【作者】 罗磊；

【导师】 孙国杰；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的：以线粒体损伤机制为中心，从线粒体超微结构、线粒体损伤三大途径（线粒体能量代谢障碍途径、线粒体动力学异常途径和线粒体通透性转换孔mPTP孔开放途径）出发，综合系统地探讨线粒体损伤机制与阿尔茨海默病发病机制之间可能存在的相关性及“益肾调督”针灸法防治AD过程中在线粒体损伤方面的可能作用机制。方法：Wistar大鼠，SPF级、雄性、体重200±20g，80只，实验前所有大鼠置于鼠笼常规适应性喂养一周；实验时，经Morris水迷宫测试筛选，剔除先天性不符合评定标准的大鼠（定位航行试验中平均逃避潜伏期较长，空间探索实验中的跨越原平台次数较少和有效区停留时间较短者评定为不合格者，予以剔除，补充相应合格者）；通过《卫生统计学》随机数字表法将80只大鼠随机分为正常组、假手术组、模型组和针灸治疗组，每组20只，分笼饲养，并用苦味酸给予染色标记编号；采用双侧海马区（前囟后3.3mm，中线左右各旁开1.5mm，深度3mm）分别注射Aβ1-42（各5μL）的模型复制方法和Morris水迷宫模型评定方法（剔除不符合评定标准者，并补充相应合格者），其中正常组、假手术组（除注射等量（双侧各5μL）的生理盐水外，其余同模型组处理方法）和模型组均进行相应处理后，正常饲养，不进行任何其它处理；针灸治疗组在模型复制成功后（已通过模型评价），先针刺“百会”穴（向前平刺3～5mm），双侧“肾俞”穴（稍向内斜刺5mm）后，选用特制艾条（Φ=0.6cm）于“百会”和“肾俞”穴位上方2～3cm处进行悬灸，使穴位表皮温度在43±1℃，持续15min，每日1次，7天为一个疗程，共计2个疗程，疗程间休息1日；运用透射电镜、免疫组化、免疫荧光、免疫印迹、RT-PCR等技术，通过观察各组大鼠海马神经元线粒体超微结构、线粒体能量代谢相关蛋白Aβ-结合性酒精脱氢酶蛋白ABAD与细胞色素氧化酶Ⅳ（COXⅣ）、线粒体动力学相关蛋白线粒体分裂蛋白1（Fis1）与内膜视神经萎缩1蛋白（Opa1）、线粒体通透性转换孔相关蛋白亲环蛋白D（CypD）等指标的变化，探讨“益肾调督”针灸法对线粒体损伤三大途径的影响。整个实验过程严格遵守国家科技部发布的《关于善待实验动物的指导性意见》相关规定。结果：（1）“实验一”结果显示，模型组大鼠毛发光泽度降低，部分可见脱毛伴行动迟缓，进食饮水量有所减少，对外界刺激反应降低；透射电镜观察其海马CA1区神经元线粒体，可见线粒体形态呈卵圆形，但其数目较少，且分布不均，内嵴大部分排列紊乱，部分可见内嵴溶解消失，基质疏松，线粒体肿胀明显且部分空泡变性；周边内质网扩张，与正常组和假手术组具有明显的差异；而针灸治疗组大鼠皮毛尚有光泽，进食饮水量正常，对外界刺激反应尚可；透射电镜观察可见线粒体形态呈卵圆形，数目尚可，且分布均匀，内嵴尚清晰且排列较整齐，少数可见嵴内腔轻微扩张及内嵴轻度溶解，基质尚均匀，未见线粒体肿胀及空泡变性；周边内质网轻度扩张，总体较模型组有所改善。（2）“实验二”结果显示，模型组大鼠海马神经元线粒体中ABAD的含量明显高于正常组和假手术组（P<0.01），COXⅣ的水平明显低于正常组和假手术组（P<0.01）；而针灸治疗组海马神经元线粒体中的ABAD的水平明显低于模型组（P<0.01），而COXⅣ的活性明显高于模型组（P<0.01），且与正常组和假手术组之间无明显的差异（P>0.05），提示“益肾调督”针灸法具有抑制ABAD的过表达，提高COXⅣ活性的作用；（3）“实验三”结果显示，模型组大鼠海马神经元线粒体融合相关蛋白Opa1的水平明显低于正常组和假手术组（P<0.01），线粒体分裂相关蛋白Fis1的水平明显高于正常组和假手术组（P<0.01）；而针灸治疗组海马神经元线粒体中Opa1的水平明显高于模型组（P<0.01），Fis1的水平明显低于模型组（P<0.01），且与正常组和假手术组比较无统计学意义（P>0.05），提示“益肾调督”针灸法具有抑制Fis1的过表达，上调Opa1水平的作用；（4）“实验四”结果显示，模型组大鼠海马神经元线粒体中CypD的表达明显高于正常组和假手术组（P<0.01）；而针灸治疗组可明显抑制大鼠海马神经元线粒体中CypD的过表达，与模型组比较具有明显的差异（P<0.01），提示“益肾调督”针灸法具有下调CypD含量的作用。结论：（1）“益肾调督”针灸法不仅整体上能够改善AD发病过程中的行为学变化，而且还能够在一定程度上改善AD发病过程中海马神经元线粒体超微结构的损伤；（2）“益肾调督”针灸法能够通过抑制Aβ-结合性酒精脱氢酶蛋白ABAD的过表达，有效地提高细胞色素氧化酶Ⅳ（COXⅣ）的活性，达到改善线粒体能量代谢的目的；（3）“益肾调督”针灸法能够有效地抑制线粒体分裂蛋白Fis1的过表达，提高线粒体融合蛋白Opa1的水平，从而调节线粒体分裂与融合的平衡，达到改善线粒体动力学异常的目的；（4）“益肾调督”针灸法能够有效地抑制线粒体通透性转换孔mPTP的必需成分之一，即亲环蛋白酶D（CypD）的过表达，从而抑制线粒体通透性转换孔的异常开放，达到改善线粒体膜内外渗透失衡的目的。因此，海马神经元线粒体损伤与阿尔茨海默病发病机制之间具有密切的相关性，且“益肾调督”针灸法可能通过改善海马神经元线粒体超微结构的病理改变及调节线粒体损伤三大途径的异常，进而改善线粒体损伤，达到防治AD的目的。更多还原

【Abstract】 Objective: To discuss the relevance between the mechanismsmitochondrial damaged and the mechanisms Alzheimer’s disease illnessed, inthe center of mitochondrial damaged, through the mitochondrialultrastructure and three approach mitochondrial damaged(Mitochondrialenergy metabolism、Mitochondrial dynamics、Mitochondrial permeabilitytransition pore). Meantime, to discuss the possible mechanisms in themitochondrial damaged on the prevention of AD by the Kidney-reinforcingand Du Meridian-regulating Acupuncture-Moxibustion.Methods: Wistar rat,SPF, male,200±20g,80rats.Before theexperiment, all rats adaptive feeded about week in the squirrel cage; Theexperiment beginning, by the Morris water maze(MWM) test screened, toexclude the congenital rats which do not meet the assessment