【作者】 徐冰；

【导师】 徐江平；

【作者基本信息】 南方医科大学 ， 药理学， 2010， 硕士

【摘要】 目的：观察白藜芦醇苷对大鼠局灶性脑缺血再灌注损伤后急性损伤及长期给药后运动功能康复的影响。并在此基础上探讨白藜芦醇苷的作用机制。体外培养PC12细胞,建立氧糖剥夺损伤模型(oxygen-glucose deprivation,OGD),观察白藜芦醇苷对细胞形态及细胞活力的影响。方法：(1)采用栓线法制备大鼠急性局部脑缺血再灌注损伤模型(MCAO)。在剂量研究中,将大鼠随机分成7组：模型组、假手术组、阳性对照组、白藜芦醇苷各剂量组(5mg/kg、10mg/kg、20mg/kg、40mg/kg)。观察白藜芦醇苷对大鼠缺血大脑的梗塞面积及含水量的影响。(2)较长周期给药实验中,将大鼠随机分成6组：模型组、假手术组、阳性对照组、白藜芦醇苷高、中、低各剂量组,造模成功后第2、第4、第6、第8天,称量体重,分别采用姿势反射试验、肢体不对称试验两种方法作为行为学评价指标,观察白藜芦醇苷对大鼠缺血大脑的保护作用。(3)体外培养PC12细胞,应用化学方法建立细胞OGD损伤模型,观察细胞形态学改变,并且应用四甲基偶氮唑蓝法(methyl thiazolyl tetrazolium, MTT)观察不同浓度白藜芦醇苷对细胞活力的影响,测定细胞外液乳酸脱氢酶(Lactate dehydrogenase, LDH)浓度,一氧化氮(NO)及丙二醛(MDA)含量,超氧化物歧化酶(SOD)活性。结果：(1)与大鼠模型组(28.30±7.92)比较,应用白藜芦醇苷后大鼠脑梗塞面积逐渐降低呈浓度依赖趋势分别为19.21±14.72、15.32±13.53、14.47±10.85、13.74±12.89。与大鼠模型组(65.81±1.75)比较,应用白藜芦醇苷后大鼠水肿程度逐渐降低呈浓度依赖趋势分别为64.27±1.45、63.91±1.53、63.68±1.80、63.57±1.65。白藜芦醇苷5mg/kg组有改善效果但无统计学意义。(2)较长周期给药后体重变化率及行为学评价结果显示：i体重变化率：假手术组除术后第1天体重下降,其余每天体重呈增长趋势。模型组由于缺血再灌注引起的损伤,造成体重呈下降趋势。(各时间点与给药前比较)体重变化率,与假手术组同时间点(1.26±8.40、4.44±6.84、7.22±6.18、10.80±5.81)比较,模型组造模后第2天、4天、6天和8天有显著性增加(-7.61±5.71、-10.57±10.49、-12.40±12.29、-12.05±13.52),有显著性差异(P＜0.01)。白藜芦醇苷低、中、高剂量组(5、10、20mg/kg)给药后可改善缺血再灌注引起的体重下降。同时间点体重变化率与模型组比较,中剂量组造模后第2天、4天和6天有显著性降低(-9.07±3.85、-9.44±11.97、-10.90±15.82),有显著性差异(P＜0.05)；高剂量组造模后第4天和6天有显著性降低(-12.15±12.80、-13.25±13.89),有显著性差异(P＜0.05)；阳性对照组给药可改善缺血再灌注引起的体重下降(-9.58±3.09、-9.12±6.56、-9.43±7.67、-7.50±8.46),且有显著性差异(P＜0.05)。ii姿势反射试验：造模后第2天、4天、6天和8天各时间点与假手术组(0±0)比较,模型组有显著性增加(1.42±0.48、1.36±0.33、1.29±0.51、0.91±0.30),具有显著性差异(P＜0.01)。白藜芦醇苷低、中、高剂量组(5、10、20mg/kg)给药后可改善缺血再灌注引起的肢体功能异常。与模型组比较,高剂量组能显著性降低缺血再灌注引起的肢体功能异常(1.01±0.37、0.78±0.42、0.77±0.28、0.59±0.39),具有显著性差异(P＜0.01)。与模型组比较,低、中剂量组在造模后第4天能显著性降低缺血再灌注引起的肢体功能异常(1.06±0.29、1.01±0.30),有显著性差异(P＜0.05)；阳性对照组给药可显著性降低缺血再灌注引起的肢体功能异常(1.08±0.31、0.78±0.31、0.87±0.63、0.64±0.44),且有显著性差异(P＜0.05)。iii肢体不对称试验：与假手术组(9.5±21.51、-0.75±25.77、4.25±23.64、4±24.42)比较,造模后第2天、4天、6天和8天各时间点模型组前肢不对称应用评分均显著升高(66.43±32.78、69.29±23.69、50.36±17.81、51.79±40.03),具有显著性差异(P＜0.05)。白藜芦醇苷低、中、高剂量组(5、10、20mg/kg)给药后可改善缺血再灌注引起的左前肢触及桶壁频率较少,右前肢触及桶壁频率较多。与模型组比较,高剂量组在造模后第2天、4天和6天能显著下降前肢不对称应用评分(35.67±34.69、29±27.33、28.33±27.82),具有显著性差异(P＜0.05)。与模型组比较,低、中剂量组在造模后第4天能显著下降前肢不对称应用评分(47.5±18.60、46.88±19.07),具有显著性差异(P＜0.05)；阳性对照组在造模后第2天、4天和6天能显著下降前肢不对称应用评分(26.56±29.98、44.38±28.57、33.13±27.5),且有显著性差异(P<0.05)。白藜芦醇苷能显著改善缺血引起的大鼠肢体活动障碍。(3)氧糖剥夺损伤后,PC12细胞形态发生了一定的改变,白藜芦醇苷12.5μM, 25μM,50μM三个剂量组显著降低了氧糖剥夺对PC12细胞成的损伤：PC12细胞的A值由模型的70.45±2.94增加到74.2±3.88、92.55±5.08、90.3±4.55；且有显著性差异(P<0.05)。与模型组705.85±30.65相比,应用白藜芦醇苷后PC12细胞各组细胞外液LDH活力(U/L)也逐渐降低呈浓度依赖趋势LDH分别为630.76±31.28、619.72±23.91、599.34±37.54,且有显著性差异(P＜0.05)。与模型组11.09±1.1相比,应用白藜芦醇苷后PC12细胞各组细胞外液MDA含量(nmol/mg)也逐渐降低呈浓度依赖趋势MDA分别为9.43±0.53、9.27±0.84、7.82±0.63，且有显著性差异(P＜0.05)。与模型组16.69±0.3相比,应用白藜芦醇苷后PC12细胞各组细胞外液SOD活性(U/mg)也逐渐增高呈浓度依赖趋势SOD分别17.67±0.23、18.79±0.26、18.68±0.15,且有显著性差异(P＜0.05)。与模型组42.25±0.92相比,应用白藜芦醇苷后PC12细胞各组细胞外液NO含量(μmol/g)也逐渐降低呈浓度依赖趋势NO分别为31.25±2.11、25.88±2.06、25.38±2.73,且有显著性差异(P＜0.05)。结论：白藜芦醇苷对大鼠缺血大脑有保护作用,能明显减少大脑的梗塞面积和水肿程度,改善缺血引起的肢体行为障碍；离体细胞实验表明白藜芦醇苷对PC12细胞氧糖剥夺损伤具有保护作用。更多还原

