回药扎里奴思方对脑缺血再灌注痰热腑实模型大鼠神经保护作用及炎性级联反应的影响

发布时间：2018-04-27 18:06

本文选题：脑缺血再灌注 + 痰热腑实　；参考：《宁夏医科大学》2017年硕士论文

【摘要】：脑梗死造成的神经功能损害对人类的健康有着严重的影响,使患者出现残疾的几率已位居第一,病死率位居第三,仅低于癌症和心肌梗死。据世界卫生组织预计在每年大约有1500万患有脑血管相关疾病,缺血性脑血管病是其发病率的4/5。脑梗死的患病已经逐渐年轻化,其复发、致残和死亡的机率均较高,不但使患者的生活质量下降,也给家庭和社会造成了沉重的负担,给我国医疗卫生产生了巨大压力。脑部血流减少后,脑组织会产生一系列炎性级联效应,包括炎症因子的表达、氨基酸毒性的兴奋、神经元细胞死亡、周围去极化四个不同的机制,且各机制在发生时间上可相互影响、互成因果。其中炎症反应是损伤级联反应发生过程中氧自由基、细胞因子及促炎酶共同促发的结果。细胞因子既可通过作用于内皮细胞,将受体激活,从而使白细胞进入脑内,又可以直接刺激小胶质或星形胶质细胞释放有害成分,最终形成脑损伤。病与证结合既体现了中医的基本理论,又结合了西医学理论与动物实验学的知识,运用西医学的病因理论制备了动物的疾病,同时将中医的证候建立在疾病的模型中,使动物模型中的证候与疾病相融合,使其具有较好的稳定性和可靠性,并且可以动态的分阶段的、系统的进行观察与研究,因此病证结合动物模型的研究已成为现代中医药研究模式的一个重要发展趋势,对脑梗死的疾病发病机制及用药探索有着重大的实际应用意义。目的:1.通过检测痰热腑实脑缺血再灌注模型大鼠血脂血糖等指标,明确脑缺血痰热腑实模型应激性代谢功能的变化,观察扎里奴思方对脑梗死代谢功能紊乱的调节作用。2.通过观察脑缺血再灌注后痰热腑实型大鼠神经功能和神经元细胞的病理变化,明确扎里奴思方对神经细胞的保护作用。3.通过观察脑缺血再灌注痰热腑实型模型大鼠经再灌注后炎性相关因子表达的情况,从扎里奴思方对其变化的影响,明确其可能的作用机制;方法:Sprague Dawley大鼠,雄性,250只,SPF级,将其随机分为正常组、假手术组、单纯脑梗死组、痰热腑实组、尼莫地平组、扎里奴思方组共六个组,其中正常组10只,其余各组分为1天、3天、7天3个取材时间点每个时间点15只大鼠;正常组、假手术组、单纯脑梗死组给予生理盐水灌胃,其余各组大鼠通过药物灌胃制备痰热腑实证候模型,痰热腑实模型完成后行大脑中动脉阻塞(MCAO)手术,术后2小时进行缺血再灌注;对模型大鼠的宏观表现进行观察,对大鼠的神经功能采用Longa 5级4分法进行评分,大鼠脑组织神经元细胞采用苏木精-伊红(Hematoxylin and Eeosin staining-HE)染色法观察;检测模型大鼠总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、血糖(glucose,GLU)、低密度脂蛋白(low-density lipoprotein,LDL)等能量代谢指标的变化;运用免疫组化测定各取材点缺血侧脑组织中TNF-α、白介素6、VCAM-1、ICAM-1阳性细胞数,实时荧光定量(Real-time PCR,RT-PCR)法测定各因子m RNA的表达,探讨扎里奴思方对痰热腑实脑缺血模型大鼠炎性级联反应及能量代谢的影响及其神经保护机制。结果:1.痰热腑实模型大鼠在脑缺血再灌注后血脂及血糖等指标的变化及扎里奴思方对其的影响相比正常组,单纯脑梗死组大鼠甘油三酯、血糖均有显著的增高(P0.05);痰热腑实模型组大鼠血糖较假手术组和单纯脑梗死组升高(P0.05);总胆固醇、甘油三酯、低密度脂蛋白相比假手术组明显上升(P0.05);与痰热腑实1天组比较,尼平组TC、TG、LDL-C显著降低(P0.05);痰热腑实3天组比较尼莫地平3天组TC、TG、LDL-C差异显著(P0.05);扎方组1天、3天、7天组与痰热腑实组相比TC、TG、LDL-C均有显著降低(P0.05);HDL-C有明显的升高(P0.05);GLU在尼平组和扎方组3天、7天时显著减少(P0.05)。2.痰热腑实脑缺血模型大鼠再灌注后神经细胞损伤及扎里奴思方的保护作用假手术组大鼠未出现评分升高的表现,经过MCAO手术后的大鼠较假手术组和正常组相比,神经功能评分明显升高;单纯脑梗死组及痰热腑实组大鼠与假手术组相比,1天、3天、7天的神经功能评分均明显升高(P0.01),尤以1天组大鼠上升最为明显,且痰热腑实模型组神经功能缺损较单纯脑梗死组明显;给药后,扎里奴思方组和尼莫地平组大鼠在各时间点神经功能评分均开始降低,并随给药时间延长,评分下降明显(P0.05);尼莫地平组神经功能评分较扎里奴思方组略高。3.痰热腑实模型大鼠脑缺血再灌注后脑组织神经元细胞形态的改变及扎里奴思方对其的影响扎里奴思方对痰热腑实脑缺血再灌注大鼠缺血侧脑细胞的影响:HE染色结果显示,与单纯脑梗死组相比,假手术组海马组织结构排列整齐,胞体、核仁清晰,染色均匀,神经元细胞排列紧密;模型组海马组织细胞结构松散,排列散乱,神经元细胞消失较多,部分神经细胞胞核变小,细胞间质内可见大小不等的空泡,细胞核深染且核仁消失,皱缩,界限不清,大部分有退形性改变,神经细胞有嗜酸性改变;扎方组和尼平组神经细胞排列稍乱,仍可见细胞固缩,其周边可见神经细胞轻度肿胀,胞浆、胞膜、胞核尚能分清,程度较模型组明显减轻,扎方组较尼莫地平组改善明显。4.痰热腑实模型大鼠脑缺血再灌注后炎症因子的表达及扎里奴思方对其的影响4.1扎里奴思方对TNF-α表达的影响TNF-α在假手术组中表达较少,单纯脑梗死组与假手术组相比阳性细胞数增多明显(P0.01);随着时间的延长,TNF-α阳性细胞数呈下降趋势,与假手术组相比仍较高;尼平组与扎方组相对痰热腑实组来说,TNF-α阳性细胞数都有所下降(P0.01),以扎里奴思方1d,3d时下降更为明显(P0.05);模型组各时间点比假手术组TNF-αm RNA表达升高显著(P0.01);尼平组和扎方组相比模型组TNF-αm RNA表达降低明显(P0.01);扎里奴思方组较尼莫地平各组TNF-αm RNA表达有所降低,其中扎里奴思方1、7天降低明显(P0.05)。4.2扎里奴思方对IL-6β表达的影响单纯脑梗死组IL-6阳性细胞数比假手术组增多明显(P0.05);扎里奴思方组各时间点与单纯脑梗死组及痰热腑实组比较差异亦均有显著性(P0.05);与尼平组比较扎方组在1天、3天时均具有显著差异(P0.05);在假手术组中IL-6 m RNA表达较低,模型组各时间点相比假手术组,IL-6 m RNA表达显著升高(P0.01),在3天时达到高峰;尼莫地平组和扎里奴思方组相比模型各组,IL-6 m RNA表达明显降低(P0.01);扎方各组相比尼平1天、3天、7天组,IL-6 m RNA表达降低明显(P0.05)。4.3扎里奴思方对ICAM-1表达的影响假手术组中ICAM-1阳性细胞较少表达,单纯脑梗死组相比,假手术组大鼠缺血侧脑组织中阳性细胞表达升高明显(P0.01);在术后1天表达最多,扎方组与尼平组大鼠缺血侧脑组织中阳性细胞表达减少且与模型组各组相比差异明显(P0.01);扎方组相比尼平组,在1天、3天时减少明显;脑缺血后1天、3天、7天,模型组ICAM-1 m RNA的表达明显升高,3天后开始减少(P0.01);痰热腑实组在7天时较单纯脑梗死组表达稍有增多;扎方各组和尼平各组与非给药组比较,ICAM-1 m RNA的表达都明显降低,(P0.01,P0.05);扎方各组与尼平1天、3天、7天组比较ICAM-1 m RNA表达降低明显(P0.05)。4.4扎里奴思方对VCAM-1表达的影响假手术组中VCAM-1阳性细胞表达较少,单纯脑梗死组与假手术组相比,各时间点VCAM-1细胞阳性表达明显增多(P0.05);扎方组和尼莫地平组各时间点VCAM-1细胞阳性表达明显减少(P0.05);扎里奴思方与尼莫地平组相比,1天组VCAM-1细胞阳性表达降低尤其显著(P0.05);VCAM-1 m RNA在假手术组中已有少量表达,模型组各时间点假手术组相比,VCAM-1 m RNA显著升高(P0.01);相比非用药组,扎方组和尼平组VCAM-1m RNA表达明显降低(P0.01,P0.05);扎方各组相比尼平1天、3天、7天组VCAM-1 m RNA表达明显降低(P0.05)。结论:1.痰热腑实模型大鼠在脑缺血再灌注反应后应激性出现血糖、TG、胆固醇、LDL的升高,HDL的降低,应用扎里奴思方后大鼠血糖及血脂水平均有不同程度的改善,说明其对梗死后脂质的代谢有促进作用。2.痰热腑实模型大鼠在脑缺血损伤后神经细胞出现不同程度的损伤并表现出神经功能缺失症状,扎里奴思方可促进受损细胞的恢复并改善其神经功能缺损程度。3.大鼠经脑缺血再灌注后炎性因子的表达明显增多,运用扎里奴思方后可明显降低其表达,说明其可能是通过抑制炎性级联反应发挥脑保护作用。
