ABSTRACT
Objective:To investigate the role of platelets aggregation in the developing process of ductus arteriosus closure of newborn pups, and the effect of platelet membrane glycoprotein Ⅱb-Ⅲa (GPⅡb-Ⅲa) receptor antagonist (tirofiban).Methods:Four 24-month-old Beagle bitches were selected and numbered 1, 2, 3, and 4 respectively, and their pups were removed by cesarean section in two batches 1-2 days before the expected date of delivery. Bitches 1 and 2 were the first batch. Eighteen newborn pups were removed after cesarean section as the control group. They were divided into three subgroups: 1-hour subgroup, 4-hour subgroup, and 12-hour subgroup according to postnatal time point, with 6 pups in each subgroup. The newborn pups were injected with normal saline 10 mL/kg via jugular vein immediately after birth. Bitches 3 and 4 were the second batch. Nineteen newborn pups were removed by cesarean section as tirofiban group. They were also divided into three subgroups: 1-hour subgroup ( n = 6), 4-hour subgroup ( n = 6), and 12-hour subgroup ( n = 7) according to the postnatal time point. The newborn pups were injected with tirofiban hydrochloride injection 10 mL/kg (10 mL injection including 2.5 mg of tirofiban) via jugular vein immediately after birth. The diameter of ductus arteriosus was measured by echocardiography. Ductus arteriosus was removed by surgical dissection and divided into two parts. Western blotting and immunohistochemistry were used to detect the expression of platelet membrane GPⅡb-Ⅲa, respectively. Results:In the control group, 1 newborn pup died at 0.5 hour after birth in the 1-hour subgroup. The experiment was completed by 19 in the tirofiban group. Ductus arteriosus of all pups were not closed in 1-hour subgroups of the two groups, and there was no significant difference in the diameter of ductus arteriosus between the control group and the tirofiban group (mm: 1.72±0.08 vs. 1.70±0.11, P > 0.05). Ductus arteriosus of 1 newborn pup in 4-hour subgroup of the control group was closed, but the ductus arteriosus of all the newborn pups in 4-hour subgroup of the tirofiban group were not closed. The diameter of ductus arteriosus of the tirofiban group was significantly larger than that of the control group (mm: 1.52±0.15 vs. 0.95±0.48, P < 0.05). Ductus arteriosus of all pups were closed in 12-hour subgroup of the control group, but the ductus arteriosus of 2 pups of the tirofiban group were still not closed, with the diameter of ductus arteriosus of 1.0 mm and 1.1 mm, respectively. Western blotting showed that at 1-hour, 4-hour and 12-hour after birth, the expression of platelet membrane GPⅡb-Ⅲa was gradually increased in ductus arteriosus of newborn pups of the two groups. The expression of GPⅡb-Ⅲa in 1-hour subgroup of the tirofiban group was significantly lower than that in the control group (GPⅡb-Ⅲa/β-actin: 0.67±0.07 vs. 0.84±0.16, P < 0.05). The expression of GPⅡb-Ⅲa in 4-hour and 12-hour subgroups of the tirofiban group were slightly lower than those in the control group (GPⅡb-Ⅲa/β-action: 0.85±0.12 vs. 0.95±0.11 in 4-hour subgroup, 1.04±0.16 vs. 1.09±0.17 in 12-hour subgroup, both P > 0.05). Immunohistochemistry showed that the change trend of platelet membrane GPⅡb-Ⅲa in ductus arteriosus of newborn pups in both groups was similar to the results of Western blotting. Conclusions:The ductus arteriosus of newborn pups begin to close 1-4 hours after birth, and all closed at 12 hours after birth. The expression of platelet membrane GPⅡb-Ⅲa in ductus arteriosus increase gradually after birth, and the platelet aggregation may participate in and promote ductus arteriosus closure to some extent. Tirofiban, a platelet membrane GPⅡb-Ⅲa receptor antagonist, may delay ductus arteriosus closure of newborn pups to some extent by inhibiting platelet aggregation.
ABSTRACT
Objective To investigate if mitochondrial KATP channels are involved in the cardioprotective effects of astragalus membranaceus in immature rabbit hearts. Methods Twenty-four New Zealand white rabbits of both sexes aged 14-21 days weighing 300-350 g were anesthetized and heparinized. The hearts were rapidly removed after thoracotomy and mounted on a Langendorff apparatus via ascending aorta and perfused with oxygenated (95% O2-5% CO2) Krebs-Hensleit buffer (KHB) solution at 60 cm H2O (perfusion pressure) and 38 ℃ . A fluid-filled latex balloon was inserted into left ventricle via left atrium for measurement of left ventricular developed pressure ( LVDP) , Global myocardial ischemia was induced by suspension of perfusion for 30 min followed by 45 min reperfusion. The animals were randomly divided into 3 groups with 8 animals in each group : group A control;group B astragalus and group C astragalus +- 5-HD (a selective inhibitor of the mitochondrial KATP channel). In group B the hearts were perfused with astragalus 40 g? L-1 for 15 min before ischemia. In group C the hearts were perfused with 5-HD 100?mol?L-1 for 5 min followed by 10 min astragalus perfusion before ischemia. In control group the hearts were perfused with only KHB before ischemia. Coronary flow (CF), HR, LVDP and ? dp/dtmax, were measured at 5, 10, 15, 30 and 45 min of reperfusion and recorded. Coronary effluent was collected at 10 min of reperfusion for determination of CK-MB, LDH and CK levels . At the end of 45 min reperfusion the isolated heart was removed for determination of myocardial ATP and iNOS levels and electron microscopic examination.Results In astragalus group (B) LVDP ? dp/dtmax and CF recovered significantly better and myocardial ATP content was significantly higher as compared with group A and C (P