摘要
毛冬青甲素是从毛冬青中提取的经半合成的一种五环三萜类化合物，其结构不同于目前国内外报道的毛冬青成分结构，属我国首创新药。临床试用于治疗缺血性脑血管病，冠心病，周围血管病，疗效显著。目前国内外大多对毛冬青甲素药理作用的研究都集中在上述几方面，但对其在病理情况下，如急性心肌缺血梗死时对心肌电生理情况的影响的研究和相关报道比较少。本实验结扎大鼠前降支制作心梗模型，测定毛冬青甲素对受损心肌的动作电位和钙离子通道的影响；同时也测定了其对心肌心电，血压和心率等相关参数的影响，为进一步研究毛冬青甲素对心血管系统的药理作用，为不远将来广泛的临床应用提供实验室依据。
本工作目的是拟通过及结扎大鼠前降支制作心梗模型，测定毛冬青甲素对受损心肌的动作电位和钙离子通道的影响；同时也测定了其对心肌心电，血压和心率等相关参数的影响，为进一步研究毛冬青甲素对心血管系统的药理作用提供实验室依据。
按文献报道的方法，将大白鼠300-400g用10%的水合氯醛麻醉，做气管插管，股静脉和主动脉插管，记录正常血压、心电和动作电位。然后给予人工呼吸、并开胸，于左冠状动脉前降支起始处用7#手术线结扎，造成心梗模型。与硝酸甘油对照共分四组，分别测量存活时间，动作电位和受损心肌的钙离子通道等数据。
结果显示：
一、在正常对照组，心脏的存活时间大于20.0个小时。当冠状动脉前降支起始处用7#手术线结扎后其心脏的存活时间5.6±1.2小时。提前注射毛冬青甲素和硝酸甘油，同样做冠状动脉前结
扎，心脏的存活时间，对照组存活时间：5.6±1.2点注毛甲素组：15.8±1.8点注硝酸甘油组：14.2±1.6。与对照组和硝酸甘油组比较，P＜0.05有统计学意义，表明毛冬青甲素可以明显延长缺血大鼠的存活时间。
二、提前注射毛冬青甲素和硝酸甘油，同样做冠状动脉前结扎，10-20分中做同样的记录，心率和血压分别为254.50±32.07、86.83±7.57mmHg和244.67±24.65、49.50±9.81mmHg与心梗对照组和硝酸甘油组比较，毛冬青甲素可以明显升高血压（P＜0.01）但心率变化不明显，无显著性差别（P＞0.05）。
三、记录在体心室肌细胞动作电位幅值（APA）、最大舒张电位（MDP）、最大除极速度（Mmax）、动作电位发放频率（APF）、复极10%、50%和90%时的时程（APD10、APD50、APD90）。结果见下表。
毛冬青甲素对缺血缺氧大鼠心室肌动作电位的影响
APA（mV）
MDP(mV)
Mmax(V/s)
APD10(mS)
APD50(mS)
APD90(mS)
空白对照组
76.33±13.50
53.5±10.07
182.16±11.22
26.16±4.35
94.50±7.00
126.83±6.94
毛冬青甲素组
69.66±9.13
49.00±7.64
174.33±15.36
31.00±2.60*
109.66±12.45*
135.16±6.2
硝酸甘油组
54.50±5.90
46.33±2.893
167.83±15.70
25.16±4.26
81.17±11.34
119.33±12.7
注：*表示P＜0.05，n=15 说明毛冬青甲素可延长心肌动作电位时间。
四、在应用缺血缺氧液条件下，测得心室肌细胞上的L-型钙通道在0mV时的峰值电流为0.153±0.03，与正常对照组相比，p<0.001。而在缺血缺氧的单个心室肌细胞上和加入毛冬青甲素（20mg/Kg）3分钟后，做同样的记录。其钙通道电流幅值在0mV为（-0.195±0.02nA，p<0.001，n=6），具有统计学意义，表明毛
冬青甲素可以促进缺血心肌细胞的钙离子内流。
近年来的研究表明心肌细胞内钙离子的活动与环核苷酸有密切关系。细胞膜上功能性慢钙通道的数量和开放的可能性均受到cAMP水平的调节。cAMP可以引起钙通道开放，钙离子内流增加。本实验发现毛冬青甲素可以延长ADP，但动作电位峰值变化不大。另有实验用钙拮抗剂硝苯吡啶可全部阻断毛冬青甲素延长ADP的效应，因此可推断其引起钙离子内流，延长了平台期时段，进而延长了ADP。
本实验说明在病理情况下毛冬青甲素对缺血大鼠心肌同样具有该作用，延长心肌动作电位时间，促进心肌钙离子内流。心肌细胞内钙离子的增加可以引起心肌收缩力的增加，钙离子的增加有两种途径，一是通过兴奋肾上腺素能受体，引起L型钙通道开放增加，进而钙离子内流增加。在体的心脏存在受到肾上腺素影响的可能，但肾上腺素所引起的心肌收缩的增强，同时应该并存心率的增快，血压的升高等生理现象。本实验结果显示在毛冬青甲素作用下，心率略减慢，收缩压升高，而舒张压变化不明显。故其通过该种途径引起钙离子内流增加的可能性不大。有实验以离体心肌为研究，结果显示毛冬青甲素同样增加其收缩功能，也不支持其通过神经、体液的因素影响心肌收缩力。另一途径是通过增加心肌细胞内cAMP的，引起钙离子通道开放增多，内流增加所致。已有实验证明毛冬青甲素可以引起细胞内的cAMP的增加，说明该种机制可能性较大。
结论：毛冬青甲素可促进缺血大鼠心肌细胞的钙离子内流，延长ADP，但动作电位峰值变化不大。在临床上，对急性缺血性
心脏病可能具有抗心律失常、加强心肌收缩力作用。毛冬青甲素也明显升高实验鼠心梗后血压，稳定心率，从而保证了心梗后冠状动脉血流，提高麻醉鼠心
Holly is dehydrated root of the Ilex plant which is widespreaded in the Guangdong and Guangxi province. It is ofen used as the traditional Chinese medicinesed in the South in China. it’s alias is fur tree, tast hard, sex is mini-cold. long, longago,people found it have the effect that can make blood flow quikely, recede swelling, relieve pain, schedule hot and detoxicate.These 20 years,there are many I literature reports about chemistry, pharmacology and clinic reseach of IA. IA is a terpenoid that is extacted from Holly dehydrated root and purificated and process through chemical reaction. It is worked out firstly by our country. Resent study showed it have anti-thrombosis, anti-myocardial ischemia physio-activity. On trial,it is proved that IA have significant effect on idchemia cerebral vessels, coronary heart disease and peripheral angiopath.