【Abstract】 Objective:To study the effect of resveratrol on the cerebral infarction and behavior in rats after focal cerebral ischemia-reperfusion injury. Based on this model, we also explore the possible mechanisms of the effect. Moreover, to investigate the effect of resveratrol on the viability and morphology of cell by establishing the oxygen-glucose deprivation (OGD) model in PC 12 cells.Methods:(1)Rat model of focal cerebral ischemia reperfusion injury made by occlusion of middle cerebral artery was used. In the dose-dependence study, rats were randomly divided into 7 groups:model group, sham-operated group, positive control group, and resveratrol-treated groups (5mg/kg, 10mg/kg,20mg/kg and 40mg/kg). The size of cerebral infarction, the extent of edema and the water content were measured in the rats. (2)In the long-term experiment, the rats were randomly divided into 6 groups:model group, sham-operated group, positive control group, and resveratrol-treated groups of high, medium and low doses. Postural reflex test and limb use asymmetry test were performed as behavioral indexes on 2th,4th,6th and 8th day after the rats were subjected to focal cerebral ischemia-reperfusion injury for evaluating the protective effect of resveratrol on the brain damage. (3) By establishing oxygen-glucose deprivation (OGD) model of PC 12 cells in vitro, we observed the change of the cells. At the same time, MTT assay was applied to evaluate the cell’s viability and biochemical method was used to determine LDH activity.the contents of nitric oxide(NO) and the malondialdehyde(MDA). the activity of superoxide dismutase(SOD).Results:(1) Compared with model group(28.30±7.92), cerebral infarction area decreased obviously:19.21±14.72,15.32±13.53,14.47±10.85,13.74±12.89. Compared with model group(65.81±1.75),the extent of brain edema decreased obviously:64.27±1.45、63.91±1.53、63.68±1.80、63.57±1.65.The 5mg/kg resveratrol-treated group had no significant effect.(2) Behavior evaluation results from the relative long-term treatment study showed that resveratrol treatment remarkably improved the limb movement of rats with cerebral ischemia-reperfusion injury with statistical significance.i the weight variation ratio:The weight of rats in the sham-operated group were significantly increased,except On dl, The weight of rats in the model group were significantly decreased.compared with sham-operated group in same time, On 2th、4th、6th and 8th day the weight variation ratio after focal cerebral ischemia and reperfusion injury in the model group were significantly increased (-7.61±5.71,-10.57±10.49,12.40±12.29,12.05±13.52), compared with the model group,On 2th、4th and 6th day