5,7,3'-三甲基橙皮素的抗氣喘作用與初、後期氣喘 動物模式之探討

Anti-asthmatic action of hesperetin-5,7,3'-O-trimethylether and investigation of asthmatic animal model with early- and late-phases of airway hyperresponsiveness

王文弘(Wen-Hung Wang)

指導教授 ： 柯文昌

臺北醫學大學/醫學院/藥理學研究所/碩士(2006年)

摘要

PART1 橙皮素 (hesperetin) 是一種選擇性PDE4的抑制劑，我們曾報告對氣喘治療具有潛力，我們為提高其PDE4H/PDE4L比，所以合成 5,7,3'-三甲基橙皮素 (hesperetin-5,7,3'-O-trimethylether, HTME) ，並研究其抗氣喘的效果。 將雌 BALB/c 小鼠腹腔內注射卵蛋白 (ovalbumin, OVA) ，使其敏感化，再以氣化的卵蛋白 (OVA) 二次激釁 (secondary challenge)，利用整體體積描述器來分析因 methacholine (MCh) 引起的氣道過度反應 (airway hyperresponsiveness; AHR)，結果顯示 HTME 能抑制卵蛋白 (OVA) 引起的氣道過度反應 (AHR)，HTME (10~100 μmol/kg, p.o.)能劑量依存性且有意義地 (P < 0.05) 減少 MCh (25~50 mg/ml) 引起的 enhanced pause (Penh) 值增加。HTME (10~100 μmol/kg, p.o.) 有意義地 (P < 0.05) 減少肺泡灌洗液中總發炎細胞數、嗜中性血球、嗜伊紅白血球、淋巴球及巨噬細胞，但最低劑量不能有意義地減少嗜中性白血球及巨噬細胞是例外外。HTME (10~100 μmol/kg, p.o.) 也會有意義地 (P < 0.05) 降低肺泡灌洗液中 IL-2, IL-4, IL-5, IFN-? 和TNF-? 的釋放，也會有意義地 (P < 0.05) 減少血清和肺泡灌洗液中的total- 和OVA-specific IgE，但最低劑量不能減少血清中total IgE的量是例外。 HTME 會濃度依存性地抑制PDE1、PDE3及PDE4，其IC50值分別為18.86、14.38、9.39 μM。HTME取代結合在敏感化天竺鼠全腦細胞顆粒HARBS之 [3H]-rolipram的EC50值為171.5 μM，因此HTME的PDE4H/PDE4L比值約為18.2。由 Lineweaver-Burk 分析發現HTME 對PDE1、PDE 3及PDE4呈競爭性的抑制，其Ki值分別為40.3, 21.9, 及7.6 μM，對PDE3和PDE4的Ki值雖然彼此間無意義差，但兩者均有意義地小於對PDE1的Ki值，顯示對PDE3及PDE4的親和力最好，其次是PDE1。 HTME (10~30 ?M) 會有意義地鬆弛敏感化離體天竺鼠氣管的基本張力，也有意義地抑制累加 OVA (10~100 ?g/ml) 引起的敏感化離體天竺鼠氣管之收縮，甚至於HTME 3 μM 能有意義地抑制OVA 100 μg/ml 引起的收縮。 結論，HTME對PDE4的親和力最高，具有選擇性且競爭性地抑制 PDE4，也同時抑制PDE3，因此有加強的效果，又因其PDE4H/PDE4L比是18.2，所以推測此藥物極具治療氣喘的潛力。 PART 2 本篇藉由methylprednisolone、pyrilamine、cromolyn三種臨床上使用的藥物，來探討具有初、後期氣道過度反應的氣喘動物模式之可用性。 將雌 BALB/c 小鼠腹腔內注射卵蛋白 (ovalbumin; OVA) ，使其敏感化，再以卵蛋白 (OVA) 氣化噴霧激釁，利用整體體積描述器來分析因 methacholine (MCh, 50 mg/ml) 引起的氣道過度反應 (airway hyperresponsiveness ; AHR)，結果顯示，methylprednisolone (10 mg/kg, s.c.)、pyrilamine (25 mg/kg, s.c.)或cromolyn (2%, inhalation) 在氣道過度反應初期會有意義地抑制 MCh所引起的enhanced pause (Penh) 值的增加。而在氣道過度反應後期，則只有methylprenisolone、pyrilamine有意義地抑制MCh所引起的Penh值增加。Methylprednisolone、pyrilamine、cromolyn和對照組比較沒有意義地減少肺泡灌洗液中的發炎細胞和細胞介素。Methyprednisolone、pyrilamine、cromolyn有意義地 (P < 0.05) 減少肺泡灌洗液和血清中的total IgE，methylprednisolone和cromolyn，但非pyrilamine，能有意義地 (P < 0.05)抑制肺泡灌洗液中的OVA-specific IgE，然而三者都不能有意義地減少血清中的OVA-specific IgE。 本實驗結果，由於 (1) methylprednisolone 無法抑制血清中的 OVA-specific IgE，與臨床效果不同; (2) 此三種藥物無法有意義地抑制發炎細胞的增加; (3) 對照組都無法使IL-2、IL-4及TNF-α增加，因此我們對此動物模式持保留態度，不加以推薦。

