大豆异黄酮对局灶性脑缺血再灌注损伤大鼠血脑屏障通透性的影响及其机制

Effect of soy isoflavones on blood-brain barrier permeability and its mechanism in rats with focal cerebral ischemia-reperfusion injury

目的探讨大豆异黄酮(SI)对局灶性脑缺血再灌注大鼠的血脑屏障(BBB)通透性的影响及相关机制。方法72只健康成年SD大鼠均分为假手术组(Sham组)、缺血再灌注组(I/R组)和SI预处理组(SI组),每组各24只。SI组每天按120 mg/kg体质量给予12 mg/mL SI灌胃1次,Sham组和I/R组每天用等体积生理盐水灌胃1次,均连续灌胃21 d。在实验第22天,I/R组和SI组大鼠用线栓阻断右侧大脑中动脉血流制作大脑中动脉缺血再灌注动物模型;Sham组不插入线栓,未阻断血流,其余手术操作与I/R和SI组一致。I/R组和SI组大鼠缺血2 h后,拔出阻塞的线栓恢复血流,再灌注24 h。根据Zea longa评分,选择造模成功的大鼠。采用2,3,5—氯化三苯基四氮唑(TTC)检测大鼠脑梗死体积,干湿重法检测脑组织含水量,伊文思蓝(EB)示踪法检测BBB通透性,Western blot检测脑组织MMP-9、TIMP-1蛋白表达。比较三组大鼠的测量数据,分析SI预处理对以上观测指标的影响。结果SI组和I/R组大鼠的神经功能评分、脑梗死体积、脑组织含水量和脑组织内EB含量均高于Sham组,而SI组低于I/R组,分别为(1.50±1.13)、(2.33±0.82)和(0.00±0.00)分,12.5%±2.34%、37.50%±2.28%和0.07%±0.03%,81.75%±0.86%、84.17%±0.54%和78.24%±0.41%,(46.50±1.41)、(55.44±1.37)和(3.83±1.49)μg/g,差异均有统计学意义(P值均<0.01)。SI组和I/R组MMP-9、TIMP-1表达均明显高于Sham组,分别为0.81±0.23、1.15±0.14、1.15±0.14和2.31±0.20、1.62±0.21、1.62±0.21,差异均有统计学意义(P值均<0.01);与I/R组相比,SI组MMP-9表达减少、TIMP-1表达增加,差异均有统计学意义(P值均<0.01)。结论SI可能通过下调缺血再灌注后脑组织MMP-9蛋白表达和上调TIMP-1蛋白表达,降低BBB的通透性,减轻缺血再灌注后脑水肿的程度和脑组织EB的含量。

Objective To investigate the effect of soy isoflavones(SI)on blood-brain barrier(BBB)permeability in rats with focal cerebral ischemia reperfusion and the related mechanism.Methods A total of 72 healthy adult SD rats were randomly divided into sham operation group(Sham group),ischemia-reperfusion group(I/R group),and SI pretreatment group(SI group),with 24 rats in each group.The SI group was intragastrically administered with SI 120 mg/kg according to body weight once a day for 21 days,and the other two groups were intragastrically administered with an equal volume of normal saline once a day for 21 days.On day 22,the rat model of middle cerebral artery ischemia/reperfusion was established by blocking the blood flow of the right middle cerebral artery with thread embolism in the I/R and SI groups.In the Sham group,no thread embolism was inserted and blood flow was not blocked.The remaining steps were consistent with the I/R and SI groups.Two hours after ischemia of the right middle cerebral artery,the thread embolism was removed,the blood flow was restored,and the middle cerebral artery was reperfused for 24 hours.According to the Zea longa score,rats with successful modeling were selected.2,3,5-triphenyltetrazolium chloride(TTC)was used to detect the volume of cerebral infarct and the water content of brain tissue was measured by dry/wet weight method.The permeability of BBB was detected by the Evans blue(EB)tracer method.The matrix metalloproteinase(MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)protein expressions were determined with Western blot.The measured data of the three groups of rats were compared,and the influence of SI pretreatment on the above observations was analyzed.Results The neurological function score,cerebral infarction volume,the content of water and EB of brain tissue in SI group and I/R group were higher than those in Sham group,while SI group was lower than I/R group[(1.50±1.13),(2.33±0.82)and(0.00±0.00)scores;12.5%±2.34%,37.50%±2.28%and 0.07%±0.03%;81.75%±0.86%,84.17%±0.54%and 78.24%±0.41%;(46.50±1.41),(55.44±1.37)and(3.83±1.49)μg/g,respectively].The differences were statistically significant(all P values<0.01).The expressions of MMP-9 and TIMP-1 in SI group and I/R group were significantly higher than those in Sham group(0.81±0.23,1.15±0.14,1.15±0.14 and 2.31±0.20,1.62±0.21,1.62±0.21,respectively).The differences were statistically significant(all P values<0.01).Compared with the I/R group,the expression of MMP-9 was decreased and the expression of TIMP-1 was increased in the SI group.The differences were statistically significant(all P values<0.01).Conclusions SI may down-regulate MMP-9 protein expression and up-regulate TIMP-1 protein expression in brain tissue after ischemia-reperfusion,and decrease permeability of BBB and reduces the degree of brain edema and EB content of brain tissue after ischemia-reperfusion.