电针对缺血再灌注脑损伤大鼠炎症细胞因子IL-4、TNF-α的影响

发布时间：2018-05-25 16:10

本文选题：电针 + 脑缺血再灌注　；参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:通过观察电针对脑缺血再灌注大鼠模型炎症反应相关因子TNF-α、IL-4的调节作用,探讨电针改善脑缺血后大鼠炎症反应的作用机制,以期进一步深化电针治疗缺血性脑病作用机理的认识,为临床治疗提供科学依据。方法:雄性清洁级SD大鼠,采用数字表法随机抽取24只作为假手术组,其余剩下的动物采用改良Longa线栓法制备大鼠右侧大脑中动脉脑缺血模型,根据Zea-longa5级4分法的评分标准,初步判断造模成功与否,将成功的模型随机分为模型组和电针组,每组有效例数为24只。假手术组只分离血管不穿插线栓;模型组用线栓阻塞中动脉2h后拔出线栓实现再灌注,之后不做任何治疗措施;电针组成功造模后选取"百会"和"大椎"穴进行针刺,连接韩式HANS-200型电针仪治疗30min,波形为疏密波,刺激频率为2/15Hz。各组于缺血再灌注24h后,参照Julio氏18分制评分法行神经行为学评分;采用TTC染色法观察脑缺血组织形态学改变和脑梗死面积百分比;采用苏木素-伊红(HE)染色法观察缺损侧脑组织细胞病理变化;采用免疫组化染色法检测缺损侧皮层组织IL-4、TNF-α的蛋白表达;采用 qRT-PCR 检测 IL-4mRNA、TNF-αmRNA 表达的变化。结果:(1)神经功能行为学评分:脑缺血再灌注损伤大鼠的神经功能行为学评分明显降低,与假手术组相比差异有统计学意义(P0.01);与模型组比较,电针干预治疗能有效提高神经行为学评分,差异有统计学意义(P0.05)。(2)脑组织形态学变化及梗死面积百分比:经TTC染色,假手术组脑组织均匀染色呈现暗红色,模型组大脑右侧缺血脑组织呈现白色,电针组染色缺失区较模型组减少。模型组和电针组脑梗死面积百分比均高于假手术组(P0.01和P0.05),与模型组比较,电针组脑梗死面积明显降低(P0.05)。HE染色,模型组见大量皱缩的细胞聚集,电针组的皱缩细胞较模型组减少。(3)缺损侧大鼠大脑皮层IL-4、TNF-α蛋白表达:IL-4蛋白的表达,与假手术组相比,模型组和电针组蛋白表达量均明显增多,差异均有统计学意义(P0.01);相比模型组,电针组蛋白表达量升高,差异有统计学意义(P0.05)。TNF-α蛋白表达量,与假手术组相比,模型组蛋白表达量明显上升,差异具有统计学意义(P0.01),电针组蛋白表达量虽略有升高,但差异无统计学意义(P0.05);与模型组相比,电针组蛋白表达明显偏低,差异具有统计学差异(P0.05)。IL-4/TNF-α蛋白量比值,与假手术组比较,模型组比值略有降低,但差异无统计学意义(P0.05),而电针组比值升高明显(P0.01);电针组与模型组比较,差异亦有统计学意义(P0.01)。(4)缺损侧大脑皮层IL-4mRNA、TNF-αmRNA的表达量:IL-4mRNA的表达,与假手术组比较,模型组、电针组均显著增多,差异有统计学意义(P0.05和P0.01);与模型组相比,电针组表达量明显上升,差异具有统计学意义(P0.05)。TNF-αmRNA的表达量,相比假手术组,模型组表达量升高,差异有统计学意义(P0.01),电针组的差异则无明显统计学意义(P0.05);与模型组相比,电针组降低,差异有统计学意义(P0.05)。IL-4mRNA/TNF-αmRNA的比值,与假手术组比较,模型组比值略低但无统计学意义(P0.05),而电针组比值升高显著,有统计学意义(P0.01);电针组与模型组比较,差异亦有统计学意义(P0.01)。结论:电针对缺血再灌注脑损伤大鼠IL-4、TNF-α炎症因子具有调控作用,提升抗炎细胞因子,下调促炎细胞因子,提升IL-4/TNF-α的比值,从而减轻缺血再灌注区的炎症反应,降低梗死面积,改善神经功能。
[Abstract]:Objective: To observe the effect of Electroacupuncture on the inflammatory response related factors TNF- alpha and IL-4 in the rat model of cerebral ischemia reperfusion, and to explore the mechanism of electroacupuncture to improve the inflammatory response in rats after cerebral ischemia, in order to further deepen the understanding of the mechanism of the treatment of ischemic encephalopathy by electroacupuncture and provide a scientific basis for clinical treatment. The SD rats were randomly selected as the sham operation group by the digital table method. The remaining animals were prepared by modified Longa thread embolus method to prepare the right cerebral artery cerebral ischemia model of the right middle cerebral artery in rats. According to the score standard of the Zea-longa5 grade 4 method, the model group and the electroacupuncture group were randomly divided into the model group and the electroacupuncture group. The effective examples of each group were randomly divided into each group. The number is 24. The sham operation group only separated the blood vessels and did not insert the thread plug; the model group used the thread plug to block the middle artery 2h after pulling out the thread to realize the reperfusion, and then did not do any treatment. The electroacupuncture group successfully selected "Baihui" and "Da Zhui" point for acupuncture, connected with the Korean HANS-200 electroacupuncture apparatus to treat 30min, the waveform was the density wave, the stimulation frequency was 2/15 Hz. after ischemia and reperfusion for 24h, the neurobehavioral score was scored by Julio's 18 score scoring system. TTC staining was used to observe the morphological changes of cerebral ischemia and the percentage of cerebral infarction area, and the pathological changes of the defect side brain tissue were observed with hematoxylin eosin (HE) staining, and the defect lateral cortex was detected by immunohistochemical staining. Tissue IL-4, TNF- alpha protein expression; changes in the expression of IL-4mRNA and TNF- alpha mRNA by qRT-PCR. Results: (1) neurological functional behavioral score: the neurological functional behavior score of rats with cerebral ischemia-reperfusion injury was significantly reduced, compared with the sham operation group (P0.01); compared with the model group, the electroacupuncture intervention