利用動物及細胞模式探討牛樟芝功效成份Ergostatrien-3β-ol對肝臟缺血再灌流損傷之抗氧化及抗發炎機制

Abstract

牛樟芝被認為是具有醫療功效的珍貴中藥材，常被用來改善食物藥物中毒、腹瀉、高血壓以及肝癌。近來研究發現，牛樟芝的生物活性成分具有多種功效，其中護肝之效果尤其顯著。Ergostatrien-3β-ol (EK100)為牛樟芝當中的有效活性成分之一，子實體及菌絲體中皆存在，然而其相關研究甚少。本實驗利用動物模式模擬缺血再灌流所造成的肝損傷 (hepatic ischemia and reperfusion injury；IR injury)，並藉由牛樟芝之有效成分 EK100 可護肝、抗發炎的特性，觀察是否能減少肝臟缺血再灌流所產生的傷害。於臨床上，心肌梗塞、中風、大面積的創傷或組織器官移植都有可能產生缺血再灌流損傷，在阻斷血流及氧氣的供應下，對於組織器官產生不可逆的損傷，當再恢復血流供應時，頓時有大量血液再次供給器官組織氧氣及營養，然而此時因為大量含氧血瞬間由血管進入組織器官中，同時產生大量的活性氧化物，造成細胞更大量的損傷，進一步導致細胞發炎，造成惡性循環。本研究在動物實驗中發現EK100可有效降低肝臟缺血再灌流損傷所造成的ALT、AST的上升，並可使促發炎物質TNF-α、IL-6及IL-1 β下降。對於肝臟中iNOS mRNA的表現量也有顯著降低的效果，在抗氧化方面，EK100可造成肝臟抗氧化酵素SOD的上升。因此進一步以細胞模式分析EK100的抗發炎和抗氧化機制。發現EK100可藉由促進Nrf2進入細胞核提升反應活性，進一步活化下游基因HO-1及NQO1 mRNA的表現量，並在濃度10 μM 達最顯著效果，時間則以17小時效果最佳。另外在人類肝癌細胞HepG2以H2O2誘導氧化壓力處理6小時，HO-1 mRNA表現量增加約3倍，給予EK100前處理17小時的組別，HO-1 mRNA表現量則增加至約7倍，但在12小時所有組別之HO-1 mRNA表現量與控制組間則無顯著差異。給予小鼠巨噬細胞Raw264.7預處理E100，並利用LPS (1 μg/ml)誘導產生發炎反應，EK100可減少iNOS之mRNA表現並且降低NO產量，其可能是調控HO-1之表現增加達到此效果，並非藉由調控轉錄因子NF-κB達到抗發炎之功效。由本實驗結果可知EK100具有良好的抗發炎及抗氧化功效，藉由此功效可有效降低大鼠缺血再灌流損傷，未來極具發產潛力。

Antrodia camphorate has been considered as one of most efficient and precious Chinese herbal medicines, and it has been used as remedy for curing food poisoning, diarrhea, stomachache, hypertension, itchy skin, and liver cancer. Recently, liver protecting characteristic of Antrodia camphorata was believed as the most important bioactivity. Ergostatrien-3β-ol (EK100), which exists in both fruiting body and mycelia, is one of compounds with high liver-protecting property； however, investigation for this compound is scarce. In the present research, animal model with hepatic ischemia and reperfusion injury (IR injury) was created, and EK100, which is asserted to possess liver-protecting and anti-inflammatory effects, was used for decreasing live damages resulted from IR injury. Clinically, IR injury occurs in patients with myocardial infraction, stroke, or large trauma, in which tissue is injured irreversibly by deficient supply of oxygen due to blood flow blocking, and harmed again by violet inflammatory response brought from fast and massive accumulation of reactive oxygen species after blood is resupplied. In our animal study, EK100 decreased ALT and AST, which is mainly come from liver injury, and it also lowered the proinflammatory cytokines such as TNF-α, IL-6, as well as IL-1. In addition, EK100 significantly reduced the mRNA expression of iNOS, and increased SOD, which is an antioxidant enzyme in liver. A further investigation in anti-inflammatory and antioxidant mechanisms of EK100 was conducted by cell model. EK100 improved the translocation of Nrf2 into nuclei, activating the downstream genes of HO-1 and NQO1. The most efficient dosage and effect time were observed at 10 μM and 17 hours.