【作者】 赵利华；

【导师】 张家维；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2009， 博士

【摘要】 研究目的:随着我国人口老龄化的加剧,延缓衰老成为医学工作者的重要任务,特别是对脑功能减退的改善是延缓衰老的重要研究方向。本课题通过D-半乳糖注射建立小鼠衰老模型,探讨艾炷灸延缓脑衰老的作用机制。方法:先后两批次用Morris水迷宫筛选三月龄体重30±2克的健康雄性小鼠72只。编号后,分为六组进行水迷宫实验。实验造模时,按随机区组设计将72只小鼠分为:生理组盐水组(简称生理组)、造模组、艾炷灸足三里、悬钟穴组(简称艾灸1组)、艾炷灸关元、百会穴组(简称艾灸2组)、电针足三里、悬钟穴组(简称电针组)、尼莫地平灌胃组(简称尼莫地平组)六组。造模组和各干预治疗组以D半乳糖按120mg/kg/d颈背部皮下注射,连续42天,同时生理组颈背部皮下注射等量的生理盐水42天。造模第13天开始艾柱灸、电针、尼莫地平灌胃治疗,隔天一次,各组共15次。生理组和造模组不做任何治疗性干预,但给予与各治疗组同样时间、同等程度的捉抓,造模、治疗结束后,再次行迷宫行为学检测后断头处死,取脑固定,或脑组织匀浆。另取6只18月龄自然衰老小鼠与模型组做组织形态学比较。主要观察指标有:(1)电脑采集各组小鼠造模治疗前后Morris水迷宫行为学指标,包括定位航行实验的指标:“潜伏期”、“寻求次数”、“停留时间”、“平台象限百分比(简称平台象比)”和空间探索实验指标:“原平台象限百分比(简称原象比)”、“跨台次数”;(2)各组小鼠造模治疗后大脑组织光镜下病理形态学变化、神经元密度;(3)各组小鼠造模治疗后大脑皮质电镜下超微结构变化;(4)各组小鼠造模治疗后大脑组织NO含量变化;(5)各组小鼠造模治疗后大脑组织TNOS、iNOS活力变化;(6)各组小鼠造模治疗后大脑组织总SOD、CuZnSOD活力变化;(7)各组小鼠造模治疗后大脑皮质c-fos的表达;(8)各组小鼠造模治疗后大脑皮质p16的表达;(9)各组小鼠造模治疗后大脑皮质Rb的表达;(10)各组小鼠造模治疗后大脑皮质、海马CA3区原位杂交c-fosmRNA的表达。结果:(1)造模、治疗前六组小鼠的学习记忆能力一致,无差别。实验后,①造模组与其他五组相比,“潜伏期、寻求次数、停留时间、平台象比、跨越次数、原象比”六项指标均差异有显著性意义(P＜0.01或P＜0.05),模型组“潜伏期”较生理组、艾灸1、2组、电针组、尼莫地平组延长,模型组“寻求次数、停留时间、象比、跨越次数、原象比、平台象比”较其他各组缩短、降低,②其他五组,尼莫地平组与生理组比较在潜伏期、平台象比差异有显著性意义(P＜0.05),电针组、艾灸2组在潜伏期与生理组比较差异有显著性意义(P＜0.05)。其余各项指标在五组间无差异。提示D-半乳糖造成亚急性衰老小鼠脑功能受损,学习记忆能力下降。艾炷灸、电针干预组小鼠的学习记忆能力与生理组的学习记忆能力相似,尼莫地平组学习记忆能力与生理组差异有显著性意义(P＜0.05),但与其他三个治疗组无差异。(2)光镜下大脑组织切片苏木伊红染色:①生理组大脑皮层细胞结构正常,胞核紫蓝色清晰,胞浆丰富呈浅紫蓝色,间质无水肿表现,海马CA3区锥体细胞2～3层,排列紧密,细胞核圆而大,核呈,核仁清晰。②模型组、自然衰老组可见皮层变薄,大部分神经元变性,体积变小,胞核与胞浆界限模糊,呈蓝黑色,核固缩成三角形或不规则形,核仁消失。海马CA3区锥体细胞层数减少,排列稀疏、不规则,细胞体积变小,可见核固缩现象。③艾灸1、2组、电针组和尼莫地平组组仍有不同程度的神经细胞变性,但变性神经细胞数量显著减少,零星可见部分损伤细胞,海马CA3区锥体细胞层细胞形态、排列基本正常。④各组小鼠脑皮质及海马CA3区神经元密度差异有显著性意义(P＜0.01),组间比较,生理组、_艾灸1、2组、电针组、尼莫地平组与模型组、自然衰老组神经元密度比较差异有显著性意义(P＜0.01),模型组神经元细胞密度下降。自然衰老小鼠大脑皮质神经元密度与模型组无差异,但在海马CA3区自然衰老组神经元密度较模型组减少明显(P＜0.05)。生理组、艾灸1、2组、电针组之间神经元密度无差异,尼莫地平组与前四组比较在皮质区神经元密度差异有显著性意义(P＜0.01或P＜0.05),在海马区仅与生理组神经元密度差异有显著性意义(P＜0.05)。(3)电镜下:①生理组组织结构较好,神经细胞结构清晰,细胞为锥体形或多边形,胞浆较宽,细胞器丰富,可见较多的线粒体、粗面内质网、高尔基体、核糖体等,核染色质疏松,核仁清楚及少量吞噬溶酶体。未见明显水肿等其他变化。轴索结构清楚,微丝微管丰富,排列整齐。②造模组神经细胞有变性,变性细胞程度不一,少数神经细胞轻度变性,但普遍程度较重。线粒体数量增多、增大或空泡变性,线粒体脊模糊,核糖体脱落。轴索肿胀空泡化,微丝微管数量减少,排列散乱。部分髓鞘结构松解或脱失,出现较多的吞噬溶酶体(脂褐素)。部分小血管内皮细胞有变性,血管周隙扩大,星形细胞空泡变性或溶酶体增多,足突破坏较明显。③其它各干预治疗组依病变程度从重到轻排序:尼莫地平组,艾灸2组,电针组和艾灸1组。(4)六组小鼠的大脑组织NO含量差异有显著性意义(p＜0.01),两两比较:模型组的大脑组织NO含量较其他五组显著升高(p＜0.01),其他五组间脑组织NO含量比较无明显差异。(5)六组小鼠的大脑组织总NOS(TNOS)活力和诱导型(iNOS)活力具有显著性差异(p＜0.01),两两比较:模型组的脑组织TNOS、iNOS活力较其他五组显著升高(p＜0.01),其他五组脑组织TNOS、iNOS活力比较无明显差异。(6)六组小鼠的大脑组织总SOD活力和铜锌SOD(CuZnSOD)活力具有显著性差异(p＜0.01),两两比较:模型组脑组织总SOD活力和CuZnSOD活力较其他五组显著降低(p＜0.01),其他五组脑组织总SOD活力无明显差异;尼莫地平组、电针组脑组织CuZnSOD活力与艾灸2组比较差异有显著性意义(p＜0.01,p＜0.05),尼莫地平组CuZnSOD活力与艾灸1组比较差异有显著性意义(p＜0.05),其他各组间比较无差异。(7)各组大脑皮质均有c-fos、p16、磷酸化Rb(pRb)的蛋白表达,模型组c-fos、pRb蛋白表达细胞较少、染色浅,生理组、艾炷灸、电针、尼莫地平灌胃治疗组c-fos、pRb蛋白表达细胞较多、染色较深。模型组p16蛋白表达细胞较较多、染色较深,生理组、艾炷灸、电针、尼莫地平组p16蛋白表达细胞较少、染色较浅。各组c-fos、p16、pRb的蛋白表达差异有显著性意义(p＜0.01)。组间比较:造模组c-fos、pRb蛋白阳性表达神经元密度较其他五组降低(p＜0.01),造模组p16蛋白表达神经元密度较其他五组升高(p＜0.01)。尼莫地平组c-fos阳性表达神经元密度与生理组、艾灸1组比较差别均具有显著性意义(p＜0.01,p＜0.05)。其余指标在五组间无差异。(8)各组大脑皮质、海马CA3区均有c-fosmRNA的表达,模型组c-fosmRNA的表达细胞排列松散、染色浅,生理组和各治疗组c-fosmRNA的表达细胞较紧密、染色较深,各组c-fosmRNA的蛋白表达差异有显著性意义(p＜0.01)。组间比较:造模组大脑皮质、海马CA3区c-fosmRNA阳性表达神经元密度较其他组降低(p＜0.01,p＜0.05),其他五组在大脑皮质c-fosmRNA阳性表达神经元密度无差别;尼莫地平组海马CA3区c-fosmRNA阳性神经元表达密度与艾灸1组比较差别具有显著性意义(p＜0.05)。其余4组间海马CA3区c-fosmRNA阳性神经元表达密度无差异。结论:(1)艾炷灸干预疗法能较好改善D-半乳糖衰老小鼠学习记忆能力。(2)艾炷灸干预疗法能较好保护D-半乳糖衰老小鼠大脑皮质组织形态及超微结构,减轻氧化损伤,保护神经元细胞。(3)艾炷灸干预疗法能较好抵抗衰老脑组织自由基氧化损伤,增强脑组织的抗氧化能力。(4)艾炷灸干预疗法能促进学习记忆相关脑区大脑皮质、海马CA3区即刻早期基因c-fos、c-fosmRNA的表达。(5)艾炷灸干预疗法能影响细胞周期调控基因:抑制大脑皮质神经元细胞中与衰老相关基因P16的表达,而促进与P16呈负相关的磷酸化Rb基因的表达,进而也促进了大脑皮质c-fos、c-fosmRNA的表达。更多还原

