电针对高血脂合并脑缺血大鼠缺血皮质区Caspase-3蛋白表达的影响

发布时间：2018-06-09 12:02

本文选题：Caspase-3 + 脑缺血　；参考：《北京中医药大学》2017年硕士论文

【摘要】：目的:本实验采用高血脂合并脑缺血大鼠模型,观察脑缺血灶形态学(TTC染色和HE染色)、血脂四项(胆固醇、甘油三酯、高密度脂蛋白和低密度脂蛋白)含量和缺血皮质区Caspase-3蛋白表达的变化,探讨电针对高血脂合并脑缺血大鼠行为学、血脂四项、肝脏和脑组织形态学、缺血灶梗死体积和缺血皮质区Caspase-3表达的影响,为阐明电针抗凋亡机制提供基础研究。方法:分组:105只SD大鼠适应性饲养1周后,随机分为正常组、假手术组、模型组、电针Ⅰ组、电针Ⅱ组,每组21只,其中每组按随机数字表抽取7只做TTC染色;剩余每组14只随机分为1天组和7天组,各7只,做HE染色、血脂四项检测和Caspase-3蛋白检测。除正常组外,其余各组以高脂饲料喂养6周制备高血脂症模型。6周后电针Ⅰ组开始电针双侧"丰隆" "三阴交",1次/日,每次20 min,连续干预7天,模型组、电针Ⅱ组不干预。7天后模型组、电针Ⅰ组、电针Ⅱ组,采用FeCl3化学诱导血栓性闭塞大脑中动脉模型,假手术组除不滴加FeCl3溶液外,其余手术步骤同模型组。造模后电针Ⅰ、Ⅱ组电针双侧"丰隆"、"三阴交"及针刺"水沟""百会",1次/日,每次20min,分别干预1、7天。造模后1、3、7天分别对各组大鼠进行神经行为学评分;造模后1天TTC染色,观察脑梗死体积;造模后1、7天腹主动脉取血,检测血清中胆固醇(CHO)、甘油三酯(TG)、高密度脂蛋白(HDL-C)和低密度脂蛋白(LDL-C)含量,HE染色观察大鼠肝脏和脑组织病理变化,免疫组化方法检测缺血皮质区Caspase-3蛋白的表达。结果:1.电针对各组大鼠神经行为学的影响大鼠脑缺血造模后1、3、7天神经功能评分:与同期正常组、假手术组比较,模型组、电针Ⅰ组和电针Ⅱ组神经功能评分造模后1、3、7天均有明显差异(P0.05);与同期模型组比较,电针Ⅰ、Ⅱ组造模后1、3、7天均有明显差异(P0.05);与同期电针Ⅱ组比较,造模后1、3天,电针Ⅰ组评分有明显差异(P0.05),造模后7天,电针Ⅰ、Ⅱ组无明显差异(P0.05)。2.电针对各组大鼠血脂四项(胆固醇、甘油三酯、高密度脂蛋白和低密度脂蛋白)含量的影响血脂四项结果显示,与正常组比较,假手术组、模型组造模后1、7天,CHO、TG、HDL-C和LDL-C含量均有明显差异(P0.05);与同期模型组比较,电针Ⅰ组造模后1、7天,CHO、TG、HDL-C和LDL-C含量均有明显差异(P0.05),电针ⅡI组造模后1天,TG含量有明显差异(P0.05),CHO、HDL-C和LDL-C含量无明显差异(P0.05),造模后7天,CHO、TG、HDL-C和LDL-C含量均有明显差异(P0.05);与同期电针Ⅱ组比较,电针Ⅰ组造模后1天,CHO、HDL-C和LDL-C含量均有明显差异(P0.05),TG含量无明显差异(P0.05),造模后7天,CHO、TG、HDL-C和LDL-C含量均有明显差异(P0.05);两电针Ⅰ组比较,CHO、HDL-C和LDL-C含量有明显差异(P0.05),TG含量无明显差异(P0.05);两电针Ⅱ组比较,CHO、TG和LDL-C含量均有明显差异(P0.05),HDL-C含量无明显差异(P0.05)。3.电针对各组大鼠肝组织形态学的影响肝组织HE染色结果显示,造模后1、7天正常组和假手术组肝细胞未见变性坏死,细胞排列整齐,结构清晰。模型组造模后1天,可见淋巴细胞聚集,肝板索状结构被破坏,肝血窦小而少,偶见点状坏死灶;造模后7天,肝细胞肿胀明显,肝窦结构不清,可见脂肪变性,典型表现有广泛的气球样变性,病理表现明显比1天模型组严重。电针Ⅰ、Ⅱ组造模后1、7天,肝细胞病变较轻,异型性低,肝板、肝窦、肝小叶结构稍不清,炎性细胞比同期模型组有明显减少。4.电针各组大鼠脑梗死体积的影响TTC染色结果显示,大鼠脑缺血造模后1天,缺血部位均呈典型的缺血性改变,表明该模型制备成功。与正常组、假手术组比较,模型组、电针Ⅰ、Ⅱ组梗死体占比均有明显差异(P0.05);与模型组比较,电针Ⅰ组梗死体积占比有明显差异(P0.05),电针Ⅱ组梗死体积占比无明显差异(P0.05);与电针Ⅱ组比较,电针Ⅰ组梗死体积占有明显差异(P0.05)。5.电针对各组大鼠脑组织形态学的影响脑组织皮质区HE染色结果显示:正常组和假手术组神经细胞基本正常,排列整齐,核仁清晰。模型组造模后1天,可见胶质细胞轻度增生,神经元轻度肿胀,造模后7天可见间质细胞水肿明显,大量死亡神经元,呈筛状坏死,坏死组织周围可见炎性细胞浸润;海马CA1区染色结果显示:正常组和假手术组海马CA1区结构致密细胞核数量较多,细胞核形态基本正常。模型组造模后1天,整体未见软化灶,海马细胞萎缩较少,造模后7天海马区神经细胞数量减少,细胞核固缩,层次紊乱。电针Ⅰ、Ⅱ组神经细胞受损情况较模型组明显减轻,存活的神经细胞数量增多,海马结构排列较整齐。6.电针各组大鼠缺血皮质区Caspase-3蛋白表达的影响正常组和假手术组大鼠缺血皮质区Caspase-3蛋白呈弱阳性表达。与正常组比较,假手术组术后1、7天,Caspase-3阳性细胞数量和平均光密度值均无明显差异(P0.05);与同期假手术组比较,模型组造模后1、7天,Caspase-3阳性细胞数量和平均光密度值均有明显差异(P0.05);与同期模型组比较,电针Ⅰ组造模后1、7天,Caspase-3阳性细胞数量和平均光密度值均有明显差异(P0.05),电针Ⅱ组造模后1天Caspase-3阳性细胞数量无明显差异(P0.05),平均光密度值有明显差异(P0.05),电针Ⅱ组造模后7天,Caspase-3阳性细胞数量和平均光密度值有明显差异(P0.05);与同期电针Ⅱ组比较,电针Ⅰ组造模后1、7天,Caspase-3阳性细胞数量和平均光密度值均有明显差异(P0.05);两电针Ⅰ组比较,Caspase-3阳性细胞数量和平均光密度值有明显差异(P0.05);两电针Ⅱ组比较,Caspase-3阳性细胞数量和平均光密度值有明显差异(P0.05)。结论:1.电针可以降低神经功能评分,脑缺血早期干预对改善神经功能症状作用明显,即刻的针刺效应对神经功能改善也有一定作用。2.电针可以降低血清CHO、TG和LDL-C含量,升高HDL-C含量,随着脑缺血时间延长脑受损情况加重,电针对HDL-C作用逐渐不明显。3.脑缺血可以加重高血脂症诱导的肝损伤,电针对肝组织病理形态学变化有改善作用,可能通过减少炎性细胞降低肝损伤。4.电针对缺血区脑组织病理形态学变化有改善作用,可能通过减轻神经元变性脱失现象,神经细胞肿胀和炎性细胞浸润,减轻脑损伤。5.早期电针干预可以减少大鼠缺血部位梗死体积,降低脑组织损伤。6.电针可以下调高血脂合并脑缺血大鼠缺血皮质区Caspase-3蛋白的含量,且提前电针干预下调作用明显,说明其存在抗凋亡的作用,下调Caspase-3蛋白含量的表达可能是电针起效机制之一。
