Study Of The General Pharmacology And Synergic Effect On DAA-I With Danshensu

Purpose:Based on the general pharmacology experiment of Des-Asp-ANG I (DAA-Ⅰ), observe the effects on animal's vital sign by applying low, medium, and high doses of DAA-I under normal conditions, to evaluate the safety and application of the medicine. Also, observe the influence on rats' mortality rate of experimental chronic heart failure (CHF) by applying DAA-Ⅰalone and applying with Danshensu respectively, to evaluate the safety of applying the two together, so as to assess the advantage of the combination. Hopefully the experiment would provide scientific experimental basis for new drug research and development.Method:1. Establish four groups of mouse:high dose DAA-I group (4053pmol/ml), medium dose DAA-Ⅰgroup (1013pmol/ml), low dose DAA-Ⅰgroup (506pmol/ml), and saline control group with completely randomized design. Respectively conduct intraperitoneal injection to four groups of mouse; observe the general activity change of the mouse after 15,30,60, and 120 minutes after the injection respectively; record the change in spontaneous activity with SMART-1 analysis system for animal observation, and observe the influence of DAA-Ⅰon mouse's coordination exercises with pole test.2. Establish four groups of rats:high dose DAA-Ⅰgroup (4053pmol/ml), medium dose DAA-Ⅰgroup (1013pmol/ml), low dose DAA-I group (506pmol/ml), and saline control group with completely randomized design. After narcotizing the rats.conduct tracheal intubations, and carotid artery intubations; afterwards, link pressotransducer, tonotransducer, and electrocardiogram lead with BL-420E biological experiment system after the anesthesia of the rats; Conduct intraperitoneal injection to four groups of rats; record the change in electrocardiogram (ECG), heart rate(HR), respiratory rate, breathing movement, Systolic blood pressure(SBP), and diastolic blood pressure(DBP) of the rats after 15,30,60, and 120 minutes after the injection respectively.3. Establish five groups of rats:group, medium dose DAA-I group (1013pmol/ml), DAA-I+ Danshensu group, pure CHF model group, and saline control group with completely randomized design. Give intraperitoneal injection of Celiac injection-used adriacin(ADR) (diluted with saline water; 2mg/kg) to rats expect for those in saline control group; apply the same injection every three days; apply the same injection every seven days after five times in a row, with total amount of 20mg/kg; apply the same amount of saline water for saline control group in the same pattern, accumulating to seven weeks. Handling with ADR, give rats in Danshensu group(50mg/kg.d), medium dose DAA-Ⅰgroup (1013pmol/ml), and DAA-I+ Danshensu group(Danshensu 50mg/kg.d, DAA-Ⅰ1013pmol/kg.d) intraperitoneal injection till the fourth week, marking the end of the experiment. Observe the change in feed intake, urine volume, and general behaviors, and also the mortality rate. The whole observation lasts for eight weeks.Results:1. General activities of mouse:in high dose DAA-I group, medium dose DAA-I group, low dose DAA-Ⅰgroup and saline control group, all the mouse have smooth fur and regular size of pupils; can rotate flexibly, move freely, and walk normally; no abnormal behaviors including salivation, fibrillation of muscles, scuttling and leaping in panic, or an obvious increase in excretion is observed.2. Spontaneous activity of mouse:a difference in tranquillization is observed among saline control group, high dose DAA-Ⅰgroup, and low dose DAA-Ⅰgroup. Mouse in saline control group have longer time of tranquillization(a=0.05, P＜0.05); while the mouse in the other two groups reflect no obvious difference(a=0.05, P＜0.05).3. Coordination exercises of mouse:mouse in high dose DAA-Ⅰgroup, medium dose DAA-Ⅰgroup, low dose DAA-Ⅰgroup, and saline control group all can reach 0-degree criterion; no abnormal behaviors in coordination exercises including coasting down, drooping out, and righting reflex is observed.4. Electrocardiogram (ECG), heart rate (HR), respiratory rate, and breathing movement of rats:rats in the experimental groups reflect no significant difference in the four criterions comparing(a=0.05, P＜0.05).5. Systolic blood pressure (SBP) of rats:SBP of rats in medium dose DAA-Ⅰgroup and high dose DAA-Ⅰgroup decrease significantly; SBP of rats in medium dose DAA-Ⅰgroup and saline control group indicates a difference (α=0.05, P＜0.05); SBP of rats in high dose DAA-I group indicates a significant difference (a=0.05, P＜0.01); pair-wise comparisons among