【作者】 翟煦；

【导师】 王宏才； 荣培晶；

【作者基本信息】 中国中医科学院 ， 针灸推拿学， 2013， 博士

【摘要】 褪黑素(Melatonin,MLT)又称为褪黑激素,在哺乳动物中,一般认为主要由松果体分泌,其分泌具有昼夜节律特点。从褪黑素被发现到现在已有近100年的时间。随着研究的深入,人们对它作用的认识愈加广泛,除了与昼夜、季节、光照、生理周期、甚至生命周期相关的一些生理反应与生理功能外,褪黑素还在缓解疼痛、调节血糖、减肥、抗抑郁等方面被深入地探究,且研究者对褪黑素的研究热情经久不衰。相对于人类对于褪黑素近百年的研究,中国的传统医学理论可以被追溯至两千年前,而针刺疗法则更加久远。在中医理论形成后,针刺的作用机制被概括为“调和阴阳”,而究竟如何“调和”,传统理论并未给出一个令人信服的答案,而“阴、阳”二字的由来确是因为古人对光的观察,继而推演到对自然界昼夜、季节的更替阐释并联系到人自身的生理、病理。褪黑素作为一种跟光调节密切联系的生物信号载体,并可以产生广泛作用的激素,成为联系自然光照变化节律与人体生物节律的一个纽带。针刺的一些适应病症也恰与褪黑素缺乏类的病症相重合,这不由的让我们大胆联想,调节褪黑素分泌可能是产生针刺复合效应的机制之一。我们前期的实验发现,耳甲区存在迷走神经耳支传入纤维的分布,恰当的电刺激能够产生迷走神经刺激的效果(VNS),继而产生镇痛、调节血糖、抗癫痫、抗抑郁、抗炎、降血压等功效,并观察到刺激耳甲区所产生的中枢神经核团的放电及神经递质和相关蛋白的改变。另外,文献记载了研究者对针刺疗法和外源褪黑素协同作用的研究,认为外源褪黑素与针刺效应有相互促进的作用。我们的预实验重复了耳甲区电刺激对于病理模型大鼠镇痛、降糖及抗抑郁效应的观察,发现耳甲区电刺激后,病理模型大鼠血浆褪黑素浓度的提高。这些线索让我们联想到,耳迷走神经电刺激可以缓解病理疼痛模型大鼠的痛阈,降低糖尿病大鼠模型的血糖并促进其胰岛素分泌,缓解慢性应激大鼠模型的抑郁症状,这些情形可能有褪黑素的参与,并且耳迷走神经电刺激促进了褪黑素的分泌,提高了血浆褪黑素的浓度,从而产生或者加强了上述效应。本实验中,我们选用了Zucker Diabetes Fat (ZDF)大鼠作为模型。这种模型大鼠具有(fa/fa)肥胖基因,并有遗传性,在特殊饲料诱导下逐渐自发高血糖、高血脂、胰岛素抵抗等2型糖尿病(T2DM)的典型症状,肥胖,并发感觉神经痛等T2DM并发症,在国际上广泛用于T2DM及其并发症以及肥胖、代谢类疾病的研究,比传统胰岛β细胞损毁或部分损毁配合高脂饲料诱导的T2DM模型有着无可比拟的优势。这种模型在我们耳-迷走反射针刺效应系列研究中的应用尚属首次,耳迷走神经调节褪黑素分泌来产生针刺效应也是一个较新的研究思路。实验流程：1耳甲区电针对ZDF大鼠体重、摄食的影响1.1动物分组实验采用雄性6-8周龄Zucker Diabetes Fat (ZDF)大鼠及Zucker Lean(ZL)。ZL大鼠予以普通颗粒大鼠维持饲料喂养,ZDF大鼠予以Purina#5008(粗蛋白23.5%、粗脂肪6.5%)颗粒料饲养以诱导T2DM。所有大鼠自由饮水,维持8：00-20：00光照、20：00-8：00黑暗交替的行为学实验独立动物房中适应性饲养3天,实验室温度22±1℃,湿度50±5%,每笼4-6只。分组情况如下：1.2实验结果1.2.1体重与内脏脂肪比1)体重同周龄ZL大鼠与ZDF大鼠在实验前相比较,ZL大鼠平均比ZDF大鼠体重轻9%左右(P<0.05)。经过30天电针干预后,ZL大鼠和ZDF大鼠分别与各自的空白对照组相比,耳甲区电针组(EA-ACR)体重增长较低,MLT腹腔注射组.