一、低濃度 AVLE 對內皮 (endothelium) 依存性及對於大鼠主 動脈血管環舒張 (relaxation)之影響。
在內皮完整狀態下 (使用 CCh 3×10-6 M 測定內皮作用活性) 的離 體大鼠主動脈血管環中，給予前置收縮劑 PE 10-6 M，後給 AVLE 10 μg/ ml，離體大鼠主動脈血管環能行使舒張的效應。如結果圖 12 B.。
在去內皮的狀態下， ( 使用皂素 saponin 100 μg/ ml ; 將離體大 鼠主動脈血管環去內皮 )，會使 AVLE 10 μg/ ml 的舒張效應消失，如 圖 12 D.。與在 PE 10-6 M 之前使用一氧化氮合成酶抑制劑 L-NAME 3×10-4 M，則會取消低劑量 AVLE 舒張之效應。如圖 12 C.。
統計如 (圖 12 E.)
Fig. 12
Tension %
0 20 40 60 80 100
PE CCh AVLE d.E +AVLE
*** ***
0.5 g
10 min
A . C.
+E +E
D.
B.
-E +E
E.
Fig. 12 AVLE- induced vaso-relaxation in rat aortic rings. A. PE
control. Tracing of phenylephrine ( PE ) 10-6 M. B. The tracing showing the effect of AVLE 10 µg/ml on contractile responses of rat aortic rings with intact endothelium in response to phenylephrine 10-6 M (PE; n= 11, from 3 rats). The CCh 3×10-6 M, the endothelium functional test of the control in experiment in response to pretreated PE 10-6 M. C. Tracings showing that L-NAME prevented the relaxant effect of AVLE 10 µg/ml on PE 10-6 M contracted rat aortic rings. D. Tracing of endothelium denuded aortic contraction with compared to that endothelium intact when they were contracted with PE 10-6 M followed by addition of AVLE 10 μg/ml. E. Data summary of the relaxant effect of AVLE 10 µg/ml on the contractile responses of rat aortic rings with intact endothelium in response to phenylephrine 10-6 M (PE; n = 11). And with denuded endothelium (abbreviated as d.E) of rat aortic rings abolished the AVLE 10 µg/ml relaxation effect on phenylephrine 10-6 M contractile response.The result were compared to the relaxant effect of CCh 3×10-6 M under the same experimental condition. *** Statistically significant ( P < 0.001) compared to PE control.
二、去內皮與完整內皮之靜置張力下；AVLE 濃度累加至高劑量恢復 收縮之作用。
低劑量 AVLE 10 µg/ml 時能產生最大的內皮調節產生 NO 血管 舒張作用效應。 ( NO-mediated and Endothelium-depend manner )。而 當實驗繼續累積至高劑量 30 µg/ml、90 µg/ml 以上時，血管舒張的效 應減弱了，反而出現逆轉舒張；逆轉舒張之現象 (圖 13 B., C.)。當累 積劑量至 90 µg/ml, 甚至超過 90 µg/ml 時，AVLE 會減少離體大鼠血 管環舒張之效應，使張力水平恢復至前置血管收縮劑 PE 10-6 M 之收 縮高度。
在離體大鼠主動脈血管環去內皮的狀態下 (圖 13 D.)，則看不出 此逆轉舒張之現象。因為低劑量舒張的作用被取消了，因此看不出逆 轉舒張之反應高度。
因此，實驗中以去內皮與否之離體大鼠主動脈血管環；前置低劑 量血管收縮劑 PE 3×10-8 M，後累加 AVLE 至高劑量，作為觀察逆轉 舒張；逆轉舒張反應的張力程度之實驗設計 (圖 14 A., B.)。以及前置 使用 L-NAME 3×10-4 M 作為觀察 AVLE 高劑量之逆轉舒轉; 逆轉舒 張反應之張力現象 (圖 14 C.)。統計圖如圖 14.E。
在實驗中，在靜置水平張力下之離體大鼠血管環，給予 AVLE 以 劑量累加之方法至高劑量。結果顯示十個離體大鼠動脈血管環中占有 四成比例之血管環數量，具有「spike activity」跳動活性之現象，而 且具有重複性。 (如圖 15.)。
Fig. 13
+E D.
-E C.
+E B.
+E A.
0.5 g
10 min
E.
Fig. 13 High dose AVLE restored- contraction effect in rat aortic rings. A. PE control. Tracing of PE 10
-6 M. B. Typical tracing of concentration-dependence of AVLE 10, 30 μg/ml on PE contractions compared with control. C. Typical tracing of concentration-dependence of AVLE 10, 30, 90 μg/ml on PE contractions compared with control. D.tracing of concentration-dependence of AVLE 10, 30, 90 μg/ml on PE contractions with endothelium-denuded aortic rings. Typical tracing of concentration-dependence of AVLE on PE 10-6 M contracted aortic rings (-E, endothelium-denuded; +E, endothelium intact ). Removal of endothelium was achieved by incubating the aortic ring with Saponin 200 μg/ml for 5 minutes and assessed as the diminishment of relaxation induced by CCh 3×10-6 M. E. Summary of data obtained under conditions as described in the legend of B. (statistically significant * , P < 0.05 ; ***, P < 0.01)(n = 5, from 3 rats)
Fig. 14
A.
0.5 g
10 min
+E
B.
-E
C.
+E
D.