關鍵字

甲基橙皮素 ； 抗氣喘

並列摘要

PART 1 Hesperetin, a selective PDE4 inhibitor, has been reported to have a potential in the treatment of asthma in our laboratory. To improve its PDE4H/PDE4L ratio, we synthesized hesperetin-5,7,3’-trimethyl ether (HTME), and investigated its anti-asthmatic effects. In the sensitized and OVA-secondarily challenged BALB/c mice, an asthmatic animal model, the airway hyperresponsiveness (AHR) was measured in unrestrained animals by barometric plethysmography using a whole-body plethysmograph after exposure of methacholine (MCh, 6.25~50 mg/ml) and enhanced pause (Penh) values were determined. In the present results, HTME (10~100 ?mol/kg, p.o.) dose-dependently and significantly suppressed the enhancement of MCh (25~50 mg/ml)-induced Penh values. It also significantly reduced the increase of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils with an exception that at the least dose HTME did not significantly suppressed marcrophages and neutrophils, and significantly reduced the release of IL-2, IL-4, IL-5, TNF-?, and IFN-??in brochoalveolar lavage fluid (BALF). It also significantly reduced total and OVA-specific IgE in serum and BALF, with an exception that at the least dose HTME did not significantly reduced total IgE in BALF. HTME concentration-dependently inhibited PDE1, PDE3, and PDE4 with an IC50 value of 18.86, 14.38, and 9.39 ?M, respectively. HTME displaced [3H]-rolipram from high affinity rolipram binding sites (HARBS) of particulates of whole brains isolated from sensitized guinea pigs, with an EC50 value of 171.5 ?M. Therefore, the PDE4H/PDE4L ratio of HTME was 18.2. From Lineweaver-Burk analysis, HTME competitively inhibited PDE1, PDE3, and PDE4 activities, with a Ki value of 40.3, 21.9, and 7.5 ?M, respectively. Although the later two values did not significantly differ from each other, however, the former value was significantly greater than the later two values. It suggests that HTME has a high affinity to PDE3 and PDE4, then to PDE1. HTME (10~30 ?M) significantly relaxed the baseline tension and suppressed OVA (10~100 ?g/ml)-induced contractions in isolated sensitized guinea pig trachealis. HTME even at 3 ?M significantly suppressed OVA (100 ?g/ml)-induced ones. In conclusion, HTME had high affinity to PDE4, and at the same time it also competitively inhibited PDE3, which may potentiate anti-inflammatory effects of the former. The above results suggests that HTME, with PDE4H/PDE4L ratio of 18.2, may have potential in the treatment of asthma. PART 2 To investigate the possibility of asthmatic animal model with early- and late-phases of airway hyperresponsiveness (AHR), we used three kinds of clinically useful drugs, methylprednisolone, pyrilamine and cromolyn in this study. Methacholine (MCh, 50 mg/ml)-induced AHR in sensitized and challenged mice with ovalbumin (OVA) by barometric plethysmography, using a whole-body plethymograph. In the present results, methylprednisolone (10 mg/kg, s.c. ), pyrilamine (25 mg/kg, s.c. ), and cromolyn (2%, inhalation ) significantly inhibited enhanced pause (Penh) in the early-phase of AHR. In the late-phase of AHR, only methylprednisolone and pyrilamine significantly inhibited increase of MCh-induced Penh values. When compared to control group, these three drugs did not attenuate inflammatory cells and cytokines. These three drugs significantly reduced total IgE in bronchoalveoar lavage fluid (BALF) and serum. Methylprdnisolone and cromolyn, but not pyrilamine, significantly reduced OVA-specific IgE in BALF, but not in serum. Owing to the present results that (1) methylprednisolone, with a contrast to clinical effects, did not reduce OVA-specific IgE in serum; (2) these three drugs did not reduce inflammatory cells; and (3) IL-2, IL-4 and TNF-α did not increase in BALF of control group, we do not recommend this animal model with a conservative manner.

並列關鍵字

hesperetin-5,7,3''-O-trimethylether ； asthma

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