探討糖化終產物誘導之內皮-間質轉換在糖尿病相關胰島纖維化之角色

The role of advanced glycation end-products-induced endothelial-mesenchymal transition in diabetes-related islet fibrosis

蔡佩珊(Pei-Shan Tsai)

指導教授 ： 劉興華

國立臺灣大學/醫學院/毒理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602528

摘要

糖尿病(diabetes mellitus)的產生主要為胰島素分泌不足或胰島素阻抗而引發體內高血糖之現象，為一種代謝失調之慢性疾病，此外高血糖(hyperglycemia)為引發糖尿病血管併發症(diabetes vascular complications)的主要原因之一，往往影響病患之生活品質，嚴重甚至導致死亡。已有研究指出，細胞外基質大量的堆積(extracellular matrix, ECM)會導致內皮細胞纖維化，進而使血管內皮功能受損，為糖尿病血管病變的重要致病機轉。在糖尿病血管病變進程中，糖化終產物(advanced glycation end-products, AGEs)的量會明顯增加且堆積於體內。許多研究先前已證實糖化終產物會引起多種細胞之纖維化，然而其影響胰島內皮細胞之作用尚未明瞭。內皮-間質轉換過程為調節內皮細胞功能的路徑之一，為此本研究以胰島內皮細胞株(MS1)與糖尿病小鼠，探討糖化終產物是否經內皮-間質轉換過程，導致纖維化的發生及其作用機制。實驗結果顯示，糖尿病小鼠較對照組別呈現大量AGEs累積於胰島組織中，尤其在內皮細胞，並且間質指標蛋白(α-SMA)亦大量表現於其中。此外，細胞實驗結果顯示在AGEs (50 μg/ml)暴露48小時下，E-cadherin及CD31表現顯著下降，間質細胞指標蛋白(CTGF, α-SMA和Vimentin)與纖維化指標分子(Fibronectin)表現量顯著增加，並正向調控AGEs受體(RAGE)的表現。同時，50 μg/ml AGEs會增加AKT、p-38、Smad2/3及NF-κB磷酸化並抑制BMP7及p-Smad1/5/9表現量。另一方面，給予RAGE的中和抗體與AGEs阻斷劑(Alagebrium chloride, Ala-Cl)能顯著回復內皮-間質轉換的指標蛋白(E-cadherin 及α-SMA)與纖維化指標分子(Fibronectin)的表現。總結上述，AGEs可經RAGE調控p-38/Smad2/3, AKT/NF-κB及BMP7/Smad1/5/9訊息傳遞路徑誘發胰島內皮細胞的內皮-間質轉換與纖維化產生。

關鍵字

糖尿病血管併發症 ； 內皮細胞 ； 糖化終產物 ； 內皮-間質轉換 ； 纖維化

並列摘要

Diabetes mellitus (DM), a metabolic disorder, is characterized by hyperglycemia resulting from defects in insulin secretion or insulin resistant action. Hyperglycemia is related with the development and progression of diabetes vascular complications, which resulted in shortened life expectancy and decreased quality of life in DM patients. Endothelium dysfunction is a crucial pathophysiological factor in diabetic vasculopathy. Several studies demonstrated that pathological accumulation of extracellular matrix (ECM) proteins could induce endothelial cell fibrosis and eventually cause endothelium dysfunction. The diabetic vasculopathy process was associated with elevated advanced glycation end-products (AGEs) presence cause diabetic vascular complications. Previous studies indicated that AGEs could induce fibrosis in several types of cells. However, there was no evidence that AGEs can induce fibrosis in pancreatic islet endothelial cells. Moreover, the endothelial-to-mesenchymal transition (EndoMT) is regarded as the functional regulator of vascular endothelium. Therefore, in this study, we investigated the role of advanced glycation end-products-induced endothelial-mesenchymal transition in diabetes-related islet fibrosis. We found that the immunoreactivities for AGEs and α-SMA expressions were markedly increased in the pancreatic islet of diabetic mice compared with the control group. The protein expression of E-cadherin and CD31 was significantly decreased, but Vimentin, α-SMA and CTGF were significantly increased by AGEs (50 μg/ml) treatment for 48h. Moreover, AGE induced the elevated protein expression of the fibrosis marker, fibronectin. On the other hand, AGEs (50μg/ml) increased receptor of AGEs (RAGE) protein expression. AGEs up-regulated phosphorylation of Akt, p-38, smad2/3 and NF-κB, and down regulated BMP7 and p-smad1/5/9 expression. Furthermore, RAGE neutralized antibody or alagebrium chloride (Ala-Cl), a AGEs cross-link breaker, could alleviate the AGEs-induced EndoMT in MS1 cells. Taken together, this study was the first to demonstrat for the first time that AGEs induce the production of EndoMT markers expression significantly, such as CTGF, vimentin, α-SMA, fibronectin, and suppress E-cadherin expression in pancreatic islet endothelial cells via RAGE-regulated p38MAPK/smad2/3, Akt/NF-κB and BMP7/ smad1/5/9 signaling pathway.

並列關鍵字

diabetes vascular complications ； endothelial cell ； advanced glycation end-products ； endothelial-to-mesenchymal transition ； fibrosis

參考文獻

72. Liu, H. J., Liao, H. H., Yang, Z., & Tang, Q. Z. (2016). Peroxisome Proliferator-Activated Receptor-γ Is Critical to Cardiac Fibrosis. PPAR Res, 2016.

1. Sarathy, H., Henriquez, G., Abramowitz, M. K., Kramer, H., Rosas, S. E., Johns, T., Kumar, J., Skversky, A., Kaskei, F,. Melamed, M. L. (2016). Abdominal Obesity, Race and Chronic Kidney Disease in Young Adults: Results from NHANES 1999-2010. PloS One, 11(5), e0153588.

2. Shrivastava, S. R., Shrivastava, P. S., & Ramasamy, J. (2013). Role of self-care in management of diabetes mellitus. J Diabetes Metab Disord, 12(1), 1.

3. Guariguata, L., Whiting, D. R., Hambleton, I., Beagley, J., Linnenkamp, U., & Shaw, J. E. (2014). Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes Res Clin Pract, 103(2), 137-149.

4. TA, S. (2014). Diagnosis and classification of diabetes mellitus. Diabetes Care, 37, S81.