Fig. 14 High dose AVLE restored- contraction effect on pretreated
low dose PE in rat aortic rings A. Typical tracing of concentration-
dependent of AVLE 10, 30, 90 μg/ml on PE-induced contraction in rat aorta with endothelium (+E) . B. compared with that in denuded endothelium (-E) at low dose PE 3×10-8 M. The restoration of PE-contraction remains below the plateau PE response after introduction of AVLE 90 μg/ml. C. Experiment condition remains the same as in the about protocol except that L-NAME 3×10-4 M was used to inhibit the NO formation. D. Data summary of experiment as described in legend of Fig 14 A, B. (** P < 0.01, *** P < 0.001) (+E, n=6, from 3 rats) (-E, n=6, from 2 rats)Fig. 15
Fig. 15「Spike activity」of high dose AVLE effect on resting tesion in rat aortic rings「Spike activity」 of high dose AVLE 30, 90 μg/ml on
resting tension level of rat aortic rings. Upon Two tracings are control and high dose of AVLE stored contractile effect.0.5 g
10 min
三、AVLE與血管培育時間對血管收縮及舒張之影響
單劑量 AVLE 10 μg/ml 和單劑量 30μg/ml 有著舒張血管之效應 (圖 16 A., 16 B.)，而單劑 AVLE 10 μg/ml 可以重複數次而不影響血管內皮 活性。單劑 30 μg/ml 卻不能重複第二次 (圖 16 B.) (第二次作用下，
便無舒張血管之效應)，經 CCh 3×10-6 M 作用內皮顯示，當 AVLE 30 μg/ml 重複時 (固定浸浴在器官槽 10 分鐘)，內皮活性也會逐次減少 20%。
在低劑量 AVLE 10 μg/ml 「長時間」浸浴在器官槽中 (約 40 mins)，
則無法再重覆單劑低劑量 10 μg/ml 之舒張作用 (圖 16 C.)，而且 CCh 內皮作用活性測定中， 與後加的 PE，測定活性作用皆會有減少 20 % 之現象。如圖 16 E.與 圖 16 F.。顯示出 AVLE 在離體器官槽中，「低」
劑量「長」時間，與「高」劑量「短」時間，皆會對離體大鼠主動脈 產生損害之作用。
結果顯示 AVLE 之功能活性有「劑量依存性」和「時間依存性」
之特徵。
Fig. 16
Fig. 16 Damaged effect of high dose and low dose long time incubation of AVLE in rat aortic rings. A. Tracing of the effect high
AVLE (90μg/ml) on the subsequent contraction to PE 10-6 M, CCh-induced relaxation and contraction to KCl 80 mM. B. Tracing of CCh after AVLE 30 μg/ml three times repeat on PE 10-6 M and 10-7 M. C.Tracing of low AVLE (10 μg/ml) in time control manner ( incubation for
C ont rac tion to KC l (%)
40 mins ), and repeat CCh test. D. Statistics of pretreated high dose and low dose long time incubation of AVLE effect on PE. Black bar: Control contractions to PE 10-6 M. ( n=5, from 3 rats ) Gray bar: Contraction to PE 10-6 M after incubation with low AVLE 10 μg/ml for 40 minutes on rat aortic rings. ( n= 7 rings, from 2 rats ) Black gray bar : Contraction to PE 10-6 M after incubation with AVLE 30 μg/ml for 10 minutes on rat aortic rings. ( n= 6 rings, from 3 rats ) E. Statistics of pretreated high dose and low dose long time incubation of AVLE effect on CCh. Black bar:
Control contractions to PE 10-6 M. ( n=7, from 3 rats) Gray bar: The statistics of normal CCh control. ( n=5 , from 3 rats) Black gray bar: The statistics of low AVLE 10 μg/ml incubation in organ bath for 40 minutes compared with control of CCh. ( n= 7 rings, from 2 rats ) White gray bar:
Statistics of CCh after high AVLE 30 μg/ml ( each incubation for 10 mins repeat two or three times ) compared with control with CCh. ( n= 5 rings, from 3 rats) F. Statistics of pretreated high dose and low dose long time incubation of AVLE effect on KCl. Black bar: KCl control response. (n=5, from 3 rats) Gray bar: Control of KCl and low dose AVLE 10 μg/ml incubation in organ bath for 30~ 40 mins on rat aortic rings. (n=7, from 3 rats) Black gray bar: Control of KCl and KCl after high AVLE 30~ 90 μg/ml throµgh the (once high dose AVLE incubation in organ bath more than 30 minutes ) experiment. (n=6, from 3 rats) (** P < 0.01, *** P <
0.001)
四、高劑量 AVLE 對內皮和血管平滑肌作用後，完好程度之統計 圖 16 A.：正常控制組，比較圖 16 B.：高劑量 AVLE 短時間浸浴 在器官槽中和圖 16 C.：低劑量 AVLE 長時間浸浴在器官槽中；使用 CCh 檢測內皮活性作用。其中，高劑量短時間浸浴在器官槽和低劑量 長時間浸浴在器官槽，內皮功能活性與 PE 活性與正常控制組比較，
皆減少 20 %。而高劑量長時間則對 KCl 有 30 %損害影響之現象 (圖 16 F. : Black gray bar )。
五、AVLE 高劑量收縮之機轉
高劑量 AVLE 逆轉舒張之作用會被超氧化物歧化酶（superoxide
dismutase, SOD）200 U 結合過氧化氫歧化酶（catalase）200U，取消 了 AVLE 累加 30 μg/ml、90 μg/ml 逆轉舒張；逆轉舒張之作用。單劑量之 SOD ( 200 U )，也可單獨取消高劑量 AVLE 逆轉舒張 (restored- contraction )之反應。如圖 17. A 與統計圖 17. B。
Fig. 17
T en sion %
0 20 40 60 80 100
* **
Control SOD +CAT
SOD CAT
B.
A.
0.5 g
10 min