[Abstract]:The neurological impairment caused by cerebral infarction has a serious impact on human health. The risk of disability is ranked first. The mortality rate is third, only lower than cancer and myocardial infarction. According to the WHO, about 15 million have cerebrovascular related diseases each year. Ischemic cerebrovascular disease is the 4/5. of its incidence. The incidence of cerebral infarction has gradually become younger, its recurrence, disability and mortality are high, which not only reduces the quality of life of the patients, but also causes a heavy burden on the family and society, causing great pressure to the health and health of our country. After the decrease of blood flow in the brain, the brain tissue produces a series of inflammatory cascade effects, including inflammatory factors. Expression, excitatory activity of amino acid toxicity, neuronal cell death, and peripheral depolarization of four different mechanisms, and each mechanism can interact with each other in time. The inflammatory reaction is the result of oxygen free radicals, cytokines and proinflammatory enzymes in the process of injury cascade. The skin cell, which activates the receptor, enables the leukocytes into the brain, and can directly stimulate the microglia or astrocytes to release harmful components and eventually form brain damage. The combination of disease and syndrome combines the basic theory of traditional Chinese medicine and the knowledge of Western medicine theory and animal experiment, and makes use of the etiological theory of Western medicine to prepare the animal. At the same time, the syndrome of Chinese medicine is established in the model of the disease, and the syndrome in the animal model is fused with the disease, so it has good stability and reliability, and it can be observed and studied systematically. Therefore, the research of disease syndrome combined with animal model has become a model of modern Chinese medicine research. An important development trend is of great practical significance for the pathogenesis of cerebral infarction and the exploration of drug use. 1. through the detection of blood lipid and blood glucose in the model rats of phlegm heat and cerebral ischemia reperfusion model, the change of stress metabolic function of cerebral ischemia phlegm heat Fu Fu model was clearly defined, and the metabolic function of cerebral infarction was observed by Sali Nu Fang. .2. by observing the pathological changes of nerve function and neuron cell in the rat model of phlegm heat syndrome after cerebral ischemia and reperfusion, the protective effect of Zi Li Si Fang on the nerve cells was determined by observing the expression of inflammatory related factors in the rat model of cerebral ischemia reperfusion phlegm heat syndrome model rats after reperfusion, from the point of view of.2.. The influence of Fang on its change, clear its possible mechanism of action; methods: Sprague Dawley rats, male, 250, SPF grade, randomly divided into normal group, sham operation group, simple cerebral infarction