criteria(theescape latency shorter in the place navigation, the views across the originalplatform less and the active area stay longer in the space exploration assessedas unqualified.Meantime, to exclude rats unqualified and to add theappropriate qualifier rats).To injecte the Aβ1-42(each5μL) in bilateralhippocampus(after the anterior fontanelle3.3mm, he middle of each side open 1.5mm,3mm depth) about the model replication methods, and to screen on theMorris water maze(MWM) test(the escape latency shorter in the placenavigation, the views across the original platform less and the active area staylonger in the space exploration assessed as unqualified.Meantime, to excluderats unqualified and to add the appropriate qualifier rats).To divide randomly80rats into normal group, sham-operation group, model group andAcupuncture-Moxibustion group by the "Health Statistics" random numbertable,to house separately(N=20) and to give tag number stained with picricacid; To deal accordingly with the normal group, sham-operation group(theothers same with the model group addition to injection with NS) and modelgroup, with the normal feeding and without any treatment;After the success ofthe model replicated,the acupuncture-moxibustion group after treated with theacupuncture “baihui”(DU20)(forward3-5mm) and “Shenshu”(BL23)(withinobliquely5mm) was moxibustioned on “baihui” and “Shenshu” about2-3cmso that skin temperature points in43±1℃, continuous in15min,and totreat in two courses(a course of treatment with7days, once a day) oftreatment breaks the1st;To discuss the influence on the ABAD、COXⅣ、Fis1、Opa1、CypD and the mitochondrial ultrastructure about the three pathwaysmitochondrial damaged with the TEM technique、Immunohistochemistry、Immunofluorescence technique、Western blotting technique and RT-PCRtechnique by the Kidney-reinforcing and Du Meridian-regulatingAcupuncture-Moxibustion. During the experiment, the "Guiding Opinions onkind of experimental animals" which published by the Ministry of Sciencewas strictly complianced with.Results:(1)"Experiment1st" results show the hair gloss reduced、the hairremoval with sluggish partially visibled、the consumption and water intakedecreased、the external stimuli response-reduced which the model group was; Through the hippocampal CA1on the neurons mitochondria with TEM, theMitochondrial about the model group had oval shape、less quantity、unevendistribution、most derangement of the ridge which dissolved partially、theloose matrix、significant swelling which part of vacuolar degeneration andsurrounding the endoplasmic reticulum expansed, compare to the normalgroup and sham-groups had significant differences; However,the hair glosspassably lustered、the consumption and water normaled、the external stimulipassably reacted which the acupuncture-moxibustion group was; Through thehippocampal CA1on the neurons mitochondria with TEM, the Mitochondrialabout the acupuncture-moxibustion group had oval shape、passable quantity、even distribution、the ridge which was passable cleare、slight expansion、slightly dissolvedand and arrangement passable、the matrix passable uniform、the significant swelling and part of vacuolar degeneration unfound、surrounding the endoplasmic reticulum slightly expansed, compare to themodel group had the overall improvement.