[Abstract]:Objective: in this experiment, we used hyperlipidemia and cerebral ischemia rat model to observe the morphology (TTC and HE staining) of cerebral ischemia, the content of four lipids (cholesterol, triglyceride, high-density lipoprotein and low density lipoprotein) and the change of Caspase-3 protein expression in ischemic cortex. Four terms of lipid, liver and brain histomorphology, infarct volume and Caspase-3 expression in ischemic cortex provide a basic study for the anti apoptosis mechanism of electroacupuncture. Methods: 105 SD rats were randomly divided into normal group, sham operation group, model group, electroacupuncture group I, group II, group II, each group of 21 rats, of which each group was in each group. A random number table was used to extract 7 TTC stains; 14 of the remaining groups were randomly divided into 1 days group and 7 day group, each 7, HE staining, four blood lipid testing and Caspase-3 protein detection. Except the normal group, the rest groups were fed with high fat feed for 6 weeks to prepare hyperlipidemia model.6 weeks after the electroacupuncture group began to electroacupuncture bilateral "Feng long" "Three Yin cross" ", 1 / day, each. 20 min, continuous intervention for 7 days, model group, electroacupuncture group II group did not interfere with.7 days model group, electroacupuncture group I, electroacupuncture group II, using FeCl3 chemical induced thrombotic occlusion of the middle cerebral artery model, sham operation group in addition to the FeCl3 solution, the rest of the procedure with the model group. Water ditch "hundred meetings", 1 times / day, each time 20min, intervention 1,7 days respectively. After the model 1,3,7 days, the rats were graded respectively on neurobehavioral score, and the volume of cerebral infarction was observed at 1 days after modeling, and the abdominal aorta was taken on 1,7 days after modeling, and serum cholesterol (CHO), triglyceride (TG), high-density lipoprotein (HDL-C) and low density lipoprotein (LDL-) were detected on 1,7 days after modeling. C) content, HE staining was used to observe the pathological changes of liver and brain tissue in rats. Immunohistochemical method was used to detect the expression of Caspase-3 protein in ischemic cortex. Results: 1. the effect of Electroacupuncture on neurobehavioral effects of rats in each group was measured by the 1,3,7 day nerve function score of the rats after the model of cerebral ischemia: compared with the normal group, sham operation group, model group, electroacupuncture group I and electroacupuncture. In group II, there were significant differences in 1,3,7 days after modeling (P0.05). Compared with the same model group, there was a significant difference between group I and group II after making model (P0.05). Compared with group II of electroacupuncture at the same time, the score of electroacupuncture group I was significantly different (P0.05), 7 days after modeling (P0.05), and there was no significant difference (P0.05).2. electroacupuncture at 7 days after modeling. The four results of blood lipid four items (cholesterol, triglyceride, high density lipoprotein and low density lipoprotein) showed that compared with the normal group, the contents of CHO, TG, HDL-C and LDL-C in the sham operation group were significantly different (P0.05) in the model group 1,7 days after model building, and compared with the model group, 1,7 days, CHO, TG after the model group I was built. The contents of HDL-C and LDL-C were significantly different (P0.05). There were significant differences in TG content (P0.05) at 1 days after electroacupuncture group II I. There was no significant difference in CHO, HDL-C and LDL-C content (P0.05). There were significant differences in the content of TG, TG, CHO, HDL-C and LDL-C after 7 days of modeling. There was no significant difference (P0.05), and there was no significant