位置附近蛋白質表現增加；但對UVB所誘導之蛋白變性現象無改善作用。CAR (10、100、300 mM) 可減少水晶體蛋白受鈣活化calpainⅡ所誘發之蛋白質降解，同時延緩其受熱誘導所產生之水晶體混濁度上升的現象，且此保護作用與劑量呈正相關；其未加藥組及CAR (0、10、100、300 mM) 之60分鐘混濁度值分別增加0.199± 0.019、0.142± 0.019、0.043± 0.004、0.030± 0.005，相較於未加藥組均達統計上顯著差異 (p< 0.05)，而由PAGE觀察到CAR組在29kDa位置附近蛋白質表現量增加，另外在19kDa位置則無新增蛋白質表現。而高濃度之CAR則具延緩UVB或UVC所誘導之γ水晶體蛋白混濁現象形成及降解之效果：在UVB (4 J/cm2/hr) 照射6小時，其混濁度值在CAR (0、200、300 mM) 分別增加0.661± 0.024、0.295± 0.006、0.077± 0.008，相較於未加藥組均達統計上顯著差異 (p< 0.05)，由PAGE觀察到CAR組在44, 15~18kDa位置附近較無新增蛋白質表現；在UVC (14 J/cm2/hr) 照射4小時，其混濁度值在CAR (0、100、300 mM) 分別增加0.510± 0.058、0.356± 0.020、0.191± 0.003，相較於未加藥組均達統計上顯著差異 (p< 0.05)，而由PAGE觀察到CAR組其在21kDa位置附近蛋白質表現增加。結論: 本研究顯示Px對高濃度鈣離子或UVC所造成的蛋白損傷有延緩的作用；而CAR則可能對於細胞內鈣離子上升、UVB及UVC三者不同生化病理機制所造成的蛋白損傷有預防或延緩的作用，未來可利用白內障活體模式來證實其藥物活性。

關鍵字

吡諾克辛；肌肽；水晶體混濁度；鈣離子；外生性依鈣蛋白質分解酵素；紫外線

並列摘要

Cataract is a leading cause of blindness in the world. Aging and ultraviolet light (UV) exposure are common causes of cataract. This study aimed to set up a series of in vitro cataract models, and to investigate the efficacy and biochemical mechanisms of pirenoxine (Px) and carnosine (CAR). Three in vitro models including high calcium, exogenous calpainⅡ or UV-induced porcine lens protein turbidity were established. Turbidity was assayed by measuring optical density (OD) at 405nm and proteolysis was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that the turbidity of solution containing 50mg lens protein/mL and 10 mM calcium was increased from 0.135± 0.004 to 0.617± 0.002 after 5 day incubation (p< 0.05). The turbidity of solution containing 5mg lens protein/mL, 2 mM calcium and 7.5 units exogenous calpainⅡ/mg protein was increased from 0.135± 0.004 to 0.617± 0.002 after heating for 60 min (p< 0.05). PAGE showed that incubation with calpain resulted in loss of β-and α-crystallin. The turbidity of solution containing 20mg γ-crystallin/mL was increase from 0.061± 0.008 to 0.800± 0.006 after 6 hr UVB exposure (p< 0.05). PAGE showed that exposure to UVB resulted in γ-crystallin dimer formation. The turbidity of solution containing 0.4mg γ-crystallin/mL was increased from 0.055± 0.006 to 0.544± 0.006 after 4 hr UVC exposure (p< 0.05). PAGE showed that exposure to UVC resulted in loss of γ-crystallin. In 10 mM calcium-induced lens turbidity system, results showed that Px (0.03, 0.1, 0.3, 1 μM) possesses dose-dependent protective effect. The changes of OD for control and Px (0.03, 0.1, 0.3, 1 μM) following 5 day incubation were 0.482± 0.002, 0.321± 0.042, 0.272± 0.091, 0.229± 0.001, 0.202± 0.004, respectively. All above Px groups showed significant statistically differences compared to the control (p<0.05). As to UVC (14 J/cm2/hr) –induced γ-crystallin turbidity system, the changes of OD for control and Px (1 mM) following 4 hr UVC exposure were 0.489± 0.007, 0.401± 0.015, respectively. There was a significant statistically differences between control and Px group (p<0.05). PAGE showed that γ-crystallin at 21kDa maintained in Px group. However, this protective effect was not shown in UVB-induced γ-crystallin turbidity system. CAR (10, 100, 300 mM) showed protective effects in calcium activated exogenous calpainⅡ-induced proteolysis and delayed turbidity after heating at the same time. The changes of OD for control and CAR (10, 100, 300 mM) treatments following 60 min heating were 0.199± 0.019, 0.142± 0.019, 0.043± 0.004, 0.030± 0.005, respectively. PAGE showed that treatments with CAR resulted in protein expression increase at 29kDa and decrease at 19kDa. In UVB (4 J/cm2/hr)-induced turbidity system, the changes of OD for control and CAR (200, 300 mM) groups following 6 hr UVB exposure were 0.661± 0.024, 0.295± 0.006, 0.077± 0.008, respectively. PAGE showed that treatments with CAR resulted in less protein expression at 44, 15~18kDa. The changes of OD induced by UVC (4 J/cm2/hr) turbidity for control and CAR (100, 300 mM) groups following 4 hr were 0.510± 0.058, 0.356± 0.020, 0.191± 0.003, respectively. In all above turbidity study, CAR groups showed significant statistically differences compared to the controls (p<0.05). PAGE showed that γ-crystallin at 21kDa maintained in CAR group. In conclusion, our in vitro experimental results suggest that Px may delay the progression of cataract which caused by high concentration of calcium or UVC exposure. CAR may be useful in prevent or delay the progression of cataract which caused by intracellular calcium elevation or ultraviolet light exposure. In vivo effects of Px and CAR are needed to be confirmed by in vivo cataract animal models in the future.

並列關鍵字

Pirenoxine；Carnosine；lens turbidity；calcium；exogenous calpain；UV

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