槲皮素对大鼠全脑缺血/再灌注后学习记忆损伤的保护机制

Protective effect and mechanism of quercetin on learning and memory function after global ischemia/reperfusion injury in rats

目的观察槲皮素对大鼠全脑缺血,再灌注(ischemia/reperfusion,I/R)损伤后神经行为学、海马组织细胞凋亡和凋亡相关蛋白表达的影响,探讨槲皮素减轻全脑I/R损伤的机制。方法采用随机数字表法将72只SD大鼠分为4组(每组18只):假手术+vichcle组(S组)、I/R+vichcle组(I/R组)、I/R+槲皮素5mg·kg·d^-1组(Q5组)及I/R+槲皮素10mg·kg·d^-1组(Q10组),用4-VO法建立全脑I/R模型。从造模前3d开始,Q5组和Q10组分别以5mg·kg·d^-1和10mg·kg·d^-1的剂量给予0.05%、0.10%的槲皮素溶液灌胃,S组和I/R组则给予等容量的溶剂,每天1次,持续至观察结束。再灌注后第8天正式开始Morris水迷宫实验,记录各组大鼠第8天、第9天、第10天的逃避潜伏期和第11天的穿环指数,再灌注后24h TUNEL法和Annexin V-FITC/PI双染流式细胞仪检测海马CA1区细胞凋亡,Western blot检测海马组织Bcl-2、Bcl-xl、survivin、cleaved caspase-3、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)和磷酸化-Bad(phosphorylated-Bad,p-Bad)的表达。结果与I/R组比较,Q5组和Q10组大鼠逃避潜伏期缩短(P<0.05),在目标象限游泳的时间延长(P<0.05),穿环指数亦明显提高(P<0.05);I/R组海马CA1区可见大量胞核黄染的阳性细胞,Q5组和Q10组可见少量阳性细胞,凋亡细胞比例较I/R组明显降低(P<0.05);与I/R组比较,Q5组和Q10组Bcl-2、Bcl-xl、survivin、p-Akt和p-Bad表达水平明显升高(P<0.05),cleaved caspase-3表达水平则明显降低(P<0.05)。结论槲皮素增加大鼠全脑I/R脑组织中p-Akt、p-Bad、Bcl-2、Bcl-xl、survivin的表达,抑制cleaved caspase-3的表达,抑制细胞凋亡,提高全脑I/R大鼠的学习和记忆能力。

Objective To observe the effect of quercetin (Que) on neurological behavior, cell apoptosis and apoptosis-related protein expression in hippocampus after global cerebral ischemia/reperfusion (I/R) injury. Exploring the mechanism of quercetin-induced neuroprotective effect against global cerebral I/R injury. Methods Seventy two Male SD rats were randomly divided into 4 groups (n=18): sham+vehicle group (S group), I/R+vehicle group (I/R group), I/R+Que 5 mg·kg-·1 d-1group (Q5 group) and I/R+Que 10 mg·kg-1·d-1group (Q10 group). The global cerebral ischemia reperfusion model was established by a four-vessel occlusion (4-VO) ligation method, and the duration of ischemia was 15 min. Sham-operated animals were treated similarly to those in the ischemic group, but neither the vertebral arteries nor the common carotid arteries were occluded. The rats in Q5 group and Q10 group were administered with 0.05%, 0.10% of the quercetin solution by garage at the dose of 5 mg·kg-1·d-1and 10 mg·kg-1·d-1 respectively. S group and I/R group were given equal volume of vehicle. The quercetin and vehicle were administrated by gavage once per day 3 d before I/R and continued till the end of observation. The Morris maze test was performed 8 d after reperfusion. At 24 h after reperfusion, TUNEL staining and Annexin V-FITC/PI double labeled flow cytometry were used to evaluate apoptosis in hippocampus tissue, the expressions of Bcl-2, Bcl-xl, survivin, cleaved caspase-3, phosphorylated protein kinase B(p-Akt), Bad, p-Bad protein were determined by Western blot. Results In Morris water maze test, escape latency results showed that compared with I/R group, the escape latency in Q5 group and Q10 group was obviously reduced (P<0.05). Space probe test showed that the active time in the target quadrant in Q5 group and Q10 group were significantly longer than active time in the target quadrant (P<0.05). The frequency of crossing the original position platform was significantly increased (P<0.05). TUNEL assay showed that a large number of apoptotic cells in I/R group, while less positive cells could be seen in Q5 group and Q10 group. Annexin V-FITC/PI double staining flow cytometry detection results showed that the proportion of apoptotic cells in Q5 group and Q10 group were significantly reduced (P<0.05). Western blot results showed that Bcl-2, Bcl-xl, survivin, p-Akt and p-Bad expression levels were significantly increased in Q5 group and Q10 group (P<0.05) while cleaved caspase-3 expression level was significantly decreased (P<0.05). Conclusions Quercetin can up-regulate the expression of p-Akt, p-Bad, Bcl-2, Bcl-xl and survivin while down-regulate the expression of cleaved caspase-3, which leads to the inhibition of apoptosis, eventually protects neuron cells from global cerebral I/R injury and improves the learning and memory function.