【Abstract】 ObjectiveTo explore the mechanism of moxa cone moxibustion in anti-aging on cerebral with senile mice induced by D-galactose injection.MethodsAfter two separately selecting with the Morris water maze（MWM） test,72male mice（3 months old） were randomly and equally divided into the normal sodium control,model,moxa cone moxibustion 1（acupoint ST36 and GB39）,moxa cone moxibustion 2（acupoint DU20 and RN4）,electroacupunctue（acupoint ST36 and GB39）,Nimodipine medicated groups.The treatment and model groups were given with D-galactose injection for 42days,while the normal sodium control group was injected the same dosage of normal sodium as other groups for 42 days. From the 13^th day,the four treatment groups was treated for every other day until the model would had been finished.The six group were tested on Spatial Learning and Memory in the MWM again,and then take cerebral tissues for tissue fixation or tissue homogenate.Measured and observed the following the indexes.（1）The Spatial Learning and Memory before and after the modeled and treated with computer MWM system in six group.Including the place navigation with four targets that are escape latency,detention（platform） time,seeking （platform） frequency,percentage of platform quadrant（the percentage of swimming distant between the original platform and total distant）from the 2^th to 6^th day and the spacial probe test with two targets that are spanning （original） platform frequency and percentage of（original） platform quadrantin at the 7^th day.（2）The Pathomorphological of changes on the cerebral tissues and neurons density of cerebral cortex and hippocampal（CA3） with light-microscope in each group.（3）The ultrastructure change of the cerebral cortex in each group.（4）The Nitric Oxide（NO） content of the cerebral tissues in each group.（5）The total Nitric Oxide synthase（TNOS）activities and the induced Nitric Oxide synthase（iNOS） activities of the cerebral tissues in each group.（6）The total Superoxide dismutase（TSOD） activities and the CuZnSuperoxide dismutas（CuZnSOD） activities of the cerebral tissues in each group（7）The c-fos gene expression of the cerebral cortex in each group（8）The p16 gene expression of the cerebral cortex in each group.（9）The（Phospho- Retinoblastoma） pRb gene expression of the cerebral cortex in each group.