Epstein-Barr 病毒 (EBV) 是人類的皰疹病毒，與許多人類淋巴組織惡性腫瘤發生有關。先前實驗室發現虎杖乙醇萃取物藉由抑制潛伏膜蛋白質 1 (LMP1) 及減少細胞核 NF-B 表現而促進 EB 病毒腫瘤細胞走上凋亡，所以本研究將探討虎杖乙酸乙酯分離物 F3a 抗 EB 病毒腫瘤細胞的機制。實驗將以 Trypan blue 方法分析虎杖乙酸乙酯分離物 F3a 對 EB 病毒潛伏期第三型腫瘤細胞，Raji 細胞存活的影響；以即時定量 PCR 分析 EB 病毒基因的 mRNA 及 DNA 表現量；以 DCFH-DA 試劑分析活性氧物質 (ROS) 產量；以西方墨點法分析潛伏膜蛋白質1、凋亡相關蛋白質、ERK及 IB表現量；以流式細胞儀分析凋亡細胞數。結果顯示虎杖乙酸乙酯分離物 F3a 會導致 Raji 細胞死亡，其 CC50 為 12.08 g/ml ，也發現濃度 12.5 g/ml F3a 使 Raji 細胞內 EB 病毒的 BRLF1 及 BNLF1 mRNA 表現增加，潛伏膜蛋白質 1 (LMP1) 的蛋白質表現量增加，也會使 Raji 細胞內磷酸化的 ERK 及磷酸化的 IB 降低，細胞內 ROS 增加，細胞凋亡相關蛋白質，裂解的 caspase 及 PARP 產生，凋亡細胞數增加。所以虎杖乙酸乙酯分離物 F3a 具有開發成抗 EB 病毒相關腫瘤藥物的潛能。 Epstein-Barr virus (EBV) is a human herpesvirus; and is associated with several human lymphoid malignancies. Our earlier study found that the ethanolic extract of Polygonum cuspidatum root promotes apoptosis of EBV-positive cells due to its ability to inhibit the expression of latent membrane protein 1 and deplete NF-B in the nucleus. Therefore, the purpose of this study is to investigate the effects of the ethyl acetate fraction F3a from P. cuspidatum on Raji cells. Trypan blue assay was used to determine the viability of Raji cells; real-time quantitative PCR to assess the expression of EBV mRNA and EBV DNA in Raji cells; to measure ROS production using by DCFH-DA kit. The expression of LMP1, apoptotic-related proteins, ERK and IB were determined by western blotting. Flow cytometry analysis was used for the detection of apoptotic cells. Results show that F3a promotes Raji cells death, with a CC50 were of 12.08 g/ml F3a at 12.5 g/ml induces transcription of BRLF1 and BNLF1 and increases the expression of LMP1, which reduces intracellular phospho-ERK and phospho- IB expression. Meanwhile, F3a increases reactive-oxygen species, actived apoptotic-related proteins, caspase 3, cleavage of PARP, and increases the percentage of apoptotic cells. Therefore, F3a can be developed as a therapeutic drug in the treatment of EBV-related tumors.