group, phlegm hot Fu Fu Group, nimodipine group, and the group of nimodipine, 10 of the normal group, the other groups are divided into 1 days, 3 days, 7 days 3 timber time. There were 15 rats at each time point, the normal group, the sham operation group and the simple cerebral infarction group were given the normal saline for gastric perfusion, the other rats were given the phlegm heat syndrome model through the medicine gavage. After the phlegm heat syndrome was completed, the middle cerebral artery occlusion (MCAO) operation was performed, and the ischemia reperfusion was carried out at 2 hours after the operation, and the macroscopic performance of the model rats was carried out. The neural function of rats was evaluated by Longa 5 grade 4 method. The neuron cells in the rat brain were observed by hematoxylin eosin (Hematoxylin and Eeosin staining-HE) staining, and the total cholesterol (total cholesterol, TC), triglyceride (triglyceride, TG), blood glucose (glucose, GLU), low density lipoprotein (LDL), and low density lipoprotein (LDL) were detected in the rat model. The changes of energy metabolism indexes such as nsity lipoprotein, LDL), TNF- alpha, interleukin 6, VCAM-1, ICAM-1 positive cells, Real-time PCR, RT-PCR method were used to determine the expression of M RNA in each factor, and the inflammatory cascade in the rat model of phlegm heat cerebral ischemia model was investigated by the method of Real-time PCR, RT-PCR. The effect of reaction and energy metabolism and its neuroprotective mechanism. Results: 1. the changes of blood lipid and blood sugar in the model rats after cerebral ischemia reperfusion and the effect of TNU side on it were compared with that of the normal group. The triglyceride and blood sugar of the rats in the simple cerebral infarction group were significantly increased (P0.05); the blood sugar in the model group of phlegm heat Fu Fu model rats Compared with the sham operation group and the simple cerebral infarction group (P0.05), the total cholesterol, triglyceride and low density lipoprotein were significantly higher than the sham operation group (P0.05), and the TC, TG and LDL-C in the nimping group were significantly decreased (P0.05) compared with the 1 day group of phlegm heat, and the 3 day group of nimodipine was significantly different from the 3 days of nimodipine group (P0.05); the group was 1 days, 3 days, 7 days. TC, TG, LDL-C were significantly decreased (P0.05), and HDL-C had a significant increase (P0.05) compared with the phlegm heat group (P0.05); GLU in the nepin group and Zhan Fang Group 3 days, 7 days significantly reduced (P0.05).2. phlegm heat cerebral ischemia model rats after reperfusion nerve cell injury and the protective effect of Zhan Li Si Fang, the sham operation group did not appear to increase the score Compared with the sham operation group and the normal group, the nerve function score of the rats after the MCAO operation was significantly higher than that of the sham group and the sham group. The nerve function score of the rats in the 1 day, the 3 day and the 7 day was significantly higher than that of the sham group (P0.01), especially in the 1 day group, and the nerve function defect in the phlegm heat model group Compared with the simple cerebral infarction group, the nerve function score of the rats in the nimodipine group and the nimodipine group began to decrease after the