傳統中藥在消炎鎮痛機制作用的分子生物學研究

如意金黃膏是由如意金黃散製備而成的軟膏，而如意金黃散是中國古代方劑的粉末，通常使用於抗發炎及止痛方面。本研究系列針對如意金黃膏進行有關抗發炎、毒性及致敏性之實驗。在抗發炎方面：以西方墨點分析發現如意金黃膏可抑制脂多醣 Lipopolysaccharides (LPS) (1 ?nµg/mL) 所誘發巨噬細胞一氧化氮合成酶 (iNOS) 及第二型環氧化酶 (COX-2) 之表現的副作用，具抗發炎作用之潛力。其植物成員則顯示具有不同程度抑制一氧化氮合成酶及第二型環氧化酶之表現。LPS 所引起的iNOS 和COX-2 表現，已被確認能夠增加細胞激素和一氧化氮 ( NO ) 的產生，而它們在發炎反應中扮演極重要的角色。在老鼠的巨噬細胞稱 ( RAW264.7 )，如意金黃膏能夠依濃度相關性抑制 iNOS 和COX-2 表現。然而，如意金黃膏的各種中藥組成分別會導致不同的結果，包括增加或減少蛋白質的表現。在這之中薑黃 (Cu )、蒼朮 (At )、甘草 (GI )會比其他成分更能抑制發炎反應，在細胞培養15 小時之抑制效果最強。此一結果，可用於改善或重組如意金黃散處方之參考。至於在細胞活性及存活方面，同樣在6 小時及15 小時都顯示如意金黃膏會依濃度相關性產生細胞抑制表現，如意金黃膏本身在細胞培養後15 小時才呈現明顯的細胞毒性(50%)。而其中，以甘草、薑黃、蒼朮如意金黃膏之植物成員萃取物對於巨噬細胞在細胞培養後6 及15 小時呈現不同的細胞存活率或細胞毒性；薑黃 (Cu )、蒼朮 (At )之抑制作用可能是由於其細胞毒性造成，甘草 (GI )的細胞毒性較薑黃 (Cu )、蒼朮 (At )小；所以可能是如意金黃膏中最具抑制iNOS 和COX-2 表現之植物成員。至於在致敏性的探討，以細胞激素 ( IL-4 及 TNF-α）之生成和LPS 引起的TNF-α 表現而言，如意金黃膏及 croton oil 不增加血中之 IL-4 及 TNF-α，顯示如意金黃膏並無顯著之致敏性。此結果顯示在臨床應用上在疼痛患者貼用如意金黃膏可改善疼痛，它不會明顯引起免疫致敏性反應；此作用可能與一氧化氮合成酶及第二型環氧化酶之抑制與部份細胞毒性有關。在臨床使用上，如意金黃膏貼片證實可以明顯緩解因發炎導致的身體疼痛。

關鍵字

蛋白質表現；抗炎作用；細胞毒性；黃柏；薑黃；大黃；甘草；致敏性；巴豆油；脂多醣體；介白質-4 ；甲型細胞壞死因子；細胞激素；細胞生存率； MTT細胞生增測定；第二型環氧化? ；誘發一氧化碳生成? ；鼠科動物巨噬細胞細胞RAW264.7 ；一氧化碳；蒼朮；天南星；陳皮；厚朴；白芷；天花粉；如意金黃膏；如意金黃散；如意金黃貼片；西方點漬分析法；吳謙；醫宗金鑑(清朝) ；目測類比指標；經皮吸收；環釋性系統

並列摘要

Ruyi-Jinhuang Gao is an ointment prepared from Ruyi-Jinhuang San, a traditional formula of powder type Chinese Medicine, usually used in the anti-inflammatory analgesic care in China. The present studies were aimed 1) to detect the anti-inflammatory signaling of Ruyi-Jinhuang Gao exposed by an experimental model of lipopolysaccharides (LPS)-induced pro-inflammatory expression of inducible nitric oxide synthase (iNOS) and prostaglandin endoperoxide synthase (PGH synthase-2; COX-2). LPS-induced activation of iNOS and COX-2 has been recognized to increase cytokines and nitric oxide (NO), which of them play the predominant roles in inflammation. In RAW264.7 cells, Ruyi-Jinhuang Gao concentration-dependently inhibited LPS-induced iNOS and COX-2 expression. However, the herbal components of Ruyi-Jinhuang Gao displayed different spectrum of results, including increase and decrease of both protein expressions. Among them, inhibition by Curcuma Zedorarica Rhizoma (Cu), Atractylodis Lancea Rhizoma (At), and Glycyrrhizae Radix (Gl) are more potent than by others on iNOS and COX-2 inhibition at 15 hours. This variety of expression enhanced the possibility of formulation rearrangement. 2) Related to cytotoxicity or cell viability, Ruyi-Jinhuang Gao showed various degree of cellular survival rate with concentration-dependent after 6 and 15 hours of macrophage cell line incubation. These herbal components analysis revealed that inhibitions by Cu and At may be mainly related to their cytotoxicity. And, Glycyrrhizae Radix (Gl) showed less cytotoxicity than Cu and At. Presumably, GI is the most potent component of Ruyi-Jinhuang-Gao in inhibition of LPS-induced iNOS and COX-2 expression. 3) Concerning about Sensitization, Ruyi-Jinhuang Gao showed there was inhibition of pro-inflammatory cytokines in compared with the LPS-induced tumor necrosis factor-α (TNF-α)?w?z croton oil-induced cytokine formation of TNF-α and interleukin-4 (IL-4). It indicates that Ruyi-Jinhuang Gao may not significantly sensitize an immunologic response during the treatment of non-wounded inflammation on the body surface. Clinically, Ruyi-Jinhuang Patch, a patch preparation of Ruyi-Jinhuang Gao, has proven significantly to relief cutaneous inflammation-associated somatic pain with its benefit of less sensitization. The pharmacologic activities of Ruyi-Jinhuang Gao may be mainly due to blockade of iNOS and COX-2 with partial cytotoxicity.