【作者】 宫健伟；

【导师】 樊巧玲；

【作者基本信息】 南京中医药大学 ， 方剂学， 2013， 博士

【摘要】 研究目的：观察地黄饮子对大鼠脑缺血再灌注损伤模型的影响,探讨地黄饮子对实验性脑缺血再灌注动物模型脑组织损伤保护作用的机制,为全面研究地黄饮子抗脑缺血损伤提供实验依据。研究方法：1、地黄饮子对模型大鼠抗自由基氧化、抗细胞凋亡实验研究选用清洁级SD大鼠随机分为6组：假手术组、模型组、尼莫地平阳性药物组、地黄饮子高、中、低剂量组,采用结扎双侧颈总动脉方法制作大鼠脑缺血再灌注损伤模型。假手术组及模型组灌胃生理盐水,地黄饮子高、中、低剂量组灌服相应药物,每天1次,连用7d后造模,尼莫地平术前30min皮下注射。造模后进行血清、脑组织SOD、CAT、GSH-Px活性及MDA含量的测定,取脑组织进行Bax蛋白、Bcl-2蛋白、Caspase-3蛋白表达的检测。2、地黄饮子对模型大鼠促血管新生实验研究选用清洁级SD大鼠随机分为6组：假手术组、模型组、尼莫地平阳性药物组、地黄饮子高、中、低剂量组,采用结扎双侧颈总动脉方法制作大鼠脑缺血再灌注损伤模型。模型制作后第2日开始给药,假手术组及模型组灌胃生理盐水,地黄饮子高、中、低剂量组灌服相应药物,阳性药物组灌胃给予尼莫地平溶液,每天1次,连用7d。第8日灌注固定取脑,HE染色观察大脑海马形态学改变,用免疫组化法检测大鼠脑组织微血管密度(MVD)以及VEGF含量的表达。研究结果：1、地黄饮子对模型大鼠抗自由基氧化实验结果1.1大鼠血清抗自由基氧化检测结果：与模型组比较,阳性药物组和地黄饮子高、中、低剂量组均能显著升高SOD活性,地黄饮子高、中剂量组能显著降低MDA含量,地黄饮子高、中剂量组能显著升高CAT活性,各用药组对GSH-Px活性变化的影响无显著性差异。1.2大鼠脑组织抗自由基氧化检测结果：与模型组比较,阳性药物组和地黄饮子高、中剂量组均能显著升高SOD活性,阳性药物组和地黄饮子高、中、低剂量组能显著降低iDA含量,阳性药物组和地黄饮子高、中、低剂量组能显著升高CAT活性,地黄饮子高剂量组能显著升高GSH-Px活性。2、地黄饮子对模型大鼠脑组织抗细胞凋亡检测结果模型组大鼠Bax蛋白表达升高、Bcl-2蛋白表达降低、Caspase-3蛋白表达升高,与假手术组比较有统计学意义,各药物治疗组能降低Bax蛋白表达、升高Bcl-2蛋白表达、降低Caspase-3蛋白表达,与模型组比较有统计学意义。3、地黄饮子对模型大鼠大脑海马形态学改变观测结果HE染色结果：假手术组：脑组织无水肿,神经细胞、神经胶质细胞及毛细血管形态结构基本正常。模型组：海马区及其周围脑组织形成典型梗死表现。脑梗死区域可见大量神经元变性、坏死,胞体缩小变形,核固缩,噬神经元现象,胞浆呈嗜酸性,即为“红色神经元”改变；脑组织水肿,血管周围间隙增大,炎性细胞浸润。地黄饮子各剂量组：缺血范围比模型组小,病理改变较模型组轻。梗死区亦可见少量的神经元变性、坏死,可见少量的“噬神经元现象”：脑组织轻度水肿,血管轻度充血,无明显的炎性细胞浸润。4、地黄饮子对模型大鼠脑组织促血管新生检测结果模型组MVD计数与假手术组比较无统计学意义,地黄饮子高、中、低剂量组和阳性药物组MVD计数较模型组增加,差异有统计学意义：模型组VEGF含量较假手术组升高,差异有统计学意义,地黄饮子高、中剂量组和阳性药物组较模型组均能升高VEGF含量表达,差异有统计学意义。研究结论：1、地黄饮子能显著抑制模型大鼠血清SOD、CAT和脑组织中SOD、CAT、GSH-Px活性的降低以及MDA含量的升高,表明脑缺血再灌注后,脑组织内自由基产生增加,发生了明显的脂质过氧化反应,地黄饮子可以减少自由基的产生,减轻脂质过氧化反应,表现出良好的抗氧化损伤作用。2、地黄饮子可下调Bax蛋白水平、上调Bcl-2蛋白水平,调节大鼠脑组织的bax/bcl-2平衡,下调Caspase-3蛋白水平,抑制神经细胞凋亡,减轻脑组织损伤。3、地黄饮子能显著改善模型大鼠脑梗死区的病理改变,减轻脑组织水肿,减轻炎性细胞浸润等,对脑缺血区神经细胞功能的恢复表现出良好的效果。4、地黄饮子能促进模型大鼠脑组织VEGF的释放和表达,促进脑梗死大鼠脑组织缺血区新血管形成,有明显的促血管新生作用。更多还原

