| 摘要: | 由最近研究發現虎杖根部乙酸乙酯分劃物，F1、F2 及F3 抑制EB 病毒溶裂蛋白質的表現的EC50 分別為25、31 及8.8 μg/ml，而F1 分離物中含有10.4%白黎蘆醇，F3 分離物中大黃素含量68.2%，但是白黎蘆醇及大黃素抑制EB 病毒溶裂蛋白質表現的EC50 分別為5.6 及3.5 μg/ml，所以推論虎杖根部乙酸乙酯分劃物除了大黃素及白黎蘆醇外還有其他成分参與抗EB 病毒的功效。對白黎蘆醇抗EB 病毒機制，我們提出兩種作用模型，一、白黎蘆醇能抑制EB 病毒細胞內p38MAPK、ERK 及JNK 訊息傳遞，進而抑制AP-1 or ATF2 所結合至溶裂循環基因啟動子，使得EB 病毒溶裂循環基因無法轉錄作用，進而影響EB病毒溶裂循環。二、白黎蘆醇可能藉由活化EB病毒細胞中sirtuin (SIRT1) 的去乙醯酶功能，使得EB 病毒溶裂循環基因BRLF1 and BZLF1 啟動子不能進行轉錄。而對大黃素抗EB 病毒作用模型是藉由促進ROS 的產生，抑制Akt 表現使得下游基因 AP-1 也無法表現，而AP-1 無法結合至EB 病毒溶裂循環基因啟動子，也使得EB 病毒溶裂循環基因無法轉錄作用，進而影響EB 病毒溶裂循環。此外，也發現50 μg/ml 虎杖根乙醇萃取物能完全抑制EB 病毒表現潛伏膜蛋白質1 表現(LMP1)誘發EB 病毒腫瘤細胞凋亡；虎杖根部乙酸乙酯分劃物，F1 及F2 在濃度12.5 μg/ml 對潛伏膜蛋白質1 表現有些微影響，而F3 在濃度6.3 μg/ml 就能完全抑制EB 病毒表現潛伏膜蛋白質1 表現，因此推論虎杖根部乙酸乙酯分劃物應含有抑制EB病毒表現潛伏膜蛋白質1 表現而使EB 病毒腫瘤細胞凋亡的有效成分。所以本計劃分四部份：第一部份、抗EB 病毒及抗腫瘤有效成份的分離及鑑定。第二及第四部份、探討抗EB 病毒及毒殺EB 病毒淋巴腫瘤細胞有效成分之作用機制。第三部分，探討大黃素及白黎蘆醇抗EB 病毒機制。我們將以半製備逆相式高效液相色層分析法將抗EB 病毒及抗腫瘤有效成分分離純化，並且進行鑑定。以流式細胞儀法及real-time PCR 定量抗EB 病毒有效成份對病毒溶裂蛋白質表現、ROS 及EB 病毒DNA；以西方墨點法偵測p38MAPK、 ERK、JNK 及Akt 磷酸化或非磷酸化蛋白質表現、SIRT1 以及AP-1 族群JunD 及JAB1 或ATF2 蛋白質表現；也偵測SIRT1 去乙醯酶活性，以了解有效成份抑制EB 病毒感染、複製及病毒顆粒形成的機制。以細胞毒性實驗及細胞核型態學觀察探討有效成分抑制腫瘤細胞生長的影響，進而以流式細胞儀定量凋亡的細胞數。並且以免疫螢光法分析抗腫瘤的有效成分是否抑制腫瘤細胞內NF-κB 的表現。這些研究將有助於更了解虎杖根抗病毒及抗腫瘤有效成分的作用機制。
From our recent studies demonstrated that the ethyl acetate subfractions of Polygonum cuspidatum root F1, F2 and F3 inhibited the expression of Epstein-Barr virus (EBV) lytic proteins. The concentrations of F1, F2 and F3 to inhibit EBV lytic proteins expression by 50% (EC50) were approximately 25, 31 and 8.8 μg/ml, respectively. Further analysis for the constituents of the ethyl acetate subfractions of Polygonum cuspidatum root, found that F1 contains 10.4% resveratrol, F3 contains 68.2% emodin. The concentrations of resveratrol and emodin to inhibit EBV lytic proteins expression by 50% (EC50) were approximately 5.6 and 3.5 μg/ml, respectively. Therefore, I suggest that bioactive components of anti-EBV activity from the ethyl acetate subfractions of Polygonum cuspidatum root may be not only mediated by emodin and resveratrol. Furthermore, I propose two action models for anti-EB viral activity of resveratrol. The first model, Resveratrol suppresses the p38MAPK, ERK and JNK signal pathways in EBV-positive cells, and then interferes the binding of AP-1 and ATF2 to the promoter of immediate-early genes of EBV, leads to inhibit the transcription of immediate-early genes and affect EBV lytic cycle. The secondary model, the activation of resveratrol on deacetylase activity of sirtuin protein may involve in the transcriptional inhibition of EBV immediate-early genes, BZLF1 and BRLF1. I also propose the action model for anti-EB viral activity of emodin. Emodin promotes ROS production in EBV-positive cells, and inhibits Akt activity. Blocking of Akt pathway leads to disrupt the activation ability of AP-1 and ATF2 to EBV immediate-early genes. In addition, based on the preliminary studies, the ethanolic extract of Polygonum cuspidatum root, 50 μg/ml displayed the inhibitory effect on the expression of latent membrane protein 1 (LMP1) of EBV.and cause death of EBV-positive tumor cells. Moreover, the ethyl acetate subfractions of Polygonum cuspidatum root, F1 and F2 had little effect on the expression LMP1 at 12.5 μg/ml, F3 at 6.3 μg/ml completely inhibited the expression of LMP1 Therefore, I propose that the ethyl acetate subfractions of Polygonum cuspidatum root contains the bioactive components of anti-EBV-tumor activity. In this proposed study, I will to perform four parts: Part I, to isolate and identify the antiviral and anti-tumor components. Part II and Part IV to investigate the mechanism of anti-EBV and cytotoxicity to EBV-positive tumor cells. Part III to explore the action model of resveratrol and emodin. Semi-prepared reverse phase high performance liquid chromatography will be used to isolate and identify the antiviral and anti-tumor components. Using flow cytometric analysis and real-time PCR to quantitate the expression of EBV lytic proteins ROS and determine the amount of EBV DNA. Immunoblotting assay will used to determine the expression of phosphorylated and dephosphorylated p38MAPK, ERK, JNK and Akt, SIRT1 and AP-1 members (JunD and JAB) and ATF2. These results will be promoted to understand bioactive components to EBV infection, replication and virus particle production. I will perform the cytotoxicity assay and nuclei morphology observation was used to analyze whether bioactive components have the anti-proliferative activity on EBV-positive tumor cells. Indirect immunofluorescence was performing to investigate the NF-κB expression. The study is important for elucidating the antiviral and antitumor mechanism of Polygonum cuspidatum root. |
