蒙医灸疗法对老年性痴呆模型大鼠的作用机理研究

发布时间：2018-04-29 06:02

本文选题：蒙医 + 灸疗法　；参考：《北京中医药大学》2014年博士论文

【摘要】：研究目的 1.本课题根据蒙医文献记载及前期研究基础上提出蒙医灸疗法可能对老年性痴呆模型AD大鼠有保护和改善脑神经细胞损伤,改善脑神经功能回复的假设,采用蒙医灸疗法治疗AD大鼠的实验研究方法,阐明蒙医灸疗法对老年性痴呆病的治疗作用及其机理。 2.采用大鼠皮下注射D-半乳糖和双侧海马注射Aβ25-35建立大鼠老年性痴呆病理模型,以蒙医“顶会穴”、“心穴”和“胃穴”为切入点,设置正常组、假手术组、模型组、灸疗组和西药组,观察灸疗法对AD大鼠的学习记忆能力、相关胆碱、氧化、自由基酶的含量,脑组织神经细胞病理改变、脑组织相关基因的mRNA和蛋白表达的影响,阐明蒙医灸疗法治疗AD的作用及其机理。研究方法 1.动物选择及筛选方法：选用体质量220±20(g)、健康清洁级雄性SD大鼠,将大鼠分别进行MorriS水迷宫测试,剔除天生痴呆大鼠,选出合格大鼠70只。 2.大鼠分组及造模：将70只大鼠按体质量随机分为5组,正常组、假手术组、模型组、灸疗组和西药组,每组14只。前6周：模型组、灸疗组和西药组三组大鼠均颈背部皮下注射D-半乳糖(150mg/kg.d);假手术组颈部皮下注射生理盐水(150mg/kg.d),每天一次,连续6周；正常组不予处理。第7周开始：除正常组外其余四组大鼠立体定位海马,进行颅脑手术,模型组、灸疗组和西药组三组大鼠将准备好的聚集态Aβ25-35(浓度10ug/ul)缓慢注入双侧海马,每侧luL在5min内注入。假手术组双侧海马注入等剂量的无菌生理盐水；正常组不予处理。 3.模型成功检测方法：造模完成2天后进行水迷宫实验,得出造模后大鼠的平均逃避潜伏期。凡每只大鼠的平均逃避潜伏期较造模前下降1/3者为造模成功。只有模型成功者才进入下一步实验,若出现死亡或不符合条件的大鼠则予以剔除,并遵循随机原则补齐动物。 4.治疗方法：造模成功后,开始治疗。灸疗组大鼠取其“顶会穴”、“心穴”和“胃穴”,每日一穴,每次治疗15mmin,自贴式艾柱施灸,三穴交替,10天为-疗程,一疗程后休一天,再治疗,共治疗2个疗程：西药治疗组给予临床治疗老年性痴呆的药物吡拉西坦片(脑复康)0.92mg/kg灌胃,共治疗20天；模型组和假手术组大鼠与灸疗组相同的抓取和固定,并与西药组相同剂量的生理盐水灌胃；正常组不给予任何处理。 5.取材与指标检测：治疗结束后,全部大鼠进行迷宫实验、取材和指标检测。(1)每组14只大鼠均采用Morris水迷宫实验进行行为学测试,计算出每只大鼠平均逃避潜伏期、经过平台次数及经过有效区域的比例；(2)每组4只大鼠进行心脏灌注固定后进行Nissl和TUNEL染色检测海马神经元的变化,损伤情况及凋亡情况；(3)每组10只大鼠麻醉,置于冰盒上分离皮质和左侧海马,称重,冻存,采用紫外分光光度计,测定皮质和海马组织的MDA含量和SOD、ChAT、 AchE的活性含量。(4)每组6只大鼠麻醉,置于冰盒上分离右侧海马,称重,冻存,采用实时定量RT-PCR方法检测海马组织Bcl-2、Bax的mRNA转录水平；采用Western Blot法测定海马组织Bcl-2、Bax的蛋白表达。 6.统计学处理：采用SPSS19.0软件进行统计学处理,数据以均数±标准差(x±s)表示,多组间比较用F检验,组间两两比较用LSD检验,P0.05为显著性差异。结果 1.蒙医灸疗法对AD大鼠学习记忆行为学的影响 (1)治疗结束后,各组大鼠定位航行试验,“1-5天平均潜伏期”和“经过有效区域比例”比较,模型组与正常组比较“1-5天平均潜伏期”显著延长(P0.05)、“经过有效区域比例”显著减少(P0.05),模型组与假手术组比较“1-5天平均潜伏期”显著延长(P0.01)、“经过有效区域比例”显著减少(P0.01),表明AD模型制作成功；灸疗组、西药组与模型组比较,“1-5天平均潜伏期”均显著缩短(P0.01)、灸疗组与模型组比较,“经过有效区域比例”显著提高(P0.01),西药组与模型组比较,“经过有效区域比例”无显著差异(P0.05),表明蒙医灸疗和西药对AD学习记忆功能有提高作用。 (2)治疗结束后,各组大鼠空间搜索试验,“经过平台所在区的次数”、“经过有效区域比例”比较,模型组与正常组比较“经过平台所在区的次数”和“经过有效区域比例”均显著减少(P0.05)、模型组与假手术组比较“经过平台所在区的次数”和“经过有效区域比例”均显著减少(P0.01),表明AD模型制作成功；灸疗组与模型组比较,“经过平台所在区的次数”和“经过有效区域比例”均显著提高(P0.01),西药组与模型组比较,“经过平台所在区的次数”显著提高(P0.01),“经过有效区域比例”无显著差异(P0.05),灸疗组与西药组比较“经过有效区域比例”有显著增多(P0.05)。提示蒙医灸疗对AD记忆功能有显著作用,且比西药(吡拉西坦片)有略优于的趋势。 2.蒙医灸疗法对AD大鼠海马神经细胞的影响 (1) Nissl染色结果显示：与正常组和假手术组相比,模型组大鼠大脑海马区Nissl小体的数量明显减少(P0.01),染色较浅,排列不齐,表明AD模型制作成功；经吡拉西坦片治疗后,Nissl染色形态学观察指标较模型组明显改善(P0.05)；蒙医灸疗法治疗后,大鼠海马区Nissl染色较深,Nissl小体的数量明显增加(P0.01),排列也较有序；蒙医灸疗法与西药组比较,Nissl染色较深,Nissl小体的数量明显比西药组多(P0.05)。表明,蒙医灸疗法和西药治疗能减轻AD神经元的损伤,并蒙医灸疗法作用优于吡拉西坦。 (2) TUNEL染色结果显示：正常组和假手术组大鼠海马区可偶见TUNEL阳性细胞,而模型组大鼠海马区存在较多的TUNEL阳性细胞(P0.01),表明,AD模型制作成功；经吡拉西坦片治疗后,大鼠海马区阳性细胞较模型组有所减少(P0.01),蒙医灸疗组偶见阳性细胞,与模型组对照有明显减少(P0.01),蒙医灸疗组与西药组比较,TUNEL阳性细胞比西药组明显少(P0.05)。表明,蒙医灸疗法和西药治疗均能逆转AD神经细胞的凋亡,并蒙医灸疗法的作用优于吡拉西坦片。 3.蒙医灸疗对AD大鼠的脑组织MDA、SOD和ChAT、AchE的影响 (1)各组大鼠大脑皮质和海马组织内SOD、MDA的测定 ①各组大鼠皮质和海马组织的SOD活性比较,与正常组和假手术组比较,模型组皮质和海马SOD活性显著降低(P0.01),灸疗组、西药组与模型组比较,皮质和海马SOD活性均有显著提高(P0.01),西药组与灸疗组之间比较无显著差异(P0.05)；假手术组与正常组比较,皮质和海马SOD活性有显著提高(P0.01,P0.05)。 ②各组大鼠皮质和海马组织的MDA含量比较,与正常组和假手术组比较,模型组皮质和海马MDA含量均显著提高(P0.01),灸疗组、西药组与模型组比较,皮质和海马MDA均有显著降低(P0.01),西药组与灸疗组之间比较无显著差异(P0.05)；假手术组与正常组比较,皮质MDA含量有显著减少(P0.01),海马MDA含量无显著差异(P0.05)。(2)各组大鼠大脑皮质和海马组织内ChAT、AchE的测定 ①各组大鼠大脑皮质和海马组织的ChAT活性比较,与正常组和假手术组比较,模型组皮质和海马ChAT活性均显著降低(P0.01),灸疗组、西药组与模型组比较,皮质和海马ChAT活性均有显著提高(P0.01),西药组与灸疗组之间比较无显著差异(P0.05)；假手术组与正常组比较无显著差异(P0.05)：灸疗组与正常组比较,皮质ChAT活性有显著差异(P0.05)。西药组与正常组和假手术组比较,皮质和海马ChAT活性均有显著降低(P0.01)。 ②各组大鼠大脑皮质和海马组织的AchE舌性比较,与正常组和假手术组比较,模型组皮质和海马AchE活性均显著降低(P0.01),灸疗组、西药组与模型组比较,皮质和海马AchE活性均有显著提高(P0.01),西药组与灸疗组之间比较无显著差异(P0.05)；假手术组与正常组比较无显著差异(P0.05)。 4.蒙医灸疗法对AD大鼠海马组织Bcl-2、Bax的：mRNA和蛋白表达的影响 (1)各组大鼠海马组织Bcl-2的mRNA和蛋白表达比较,与正常组和假手术组比较,模型组Bcl-2的mRNA和蛋白表达水平显著降低(P0.01),灸疗组、西药组与模型组比较,Bcl-2的mRNA和蛋白表达水平有显著提高(P0.01、P0.05),西药组与灸疗组之间比较无显著差异(P0.05)；假手术组与正常组比较,无显著差异(P0.05)。 (2)各组大鼠海马组织Bax的mRNA和蛋白表达比较,与正常组和假手术组比较,模型组Bax的mRNA和蛋白表达水平显著提高(P0.01),灸疗组和西药组与模型组比较,Bax的mRNA和蛋白表达水平均有显著降低(P0.01),西药组与灸疗组之间比较无显著差异(P0.05)；假手术组与正常组比较,无显著差异(P0.05)。结论 1.本研究采用大鼠皮下注射D-半乳糖和双侧海马注射Aβ25-35建立大鼠老年性痴呆模型,能较好的模拟AD学习记忆障碍、神经元损伤、神经细胞凋亡等行为学和病理学方面的特征,更接近AD的病理表现,具有较好的定位性和实用性。 2.蒙医灸疗法能改善AD大鼠的行为学功能,提高学习和记忆能力,对AD有防治作用。 3.蒙医灸疗法能降低AD大鼠大脑海马组织神经细胞的损伤,抑制神经细胞的凋亡,对AD有防治作用。 4.蒙医灸疗法能提高AD大鼠脑组织的SOD活性,降低MDA含量,减轻脂质过氧化反应,提高抗氧化酶活性,清除自由基,抗衰老,从而防治AD病的发生和发展。 5.蒙医灸疗法能提高AD大鼠脑组织的ChAT、AchE的活性,保护脑组织酶活性,改善脑组织能量代谢障碍,从而可能改善AD的胆碱能系统功能障碍。 6.蒙医灸疗法对AD大鼠海马组织的Bcl-2的mRNA和蛋白有上调作用,对Bax的mRNA和蛋白有下调作用,对AD的脑神经细胞凋亡有很好的抑制作用。 7.对AD大鼠的蒙医“顶会穴”、“心穴”和“胃穴”实施蒙医灸疗法后,有改善学习记忆功能,减少大脑海马神经细胞的损伤,抑制神经细胞凋亡等作用,其作用机理与提高大脑组织SOD活性、减少MDA含量,提高ChAT、AchE的活性和上调海马组织中Bcl-2的mRNA和蛋白表达,下调Bax的mRNA和蛋白表达等有密切关系。 8.蒙医灸疗法与西药(吡拉西坦片)对AD大鼠的治疗作用比较,蒙医灸疗在改善学习记忆及海马神经细胞抗凋亡方面有较明显的作用,证明蒙医灸疗法对AD的作用较优于吡拉西坦片的趋势。
[Abstract]:research objective
1. based on the literature of Mongolian medical literature and the previous study, the Mongolian medicine moxibustion therapy may protect and improve the brain nerve cell damage and improve the brain function recovery hypothesis, and the Mongolian medicine moxibustion therapy is used to treat AD rats, and the Mongolian medicine moxibustion therapy for Alzheimer's disease is clarified on the basis of the Mongolian medicine AD rats. The therapeutic effect and its mechanism.