difference in TG content (P0.05). The content of CHO, TG, HDL-C and LDL-C were significantly different (P0.05) at 7 days after the model. The content of CHO, HDL-C and LDL-C was significantly different (P0.05), and there was no significant difference in the content of CHO, HDL-C and LDL-C in the two electroacupuncture group. The effect of.3. electroacupuncture on the liver histomorphology of rats in each group (P0.05) showed that the liver tissue HE staining results showed that there was no degeneration and necrosis in the normal group and the sham operation group after the model building, the cells arranged neatly and the structure was clear. 1 days after the model group, the lymphocyte aggregation, the liver plate cable structure was destroyed, the hepatic sinusoids were small and small. On the 7 day after the model, the liver cell swelling was obvious, the hepatic sinusoid structure was not clear, the fatty degeneration was seen, the typical manifestation had the extensive balloon denaturation, the pathological manifestation was obviously more serious than the 1 day model group. The electroacupuncture group I, group II, after 1,7 days, the liver cell lesion was light, the heteromorphosis was low, the liver plate, hepatic sinus, hepatic lobule structure was slightly unclear, inflammatory cells were compared with the same The effect of the model group on the cerebral infarction volume of the.4. electroacupuncture group was significantly reduced by TTC staining. The results showed that 1 days after the cerebral ischemia, the ischemic parts were all typical ischemic changes, which showed that the model was successfully prepared. Compared with the normal group, the sham operation group, the model group, the electroacupuncture I, and the group II were significantly different (P0.05). Compared with group I, there was a significant difference in the infarct volume ratio in the electroacupuncture group (P0.05), and there was no significant difference in the infarct volume in the electroacupuncture group (P0.05). Compared with the electroacupuncture group, the infarct volume in the group I was significantly different (P0.05). The effect of.5. electroacupuncture on the brain histomorphology of the rats in each group showed that the result of HE staining in the cortex area of the cerebral cortex showed that the normal group and the artificial hand were the result of HE staining. The nerve cells in the operation group were basically normal, arranged neatly, and the nucleolus were clear. On the 1 day after the model group, the glial cells were slightly proliferated and the neurons were swollen slightly. The interstitial cell edema was visible on the 7 day after the model building. A large number of dead neurons were necrotic and inflammatory cells infiltrated around the necrotic tissue; the hippocampal CA1 staining results showed that the normal group was the normal group. The number of dense nuclei in the hippocampal CA1 area of the sham operation group was more than that of the model group, and the nucleus morphology was basically normal. The model group had no softening focus on the whole of the model group 1 days after the model building. The hippocampal cells atrophied less, the number of neurons in the hippocampus decreased in the 7 day after model building, the nucleus retraction and the level disorder. The damage of nerve cells in group I, group II was significantly reduced than that of the model group. The