- 學位論文
黃芩活性成分調控血管離子通道活性抑制阻力性血管平滑肌收縮及細胞增生
Active components of Huang-qin inhibit the resistance vascular smooth muscle contraction and cell proliferation via modulating vascular ionic channel activities
摘要
黃芩在亞洲及西方國家，被廣泛使用於治療各種心血管疾病、發炎症狀及癌症。黃芩之活性成分: 黃芩苷 (baicalin)、黃芩素 (baicalein) 及 漢黃芩素 (wogonin) 因為有著多樣化之藥理特性，被視為具有保護心血管之潛力藥物。本論文主要研究baicalin 及 baicalein 運用於保護血管之藥理作用機制，內容分為二個部份，分別探討藥物對於血管張力及血管平滑肌細胞增生之離子通道機轉。 論文的第一部分，探討黃芩活性成分是否能藉由活化大傳導性鈣離子活化型鉀離子通道 (large-conductance Ca2+-activated K+, BKCa) 及抑制電位相關性鈣離子通道 (voltage-dependent Ca2+ channel, VDCC) 使大鼠腸繫膜動脈 (mesenteric arteries, MAs) 產生鬆弛作用。實驗使用雙頻道肌動系統 (dual wire myograph) 及全細胞膜電位箝制技術 (whole cell patch-clamp)，分別測量血管張力及單一細胞離子電流變化。由張力實驗測知，baicalin 比 baicalein 具有較佳的抑制 80 mM KCl誘導血管收縮作用，此抑制作用不會受到 Nω-nitro-L-arginine methyl ester (L-NAME) 或 indomethacin 的加入而被影響。由 iberiotoxin (IbTX)、Bay K8644 及 PMA 誘導之血管收縮，皆可被 baicalin 所抑制。測量全細胞電流變化，可以觀察到 baicalin 劑量相關性的增加 BKCa 通道活性，其增加之電流可被 IbTX 所抑制。 除此之外，baicalin 活化之 BKCa 電流，可分別被不同的抑制劑所抑制，如: adenylate cyclase 抑制劑 SQ 22536、soluble guanylate cyclase 抑制劑 ODQ、競爭型 cAMP拮抗劑Rp-cAMP、競爭型cGMP 拮抗劑 Rp-cGMP、protein kinase A (PKA)抑制劑 KT5720 及 protein kinae G (PKG) 抑制劑 KT5823 等。使用 protein kinase C (PKC) 活化劑 PMA，可使 baicalin 活化之 BKCa 電流完全消失。除此之外，baicalin可劑量相關性的減少 VDCC 電流，並且使 VDCC 活化劑 Bay K8644 增加之鈣離子電流完全受抑制。此研究證實，baicalin 可以藉由活化 cGMP/PKG 及 cAMP/PKA 二種路徑，進而增加 BKCa 通道活性及抑制 VDCC 通道活性，使MAs 產生非內皮相關性之血管鬆弛作用。 論文的第二部分，探討黃芩活性成分對抗內皮素 (endothelin-1, ET-1) 誘發之肺動脈平滑肌細胞增生及離子通道機轉。實驗使用原代培養之大鼠肺臟內肺動脈平滑肌細胞，進行細胞計數、細胞存活檢測 (XTT assay)、西方墨點法 (Western blotting)、流式細胞儀 (flow cytometry) 及螢光分光光譜儀，分別評估細胞增生、蛋白質表現及細胞內鈣離子濃度變化情形。實驗結果顯示，baicalein 比 baicalin 具有較佳的抑制 ET-1 誘導之細胞增生作用。Baicalein 能劑量相關性的抑制 ET-1 誘導之細胞增生、細胞週期進展、細胞內鈣離子濃度增加、填充式鈣離子湧入 (capacitative Ca2+ entry, CCE)、瞬時受體電位陽離子通道1 (transient receptor potential canonical channel 1, TRPC1) 向上調節 (upregulation) 及細胞膜 PKCα 蛋白質過度表現。使用 PMA 可以反轉 baicalein 抑制ET-1 誘導 TRPC1 向上調節及 S 期增加的作用。除此之外，PKC 抑制劑 chelerythrine 可以增進 baicalein 對 G2/M 期的滯留 (arrest) 作用及 TRPC1 的抑制作用。這些結果說明，baicalein 可以藉由調控 PKC 路徑而抑制 TRPC1 的表現，產生對抗 ET-1 誘導肺動脈平滑肌細胞增生的作用。 綜合研究結果，baicalin 可以藉由活化 cAMP/PKA 及 cGMP/PKG 路徑，進而促進 BKCa 通道活性及抑制 VDCC 通道活性，使血管產生鬆弛作用。Baicalein 可以透過抑制 PKC 的表現，使 TRPC1 產生向下調節 (downregulation)，進而減少細胞內鈣離子濃度，達到抑制肺動脈平滑肌細胞增生的效果。基於以上研究結果，建議 bacalin 及 baicalein 可以被開發為血管保護藥物。
關鍵字
離子通道並列摘要
Scutellaria baicalensis (Huang-qin) has been widely used in Asia and Western world for promoting health. It is traditionally used for the treatment of diverse cardiovascular diseases, inflammation and tumors. Because of their diverse pharmacological properties, the active components of Huang-qin, baicalin and baicalein are regarded as potential agents of cardiovascular protection. This study explores the pharmacological effects of baicalin and baicalein on vessels and the underlying mechanisms of these effects. This study is divided into 2 sections to discuss the effect of the ionic mechanisms of baicalin and baicalein on vascular tension and proliferation of vascular smooth muscle cells, respectively. In the first section, we investigated whether the active components of Huang-qin relax mesenteric arteries (MAs) via the activation of large-conductance Ca2+-activated K+ (BKCa) channels and the inhibition of voltage-dependent Ca2+ channels (VDCCs). MAs were obtained from Sprague-Dawley rats. The contractility of the MAs was determined by using a dual wire myograph