陳皮粗萃物保護乙醯胺基苯酚誘導肝損傷之潛力評估與Nrf2路徑/表觀遺傳調控機制之探討

肝臟為人體中主要執行解毒及代謝作用的重要器官，當肝功能損傷時會造成代謝障礙並影響到其他臟器的功能，嚴重則危及生命。乙醯胺基苯酚 (acetaminophen, APAP) 為國人常用解熱鎮痛藥物的主要成分，而近年來許多研究發現食用過量 APAP 易造成嚴重的肝腎損傷。本研究乃針對可能具護肝潛力之中藥食材陳皮 (Chenpi, CP) 進行肝臟保健功效評估與其機轉作用探討。首先以 HepG2-C8 細胞評估陳皮熱水粗萃物 (water extract, WE)、乙醇粗萃物 (ethanolic extract, EE) 及熱水萃取後殘渣乙醇粗萃物 (water extraction residue ethanolic extract, WREE) 誘導 ARE-luciferase 活性之能力，其中 CP-WREE (100 μg/mL) 最能有效提升 Nrf2路徑活性，且含有較多的總酚、總黃酮、多甲氧基類黃酮 (橘紅素、川陳皮素和4'-demethyltangeretin) 等化合物含量。CP-WREE 對 APAP 誘導小鼠 AML-12正常肝細胞損傷具有顯著性的保護作用，能降低 APAP 誘導脂質過氧化情形。CP-WREE 顯著增加 AML-12 細胞內 Nrf2 與其下游抗氧化/解毒代謝酵素 (HO-1、NQO1 及 UGT1A) mRNA 和蛋白質表現量，並影響表觀遺傳學修飾酵素 (包括 Dnmts、HDACs 及 KDMs) 之蛋白質表現量。此外，CP-WREE 能有效降低體外 DNA 甲基轉移酶 (M.SssI) 活性和增加 Nrf2 啟動子非甲基化程度。綜合本研究結果得知，CP-WREE 對 APAP 誘導 AML-12細胞損傷的保護作用的機制之一是透過表觀遺傳調控 Nrf2/抗氧化路徑，且推測其功效可能與組成分包括總酚、總黃酮、多甲氧基類黃酮等化合物有關，故本研究結果可供日後保健食品開發和生物醫學研究之重要參考。

關鍵字

乙醯胺基苯酚；中藥食材；陳皮；表觀遺傳學；肝臟； Nrf2

並列摘要

The liver is a critical organ responsible for the detoxification and metabolism in the human body. Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but excessive consumption of APAP can cause severe liver and kidney damage. Aged citrus peel (Chenpi, CP) is a Chinese herb with hepatoprotective potential to be usually used as foods in Taiwan. Three extracts of CP were prepared in this study, including water extract (CP-WE), ethanolic extract (CP-EE), and water extraction residue ethanolic extract (CP-WREE). Among these extracts, we found 100 μg/mL of CP-WREE, containing higher total phenolic, total flavonoid, and polymethoxylated flavonoid (PMFs such as tangeretin, nobiletin, and 4'-demethyltangeretin) contents, effectively enhanced the Nrf2 pathway in HepG2-C8 cells stably transfected with ARE-luciferase reporter gene. CP-WREE (50 μg/mL) significantly minimized APAP-induced cell damage and lipid peroxidation in mouse AML-12 hepatocytes. In normal AML-12 cells, CP-WREE also increased mRNA and protein levels of Nrf2 and Nrf2 downstream enzymes (HO-1, NQO1, and UGT1A), regulated the protein expressions of some epigenetic modifying enzymes (Dnmts, HDACs, and KDMs), and increased the level of unmethylated Nrf2 promoter according to the results of methylation-specific PCR (MSP) assay. We futher found that CP-WREE blocked in vitro DNA methyltransferase (M.SssI) activity. These results suggested that polyphenols, flavoid, and PMFs-rich CP-WREE could protect liver from APAP-induced injury through the epigenetic regulation of Nrf2 pathway, which would open new avenues of approaches for the prevention of liver disease in human.