Ento-Ⅰ涂膜剂对大鼠脑缺血再灌注损伤的保护作用

Protective effect of Ento-Ⅰ plastic against cerebral ischemia-reperfusion injury in rats

目的研究Ento-Ⅰ涂膜剂对大鼠脑缺血再灌注损伤的预防作用,并评价其对小鼠的镇痛效果和抗凝活性。方法采用线栓法建立SD大鼠局灶性脑缺血再灌注损伤模型,雄性大鼠56只,随机分为假手术组、空白基质组、生理盐水组、Ento-Ⅰ涂膜剂预防给药组(6.67、3.33、1.67 mg/kg)、奥扎格雷钠注射液组(8.3 mg/kg,ip)共7组,通过神经病学评分、TTC染色计算脑梗死面积评价Ento-Ⅰ涂膜剂的抗脑缺血作用;通过醋酸扭体实验测定Ento-Ⅰ涂膜剂的镇痛作用:将60只昆明种小鼠随机分成空白基质组,阿司匹林灌胃组(80 mg/kg)、阿司匹林涂膜剂组(80 mg/kg)、Ento-Ⅰ涂膜剂给药组(5、10和20 mg/kg),末次给药后1 h腹腔注射0.7%醋酸溶液,观察并记录小鼠扭体反应和扭体次数;并采用毛细玻管法测定小鼠体外凝血时间,以评价Ento-Ⅰ涂膜剂的抗凝活性。结果醋酸扭体实验结果显示,与空白基质组小鼠扭体次数相比,Ento-Ⅰ涂膜剂各剂量组(5、10、20 mg/kg)能显著降低醋酸致小鼠扭体次数并提高扭体抑制率(抑制率分别是21.79%、48.89%、56.15%),且呈剂量效应趋势;毛细玻管法测定小鼠凝血时间,其中Ento-Ⅰ涂膜剂10和5 mg/kg的凝血时间分别为(155.20±54.19)和(155.80±73.84)s,与生理盐水组(92.10±24.61)s比较,能明显延长小鼠体外凝血时间(P<0.05);与空白基质组(80.40±48.09)s比较,其差异也有统计学意义(P<0.01);线栓法致大鼠脑缺血再灌注损伤实验结果显示,在脑缺血再灌注24 h时,与生理盐水组的大鼠神经病学评分(2.33±0.52)比较,Ento-Ⅰ涂膜剂3.33 mg/kg大鼠的神经病学评分(1.00±0.00)明显改善(P<0.01);据TTC染色后脑梗死面积计算结果显示,生理盐水组和空白基质组的脑梗死率分别为(24.89±7.24)%和(27.72±7.89)%,Ento-Ⅰ涂膜剂6.67和3.33 mg/kg的脑梗死率则分别为(14.01±2.65)%和(14.73±4.94)%,与2个模型组比较,Ento-Ⅰ3.33和6.67 mg/kg均能显著降低大鼠脑缺血再灌注损伤后的脑梗死率(P<0.01)。结论 Ento-Ⅰ涂膜剂有较强的镇痛作用和抗凝活性,并能降低大鼠脑缺血再灌注损伤后神经病学评分、显著降低脑梗死率,可能以此实现其抗脑缺血作用。

Objective To research the protective effect of Ento-Ⅰ against cerebral ischemia-reperfusion injury in rats,and to evaluate its analgesic and anticoagulating effects in mice. Methods The ischemic model was established with line embolism to block the middle cerebral artery of male rats. The 56 rats were randomly assigned into 7 groups of sham-operation,blank-matrix,normal saline,Ento-Ⅰplastic of 3 doses（6.67,3.33,1.67 mg/kg）,and ozagrel sodium（8.3 mg/kg,ip）. The effect of Ento-Ⅰplastic on anti-cerebral ischemia was measured by nervous function scores and the areas of cerebral infarction were determined by TTC staining for the calculation of cerebral infarction rates. The analgesic effect of Ento-Ⅰplastic was determined with acetic acid-induced twisting experiment. Sixty KM mice were randomly allocated into blank-matrix,aspirin,aspirin-plastic,and Ento-Ⅰplastic of 3 doses（5,10 and 20 mg/kg）,the number of mouse twisting were recorded right after intraperitoneal injection of 0.7% acetic acid solution at the time of 1 h after the last administration. Moreover,the anticoagulant activity of Ento-Ⅰplastic was tested by glass capillary method. Results The results of acetic acid-induced twisting experiment displayed that Ento-Ⅰplastic of all 3 dose groups（5,10 and 20 mg/kg）could significantly reduce the number of body torsion and increase the inhibitory rates of twisting,compared with that of blank matrix group（the inhibitory rates of twisting for 3 dose groups were 21.79%,48.89%,and 56.15%,respectively）,with dose-response manner. According to the results of glass capillary test,the clotting time of mouse blood could be significantly prolonged by mid-（10 mg/kg）and low-dose（5 mg/kg）of Ento-Ⅰplastic with corresponding clotting time of（155.20±54.19）s and（155.80±73.84）s,compared with normal saline group at（92.10±24.61）and blank-matrix group at（80.40±48.09,P0.05）. The experiment results of the ischemia-reperfusion injury by line embolism method in rats exhibited that Ento-Ⅰplastic in mid-dose（3.33 mg/kg）could significantly reduce the neurological scores after 24 h of reperfusion injury,from（2.33±0.52）of normal saline group to（1.00±0.00）of mid-dose group（P0.01）. The results from TTC staining revealed that the cerebral infarction rates of normal saline group and blank-matrix group were（24.89±7.24）% and（27.72±7.89）%,respectively,whereas those of 6.67 mg/kg and 3.33 mg/kg group of Ento-Ⅰplastic were（14.01±2.65）% and（14.73±4.94）%,respectively. Compared to the 2 negative-control groups,both the high- and mid-dose of Ento-Ⅰplastic could significantly reduce the cerebral infarction rates after ischemic reperfusion injury in rats（P0.01）. Conclusion Ento-Ⅰplastic demonstrates strong analgesic and anticoagulant effects,and could substantially reduce the neurological scores and reduce cerebral infarction rates for ischemia-reperfusion injured rats. These are likely to be the mechanism of action for Ento-Ⅰplastic realizing its anti-cerebral ischemia effect.