越南巴豆 (Croton tonkinensis Gagnep.) 是一種原產於越南北部的熱帶灌木，並在當地常用於治療不同種類的疾病。近期研究發現，越南巴豆的萃取物可抗發炎和癌症化學預防活性。根據先前的植物化學研究，越南巴豆含有豐富的二萜類化合物 (diterpenoids)。然而，對於越南巴豆純化的化合物在擾亂細胞膜上通道的活性尚未清楚。因此，在本篇的研究目的主要是想探討這些二萜類化合物對於離子電流振幅和閘門控制的影響，特別是電壓閘門控制型的鉀離子通道。在本篇研究選用微膠細胞 (microglial cells) 來實驗，微膠細胞是一種常見於中樞神經系統的巨噬細胞，並具有三種不同種類的鉀離子電流，分別為向內糾正型鉀離子電流 (inwardly rectifying K+ current, IK(IR))、延遲糾正型鉀離子電流 (delayed-rectifier K+ current, IK(DR)) 以及鈣離子活化鉀離子電流 (Ca2+-activated K+ current, IK(Ca))。然而這些電流在微膠細胞上的研究人有大部分程度尚未明瞭，但可以針對這些鉀離子電流的影響，提供微膠細胞在神經發炎疾病上一個重要的方法。因此，我們利用越南巴豆的化合物，croton-01, croton-02和croton-03影響微膠細胞 (SM826) 上鉀離子電流之變化，即瞭解這些化合物對於微膠細胞上內糾正型鉀離子電流 (IK(IR)) 和延遲糾正型鉀離子電流 (IK(DR)) 的生物物理學以及藥理學性質的比較。越南巴豆以濃度依賴的方式產生差異性的內糾正型鉀離子電流 (IK(IR)) 和延遲糾正型鉀離子電流 (IK(DR)) 之抑制，越南巴豆藥物(croton-01, croton-02 and croton-03)一半抑制效力的濃度 (IC50) 大約分別為8.3、14.7 和 8.1 µM。在微膠細胞中觀察到內糾正型鉀離子電流 (IK(IR)) 未活化反應出去極化的增加，不過在鈣離子活化鉀離子電流 (Ca2+-activated K+ current, IK(Ca)) 活性上則未受影響。在β-澱粉樣蛋白 (β-amyloid) 處理中，這些鉀離子電流的抑制情況也是相同的。特別的是，croton-03 能以濃度、時間和狀態依賴的方式修飾微膠細胞中內糾正型鉀離子電流 (IK(IR)) 和延遲糾正型鉀離子電流 (IK(DR)) 的振幅和閘門控制。在越南巴豆處理過後的微膠細胞，不管是否有活化的細胞組別，在誘導型一氧化氮合酶 (iNOS) 和內皮型一氧化氮合酶 (eNOS) 在蛋白質的表現都有上升的趨勢。這可能代表著在處理越南巴豆下的細胞組別，對於某些發炎因子(如一氧化氮 [NO]) 的產生會有抑制的效果。可是，在 Kir 與 KCa的訊息核糖核酸 (mRNA) 和蛋白量的表現，則越南巴豆處理後並無顯著差異。可能代表著越南巴豆影響的是鉀離子通道的電流功能，而不是訊息核糖核酸和蛋白量的多寡。這項研究提供一些ent-kraurane 型二萜類化合物之生物作用。因此預期如果在體內產生相似的作用，可利用二萜類化合物對微膠細胞在KV 與 Kir 通道有阻斷的作用，會有助於這些化合物在藥理學或治療作用的參考。

The Croton tonkinensis Gagnep. (Euphorbiaceae) is known as a tropical shrub native to northern Vietnam and has been used commonly in Vietnam to treat different types of disorders. Anti-inflammatory and cancer chemopreventive activities of C. tonkinensis extracts have been recently described. Previous phytochemical investigations have shown that C. tonkinensis contains a rich source of diterpenoids. However, none of reports have demonstrated that these compounds purified from Croton were effective at perturbing the activity of membrane ion channels. Therefore, effects of these diterpenoid compounds on the amplitude and gating of ionic currents, particularly voltage-gated K+ currents, were extensively investigated in this study. Specifically, microglial cells are brain-resident macrophage and have been clearly described to express three different types of K+ currents, namely, inwardly rectifying K+ (IK(IR)), delayed-rectifier K+ (IK(DR)) and Ca2+-activated K+ (IK(Ca)) currents. Although the functional role of these channels in microglia remains to be largely unexplored, targeting these K+ channels provides an important alternative used to influence neuroinflammatory activities of microglia. Therefore, the purpose of this work was to test whether the fractions purified from C. tonkinensis, namely croton-01, croton-02 and croton-03 could exert any perturbations on ionic currents existing in microglia SM826 cells. The biophysical and pharmacological properties of ionic currents including IK(IR) and IK(DR) in these cells were characterized and effects of these compounds were then compared. Croton differentially produced inhibition of IK(DR) and IK(IR) in a concentration-dependent manner. The IC50 values required for croton-01, croton-02 and croton-03-induced inhibition of IK(DR) were 8.3, 14.7 and 8.1 µM, respectively. The increase of IK(DR) inactivation in response to depolarization was observed in SM826 microglial cells. These had little or no effect on Ca2+-activated K+ currents. In SM826 microglial cells differentiated with β-amyloid, inhibitory action on these K+ currents remained unaltered. Particularly, the present results highlight that croton-03 is capable of modifying the amplitude and gating of both IK(IR) and IK(DR) in these microglial cells in a concentration-, time-, and state-dependent manner. The expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) was increased in microglial cells treated with crotons, regardless of whether cells have been activated with lipopolysaccharide or -amyloid. This may represent the inhibitory effect on the production of inflammatory factors - nitric oxide (NO) in these cells as they are exposed to crotons; however, there was no significant difference in the expression of mRNA and protein in both Kir and KCa. Therefore, the effect of crotons is directed at potassium current function, rather than the mRNA or the protein amount. Findings from this study provide important insights on the still mysterious biological actions of these ent-kraurane-type diterpenoids. It is anticipated that block of microglial KV and Kir channels caused by these diterpenoids contributes to their pharmacological or therapeutic actions if similar actions occur in vivo.

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