評估臺灣紫芝萃取物於 PM2.5 誘發 ROS 及細胞傷害之保護效果

Abstract

PM2.5 為新興的空氣汙染指標，所謂 PM (Particulate matter) 即為懸浮微粒，根據其粒徑大小而有所稱呼，而直徑小於或等於 2.5 微米即稱為 PM2.5，由於粒徑很小，可以進入肺且深入肺泡組織，有研究顯示長期暴露於 PM2.5 會影響肺功能，增加肺癌的發生率與死亡率，並導致氣喘、COPD 及心血管相關疾病。氧化壓力被認為是 PM2.5 引起肺部疾病之最主要因素，會藉由細胞凋亡、自噬或者壞死的方式使肺部細胞死亡。靈芝自古以來被稱為治百病的藥草，不僅對人體健康具有保健功能，對許多疾病更是有預防及緩解的效果。臺灣紫芝 (Ganoderma formosanum) 為臺灣特有種靈芝，已由文獻指出，具有良好的清除自由基與調節免疫等功效，故本研究為利用液態發酵的臺灣紫芝菌絲體酒精萃取物 (Ganoderma formosanum extracts, GFE)，探討其減緩 PM2.5 誘發之自由基的產生，並釐清此作用機轉。首先，臺灣紫芝菌絲體酒精萃取物經不同溶劑萃取，初步篩選後獲得兩有效劃分層 butanol fraction (GFE-BuOH) 與 ethyl acetate fraction (GFE-EA)。結果顯示，本研究使用之 Sigma PM2.5 於1,000 ug/ml 濃度下會導致肺部細胞 MRC-5 存活率下降，以及 ROS (Reactive oxygen species, ROS) 的產生，其中藉由細胞凋亡之機制導致細胞死亡。若分別給予臺灣紫芝菌絲體萃取物有效劃分層 GFE-BuOH 與 GFE-EA 以 200 及 100 ug/ml 濃度預處理後，可減緩 PM2.5 對細胞造成的傷害，且可能透過抑制細胞凋亡的方式來達到保護的效果。此外，PM2.5 誘發 ROS 產生之能力亦於斑馬魚動物模式上證實，且加入200 ug/ml 之 GFE-BuOH 與 GFE-EA 後也有減緩的效果。總體而言，GFE-BuOH 及 GFE-EA 可以保護肺部細胞 MRC-5 對抗 PM2.5 誘導之傷害，且於斑馬魚模式上也證實了減緩 ROS 之活性。

Fine particulate matter (PM2.5) is a type of suspended air pollutant with a diameter of ≤ 2.5 um, which makes it possible to pass through the respiratory tract and travels deep into alveolar tissue. Studies have indicated that long-term exposure to PM2.5 could reduce pulmonary function, increase morbidity and mortality of lung cancer and exacerbate asthma and COPD. One of the potential mechanisms of the impact from PM2.5 on the pathogenesis of diseases is oxidative stress, and cause cell death by ways of apoptosis, autophagy or others. Ganoderma is one of the most valued Chinese traditional medicines and G. formosanum is an endemic species of Ganoderma in Taiwan. Its bioactivities about free radical scavenging and immunostimulatory ability have been demonstrated, hence we used human lung fibroblast MRC-5 to investigate the effect of G. formosanum extracts against PM2.5-induced ROS generation and its possible mechanism. The results showed that cell viability was decreased with increased ROS generation both in a dose-dependent manner after exposed to different concentration of PM2.5, and the cell death caused by PM2.5 was related to apoptosis pathway. After 24hrs pretreatment, butanol (BuOH) and ethyl acetate (EA) fraction of G. formosanum ethanol extract (GFE) at 200 ug/ml and 100 ug/ml could significantly increase the cell viability of MRC-5 after exposed to 1,000 ug/ml PM2.5, respectively, by cell apoptosis inhibition. Moreover, the effects of the two fractions on decreasing ROS generation were also demonstrated in zebrafish. As a result, our data indicated that GFE-BuOH and GFE-EA fraction could alleviate PM2.5-induced cell death and ROS generation.