| 摘要: | 近年來國人罹患糖尿病的人數與日俱增，糖尿病會導致嚴重的併發症，例如：肝病、動脈粥狀硬化、肺部纖維化及泌尿系統感染等。長期於高糖環境下，體內的甲基乙二醛 (methylglyoxal, MG) 會累積形成糖化壓力，而乙二醛?系統 (glyoxalase system, GLO system) 的活性降低會導致MG及糖化最終產物 (advanced glycation end products) 的積累，進而導致糖尿病、心血管疾病和肺纖維化疾病的惡化。過去研究結果顯示，身體長期屬於糖尿病環境下，糖尿病患者體內甲基乙二醛濃度大量上升，會促使未折疊蛋白反應 (unfolded protein response)產生，且誘發內質網壓力 (endoplasmic reticulum stress, ER stress) 進而造成內皮細胞損傷。目前已知ER stress會刺激肺上皮細胞的上皮-間質轉化 (EMT)，促使肺部纖維化及肋膜纖維化產生。乙二醛?系統中GLO1是體內MG及其他活性雙羰基化合物代謝的速率決定酵素，研究發現MG血中濃度的降低或GLO1表現增加，可有效減緩MG對細胞造成的EMT及ER stress，因此有望改善與糖尿病相關之老化與代謝性疾病。
本研究利用GLO1 promoter活性試驗測試超過一千兩百種臨床藥物後發現，enalapril及ketotifen為強效之GLO1 promoter刺激劑，鑒於enalapril及ketotifen減緩高血糖加重肋膜間皮纖維化機制尚未釐清，因此本研究以細胞及動物實驗模式，探討此兩項藥物能否有效減緩高血糖惡化的肋膜間皮纖維化，並進一步探究其相關機制。細胞實驗中，以西方墨點法分析證實，給予MG (250 - 750 μM)後明顯影響肋膜間皮細胞 (Met-5A細胞株) EMT及ER stress產生，在EMT標記蛋白中有意義地刺激N-cadherin及Snail與抑制E-cadherin及CLDN1的表現，且明顯的刺激ER stress標記蛋白IRE1α、Bip及ERO1α的表現；而在給予enalapril 20 μM及ketotifen 20 μM後可有效地抑制MG刺激Met-5A細胞中EMT及ER stress的產生。給予enalapril及ketotifen可明顯誘發GLO1於Met-5A細胞及小鼠肺組織中的蛋白質表現。此外，動物實驗結果進一步證實了，口服餵食enalapril (50 mg/kg/day) 及ketotifen (5 mg/kg/day)兩週，可有效改善糖尿病惡化肺部纖維化小鼠模式中肺纖維化及肋膜纖維化的產生。實驗結果指出enalapril和ketotifen可能透過刺激肋膜間皮細胞表現GLO1來抑制MG引發的EMT及ER stress產生，進而改善糖尿病誘發肺纖維化之病程。研究成果提供了糖尿病併有肺纖維化病患之臨床用藥依據，且GLO1活化劑的開發可能是有潛力的糖尿病併發症治療策略。
In recent years, the number of people suffering from diabetes has increased daily. Diabetes can lead to serious complications, such as liver disease, atherosclerosis, pulmonary fibrosis, and urinary system infection. Long-term high-glucose environment, methylglyoxal (methylglyoxal, MG) in the body will accumulate to form glycation stress. The activity of the glyoxalase system (glyoxalase system, GLO system) will be reduced, leading to MG and the end product of glycation (advanced glycation end products), leading to the exacerbation of diabetes, cardiovascular disease, and pulmonary fibrosis. Past research results have shown that the body has been in a diabetic environment for a long time, and the concentration of methylglyoxal in diabetic patients increases significantly, which will promote the unfolded protein response (unfolded protein response) and induce endoplasmic reticulum stress (ER stress ) and cause damage to endothelial cells. It is known that ER stress can stimulate the epithelial-mesenchymal transition (EMT) of lung epithelial cells and promote pulmonary fibrosis and pleural fibrosis. GLO1 in the glyoxalase system is a rate-determining enzyme for metabolizing MG and other active dicarbonyl compounds in the body. Studies have found that the reduction of MG blood concentration or the increase of GLO1 expression can effectively slow down the EMT and ER stress caused by MG to cells, so it is expected Improve aging and metabolic diseases related to diabetes.
This study used the GLO1 promoter activity test to test more than 1,200 clinical drugs and found that enalapril and ketotifen are powerful GLO1 promoter stimulators. Since the mechanism of enalapril and ketotifen slowing down hyperglycemia and aggravating pleural mesothelial fibrosis has not yet been clarified, this study uses cell and animal experimental models to explore whether these two drugs can effectively slow down the pleural mesothelial fibrosis exacerbated by hyperglycemia, and further explore its related mechanisms. In the cell experiment, Western blot analysis confirmed that administration of MG (250 - 750 μM) significantly affected the production of EMT and ER stress in pleural mesothelial cells (Met-5A cell line), and significantly stimulated N-cadherin and Snail in EMT marker proteins Inhibit the expression of E-cadherin and CLDN1, and significantly stimulate the expression of ER stress marker proteins IRE1α, Bip and ERO1α; And after administration of enalapril 20 μM and ketotifen 20 μM, they can effectively inhibit the production of EMT and ER stress in Met-5A cells stimulated by MG. Administration of enalapril and ketotifen can significantly induce the protein expression of GLO1 in Met-5A cells and mouse lung tissue. In addition, the results of animal experiments further confirmed that oral feeding of enalapril (50 mg/kg/day) and ketotifen (5 mg/kg/day) for two weeks can effectively improve pulmonary fibrosis and the production of pleural fibrosis. The experimental results indicated that enalapril and ketotifen may inhibit MG-induced EMT and ER stress by stimulating the expression of GLO1 in pleural mesothelial cells, thereby improving the course of diabetes-induced pulmonary fibrosis. The research results provide a basis for clinical medication in diabetic patients with pulmonary fibrosis, and the development of GLO1 activators may be a potential treatment strategy for diabetic complications. |
