地黄饮子对脑缺血再灌注损伤大鼠保护作用及其机制

Protective Effect of Dihuang Yinzi on Cerebral Ischemia-reperfusion Injury in Rats and Its Mechanism

目的:探讨地黄饮子对脑缺血再灌注损伤大鼠的保护作用及其初步机制。方法:建立大脑中动脉栓塞(MCAO)大鼠模型,随机分为6组,分别为假手术组,模型组,尼莫地平组(0.01 g·kg^(-1)),地黄饮子高、中、低剂量组(38.80,19.40,9.70 g·kg^(-1)),每组20只,连续给药28 d;术后第7,14,28天通过改良神经功能缺损评分(m NSS),第28天行2,3,5-氯化三苯基四氮唑(TTC)染色,苏木素-伊红(HE)染色观察地黄饮子的保护作用,应用免疫荧光技术观察侧脑室室管膜下区(SVZ)的5-溴脱氧尿嘧啶核苷(Brd U)阳性细胞数(第28天)以分析神经干细胞(NSCs)增殖情况,采用实时荧光定量聚合酶链式反应(Real-time PCR)观察Notch1,Jagged1,Hes1 mRNA的表达情况。结果:术后第7,14,28天地黄饮子高、中剂量组,尼莫地平组的m NSS明显低于同时间段的模型组(P<0.05,P<0.01);术后第28天地黄饮子高、中剂量组和尼莫地平组的脑梗死体积占脑组织体积的百分比均低于模型组(P<0.01); HE染色显示地黄饮子高、中剂量组,尼莫地平组大鼠脑组织坏死和软化灶不明显,并可见神经元细胞和神经胶质细胞增生;术后第28天,上述3组SVZ的Brd U阳性细胞数显著高于模型组(P<0.05,P<0.01),地黄饮子高剂量组Brd U阳性细胞数高于尼莫地平组(P<0.05);术后28 d地黄饮子高、中剂量组,尼莫地平组SVZ的Notch1,Jagged1,Hes1 mRNA表达水平明显高于模型组(P<0.05,P<0.01);尼莫地平组Hes1 mRNA水平高于地黄饮子高、中、低剂量组(P<0.05,P<0.01)。结论:地黄饮子能改善MCAO大鼠模型的神经功能缺损,减小脑梗死体积,减轻脑组织病理改变,从而对脑缺血再灌注损伤大鼠起到保护作用,可能与激活Notch信号通路,上调Notch1,Jagged1,Hes1 mRNA的表达,从而促进NSCs增殖有关。

Objective: To explore the protective effect and the preliminary mechanism of Dihuang Yinzi on cerebral ischemia-reperfusion injury in rats. Method: The middle cerebral artery occlusion ( MCAO) model was established. Totally 90 SD rats were randomly divided into 6 groups: sham operation group,model group, nimodipine group ( 0. 01 g·kg - 1 ) and high,medium, low-dose Dihuang Yinzi groups ( 38. 80,19. 40, 9. 70 g·kg - 1 ) ,with 20 rats in each group. The modified neurological severity score ( mNSS) was assayed at the 7th,14th,28th days after operation,and the volume of cerebral infarction,pathological changes of brain tissue,the BrdU positive cells and mRNA levels of Notch1,Jagged1 and Hes1 in subventricular zone ( SVZ) were observed respectively by triphenyl tetrazolium chloride ( TTC) stain,htorylin eastin ( HE) stain,immunofluorescence technique and reverse transcriphase polymerase chain reaction ( Real-time PCR) methods at the 28th day after the operation. Result: The mNSS on the 7th,14th,28th days of high,medium-dose Dihuang Yinzi groups and nimodipine group were significantly lower than that of model group ( P < 0. 05,P < 0. 01) . On the 28th day,the percentage of cerebral infarction volume in brain tissue volume of high,medium-dose Dihuang Yinzi groups and nimodipine group were smaller than that of model group ( P < 0. 01) . HE staining showed that the necrosis and softening lesions of brain tissue were not obvious in rats of high,medium-dose Dihuang Yinzi groups and nimodipine group,with neuronal cell and neuroglial cell proliferation. On the 28th day,the BrdU positive cells in SVZ of the above 3 groups were significantly higher than model group ( P < 0. 05,P < 0. 01) ,and the high-dose Dihuang Yinzi group was significantly higher than nimodipine group ( P < 0. 05) . On the 28th day,the mRNA levels of Notch1,Jagged1 and Hes1 of high,medium-dose Dihuang Yinzi groups and nimodipine group were significantly higher than those of model group ( P < 0. 05,P < 0. 01) ,the mRNA level of Hes1 of nimodipine group was higher than that of high,medium, low-dose Dihuang Yinzi groups ( P < 0. 05,P < 0. 01 ) . Conclusion: Dihuang Yinzi can improve the nerve function defect of MCAO rat model,and reduce the volume of cerebral infarction and the pathological changes of brain tissue,thus playing a protective role in cerebral ischemiareperfusion injury rats. Its mechanism may be related to the activation of the Notch signaling pathway,and the upregulation of expressions of Notch1,Jagged1 and Hes1 mRNA,thus promoting the proliferation of NSCs.