Rac1在糖尿病大鼠脑缺血再灌注损伤中的作用:与线粒体自噬的关系

Role of Rac1 in cerebral ischemia-reperfusion injury in diabetic rats:the relationship with mitopaghy

目的评价Rac1在糖尿病大鼠脑缺血再灌注损伤中的作用及其与线粒体自噬的关系。方法SPF级健康成年雄性SD大鼠,8周龄,体重250~280 g,腹腔注射链脲佐菌素制备糖尿病模型。糖尿病模型制备成功大鼠48只,采用随机数字表法分为4组(n=12):假手术组(sham组)、缺血再灌注组(I/R组)、缺血再灌注+慢病毒抑制Rac1组(I/R+shRac1组)和缺血再灌注+阴性慢病毒组(I/R+NC组)。I/R组采用阻塞大脑中动脉90 min,再灌注24 h的方法制备大鼠脑缺血再灌注损伤模型。I/R+shRac1组和I/R+NC组分别于模型制备前7 d,经右侧脑室注射Rac1 shRNA慢病毒载体或慢病毒阴性对照载体10μl。于再灌注24 h时断头取脑,确定脑梗死体积;采用Western blot法检测BNIP3、P62、LC3Ⅰ和LC3Ⅱ表达,并计算LC3Ⅱ/LC3Ⅰ比值。结果与sham组比较,余3组脑梗死体积增加(P<0.05);与I/R组比较,I/R+shRac1组脑梗死体积降低,LC3Ⅱ/LC3Ⅰ比值升高,BNIP3表达上调,P62表达下调(P<0.05或0.01),I/R+NC组各指标差异无统计学意义(P>0.05);与I/R+NC组比较,I/R+shRac1组脑梗死体积降低,LC3Ⅱ/LC3Ⅰ比值升高,BNIP3表达上调,P62表达下调(P<0.05或0.01)。结论抑制Rac1可减轻糖尿病大鼠脑缺血再灌注损伤,其机制与增强线粒体自噬有关。

Objective To evaluate the role of Rac1 in cerebral ischemia-reperfusion(I/R)injury and the relationship with mitopaghy in diabetic rats.Methods SPF healthy adult male Sprague-Dawley rats,aged 8 weeks,weighing 250-280 g,in which diabetes mellitus was induced by intraperitoneal streptozotocin,were used in this study.Forty-eight rats with diabetes mellitus were divided into 4 groups(n=12 each)using a random number table method:sham operation group(sham group),cerebral I/R group(I/R group),I/R plus lentivirus inhibiting Rac1 group(I/R+shRac1 group),and I/R plus lentivirus-negative control group(I/R+NC group).Cerebral I/R was induced by 90-min middle cerebral artery occlusion followed by 24-h reperfusion.In I/R+shRac1 and I/R+NC groups,Rac1 shRNA lentivirus vector and lentivirus negative control vector 10μl were injected via the right lateral cerebral ventricle at 7 days before establishing the model,respectively.Rats were sacrificed at 24 h of reperfusion,and brains were removed for determination of cerebral infarct size,expression of BNIP3,P62,LC3Ⅰand LC3Ⅱ(by Western blot).The LC3Ⅱ/LC3Ⅰratio was calculated.Results Compared with sham group,the cerebral infarct size was significantly increased in the other three groups(P<0.05).Compared with I/R group,the cerebral infarct size was significantly decreased,LC3Ⅱ/LC3Ⅰratio was increased,the expression of BNIP3 was up-regulated,and the expression of P62 was down-regulated in group I/R+shRac1(P<0.05 or 0.01),and no significant change was found in each index in group I/R+NC(P>0.05).Compared with I/R+NC group,the cerebral infarct size was significantly decreased,LC3Ⅱ/LC3Ⅰratio was increased,the expression of BNIP3 was up-regulated,and the expression of P62 was down-regulated in group I/R+shRac1(P<0.05 or 0.01).Conclusion The mechanism by which Rac1 reduces cerebral I/R injury is related to enhancing mitophagy in diabetic rats.