of the weight test, the resveratrol middle dose (10mg/kg) group were significantly decreased(-9.07±3.85,-9.44±11.97,-10.90±15.82,), On 4th and 6th day of the weight test, the resveratrol high dose (20mg/kg) group were significantly decreased(-12.15±12.80,-13.25±13.89,),the Positive control group were significantly decreased (-9.58±3.09,-9.12±6.56,-9.43±7.67,-7.50±8.46). ii Postural reflex test:On the Postural reflex test neurological score,compared with sham-operated group(0±0), On 2th、4th、6th and 8th day the model group were significantly increased(1.42±0.48,1.36±0.33, 1.29±0.51,0.91±0.30). compared with the model group,the resveratrol high dose (20mg/kg) group were significantly decreased(1.01±0.37,0.78±0.42,0.77±0.28, 0.59±0.39,). On 4th day the resveratrol low dose (5mg/kg) and middle dose(10mg/kg) group were significantly decreased(1.06±0.29,1.01±0.30),the Positive control group were significantly decreased (1.08±0.31,0.78±0.31,0.87±0.63,0.64±0.44).iii limb use asymmetry test:On limb use asymmetry test score, compared with sham-operated group(9.5±21.51,-0.75±25.77,4.25±23.64,4±24.42),On 2th、4th、6th and 8th day the model group were significantly increased (66.43±32.78,69.29±23.69, 50.36±17.81,51.79±40.03),compared with the model group, On 2th、4th and 6th day the resveratrol high dose (20mg/kg) group were significantly decreased(35.67±34.69, 29±27.33,28.33±27.82), On 4th day the resveratrol low dose (5mg/kg) and middle dose (10mg/kg) group were significantly decreased(47.5±18.60,46.88±19.07), On 2th、4th and 6th day the Positive control group were significantly decreased (26.56±29.98,44.38±28.57,33.13±27.5). (3) In the cell test, the cellular morphology of PC12 cells were altered after OGD. resveratrol (12.5μM,25μM,50μM) could significantly decrease the injury of PC 12 by OGD. compared with the model group (70.45±2.94), the MTT value of resveratrol group increased obviously:74.2±3.88, 92.55±5.08,90.3±4.55. In PC12 cells, compared with the model group (705.85±30.65), LDH leakage of resveratrol group decreased obviously: 630.76±31.28,619.72±23.91,599.34±37.54. In PC12 cells, compared with the model group (11.09±1.1), MDA leakage of resveratrol group decreased obviously: 9.43±0.53、9.27±0.84、7.82±0.63. In PC12 cells, compared with the model group (16.69±0.3), SOD leakage of resveratrol group increased obviously:17.67±0.23、18.79±0.26、18.68±0.15. In PC12 cells, compared with the model group (42.25±0.92), NO leakage of resveratrol group decreased obviously:31.25±2.11、25.88±2.06、25.38±2.73.Conclusion:Resveratrol has protective effects on focal cerebral ischemia-reperfusion injury,which was demonstrated by significantly reducing the infarct size and brain edema and improves physical disabilities caused by ischemia. And resveratrol could exert a protective action on oxygen-glucose deprivation in PC12 cells.更多还原