system. The activity of BKCa channels and VDCCs was measured by using whole-cell patch-clamp recordings in single myocytes. The results showed that baicalin had a better effect than baicalein in attenuating the 80 mM KCl-induced MAs contraction. Baicalin attenuated the contraction of the MAs induced by 80 mM KCl in a concentration-dependent manner. Nω-nitro-L-arginine methyl ester (L-NAME) and indomethacin had only a slight effect on baicalin-induced vasorelaxation. Contractions induced by iberiotoxin (IbTX), Bay K8644 and phorbol 12-myristate 13-acetate (PMA) were abolished by baicalin. In the single myocytes, baicalin activated BKCa channels in a concentration-dependent manner. Baicalin-Increased BKCa currents were abolished by IbTX. Baicalin-enhanced BKCa current was significantly attenuated by SQ 22536 (an adenylate cyclase inhibitor), ODQ (a soluble guanylate cyclase inhibitor), competitive antagonists of cAMP and cGMP (Rp-cAMP and Rp-cGMP), cAMP- and cGMP-dependent protein kinase inhibitors (KT5720 and KT5823). The perfusate with PMA abolished baicalin-enhanced BKCa currents. In addition, baicalin reduced the amplitude of VDCC currents in a concentration-dependent manner and abolished Bay K8644-enhanced VDCC currents. These results demonstrate that baicalin relaxes MAs by activating BKCa channels, as well as inhibiting VDCCs via endothelium-independent mechanisms and stimulation of cGMP/PKG and cAMP/PKA pathways. In the second section, we investigated the antiproliferative effects of the active components of Huang-qin on the proliferation of pulmonary artery smooth muscle cells (PASMCs) mediated by endothelin-1 (ET-1) and the mechanisms underlying these effects. Intrapulmonary artery smooth muscle cells were obtained from Sprague-Dawley rats and cultured. The proliferation of PASMCs was quantified by cell counting and performing an XTT assay. The expression of transient receptor potential canonical channel 1 (TRPC1) and membrane protein kinase Cα (PKCα) was determined by Western blotting. The cell cycle pattern was determined by flow cytometry. The intracellular calcium concentrations ([Ca2+]i) were measured by using the spectrofluorophotometer and flowcytometry. The results showed that baicalein had a better effect than baicalin in inhibiting the ET-1-induced PASMCs proliferation. Baicalein inhibited PASMCs proliferation, promoted cell cycle progression, enhanced [Ca2+]i levels, increased capacitative Ca2+ entry (CCE), up-regulated the TRPC1 and membrane protein kinase Cα (PKCα) expression induced by ET-1. PMA, a PKC activator, reversed the inhibitory effects of baicalein on ET-1-induced TRPC1 upregulation and S phase accumulation, whereas chelerythrine, a PKC inhibitor, stimulated baicalein-mediated G2/M phase arrest and TRPC1 channel inhibition. These findings suggest that baicalein protects against ET-1-induced proliferation of PASMCs via the modulation of PKC-mediated TRPC1 channels. In summary, the vasorelaxant effects of baicalin can be attributed to VDCC inhibition and BKCa channel activation through the cAMP/PKA and cGMP/PKG pathways. The antiproliferative effect of baicalein is related to decreased in [Ca2+]i by downregulation of the TRPC1 channel through PKC inhibition. Based on our findings, baicalin and baicalein are suggested to be developed as vascular protective agents.