(2)"Experiment2st" results show,compare to the normal group and sham-groups,the model group had morecontents with ABAD(P<0.01) and less contents with COX Ⅳ(P<0.01);However, compare to the model group, the acupuncture-moxibustiongroup had less contents with ABAD(P<0.01) and more contents with COXⅣ(P<0.01),and had no significant difference between the normal group andsham-group(P>0.05).It prompted that the overexpression on ABAD could beinhibited and the activity on COX Ⅳ could be increased by theKidney-reinforcing and Du Meridian-regulating Acupuncture-Moxibustion.(3)"Experiment3st" results show, compare to the normal group andsham-groups,the model group had less level with Opa1(P<0.01) and morelevel with Fis1(P<0.01);However, compare to the model group, theacupuncture-moxibustion group had more level with Opa1(P<0.01) and lesslevel with Fis1(P<0.01),and had no significant difference between the normal group and sham-group(P>0.05).It prompted that the overexpression on Fis1could be inhibited and the level on Opa1could be increased by theKidney-reinforcing and Du Meridian-regulating Acupuncture-Moxibustion.(4)"Experiment4st" results show, compare to the normal group andsham-groups,the model group had more level with CypD(P<0.01);However,compare to the model group, the acupuncture-moxibustion group had lesslevel with CypD(P<0.01).It prompted that the level on CypD could bedecreased by the Kidney-reinforcing and Du Meridian-regulatingAcupuncture-Moxibustion.Conclusion:(1) The Kidney-reinforcing and Du Meridian-regulatingAcupuncture-Moxibustion could improve effectively the Learning-memoryand improve the damage on mitochondrial ultrastructure of hippocampalneurons.(2) The Kidney-reinforcing and Du Meridian-regulatingAcupuncture-Moxibustion could inhibit the overexpression of ABAD,increase the activity of COXⅣ and improve the obstacle on dysfunction ofmitochondria energy metabolism.(3) The Kidney-reinforcing and DuMeridian-regulating Acupuncture-Moxibustion could inhibit effectively theoverexpression of Fis1, increase the level of Opa1, adjust the balance betweenfission and fusion and improve the abnormal on mitochondrial dynamics.(4)The Kidney-reinforcing and Du Meridian-regulating Acupuncture-Moxibustion could inhibit effectively the overexpression of CypD, one of theessential ingredients in the mPTP, inhibit the opening on mPTP and improvethe imbalances on the internal and external permeability. Therefore, It has aclose correlation between the hippocampal neurons mitochondrial damagedand the pathogenesis of Alzheimer’s disease.Meantime, TheKidney-reinforcing and Du Meridian-regulating Acupuncture-Moxibustioncould improve the pathological changes in the hippocampal neurons mitochondrial ultrastructure and the abnormal with three approach of themitochondrial damaged, and in order to improve mitochondrial damaged andprevent-treat the AD.更多还原