2. the rats were injected subcutaneous injection of D- galactose and bilateral hippocampal injection of A beta 25-35 to establish the pathological model of Alzheimer's disease in rats. The normal group, sham operation group, model group, moxibustion group and Western medicine group were set up in the Mongolian medicine "top cavern", "heart point" and "gastric acupoint". The learning and memory ability of moxibustion therapy on AD rats was observed, choline and oxidation were observed. The effects of the content of free radical enzymes, the pathological changes of brain tissue, the mRNA and protein expression of brain tissue related genes, and the effect of Mongolian medicine moxibustion therapy on the treatment of AD and its mechanism.
research method
1. animal selection and screening methods: the body mass of 220 + 20 (g), healthy and clean male SD rats, the rats were tested by MorriS water maze, and the rats were eliminated, and 70 qualified rats were selected.
2. rats were divided into groups and models: 70 rats were randomly divided into 5 groups according to body mass, normal group, sham operation group, model group, moxibustion group and Western medicine group, 14 rats in each group. The model group, moxibustion group and Western medicine group were injected with D- galactose (150mg/kg.d) subcutaneously in the neck and back of the moxibustion group and the western medicine group, and the sham operation group was subcutaneously injected with saline (150mg/kg.d), daily neck saline (150mg/kg.d), daily neck subcutaneous injection (150mg/kg.d), every day. At one time for 6 weeks, the normal group did not handle the treatment. Seventh weeks began: the other four groups of rats, except the normal group, stereotaxic hippocampus, performed craniocerebral operation, model group, moxibustion group and Western medicine group three rats, the prepared A beta 25-35 (concentration 10ug/ul) was slowly injected into the bilateral hippocampus, each side luL was injected into the 5min. The bilateral hippocampus injection in the sham operation group was injected into the hippocampus. The rats in the normal group were not treated with equal dose of sterile saline.
3. model successful detection method: the water maze experiment was carried out after the model was completed after 2 days. The average escape latency of rats after the model was obtained. The average escape latency of each rat was more successful than that of 1/3 before the model. Only the model successes entered the next experiment, and the rats who died or did not conform to the conditions were eliminated, And follow random principles to complement animals.
4. treatment methods: after the success of the model, start the treatment. The moxibustion group took the "cacupoint point", "heart point" and "stomach point", one acupoint daily, each treatment 15mmin, the Three Acupoints alternately, 10 days as a course of treatment, one day after a course of treatment, and then treated for 2 courses of treatment: the western medicine treatment group was given clinical treatment of Alzheimer's disease. The drug Piracetam Tablets (brain Fukang) 0.92mg/kg was administered to the stomach for 20 days. The rats in the model group and the sham operation group were captured and fixed with the moxibustion group, and the same dose of saline was given to the western medicine group. The normal group did not give any treatment.
5. material and index test: after the treatment, all rats were tested by labyrinth experiment, material and index test. (1) 14 rats in each group were tested by Morris water maze test. The average escape latency of each rat was calculated, and the number of platform times and the proportion of effective areas were passed. (2) 4 rats in each group were perfused with heart perfusion. After fixation, Nissl and TUNEL staining were performed to detect the changes in hippocampal neurons, damage and apoptosis. (3) 10 rats in each group were anesthetized, placed on the ice box and left hippocampus, weighing, frozen, and using ultraviolet spectrophotometer to determine the MDA content of cortex and hippocampus and the activity of SOD, ChAT, AchE. (4) 6 rats in each group After anesthesia, the right hippocampus was separated on the ice box, weighed and frozen. The mRNA transcriptional level of hippocampal Bcl-2, Bax was detected by real-time quantitative RT-PCR, and the protein expression of Bcl-2 and Bax in hippocampus was measured by Western Blot.
6. statistical processing: SPSS19.0 software was used for statistical processing. The data were expressed with mean number + standard deviation (x + s). F test was used in multiple groups, and 22 of groups were compared with LSD test, and P0.05 was significant difference.
