摘要
糖尿病腎病變是糖尿病病患嚴重的併發症和主要死因之一。其病理變化包括早期腎臟細胞增生、晚期細胞肥大和細胞外間質(例如膠原蛋白)堆積，最後導致腎臟纖維化。非酵素性糖化作用所產生的高度糖化終產物(advanced glycation end-products；AGE)則與糖尿病腎病變的致病機轉有密切的關係。而高度糖化終產物所誘發的訊息傳遞路徑及生物效應，可能與PKC、MAPK及JAK2有關。而近期有人發現，wnt-4訊息傳遞路徑可能與腎臟纖維化有關。因此我們以近端腎小管LLC-PK1細胞為對象，探討wnt訊息傳遞路徑在高度糖化終產物所誘發LLC-PK1近端腎小管細胞之生物效應中所扮演的角色。實驗結果顯示，高度糖化終產物在刺激近端腎小管細胞2天後，不僅能抑制細胞分裂，而且能刺激膠原蛋白的製造，但於西方點墨法實驗中則發現wnt-4蛋白質表現量無顯著改變，而wnt訊息傳遞路徑的下游蛋白─乙型連環素(β-catenin)表現則明顯降低，且受乙型連環素之調控且與細胞生長有關的下游蛋白質cyclin-D1之表現量亦顯著減少，故我們推論高度糖化終產物可能經由抑制乙型連環素訊息傳遞路徑抑制細胞增生。同時高度糖化終產物也能刺激膠原蛋白的製造，又在wnt訊息傳遞路徑中，GSK-3主要調控乙型連環素的分解，故我們進一步假設高度糖化終產物藉由活化GSK-3來減少乙型連環素的表現。為證實以上論點，我們在給予高度糖化終產物時，同時加入GSK-3抑制劑─SB216763，結果顯示加入GSK-3抑制劑後，逆轉了高度糖化終產物所抑制的細胞增生、乙型連環素、cyclin-D1及膠原蛋白IV的表現，也逆轉了高度糖化終產物所刺激的膠原蛋白製造。而AG490(JAK2抑制劑)、Wortmanin (PI3K抑制劑)、Staurosporine (PKC抑制劑) 及PD98059和SB2030580 (MAPK抑制劑)都能逆轉高度糖化終產物所抑制的乙型連環素，但只有AG490和SB2030580能逆轉高度糖化終產物所抑制的cyclin-D1蛋白質表現。另外在動物實驗中亦發現STZ所誘導的糖尿病大白鼠中，在第3個月時腎臟組織有乙型連環素減少的情形。綜合上述結果證實，高度糖化終產物是藉由活化GSK-3以抑制β-catenin及cyclin-D1，進而抑制細胞增生，因此我們推測可能和糖尿病腎病變有關。再者PI3K、PKC、MAPK和JAK/STAT等訊息傳遞路徑在高度糖化終產物培養的LLC-PK1細胞中和β-catenin及cyclin-D1的調節有著重要的交互作用。
並列摘要
In Taiwan, diabetes mellitus and end-stage renal disease is the 5th and 7th leading cause of death, respectively. Furthermore, diabetic nephropathy (DN) is a major cause of morbidity and mortality, which occurs in 20-40﹪of the diabetic patients. Advanced glycation end-product (AGE) is important in the pathogenesis of diabetic nephropathy, which is characterized by cellular hypertrophy/hyperplasia leading to renal fibrosis. However, the signal transduction pathway of AGE remains poorly understood. Wnt signaling pathway may be associated with renal fibrosis. Although most studies focused only on glomerulopathy in DN. Thus, we studied the role of wnt signal pathway in the effects of AGE on cellular growth, protein expression and collagen synthesis in proximal tubular cell line (LLC-PK1).We found that AGE (100 μg/ml) inhibited the β-catenin (but not wnt-4) protein expression in dose- and time- dependent inhibition manner in LLC-PK1 cells. Besides, AGE also decreased cyclin-D1 protein expression and cellular mitogenesis after 48 hours treatment. In contrast, AGE increased intra-collagen synthesis and type IV collagen protein expression. GSK-3 modulated the expression of β-catenin and many DN-associated factors. Thus, we hypothesized that DN and/or AGE are/is associated with an enhanced GSK-3 activity and β-catenin pathway.We found that GSK-3 inhibitor (SB216763) reversed AGE-inhibited β-catenin、cyclin-D1 protein expression and mitogenesis. AGE-induced type IV collagen production and intra-collagen synthesis were decreased by SB216763. We also found that JAK2 inhibitor (AG490), PI3K inhibitor (Wortmanin), MAPK inhibitor (PD98059 and SB203580) and PKC inhibitor (Staurosporine) reversed AGE-inhibited β-catenin protein expression, but only AG490 can reverse AGE-inhibited cyclin-D1 protein expression. In immunohistochemistry, Streptozotocin-diabetic rats also decreased β-catenin protein expression at three months.In conclusion, AGE decreased β-catenin and cyclin-D1 protein expression dependent of GSK-3, consequently inhibited cell proliferation. PI3K、PKC、MAPK and JAK/STAT signal pathways may have significance associating with β-catenin signal pathway in AGE-induced effects.