could be used. The difference of neurobehavioral score was statistically significant (P0.05). (2) the morphological changes of brain tissue and the percentage of infarct area: after TTC staining, the uniform staining of the brain tissue in the sham operation group was dark red, the right cerebral ischemic brain tissue in the model group was white, the dyed area in the electroacupuncture group decreased compared with the model group. Model group and electroacupuncture group had cerebral infarction. The area percentage was higher than that of the sham operation group (P0.01 and P0.05). Compared with the model group, the area of cerebral infarction in the electroacupuncture group was significantly reduced (P0.05).HE staining. The model group saw a large number of crumpled cells aggregated, and the contraction cells in the electroacupuncture group decreased compared with the model group. (3) the IL-4, TNF- alpha protein expression of the cerebral cortex of the defected side rats: the expression of IL-4 protein and the sham operation group. The expression of protein in the model group and electroacupuncture group increased significantly, and the difference was statistically significant (P0.01). Compared with the model group, the expression of protein in the electroacupuncture group increased, and the difference was statistically significant (P0.05).TNF- alpha protein expression. Compared with the sham operation group, the amount of the model group was significantly increased, the difference was statistically significant (P0.01), the electroacupuncture group eggs were statistically significant (P0.01). Although the white expression was slightly higher, the difference was not statistically significant (P0.05). Compared with the model group, the protein expression in the electroacupuncture group was significantly lower, and the difference was statistically significant (P0.05).IL-4/TNF- alpha protein ratio. Compared with the sham operation group, the ratio of the model group decreased slightly, but the difference was not statistically significant (P0.05), but the ratio of the electroacupuncture group increased significantly (P0.01 The difference between the electroacupuncture group and the model group was statistically significant (P0.01). (4) the expression of IL-4mRNA in the cerebral cortex of the defect side and the expression of TNF- alpha mRNA: the expression of IL-4mRNA was significantly increased in the model group and electroacupuncture group compared with the sham operation group, and the difference was statistically significant (P0.05 and P0.01). Compared with the model group, the expression of the electroacupuncture group was obviously increased and the difference had the difference. The expression of.TNF- alpha mRNA in statistical significance (P0.05), compared with the sham operation group, the expression of the model group increased, the difference was statistically significant (P0.01), the difference in the electroacupuncture group was not significant (P0.05). Compared with the model group, the electroacupuncture group decreased, the difference was statistically significant (P0.05) the ratio of.IL-4mRNA/TNF- alpha mRNA, compared with the sham operation group, the model was compared with the sham group. The ratio of the group was slightly lower but not statistically significant (P0.05), but the ratio of the electroacupuncture group increased significantly (P0.01), and the difference between the electroacupuncture group and the model group was also statistically significant (P0.01). Conclusion: Electroacupuncture has a regulatory effect on IL-4, TNF- alpha and inflammatory cytokines in rats with cerebral ischemia-reperfusion injury, promoting anti-inflammatory cytokines and down-regulation of proinflammatory cells. It enhances the ratio of IL-4/TNF- alpha, thereby alleviating inflammatory reaction, reducing infarct size and improving neurological function in ischemia-reperfusion area.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

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