Result
1. the effect of Mongolian medicine moxibustion on learning and memory behavior of AD rats
(1) after the treatment, the navigation test of the rats in each group, the average incubation period of 1-5 days and the ratio of the effective area were compared. The model group compared with the normal group. The average incubation period of the 1-5 days was significantly prolonged (P0.05), and the "effective area ratio" was significantly reduced (P0.05). The model group was compared with the sham operation group for the average incubation period of 1-5 days. Significantly prolonged (P0.01), "through effective regional proportion" significantly reduced (P0.01), indicating the success of the AD model, moxibustion group, western medicine group and model group, "1-5 days average latency" significantly shortened (P0.01), moxibustion group and model group, "the effective area ratio" significantly improved (P0.01), western medicine group and model group, "the" There was no significant difference in the proportion of effective areas (P0.05), indicating that Mongolian medicine moxibustion and Western medicine could improve the learning and memory function of AD.
(2) after the treatment, the space search test of the rats in each group, "the number of the area in the platform", "through the effective area ratio", the model group and the normal group compared "the number of the platform area" and "the effective area ratio" significantly decreased (P0.05), the model group and the sham operation group "through the platform area" The number of times "and" the ratio of effective area "decreased significantly (P0.01), indicating that the AD model was successfully made. Compared with the model group, the number of the moxibustion group and the model group increased significantly (P0.01), and the number of the western medicine group and the model group was significantly higher (P0.01). There was no significant difference between the "effective area ratio" (P0.05). The moxibustion group and the western medicine group had a significant increase in the "effective area ratio" (P0.05). It suggested that moxibustion in Mongolian medicine has a significant effect on the memory function of AD, and is slightly better than the western medicine (Piracetam Tablets).
2. the effect of Mongolian medicine moxibustion on hippocampal neurons in AD rats
(1) the results of Nissl staining showed that compared with the normal group and the sham operation group, the number of Nissl corpuscles in the hippocampus of the model group was significantly reduced (P0.01), the staining was shallow, and the arrangement of the AD model was successful. After the Piracetam Tablets treatment, the morphological observation index of Nissl staining was significantly improved (P0.05), and the Mongolian medicine moxibustion therapy was better than that of the model group. After treatment, the Nissl staining in the hippocampus of the rats was deeper, the number of Nissl corpuscles increased obviously (P0.01) and the arrangement was more orderly. Compared with the western medicine group, the Mongolian medicine moxibustion therapy was much deeper than the western medicine group. The number of Nissl corpuscles was much more than that of the western medicine group (P0.05). It showed that the Mongolian medicine moxibustion and Western medicine treatment could reduce the injury of AD neurons, and the Mongolian medicine moxibustion therapy was superior to the moxibustion therapy of Mongolian medicine. In piracetam.
(2) the results of TUNEL staining showed that TUNEL positive cells could be seen in the hippocampus of the normal group and the sham operation group, while there were more TUNEL positive cells (P0.01) in the hippocampus of the model group, indicating that the AD model was successfully made. After the treatment of Piracetam Tablets, the positive cells in the hippocampus of the rats decreased (P0.01) and the moxibustion group in the Mongolian medicine group was even less than that of the model group (P0.01). Compared with the model group, the positive cells were significantly reduced (P0.01). Compared with the western medicine group, the moxibustion group in Mongolian medicine and the western medicine group were significantly less than the western medicine group (P0.05). It showed that the Mongolian medicine moxibustion therapy and the western medicine treatment could reverse the apoptosis of the AD nerve cells, and the effect of Mongolian medicine moxibustion was better than that of the Piracetam Tablets.
3. effect of Mongolian medicine moxibustion on MDA, SOD and ChAT, AchE in AD rats
(1) determination of SOD and MDA in cerebral cortex and hippocampus of rats in each group.
Compared with the normal group and the sham operation group, the activity of cortical and hippocampal SOD in the model group was significantly lower than that of the normal group and the sham group (P0.01). Compared with the model group, the activity of the cortex and hippocampus of the model group was significantly improved (P0.01), and there was no significant difference between the western medicine group and the moxibustion group (P0.05), and the sham operation between the western medicine group and the moxibustion group was not significant (P0.05), and the sham operation of the group was compared with the normal group and the sham operation group (P0.01). Compared with normal group, the activity of SOD in cortex and hippocampus increased significantly (P0.01, P0.05).
