α-硫辛酸延緩脂多醣誘導庫氏細胞NLRP3發炎小體活化及庫氏細胞培養液誘發小鼠FL83B肝臟細胞胰島素阻抗之研究

Abstract

第二型糖尿病的主要病因為胰島素阻抗，造成身體周邊組織無法讓血液中之葡萄糖順利進入細胞氧化產能。文獻指出，糖尿病的成因與身體的氧化壓力有高度相關性。硫辛酸(α-lipoic acid, ALA)具有抗氧化、協助身體產能代謝及協助體內抗氧化物質之還原再生等功能。本研究欲探討硫辛酸對於給予脂多糖(lipopolysaccharide, LPS)誘導不朽型小鼠庫氏細胞株(Immortal Kupffer cells)抗發炎及其改善庫氏細胞條件培養液誘發小鼠肝臟FL83B上皮細胞株胰島素阻抗之效果與可能機轉。庫氏細胞株先給予不同濃度(5, 100, 500, 2000μM)之ALA處理6小時後，再同時給予LPS 1μg/mL刺激6小時及nigericin 13.4μM 或ATP 1μM處理2小時增強發炎因子訊號，分析庫氏細胞發炎相關蛋白質表現量，並收集條件培養液進行後續實驗；其次，以庫氏細胞株條件培養液培養FL83B細胞株，並分析FL83B細胞胰島素阻抗相關蛋白質表現量。結果顯示，ALA濃度2400μM時庫氏細胞仍具有100%以上之存活率。而以LPS誘導庫氏細胞發炎之條件培養液其IL-1β產量與未誘導者相較提升了21.3倍，但是庫氏細胞株先給予ALA 2000μM處理再以LPS誘導後其IL-1β之產量降低了105倍。利用JC-1觀察ALA對LPS誘導庫氏細胞粒線體膜電位保護之效果，結果顯示ALA可以抑制LPS造成庫氏細胞粒線體膜電位失衡，並降低ROS對細胞之傷害。Western blot分析發現，ALA可以有效降低LPS誘導之庫氏細胞NF-κB、NLRP3蛋白之表現；另一方面，ALA可以明顯提升LPS處理庫氏細胞條件培養液培養FL83B細胞株胰島素傳訊蛋白p-PI3K、p-AKT及GLUT2之表現量。以上結果顯示，ALA可以減輕LPS誘導之庫氏細胞NLRP3發炎體活化，以及LPS誘導庫氏細胞條件培養液培養時FL83B細胞之胰島素阻抗。T2DM is characterized by insulin resistance that results in hyperglycemia. Previous studies indicated that the occurrence of T2DM is highly associated with high fat high carbohydrate diet, obesity and excess oxidative stress in human body. Alpha-lipoic acid (ALA) is a sulfur-containing strong anti-oxidant. The aim of this study is to investigate the effect of ALA on NLRP3 inflammasome activation in LPS-induced immortal Kupffer cells and the insulin resistance in Kupffer cells conditioned mediun incubated FL83B mouse hepatocytes. Kupffer cells were incubated with ALA (5, 100, 500, 2000μM) for 6 hrs, and then treated with LPS 1μg/mL 6 hrs and nigericin 13.4μM 1 hr or ATP 1μM 2 hr to activate the inflammasome. These Kupffer cells conditioned medium were collected for analysis for cytokines, and incubated with the FL83B mouse hepatocytes to induce insulin resistance in liver cells. The results showed that LPS increased IL-1β level by 21.3 folds, whereas 2000μM ALA decline IL-1β level by 105 folds in Kupffer cells conditioned medium. The MTT assay showed that no toxicology was abserved in a high concentration of 2400μM of ALA in Kupffer cells. ALA reduced the production of ROS from mitochondria damage in LPS-treated Kupffer cells. Results from Western blot analysis indicated that ALA decreased the NLRP3 inflammasome activation related protein expression such as NF-κB and NLRP3 in LPS-treated Kupffer cells. Moreover, ALA increased the insulin nsignaling related protein expression such as p-PI3K/p-AKT and GLUT2 in Kupffer cells conditioned medium-incubated FL83B liver cells. The above results suggest that ALA may possess potential on alleviating NLRP3 inflammasome activation in LPS-induced Kupffer cells and improving the insulin resistance in Kupffer cells conditioned medium incubated FL83B mouse hepatocytes.