（10）The c-fosmRNA expression of the cerebral cortex and hippocampal（CA3） in each groupResults（1）six groups mice were equal on spatial learning and memory before the experiment（P＞0.05）,after modeled and treated,①)The significant difference was observed between the modeled group and the other five groups on spatial learning and memory（P＜0.01或P＜0.05）,Comparing with the other five groups, the escape lantency of modeled group was prolonged and the seeking frequency, detention time,percentage of platform quadrant,the spanning（original） platform frequency,the percentage of（original） platform quadrant were decreased②There were significant difference between the Nimodipine medicated group and the normal sodium control group in escape lantency,percentage of platform quadrant（P＜0.05）,the escape lantency of the moxa cone moxibustion 1,the moxa cone moxibustion 2,the Dianzhen ones were longer than that of the normal sodium control significantly（P＜0.05）.The other indexes in five group are no differences.（2）With Hematoxylin and eosin（H·E） staining under the light-microscopeThe cerebral cortex and hippocampal neurons struacture of the normal sodium control group was normal,nucleus with Purple blue,rich cytoplasm with slight purple,without Swelling in Mesenchymal,on the other hand,the model group had similar change with the physio-senility mices,the cerebral cortex was thin with degeneration,the neurons become smaller,sparse,nuclei and cytoplasm are muddled,pyknosis showing dark blue.The Neuronal density in the model group was lower than that of other five group（p＜0.01）.The four treated groups had the neurons degeneration in varying degrees,but the neurons degeneration was lightented than that of the model group.（3）The Ultrastructure of the cerebral cortex in normal sodium control group was better with clear neurons structure,rich organelle,showing more mitochondria,rough endoplasmic reticulum,golgi apparatus,ribosomes,etc., loose chromatin,nucleolus clearly.A small amount of phagocytic lysosome without swelling and other changes.There was axonal structure clearly, tubulin and microfilament rich neatly arranged.The model group was increased in the number of mitochondriawith extended or vacuolar,mitochondria ridge vague,detached ribosomes.Axonal was swelling and vacuolization,tubulin and microfilament were reducing in the number with scattered.The Part of myelin structure was loss,showing more phagocytic lysosomes（lipofuscin）.The Part of the vascular endothelial cells have degeneration,perivascular space expansion,stellatecell vacuolar withthe foot process was damage obviously. The neurons of four treated groups had degeneration in varying degrees,with the order by lesions sorting from heavy to light being the Nimodipine medicated,the moxa cone moxibustion 2,the electroacupunctue,the moxa cone moxibustion 1.（4）There were significant difference on the NO content of cerebral tissues in six group（p＜0.01）,the NO content of cerebral tissues in model group was higher than that of in other five group（p＜0.01）.There was no difference between other five group.（5）There were significant difference on the TNOS,iNOS activities of cerebral tissues in six group（p＜0.01）,the TNOS,iNOS activitiesof cerebral tissues in model group was increased than that of in other five group （p＜0.01）.There was no difference between other five group.