Compared with the normal group and the sham operation group, the content of the cortex and hippocampal MDA in the model group was significantly increased (P0.01). Compared with the model group, the cortex and hippocampus MDA were significantly decreased (P0.01), and there was no significant difference between the western medicine group and the moxibustion group (P0.05), and the sham operation group had no significant difference (P0.01). Compared with the normal group, the content of cortical MDA was significantly decreased (P0.01), and there was no significant difference in the content of MDA in the hippocampus (P0.05). (2) the determination of ChAT and AchE in the cerebral cortex and hippocampus of rats.
Compared with the normal group and the sham operation group, the activity of ChAT in the cortex and hippocampus of the model group was significantly lower than that of the normal group and the sham group (P0.01). Compared with the model group, the activity of the cortex and hippocampus of the model group was significantly increased (P0.01), and there was no significant difference between the western medicine group and the moxibustion group (P0.05), compared with the normal group and the sham operation group (P0.01). There was no significant difference between the sham operation group and the normal group (P0.05): compared with the normal group, the activity of cortex ChAT in the moxibustion group was significantly different (P0.05). Compared with the normal group and the sham operation group, the activity of ChAT in the cortex and hippocampus decreased significantly (P0.01).
Compared with the normal group and the sham operation group, the AchE activity of the cortex and hippocampus of the model group was significantly lower than that of the normal group and the sham group (P0.01). Compared with the model group, the activity of the cortex and hippocampus of the model group was significantly increased (P0.01), and there was no significant difference between the western medicine group and the moxibustion group (P0.05), compared with the normal group and the sham operation group (P0.01). There was no significant difference between the sham operation group and the normal group (P0.05).
4. effect of Mongolian medicine moxibustion on Bcl-2 and Bax: mRNA and protein expression in hippocampus of AD rats
(1) the mRNA and protein expression of Bcl-2 in hippocampus of rats in each group, compared with the normal group and sham operation group, the mRNA and protein expression level of Bcl-2 in the model group decreased significantly (P0.01). Compared with the model group, the level of mRNA and protein expression of Bcl-2 was significantly increased (P0.01, P0.05), and there was no significant difference between the western medicine group and the moxibustion group. Difference (P0.05); there was no significant difference between sham operation group and normal group (P0.05).
(2) compared with the normal group and the sham operation group, the expression of mRNA and protein of Bax in the model group was significantly higher than that of the normal group and the sham group (P0.01). The level of mRNA and protein expression of Bax in the moxibustion group and the western medicine group were significantly lower than that of the model group (P0.01). There was no significant difference between the western medicine group and the moxibustion group (P0.01). (2) the expression of mRNA and protein in the model group was significantly higher than that of the model group (P0.01). P0.05); there was no significant difference between the sham operation group and the normal group (P0.05).
conclusion
1. the rat model of Alzheimer's disease was established by subcutaneous injection of D- galactose and bilateral hippocampal injection of A beta 25-35 in rats. It could well simulate the behavioral and pathological features of AD learning and memory impairment, neuron injury, neural cell apoptosis and so on. It is closer to the pathological appearance of AD, and has good localization and practicability.
2. Mongolian medicine moxibustion therapy can improve the behavioral function of AD rats, improve learning and memory ability, and prevent and cure AD.
3. Mongolian medicine moxibustion therapy can reduce the damage of nerve cells in the hippocampus of AD rats, inhibit the apoptosis of nerve cells, and prevent and cure AD.
4. Mongolian medicine moxibustion can improve the activity of SOD in the brain tissue of AD rats, reduce the content of MDA, reduce the lipid peroxidation, improve the activity of antioxidant enzymes, remove the free radicals and resist senescence, thus preventing and developing the occurrence and development of AD disease.
5. Mongolian medicine moxibustion can improve the activity of ChAT and AchE in the brain tissue of AD rats, protect the activity of brain enzymes and improve the energy metabolism disorder of brain tissue, which may improve the dysfunction of the cholinergic system of AD.
6. Mongolian medicine moxibustion therapy has an up-regulated effect on the mRNA and protein of Bcl-2 in the hippocampus of AD rats. It has a downregulation effect on the mRNA and protein of Bax, and has a good inhibitory effect on the apoptosis of brain nerve cells of AD.
7. after the Mongolian medicine "Ding Hui point", "Xin point" and "stomach acupuncture point" in Mongolian AD, Mongolian medicine moxibustion therapy can improve learning and memory function.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R29

【参考文献】