administration, and the score decreased with the prolongation of the time of Administration (P0.05). The nimodipine group's nerve function score was slightly higher than the.3. phlegm heat syndrome rat brain tissue after the cerebral ischemia reperfusion. The changes of neuronal cell morphology and the effect of Zi Li Si Fang on the ischemic side brain cells of rats with phlegm heat and cerebral ischemia reperfusion. The results of HE staining showed that the structure of the hippocampal structure in the sham operation group was neatly arranged, the body, the nucleolus were clear, the coloration was uniform, and the neuron cells were closely arranged. In the model group, the cell structure of the hippocampus was loose and scattered, the neuron cells disappeared more, the nuclei of some nerve cells became smaller, and the cells in the cells were visible in different size vacuoles. The nuclei were deeply dyed and the nucleolus disappeared, and the lines were indistinct. Most of the cells were degenerated, and the cells of the deity were eosinophilic, and the nerve cells in the Zhan Fang Group and the nepin group were changed. There was a slight disorder in the arrangement of the cells. The peripheral nerve cells were slightly swelling, the cytoplasm, the membrane and the nucleus were still distinct, and the extent of the cells was clearly reduced. The expression of the inflammatory factors after the cerebral ischemia reperfusion in the.4. phlegm heat model rats and the effect of zhali Nu Si Fang on it were better than the nimodipine group, and the influence of zhali Nu Si Fang on it was 4.1 zhali Nu square The expression of TNF- alpha was less in the sham operation group than in the sham operation group. The number of positive cells in the simple cerebral infarction group increased significantly (P0.01) compared with the sham operation group (P0.01). The number of TNF- alpha positive cells decreased with the prolongation of time, and was still higher than the sham operation group. The number of TNF- alpha positive cells in the niping group and the Zhan Fang group compared with the phlegm heat group, the number of TNF- alpha positive cells was all The decrease (P0.01), with zari Nu side 1D, 3D decreased more obviously (P0.05), and the expression of TNF- alpha m RNA in the model group was significantly higher than that of the sham operation group (P0.01), and the expression of TNF- alpha m RNA decreased significantly (P0.01) in the nimping group and the Zai Fang Group, and the zari Zaru side group was lower than the nimodipine group. Nu Si Fang 1,7 days decreased significantly (P0.05).4.2 Zhan Nu side effect on IL-6 beta expression in simple cerebral infarction group, the number of IL-6 positive cells increased significantly (P0.05), and the time points in the zhali Nu Sifang group were also significantly different from those in the simple cerebral infarction group and the phlegm heat group (P0.05), and the group was compared with the niping group at 1 days and 3 days. There were significant differences (P0.05); the expression of IL-6 m RNA in the sham operation group was low, and the expression of IL-6 m RNA in the model group was significantly higher than that in the sham operation group (P0.01), and reached the peak at 3 days. The expression of IL-6 m RNA was significantly lower in the nimodipine group and the zhali Nu Fang group than in the model group (P0.01); 1 days, 