| Abstract: | 我們先前的研究已測試清胃散及其組成方當歸、生地、黃連、牡丹皮及升麻等之 試管內抑制口腔菌及一般病菌之作用。小蘗鹼是從中草藥例如黃連中萃取之一種植物 鹼。在過去十年中，很多論文報導小蘗鹼及其衍生物的藥物功能治療腫瘤、高血脂、 發炎、細菌及病毒感染等。此外，一般認為小蘗鹼在臨床使用劑量並沒有基因毒性、 細胞毒性、突變性、或基因重組性。為了研究清胃散之使用性，我們預計探討小蘗鹼、 黃連及清胃散之生物及功能效應。HPLC 用來定量分析小蘗鹼在黃連及清胃散之含量。 此三年期之研究計畫將研究小蘗鹼、黃連及清胃散(1) 抗單純?疹病毒(2) 抗口腔 癌/細胞凋亡及(3) 生體內細胞激素調節之功能。第一年，單純?疹病毒第一型F 病毒 株及第二型333 病毒株將被用來測試抗病毒效果。空斑減少法用來測試病毒效價。假 如有抗病毒效果，小蘗鹼、黃連及清胃散作用在抗單純?疹病毒的目標，將用病毒吸 附與穿透法來檢視。同位素標記及定量即時PCR 用來檢測病毒DNA 及蛋白。藉由西 方墨點法，小蘗鹼、黃連及清胃散加上一種DNA 合成抑制劑PAA 用來檢測病毒晚期 蛋白。第二年，由於小蘗鹼曾被報導具有經由細胞凋亡抑制腫瘤發展之作用，因此將 研究小蘗鹼、黃連及清胃散是否可抑制口腔癌及其作用機制。口腔癌細胞TSCCa、 GNM、KB、OEC-M1 及OC-2 及口腔正常纖維母細胞用來測試。台盼蘭排除法及MTT 法用來測試細胞毒性。如果小蘗鹼、黃連及清胃散可選擇性毒殺癌細胞，其是否有時 間或劑量依賴性及其作用機制將深入探討。我們先前使用過的caspase 3, annexin V/ PI 染色、DNA 片段化及細胞週期分析將用來評估細胞凋亡。第三年，小蘗鹼對於細胞激 素調節之角色將被闡明。我們將使用Balb/c 小鼠來探討其是否有時間或劑量依賴性細 胞激素調節作用。急性及亞急性測試包括生化分析及病理檢驗都將執行。Th1 (IL-2 及 IFN-), Th2 (IL-4 及IL-10), 前發炎細胞激素(IL-8 及TNF-)，及細胞凋亡細胞激素(Fas and FasL) 都將檢測。此結果並將與我們針對復發性口腔潰瘍患者體內細胞激素之結果 比較，以了解小蘗鹼、黃連及清胃散作為口腔疾病之免疫調節劑之可行性。
Ching-Wei-San and its individual herbal components, Coptidis rhizoma, Angelicae sinensis radix, Rehmanniae radixet rhizom, Moutan radicis cortex, and Cimicifuga foetida, were tested in our lab for in vitro inhibitory effects on oral pathogenic bacteria and common pathogens. Berberine is an isoquinoline alkaloid extracted from Chinese herbs such as Coptidis rhizome. In the past decade, many papers reported the pharmacological properties of berberine and its derivatives for treating tumor, hyperlipemia, inflammation, bacterium and virus infection. Additionally, it is generally considered that berberine has neither genotoxicity, cytotoxicity, mutagenicity nor recombinogenicity at doses used in clinical applications. In order to study the usage of Ching-Wei-San, the biological and functional effects of berberine, Coptidis rhizome, and Ching-Wei-San were thus investigated. HPLC will be used to quantitative analyze the chemical constituents of berberine. This 3-year research project will study the (1) anti-herpes simplex virus effects (2) anti-oral cancer/ apoptosis, and (3) cytokine modulation in vivo of berberine, Coptidis rhizome, and Ching-Wei-San. In Year 1, herpes simplex virus type 1 strain F and type 2 strain 333 will be tested for the antiviraial effects. For virus titration, plaque reduction test will be used. If antiviral effects were exhibited, the sensitive target(s) of HSV replication to berberine, Coptidis rhizome extract, and Ching-Wei-San was examined on virus adsorption and penetration. Isotope labeling and quantitative real-time PCR were also used for detection of viral DNA and protein synthesis. By western blotting, the anti-viral late protein synthesis effects of berberine, Coptidis rhizome extract, and Ching-Wei-San with PAA, a DNA synthesis inhibitor, as positive control were assayed. In Year 2, since berberine has been reported to protect individual against tumor development through apoptotic pathway, it is interesting to study whether berberine, Coptidis rhizome extract, or Ching-Wei-San selectively inhibit oral cancer cells and to study its action mechanism. Oral cancer cell lines including TSCCa, GNM, KB, OEC-M1, and OC-2 were selected for the evaluation. Normal oral fibroblasts were also included as controls. Cells will be tested with berberine, Coptidis rhizome extract, or Ching-Wei-San by trypan blue exclusion test and MTT assay for cytotxcity. If berberine, Coptidis rhizome extract, or Ching-Wei-San exhibit selective cytotoxicity toward cancer cells, the cytotoxicity whether is time- and/or dose- dependent will be studied. Moreover, the mechamism of cytotxcity will also be investigated using caspase 3, annexin V/ PI staining, DNA fragmentation, and cell cycle analyses as parameters for apoptosis analyses which have been used in our lab before. In Year 3, the role of berberine on the expression of cytokines will be elucidated. Our previous studies have been demonstrated that cytokine modulation effects, contributed to physiological or pathological characteristics, induced by some of medicinal herbs can function in a timeand/ or dose-dependent manner. In order to clear the hypothesis that berberine exhibit modulatory effects of cytokines production, Balb/c mice will be used to study the timeand dose-dependent cytokine modulation effects in vivo. Acute toxicity test and subacute toxicity test including ALT, AST, creatinine, and BUN detections as well as pathological examination will also be performed. Th1 type (IL-2 and IFN-), Th2 type (IL-4 and IL-10), proinflammatory cytokines (IL-8 and tumor necrosis factor (TNF-)), and apoptotic cytokines (Fas and FasL) will be tested. These results will be discussed with our previous studies of serum cytokine levels in recurrent aphthous ulcer patients to examine the possibility of using bereberine, Coptidis rhizome extract, or Ching-Wei-San as immune modulatants for oral disease. |
