牡荆素通过12/15-LOX信号通路减轻大鼠脑缺血再灌注损伤的研究

目的探讨牡荆素对大鼠大脑中动脉缺血再灌注损伤的保护作用及可能机制。方法 60只雄性SD大鼠随机分为假手术组、模型组及牡荆素40、80 mg/(kg·d)组,每组15只。制作大鼠大脑中动脉缺血(90 min)/再灌注(24 h)损伤模型。术前1周及模型制作前30 min,各组分别ip相应药物,假手术组和模型组给予等量生理盐水。观察牡荆素对神经功能缺损评分、脑指数及梗死体积的影响,采用激光多普勒血流仪监测再灌注后大鼠缺血侧局部脑血流量的动态变化,测定缺血侧脑组织活性氧(ROS)水平、超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、Caspase-3活性及12羟二十烷四烯酸(12-HETE)、15羟二十烷四烯酸(15-HETE)含量;实时荧光定量PCR检测Bcl-2、Bax、ALOX15 mRNA的表达,Western blotting检测ALOX15蛋白、p38MAPK及p-p38MAPK蛋白表达。结果与假手术组相比,模型组大鼠神经缺失症状评分、脑指数及梗死体积显著升高(P<0.01),ALOX15 mRNA和蛋白的表达及p38MAPK和p-p38MAPK蛋白表达显著增加(P<0.01),同时Caspase-3活性、Bax mRNA表达明显升高(P<0.01),而Bcl-2 mRNA表达显著下降(P<0.01)。与模型组比较,牡荆素预处理能能显著改善模型大鼠神经功能缺损症状,降低脑指数,减少梗死体积,增加缺血侧大脑局部血流量,明显增加缺血侧脑组织Bcl-2m RNA表达(P<0.05、0.01),提高SOD活性(P<0.05、0.01),降低ALOX15 mRNA、Bax mRNA和ALOX15、p38MAPK和p-p38MAPK蛋白表达(P<0.05、0.01),下调Caspase-3活性、15-HETE、12-HETE的表达和MDA水平(P<0.05、0.01)及减少ROS的生成(P<0.05、0.01)。结论牡荆素对局灶性缺血大脑具有神经保护作用,并可改善缺血侧大脑血流供应。其作用机制可能为通过12/15-LOX信号抑制p38MAPK介导的细胞凋亡,同时降低氧化应激和炎症,从而减轻脑I/R损伤。

Protective effect of vitexin on cerebral ischemia-reperfusion injury in rats through 12/15 LOX signaling pathway

Objective To investigate the protective effect and possible mechanism of vitexin on middle cerebral artery ischemia-reperfusion injury in rats. Methods Sixty male SD rats were randomly divided into sham operation group, model group and vitexin 40, 80 mg/(kg·d) ] treatment groups. The middle cerebral artery ischemia (90 min) /reperfusion (24 h) injury model was established. One week before operation and 30 minutes before model making, rats in each group were administrated the corresponding drugs by intraperitoneally injected. Rats in the sham operation group and the model group were given the same amount of normal saline. The effect of vitexin on neurological deficit score, brain index and infarct volume were observed. The dynamic changes of cerebral blood flow in the ischemic side were monitored by laser Doppler flowmetry. The levels of ROS, SOD, MDA, Caspase-3, 12-HETE and 15-HETE were measured. The expression of Bcl-2, Bax, ALOX15 mRNA was detected by real-time fluorescence quantitative analysis. The expression of ALOX15, p38MAPK and p-p38MAPK protein were detected by Western blotting. Results Compared with the sham operation group, the neurological deficit symptom score, brain index and infarct volume of rats in the model group were significantly increased (P<0.01), the expression of ALOX15 mRNA and protein and the expression of p38MAPK and phospho-p38mapk protein were significantly increased (P<0.01), the activity of Caspase-3 and the expression of Bax mRNA were significantly increased (P<0.01), while the expression of bcl-2 mRNA was significantly decreased (P<0.01). Compared with the model group, vitexin pretreatment could significantly improve the symptoms of neurological deficit, reduce brain index and infarct volume, increase regional cerebral blood flow, increase Bcl-2 mRNA expression (P<0.05, 0.01), increase SOD activity (P<0.05, 0.01), and reduce ALOX15 mRNA and Bax MRNA and protein expressions of ALOX15, p38MAPK and phospho-p38mapk (P<0.05, 0.01), caspase-3 activity, 15-HETE, 12-HETE expression and MDA level were down regulated (P<0.05, 0.01), and ROS production was reduced (P<0.05, 0.01). Conclusion Vitexin has neuroprotective effect on focal cerebral ischemia and can improve the blood supply of ischemic side. The mechanism may be that Vitexin may inhibit p38MAPK mediated apoptosis through 12/15-LOX signal, and reduce oxidative stress and inflammation, thereby reducing brain I/R injury.