槲皮素对大鼠全脑缺血/再灌注损伤的保护作用

Protective effect of quercetin against global ischemia/reperfusion injury in rats

目的 观察槲皮素对大鼠全脑缺血/再灌注(I/R)损伤后海马CA1区病理学变化、神经功能、血脑屏障(BBB)损伤、活性氧(ROS)生成和炎性因子表达的影响。方法 216只SD大鼠被随机分为:假手术+vehicle组(sham组)、I/R+vehicle组(I/R组)、I/R+槲皮素5 mg/(kg · d)组(低剂量组)及I/R+槲皮素10 mg/(kg · d)组(高剂量组)。I/R组、低剂量组和高剂量组采用4-VO法建立全脑I/R模型,缺血时间为15 min,sham组仅仅予以暴露双侧椎动脉以及颈总动脉后缝合。四组大鼠均在造模前3 d开始灌胃,sham组和I/R组均以0.1%的Tween80(1 ml/100 g)灌胃,低剂量组和高剂量组分别以0.05%、0.1%的槲皮素溶液(1 ml/100 g)灌胃,1次/d,持续至观察结束当天。再灌注24 h HE染色观察海马CA 1区形态学变化;再灌注12,24,48 h利用神经功能缺陷评分(NDS)评价神经功能,干湿重法检测脑含水量,伊文氏蓝法(Evan’ s blue,EB)检测BBB通透性;再灌注2,12,24 h DCFH-DA法检测海马细胞ROS含量;再灌注2,12,24,48 h real-time PCR法检测海马组织IL-1β、TNF-α、IL-6、IFN-γ mRNA水平,ELISA法检测海马组织和血清中IL-1β、TNF-α、IL-6、IFN-γ表达。结果 与I/R组相比,低剂量组和高剂量组海马CA1区损伤减轻;NDS评分均显著高于I/R组( P <0.05);再灌注24,48 h,低剂量组和高剂量组伊文氏蓝含量和脑含水量较I/R组显著降低( P <0.05);再灌注2,12,24 h,高剂量组ROS含量均显著低于I/R组( P <0.05);高剂量组IL-1β mRNA,TNF-α mRNA,IL-6 mRNA在各时间点均较I/R组显著降低( P <0.05);低剂量组和高剂量组在24 h和48 h IFN-γ mRNA水平均较I/R组显著降低( P <0.05);与I/R组相比,低剂量组和高剂量组海马组织和血清IL-1β、TNF-α、IL-6在各时间点表达均显著降低( P <0.05);IFN-γ表达则在24 h和48 h较I/R组显著降低( P <0.05)。结论 槲皮素能够改善大鼠全脑I/R损伤后神经功能,减轻BBB损伤,减少ROS生成,抑制IL-1β、TNF-α、IL-6、IFN-γ的表达,减轻组织损伤。

Objective To observe the effect of quercetin on the the morphology of hippocampus CA1 area, neurological function, blood brain barrier(BBB), the production of reactive oxygen species(ROS) and the expression of inflammatory factors in rats after global cerebral I/R injury. Methods A total of 216 Male SD rats were randomly divided into four groups: sham+vehicle group(sham group), I/R+vehicle group(I/R group), I/R+Que 5 mg/(kg · d) group(low-dose group) and I/R+Que 10 mg/(kg · d) group(high-dose group). The global cerebral ischemia reperfusion model in I/R group, low-dose group and high-dose group was established by a four-vessel occlusion(4-VO) ligation method, and the time duration of ischemia was 15 min. The bilateral vertebral artery and carotid artery in sham group were only exposed, and then the incisions were sutured. All the rats in four groups were gavaged 3 d before the modeling once a day untill the end of the observation. The rats in both sham group and I/R group were gavaged with 0.1% Tween80(1 ml/100 g). The rats in low-dose group and high-dose group were gavaged with 0.05% and 0.1% quercetin solution (1 ml/100 g), respectively. The hematoxylin and eosin(HE) staining in the hippocampus CA1 area was performed at 24 h after reperfusion to evaluate the pathological changes. Neurological deficit score(NDS) was assessed at 12,24,48 h after reperfusion to eva-luate neurological function. Evan’s blue content was detected at 12,24,48 h after reperfusion to evaluate the permeability of BBB. DCFH-DA detection was employed to evaluate ROS content in rats hippocampal at 2, 12, 24 h after reperfusion. Real-time PCR was used to measure the mRNA levels of IL-1beta, TNF-alpha, IL-6 and IFN-gamma in rats hippocampus at 2,12,24,48 h after reperfusion. ELISA was used to detect serum and hippocampal protein levels of IL-1beta, TNF-alpha, IL-6 and IFN-gamma in rats hippocampus at 2,12,24,48 h after reperfusion. Results HE staining showed that there were less damages in the hippocampus CA1 area in low-dose group and high-dose group than in I/R group. Compared with I/R group, NDS scores were significantly increased in low-dose group and high-dose group( P <0.05), while the brain water content and EB content at 24 h and 48 h after reperfusion decreased significantly( P <0.05). ROS levels were significantly lower in low-dose group and high-dose group than that in I/R group( P <0.05). Real-time PCR showed that IL-1beta mRNA, TNF-alpha mRNA and IL-6 mRNA levels in high-dose group were significantly lower than that in I/R group at all the time points( P <0.05). IFN-gamma mRNA levels at 24 h and 48 h in low-dose group and high-dose group were significantly lower than those in I/R group( P <0.05). ELISA assay showed that compared with I/R group, IL-1beta, TNF-alpha, IL-6 were decreased significantly in the hippocampus and serum in low-dose group and high-dose group at each time point after perfusion( P <0.05). The IFN-gamma at 24 h and 48 h in low-dose group and high-dose group were significantly lower than those in I/R group( P <0.05). Conclusion Quercetin could improve the neurological function, reduce the damage of tissue, inhibit the blood brain barrier damage, reduce the production of ROS, inhibit the expression of IL-1beta, TNF-alpha, IL-6, IFN-gamma in rats after global cerebral I/R, which eventually protect neuron cells against global cerebral I/R injury.