Many Pharmacology experiments proved: firstly, it can anti- thrombosis by increasing the cAMP level in platelet and inhibiting the platelet aggretiong. scondly, Findings suggest that IA can increase the activity of myocardial cell agaist ischemia, and improve the ability of protecting cardiac muscle. Thirdly, IA can acceleration the calcium ions channels of the nomal myocardial cell. At present, most studies concentrate these methods, very few reported the Pathological conditions, for example, there is very few correlation reports about electrophysiologic after acute myocardial infarction.
The study is to determinate the effets about action potential and calcium ions channel of IA on cardiac muscle by fabricating the modle of acute myocardial infarction with deligating Rats anterior descending branch. Meanwhile,determinated the effets aout electrocardiogram, blood pressure and heart rate. The project provide laboratory data that is to help to study the effet of IA on cardiovascular system.
According to literature reports, after anesthetized with Chloral Hydrate, rats were faced upward fixed on the opetating table. Opening the chest between the left third and fourth rid. The heatt was exposed. A 0 nylon suture attached to a fine needle was placed under the left anterior descending coronary arteryu and ligated coronary artery immediately. return heart into the chest, squeeze the blood and water out and swiftly close the chest. The time opening the chest is no more than 30 seconds. Shan surgical roup placed the suture only but not ligate coronary artery. Measure the electrocardiogram as before five minutes before operation. The whole experiment is divided into two sections. Sction 1: IA is administered before deligating Rats anterior descending branch and observed the effets about action potential and calcium ions channel of IA on cardiac muscle .Sction 2: nitroglycerin was used and contrasted.
Result
Firstly,stock time of the nomal group is more than 20 hours. when A 0 nylon suture attached to a fine needle was placed under the
left anterior descending coronary arteryu and ligated coronary artery immediately. return heart into the chest, squeeze the blood and water out and swiftly close the chest.The time of the IA group and the Nitroglycerin group are elongated. Statistics data: normal group5.6±1.2 ;the IA group 15.4±1.8; the Nitroglycerin group 14.2±1.6. There is significant difference between IA group and the normal group, P＜0.05. It show that IA can significantly elongated the stock time of the AMI rate.
Secondly, before A 0 nylon suture attached to a fine needle was placed under the left anterior descending coronary arteryu, IA or Nitroglycerin was Injected, the heat rate and the blood pressur of IA group and Nitroglycerin group separately is 254.50±32.07、86.83±7.57 mmHg and 244.67±24.65、49.50±9.81 mmHg. BP of IA group is higher than Control group and Nitroglycerin group,(P<0.01). But HR is no significant difference between them,(P>.05).
Thirdly, we use the suspended microcledtrode to recorder the action potential of myocytes and analyse the parameters of action potential by computer. We found that AP were inc
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