number of surviving nerve cells increased, and the expression of Caspase-3 protein in the ischemic cortex of the rats with.6. electroacupuncture group was more orderly. The expression of Caspase-3 protein in the ischemic cortex of the normal group and the sham operation group was weakly positive. Compared with the normal group, the number of Caspase-3 positive cells and the mean light density value of the Caspase-3 positive cells in the sham operation group were 1,7 days after operation. There was no significant difference (P0.05). Compared with the sham operation group in the same period, the number of Caspase-3 positive cells and the mean light density of the model group were significantly different at 1,7 days after model building (P0.05). Compared with the model group, the number of Caspase-3 positive cells and the number of light density were significantly different at 1,7 days after the model group (P0.05), and the model of electroacupuncture group II was made by the model group (P0.05). There was no significant difference in the number of Caspase-3 positive cells in the last 1 days (P0.05), and the mean light density was significantly different (P0.05). The number of Caspase-3 positive cells and the mean light density were significantly different (P0.05) at the 7 day after the electroacupuncture group II group. Compared with the Electroacupuncture group II group, the number of Caspase-3 positive cells and the mean optical density value of the Caspase-3 positive cells after the electroacupuncture group I made the model. There were significant differences (P0.05). Two electroacupuncture group I compared the number of Caspase-3 positive cells and average optical density (P0.05); two electroacupuncture group II compared, the number of Caspase-3 positive cells and the mean light density value is significantly different (P0.05). Conclusion: 1. electroacupuncture can reduce the neurological function score, cerebral ischemia early intervention to improve nerve work The effect of symptomatic effect is obvious. The immediate acupuncture effect also has certain effect on the improvement of nerve function..2. electroacupuncture can reduce the content of serum CHO, TG and LDL-C, increase the content of HDL-C. With the prolongation of cerebral ischemia time, the condition of brain damage is aggravated. The effect of Electroacupuncture on HDL-C is gradually not obvious in.3. cerebral ischemia, which can aggravate the liver injury induced by hyperlipidemia, the Electroacupuncture of the liver. The changes of histopathology can be improved by reducing the injury of inflammatory cells and reducing the liver injury..4. electroacupuncture can improve the pathological changes of the brain tissue in the ischemic area. It may reduce the degeneration and loss of neurons, the swelling of the nerve cells and the infiltration of inflammatory cells, and reduce the brain injury in the early stage of.5. by Electroacupuncture intervention to reduce the deficiency of rats. .6. electroacupuncture can reduce the content of Caspase-3 protein in the ischemic cortex of rats with hyperlipidemia and cerebral ischemia, and the effect of early electro acupuncture intervention is obvious, indicating the effect of anti apoptosis, and the expression of Caspase-3 protein can be one of the mechanism of electroacupuncture.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

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