（6）There were significant difference on the TSOD,CuZnSOD activities of cerebral tissues in six group（p＜0.01）,the TSOD,CuZnSOD activities of cerebral tissues in model group was lower than that of in other five group （p＜0.01）.There was no difference between five others on TSOD activity.The CuZnSOD activities in the Nimodipine medicated were lower than that of the moxa cone moxibustion land2（p＜0.01,p＜0.05）,and The CuZnSOD activity in the electroacupunctue were lower than that of the moxa cone moxibustion 2 （p＜0.05）,there were no difference between others on CuZnSOD activity. （7）There were c-fos、p16、Phospho-Rb on the cerebral cortex in each group. The neurons with c-fos、Phospho-Rb protein expression on the cerebral cortex in model group was scatter and slight staining,The Cells with c-fos、Phospho-Rb protein expression were more closely and deeper staining in other fives.The neurons with p16 Protein expression on the cerebral cortex in model group was deeper staining,but in others the Cells with p16 protein expression were slightly staining.The neuronal density of c-fos,p16,Phospho-Rb on the cerebral cortex had significant difference on six groups.The neuronal density of c-fos,Phospho-Rb on the cerebral cortex on the model group were decreased compared with other groups（p＜0.01,p＜0.05）,the neuronal density of p16 on the cerebral cortex was increased compared with other groups（p＜0.01, p＜0.05）.The neuronal density of c-fos on the cerebral cortex of the Nimodipine medicated was significant difference compared with the normal sodium control group and the moxa cone moxibustion 1.There were no differences on other indexes between treatment groups.（8）There were c-fosmRNA expression on the cerebral cortex and hippocampal （CA3） in each group.The neurons with c-fosmRNA expression on the cerebral cortex and hippocampal（CA3） in model group was scatter and slight staining, in others group the cells with c-fosmRNA expression were more closely and deeper staining.The neuronal density of c-fosmRNA on the cerebral cortex and hippocampal on the model were decreased compared with other groups（p＜0.01, p＜0.05）.There were no differences on neuronal density of the cerebral cortex c-fosmRNA between five treatment groups.The neuronal density of c-fosmRNA expression on hippocampal in the Nimodipine medicated were lower than that of in the moxa cone moxibustion 1,no differences between four other treatment groups.Conclusion（1）The moxa cone moxibustion therapy on ST36,GB39 can improve the spatial learning and memory of aging mice induced by D-galactose.（2）The moxa cone moxibustion therapy on ST36,GB39 can Protect the cerebral tissues and ultrastructural morphology of aging mice induced by D-galactose, reduce oxidative damage and protect neurons.（3）The moxa cone moxibustion therapy on ST36,GB39 can resist free radicals damage in aging brain tissue,improve brain tissue antioxidant capacity. （4） The moxa cone moxibustion therapy on ST36,GB39 can inhibit p16 protein expression on the cerebral cortex,promote c-fos,Phospho-Rb protein expression on on the cerebral cortex.（5） The moxa cone moxibustion therapy on ST36,GB39 can improve c-fosmRNA expression on the cerebral cortex and hippocampal.更多还原