other groups contain no significant difference (a=0.05, P>0.05).6. Diastolic blood pressure (DBP) of rats:DBP of rats in medium dose DAA-I group and high dose DAA-Ⅰgroup decrease significantly, indicating a significant difference with saline control group (a=0.05, P＜0.01); pair-wise comparisons among other groups contain no significant difference (a=0.05, P>0.05).7. Duplicating chronic heart failure (CHF) models, rats in saline control group reflect no significant abnormality. For the other four groups, the rats start to appear multiple degrees of CHF early performance including slow-moving, diet reduce, and diarrhea from the third week. During the fourth and fifth weeks, rats in pure CHF model group and each experimental group start to have ascites, and those in pure CHF model group appear earlier; serious symptoms include cyanosis around lip, cadaverous in conjunctiva, achypnea or weak breath, trichomadesis, abnormal walking or groveling still; and individual deaths. After dosing the rats in experimental groups, death rate reduced as the time extends, and CHF-related symptoms are alleviated, especially for those receiving a combined treatment of DAA-Ⅰ+ Danshensu. Symptoms in rats of model group reflect no obvious improvement. Conduct anatomy of rats in model group after eight weeks, hepatomegaly, sclerosis, and atrophy in liver can be observed; there are also fibrocystic changes triggered by peritonitis, synechia and hyperplasia in omentum and intestinal tube, tubercle in abdominal wall, and hydroncus and congestion in intestine, sometimes even bloody ascites. The above symptoms are in conformity with the pathological changes in CHF models triggered by ADR, as reported by relevant documents.8. After eight weeks after the duplication of CHF model, the death rates are:40% in Danshensu group; 40% in medium dose DAA-I group (1013pmol/ml); 30% in DAA-I+ Danshensu group; 50% in pure CHF model group; 0% in saline control group.Conclusions:1. DAA-Ⅰhas no significant effects on mouse's normal behaviors and coordinative exercises in high, medium, and low doses. 2. DAA-Ⅰhas no significant effects on mouse's spontaneous activity in medium dose, and has certain excitability effects on zentralnerven System. It is estimated that the combination of DAA-I and AT1-receptor can result in certain biphasic regulation effects, thus activating RAS systems in both high and low doses, facilitating the generation of AngiotensinⅡ(All), stipulating adrenal cortex glomorulosa zona, precipitating the secretion of aldosterone and water-sodium retention, exciting ganglion sympathetic to increase the secretion of norepinephrine, adding sympathetic transmitter, and enhancing spontaneous activity.3. DAA-Ⅰhas no significant effects on rats' respiratory rate and breathing movement in each dose. The result proves the previous research conclusion that DAA-I is an indomethacin-sensitive-type AT1's receptor subtype, which brings antagonism Ang II and have effects on cardiovascular system, while without influencing respiratory system directly.4. DAA-Ⅰhas no significant effects on rats' heart rate(HR), and maximum value, minimal value, and peak value in ECG's three major indexes in each dose. The result is in accordance with the recently agreed DAA-Ⅰmechanism of action, i.e. ARB medicines that take effect via antagonism Ang II do not lead to the change in the change in human's heart rate(HR) and ECG.5. DAA-I leads to a significant decrease in rats' SBP and DBP in both high and medium doses, and the two doses indicate no obvious difference. In low dose, DAA-I has no significant effect on rats' SBP and DBP. The result proves the previous research conclusion that DAA-I has no significant effect in low dose, and does not appear as dose-dependent medicine in high and medium doses.6. On condition that the accumulated dose is guaranteed, the duplication of rats'CHF models with Adriamycin proves to be reliable. After the molding, the rats can appear the following CHF-related signs and symptoms:crouching and tireness; diets reduce; diarrhea; cyanosis around lip; tachypnea; hair withered; seroperitoneum; and individual deaths, etc.7. The use of medium dose of DAA-I alone, Danshensu alone, and a combined use of DAA-Ⅰand Danshensu can obviously alleviate CHF-related symptoms. Among the above solutions, medium dose of DAA-I+ Danshensu can achieve a 30% of death rate, the lowest figure; comparatively, the death rate is 40% when DAA-I or Danshensu is used alone. Thus the combination is of better effects, indicating certain coordinative impacts.