- 學位論文
對乙醯氨基酚與薑黃素使用於肝腫瘤細胞Hep G2之細胞毒性研究
Incorporate effect of Acetaminophen and Curcumin on liver cytotoxicity in Human cell line Hep G2
摘要
對乙醯氨基酚(acetaminophen, APAP)廣泛使用於解熱鎮痛，在治療劑量下使用安全性高且副作用低。因APAP使用廣泛亦取得容易，常見因過度或不正確使用、長期高劑量，或同時合併使用某些增加肝臟代謝的藥物，引發肝臟之潛在毒性。薑黃素(curcumin)是由薑科(Curcuma longa Linn.)植物所萃取的物質。在傳統中醫藥食同源的觀念，以薑黃素為主的保健食品種類日漸增多。唯薑黃素是否具有潛在肝毒性或增強APAP所引起之肝毒性，仍未被探討。 本研究即以Hep G2細胞作為肝細胞毒性對象，探討APAP和薑黃素單獨作用於Hep G2細胞所可能引發之肝細胞毒性；另一方面探討二者合併使用後，對於Hep G2細胞可能產生之毒性作用，藉以了解APAP和薑黃素作用於肝細胞毒性的表現。實驗中利用Thiazolyl blue[3-(4,5-dimethy -thiazol-2yl)-2,5-diphenyltetrazalium-bromide, MTT]與粒線體去氫酶反應之細胞活性試驗、肝臟生化酵素指標：丙氨酸轉氨酶(alanine amino transferase, ALT)、天冬氨酸轉氨酶(asparate aminotransferase, AST)及乳酸脫氫酶(lactate dehydrogenase, LDH)之酵素活性測定、肝臟微粒體酶 (liver microsomes)之 S9 fraction 藥物代謝試驗、細胞內抗氧化酵素麩胺基硫( glutathione, GSH )濃度測試及流式細胞儀分析細胞凋亡等方法進行探討。 實驗結果顯示，APAP與薑黃素均對 Hep G2細胞有不同程度的細胞毒性，其24小時細胞半致死濃度(LC50)分別為16.13 mM及25.32 mM，肝臟酵素(ALT、AST和LDH)活性於APAP 15 mM及薑黃素5mM時顯著性增加。兩者經S9作用後之代謝物對細胞毒性明顯減少(p<0.05)，但較高濃度之APAP代謝物仍有相當之細胞毒性，且影響細胞內肝臟酵素活性及抗氧化物質GSH的改變。薑黃素對APAP代謝物所誘發的細胞毒性具加強作用，引起細胞死亡率增加，但對APAP在細胞內造成的細胞凋亡及細胞壞死並無影響差異。細胞內GSH濃度於 APAP30 mM明顯降低。而薑黃素於30 mM濃度下，細胞內GSH濃渡雖略為增加，但兩者合併作用對APAP所消耗之GSH並未降低，其作用機制仍有待進一步探討。
並列摘要
Acetaminophen (APAP) is an analgesic and antipyretic agent. At therapeutic doses, APAP is relatively safe with low side effects. However, long-term, high-dose treatment with APAP or co-treatment with other medications may be potentially toxic to the liver function. On the other hand, curcumin is a yellowish extract from Curcuma Longa. root.. In traditional Chinese medicine. It is popular to use curcumin as a health food in dietary. However, whether curcumin is a potential hepatotoxic agent is not clearly defined. The aim of this study was to assess the potential effects of curcumin on APAP-induced hepatic cytotoxicity in Hep G2 cells. MTT assay was used to determine the cell viability. The enzymatic activity changes of alanine amino transferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured as the indices of liver damage. In addition, the level of intracellular GSH was assessed by ELISA and apoptosis was further analyzed by flow cytometry. Metabolites of APAP and curcumin derived from incubation with isolated liver microsomes (S9 fraction) were also evaluated. Results revealed that the median lethal concentration (LC50) of APAP and curcumin in Hep G2 cells (for 24-hr treatment) were 16.13 mM and 25.32 mM, respectively. APAP and curcumin significantly increased the activities of ALT, AST, and LDH,respectively. In combination, curcumin could significantly enhance the APAP-induced cytotoxicity and the enzymatic activities. In liver microsome (S9 fraction) metabolic assay, the APAP metabolites posed weaker cytotoxicity comparing to the parent compound. On the contrary, the cytotoxicity and enzymatic activities were significantly reduced when curcumin was metabolized. Curcumin could also significant elevate the APAP metabolites-induced cytotoxicity. Flow cytometry analysis showed that APAP induced apoptosis in a dose-dependent manner, while curcumin induced apoptosis was not evidence. The level of glutathione was significantly decreased at 30 mM of APAP treatment, but was not elevated in curcumin treatment at concentrations less than 30 mM. In conclusion, curcumin potentiated APAP-induced hepatic cytotoxicity but not apoptosis. The underlying mechanism needs to be elucidated in the future.