【Abstract】 Research objective:To observe the effect of Dihuang Yinzi on cerebral ischemia reperfusion injury in model rat, to explore the mechanism of Dihuang Yinzi on experimental cerebral ischemia reperfusion animal model of brain tissue injury, to provide the experimental basis for the comprehensive study of Dihuangyinzi against cerebral ischemia injury.Research methods:1.Dihuang Yinzi on rat model of anti free radical oxidation, resistance to apoptosis in experimental studySixty clean SD rats were randomly divided into6groups:sham operation group, model group, nimodipine positive drug group, Dihuang Yinzi high, medium and low dose group.The cerebral ischemia reperfusion was established by ligating bilateral common carotid artery. Sham operation group and model group were lavaged with normal saline, Dihuang Yinzi high, medium and low dose group fed with corresponding drugs,1times a day, used in conjunction7d after molding, nimodipine preoperative30min subcutaneous injection. The activities of SOD、CAT、 GSH-PX and the content of MDA in the serum and brain of rats were observed after cerebral ischemia reperfusion. The expression of Bax、Bcl-2and Caspase-3proteins were detected after cerebral ischemia reperfusion.2. Dihuang Yinzi on promoting angiogenesis in experimental model ratSixty clean SD rats were randomly divided into6groups:sham operation group, model group, nimodipine positive drug group, Dihuang Yinzi high, medium and low dose group.The cerebral ischemia reperfusion was established by ligating bilateral common carotid artery. Model making the second day after administration, sham operation group and model group were lavaged with normal saline, Dihuang Yinzi high, medium and low dose group fed with corresponding drugs, nimodipine group administrated with nimodipine solution,1time a day, for7d. Eighth perfusion fixation from the brain, observe the change of brain hippocampus morphology by HE staining. Microvessel density(MVD) and the expression of VEGF content were detected in rat brain by immunohistochemical method. Research results:1. Dihuang Yinzi on model rat of anti free radical oxidation experiments1.1Results of anti free radical oxidation of rat serum:compared with the model group, positive drug group and decoction of Dihuang Yinzi high, medium and low dose group can significantly increase the activity of SOD, Dihuang Yinzi high, middle dose group can significantly decrease the content of MDA, Dihuang Yinzi high, middle dose group can significantly increase the activity of CAT, no significant differences between the groups on the effect of GSH-Px activity.1.2Results of anti free radical oxidation in rat brain tissue:compared with the model group, positive drug group and Dihuang Yinzi high, middle dose group can significantly increase the activity of SOD, positive drug group and Dihuang Yinzi high, medium and low dose group can significantly decrease the content of MDA, positive drug group and Dihuang Yinzi high, medium and low dose group can significantly increase the activity of CAT, Dihuang Yinzi high dose group can significantly increase the activity of GSH-Px.2. Results of Dihuang Yinzi on resistance to apoptosis in brain tissue of ratExpression of Bax protein in model rat group increased, decreased expression of Bcl-2protein, Caspase-3protein expression increased, with statistical significance compared with the sham operation group, the treatment group can reduce the Bax protein expression, increase Bcl-2protein expression, Caspase-3protein expression was decreased, there was statistical significance compared with the model group.3. Results of Dihuang Yinzi on brain hippocampus morphology changes of model ratResults of HE staining:sham operation group:brain tissue without edema, nerve cells, glial cells and capillary morphology is basically normal. Model group:the formation of typical infarct in hippocampus and surrounding brain tissue. Cerebral infarction area shows a large number of neuronal degeneration, necrosis, reduced cell body deformation, nuclear pyknosis neurons, bite phenomenon, cytoplasm is eosinophilic, known as "red neurons" change; brain edema, blood vessels around the gap increases, the infiltration of inflammatory cells. Dihuang Yinzi groups: Ischemia range than the model group, the pathological changes were slighter than those in model group. Infarction area neuronal degeneration, necrosis were little, there were a few "bite neurons phenomenon"; brain edema, vascular mild congestion, no obvious infiltration of inflammatory cells.4. Results of Dihuang Yinzi on angiogenesis in brain tissue of ratThere was no significant difference of MVD count between the model group and sham operation group, The MVD count of Dihuang Yinzi high, medium and low dose group and positive drug group increased than those of model group, the difference was statistically significant.Content of VEGF in model group was higher than in the sham operation group. Dihuang Yinzi high, medium dose group and positive drug group can increase the content of VEGF expression, the difference was statistically significant compared with the model group.Research conclusion:1. Dihuang Yinzi can significantly inhibit the activity decrease of serum of SOD、CAT and brain tissue of SOD、CAT、GSH-Px and the increase of MDA content of rat model, showed that after cerebral ischemia and reperfusion, free radical in brain tissue is increased, the lipid peroxidation was generated, Dihuang Yinzi can reduce free radical, reduce lipid peroxidation, exhibit good antioxidant effect.2. Dihuang Yinzi can downregulate Bax protein level, upregulation of Bcl-2protein, bax/bcl-2balance regulation in rat brain tissue, downregulation of Caspase-3protein levels, and inhibited apoptosis, and relieve the injury of brain tissue.3. Dihuang Yinzi can significantly improve the pathological changes in rat cerebral infarction model, reduce cerebral edema, reduce the infiltration of inflammatory cells, the function of brain nerve cells in ischemic region recovery has shown good results.4. Dihuang Yinzi can promote the expression of brain tissue of rats with the release of VEGF, promote the brain of rats with cerebral infarction ischemic neovascularization, have the effect of angiogenesis obviously.更多还原