TM1-1，TM1-1 DP與六神花萃取物在大鼠心臟缺血-再灌流模式之心血管作用評估

Abstract

中文摘要-第一部分 組織缺血後再灌流引起的損傷，在臨床上是常見且已被證實的，病人在施行心血管手術或器官移植後、或當病人遭受心肌梗塞、冠狀動脈阻塞及缺血性中風的情況下，都會導致組織再灌流的損傷，並影響到病人的預後。 (+)-Thaliporphine是一種結構為phenolic aporphine的生物鹼，來自樟科植物，已經經實驗證實可經由抑制鈉電流 (INa)、瞬間外流鉀電流 (Ito)產生抗心律不整活性、抗氧化及提高血中NO含量等途徑，來達到保護心臟並且減少缺血再灌流的傷害。TM1-1、TM1-1 DP 這兩種化合物是 (+)-Thaliporphine的衍生物，本篇主要在評估這兩個衍生物是否具有對於心臟缺血再灌流的保護效果。 在活體心臟缺血1小時再灌流2小時模式中，使用成年雄性、重約250~350公克的Sprague Dawley (SD) 品系大鼠，來測試TM 1-1(50μg/kg)與TM 1-1 DP(50μg/kg) 的心臟保護效果，紀錄大鼠的LVSP、心跳、心電圖、心電圖、+dP/dt、SV、EF%、ESPVR、EDPVR、PRSW與心肌梗塞壞死區域(IS)的變化，且抽血測量血清中LDH、CPK、CK-MB與NO的總量；另外收取心肌缺血部位組織，利用西方點墨法測定相關蛋白質表現量。 實驗結果顯示：TM 1-1與TM 1-1DP在50μg/kg的劑量下，有減少梗塞壞死面積與保存心臟收縮功能讓心臟具有較佳調控前負荷變化的能力，並能增加血中NO的總量和活化eNOS與RISK途徑且能減少細胞凋亡前驅物質Caspase 3的表現，整體來說，TM 1-1與TM 1-1 DP在低劑量下就具有對缺血再灌流損傷心臟功能保護效果。 摘要-第二部分 先前的研究報告發現，東南亞國家與非洲國家經常使用的一種辛辣香料Spilanthes acmella（Paracress）具有利尿、抗菌和消炎等活性，且發現這種辛辣香料在很低量就具有很強的生物活性。在我們的研究中我們嘗試去評估Spilanthes acmella（Paracress）的丁醇層萃取物在大鼠心臟功能和離體心臟血管肌肉組織的心血管效應。

實驗對象為Sprague-Dawley（SD）大鼠，我們使用離體心臟和降主動脈血管平滑肌肌肉來研究直接的藥效作用，測試藥物對於肌肉收縮力的影響。研究中發現：在心臟肌肉組織（N= 7），其收縮力會隨著藥物濃度的累加積累而顯著降低，尤其是在左心房肌肉組織，可以抑制大約50%的收縮力；右心房的自主心跳速率會在某一特定濃度範圍會受到抑制。而在離體降主動脈平滑肌肉組織實驗中（N =5），血管放鬆作用會隨萃取物濃度增加而增強；如在血管實驗前先以L-NAME（10-5 M）前處理，則藥物增強血管舒張作用的效果會減弱，且透過去除血管內皮的動作，也會使藥物血管舒張作用減弱。 此外，我們探討了萃取物對phenylephrine誘導平滑肌收縮實驗的影響，發現藥物組比起對照組有較高的EC50。代表此萃取物可能會對血管放鬆有關的受體有作用(如甲型交感神經受器阻斷作用等)。為了比較動物體內和體外試驗的結果，我們注入不同劑量的萃取物（IP）發現的+dP / dt，當給予萃取物劑量為10毫克/公斤時，左心室收縮壓和心跳速率抑制效用最強。 總結來說，Spilanthes acmella的丁醇萃取物是一個強效的血管擴張劑，也表現出一些直接抑制心肌收縮力的藥效作用。六神花，這個在東南亞與非洲地區的人們日常生活經常使用的辛辣香料，可能存在很強的心血管系統抑制活性，且可能在某些特殊狀況下是存在有傷害性，這個課題值得我們深入探討研究。

Abstract-Part1 Ischemia-reperfusion injury in clinical practice is common and has been confirmed. The patients in the implementation of cardiovascular surgery or organ transplant, or coronary angioplasty will result in reperfusion injury, and affect the patient's prognosis.

(+)-Thaliporphine is a structure of phenolic alkaloid from the Lauraceae, has been reported to excert anti-arrhythmic activity through the inhibition of the sodium current of NO (INa) and transient outward potassium current (Ito), as well as to protect myocardium from I/R injury via antioxidant activity and increasing the content of NO in the blood, TM 1-1 and TM 1-1 DP are derivatives of (+)-thaliporphine. This study aimed to assess the cardiac protective activity of both compounds in ischemia/reperfusion. The adult male, weighing about 250 to 350 g Sprague Dawley (SD) strain rats were subjected to one hour cardiac ischemia followed by two hours reperfusion. TM 1-1 (50μg/kg) and TM 1-1 DP (50μg/kg) were intravenously infused at 10 minutes before the start of reperfusion. Rat’s LVSP, heart rate, electrocardiogram, +dP/dt, SV, %EF, ESPVR, EDPVR and PRSW were recorded and myocardial infarct size were measured. The blood was collected and the level of LDH, CPK, CK-MB and NO were measured; a separated tissue of myocardial ischemia zone was used to determine the expression of related proteins by Western blot. The results of this study revealed that 50 μg/kg of TM 1-1 and TM 1-1 DP reduced infarct size and increase the preload-recruitable stroke work of I/R rat heart. Biochemical study of the ischemic myocardium showed that the treatment with both agents resulted in an increase expression of p-AKT, p-eNOS, p-ERK and a reduced expression of p-P38 and Caspase 3. In conclusion, TM 1-1 and TM 1-1 DP exerts similar cardiac protection against I/R injury of rat hearts. Abstract-part2 Previous studies have found out that the Spilanthes acmella (Paracress) ,a spice which has been used as a medicine for toothaches and analgesic, has diuretic, antibacterial and anti-inflammatory activities. In our study, we try to access the cardiovascular effects of the butyl alcohol extracts of S. acmella in vivo and in vitro. The isolated heart and descending aorta muscles of Sprague-Dawley (S.D) rats were used to examine the effect of drug on muscle contractility. In the cardiac isolated muscle (n=7),the extracts dose-dependently decreased cardiac contractility with accumulated increase of drug concentrations, especially in left atrial muscle, and heart rate of isolated right heart has same result. And in the descending aorta (n=5), the vaso-relaxation effect of the extract increased concentration dependently. The vassal relaxant effect was blocked by L-NAME (10 -5 M) pretreatment and diminished in denuded smooth muscle. In addition, we examine the effect of the extract on phenylephrine-induced smooth muscle contraction. The drug groups have higher EC50 of phenylephrine than control group. Therefore, in order to compared the results in vivo and in vitro, we inject different dose of the extract (i.p.) was found that the +dP/dt, left ventricular systolic pressure and heart rate was suppressed by 10 mg/kg of the extract. To sum up, the Spilanthes acmella is a potent vasodilator, and also exhibits some direct inhibition of cardiac contractility. This result subjected that the extract from the common spicy herb used in south-east Asia may exert some inhibitory effect on cardiovascular system.