3 days, and 7 days compared with the nimodipine group. Group, IL-6 m RNA expression decreased significantly (P0.05).4.3 zhalnu side effect on the expression of ICAM-1 in sham operation group less expression of ICAM-1 positive cells, pure cerebral infarction group, compared with the sham group, the expression of positive cells in the ischemic side brain tissue in the sham operation group increased significantly (P0.01), 1 days after the operation, the most, zhfang group and the nipping group rats ischemic side brain group The expression of positive cells in the fabric decreased significantly (P0.01) compared with that in the model group (P0.01). Compared with the neping group, the expression of positive cells decreased significantly at 1 days and 3 days. The expression of ICAM-1 m RNA in the model group increased significantly after 1 days of cerebral ischemia, and decreased in 3 days (P0.01); the expression of phlegm heat group was slightly more than that in the simple cerebral infarction group at 7 days; ligation prescription The expression of ICAM-1 m RNA in each group and nping group decreased significantly (P0.01, P0.05), and the expression of ICAM-1 m RNA in the group of Zhan Fang and nping 1 days, 3 days and 7 days was significantly lower (P0.05). The expression of VCAM-1 positive cells in the sham operation group was less than that in the sham operation group, and the simple cerebral infarction group and the sham operation group were less. The positive expression of VCAM-1 cells in each time point increased significantly (P0.05), and the positive expression of VCAM-1 cells in the Zhan Fang Group and nimodipine group decreased significantly (P0.05), and the positive expression of VCAM-1 cells in the 1 day group was significantly lower than that in the nimodipine group (P0.05), and the VCAM-1 m RNA had been expressed in a small amount in the sham operation group, and the model of VCAM-1 m RNA had been expressed in a small amount. Compared with the sham operation group at all time points, VCAM-1 m RNA increased significantly (P0.01), and the expression of VCAM-1m RNA in Zhan Fang Group and neping group was significantly decreased (P0.01, P0.05), compared with non medication group (P0.01, P0.05), and the expression of VCAM-1 m RNA was obviously decreased (P0.05) in the group of Zhan Fang 1 days, 3 days and 7 days. Conclusion: 1. phlegm heat Fu Fu model rats stress after cerebral ischemia reperfusion injury. The increase of blood sugar, TG, cholesterol, LDL, the decrease of HDL, the improvement of blood sugar and blood lipid levels in the rats after the use of the Chi Nu Si Fang, shows that the lipid metabolism in the post infarction rats has a promoting effect on the.2. phlegm heat and Fu Fu model rats with different degrees of injury in the neural cells after the cerebral ischemia injury and showing the lack of nerve function. The loss of symptoms, Zaru Si Fang can promote the recovery of damaged cells and improve the degree of nerve function defect in.3. rats. The expression of inflammatory factors in rats after cerebral ischemia reperfusion is obviously increased. The expression of zari Nu side can be obviously reduced after zari Nu Fang, which indicates that it may play the role of brain protection by inhibiting the inflammatory cascade reaction.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R29

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