(MTL-ZDF)与N-ZDF组相比较也较轻(P<0.05),而耳缘电刺激组(EA-AE)与其空白对照组(Naive, N)相比无统计学差异(P>0.05)。从趋势上来看,ZL与ZDF对照组在体重增长率上(κ=(y-b)/x)有差异(P<0.05),N-ZDF体重增长较N-ZL组要快,N-ZDF与N-ZL末次体重测量有极显著差异(P<0.01)。1.2.2摄食量与N-ZL组相比,ZDF组30日均摄食量显著较大(P<0.01),而在ZL组的电针干预的两组组(EA-AE, EA-ACR)其摄食量较低(P>0.05),但这两组间没有统计学差异(P>0.05)；与N-ZDF组比较,MLT-ZDF组30日均摄食量较低(P<0.05),而ZDF电针干预的两组(EA-AE-ZDF, EA-ACR-ZDF)与N-ZDF之间没有统计学差异(P>0.05)2耳甲区电针对ZDF大鼠疼痛行为的影响2.1动物分组2.2实验结果2.2.1机械痛阈在手术前,各组比较均无显著差异(P>0.05)；手术及电针刺激30天后,与N-ZL组相比,ZDF大鼠各组机械刺激收爪时间显著下降(P<0.01),与N-ZDF相比,C-ZDF(?)且收抓时间更低(P<0.05)；而ZDF耳甲区电刺激(EA-ACR-C-ZDF)与MLT干预组(MLT-C-ZDF)较N-ZD和C-ZDF组高(P>0.05)。从各组变化趋势上来看,N-ZL组前后变化无显著差异(P>0.05),N-ZDF组30天后较之前相比显著降低(P<0.01),ZDF的3个CCI手术组在术后机械刺激收爪时间显著下降(P<0.01),EA-ACR-C-ZDF与MLT-C-ZDF丙组干预后械刺激收爪时间有持续稳定的回升,且高于N-ZDF组(P<0.05),MLT-C-ZDF组与EA-ACR-C-ZDF组之间无统计学差异(P>0.05)。2.2.2热痛阈在手术前,各组比较均无显著差异(P>0.05)；手术及电针刺激30天后,与N-ZL组相比,ZDF大鼠各组机械刺激收爪时间下降(P<0.05；C-ZDF P＜0.01) MLT-C-ZDF(?)且无统计学差异(P>0.05)：与N-ZDF相比,C-ZDF组收抓时间较低(P<0.05),MTL-C-ZDF组较高(P>0.05),EA-ACR-C-ZDF组无统计学差异(P>0.05)；与C-ZDF组相比,EA-ACR-C-ZDF组较高(P<0.05),MTL-C-ZDF组显著升高(P<0.01)；MTL-C-ZDF组与EA-ACR-C-ZDF组比较,MTL-C-ZDF组较高(P>0.05)。从各组变化趋势上来看,N-ZL组前后变化无显著差异(P>0.05),Z-ZDF组30天后较之前相比降低(P<0.05),ZDF的3个CCI手术组在术后机械刺激收爪时间下降(P<0.05；C-ZDF, MLT-C-ZDF P<0.01), EA-ACR-C-ZDF与MLT-C-ZDF两组干预后械刺激收爪时间有所回升,MLT-C-ZDF组与C-ZDF组(P<0.05)与EA-ACR-C-ZDF组(P<0.01)相比回升较为显著。3耳甲区电针对ZDF大鼠血浆相关生化指标的影响3.1动物分组3.2实验结果3.2.1血糖实验前同周龄的ZDF大鼠与ZL大鼠相比血糖水平已有差异,ZDF大鼠血糖显著高于ZL大鼠血糖水平(P<0.01)；与ZDF空白对照组相比(N-ZDF), ZDF其余三个干预组的血糖均有下降(P<0.05),且EA-ACR-ZDF组与MLT-ZDF组的血糖下降尤为显著(P<0.01)；ZL各组间血糖比较未见明显差异(P>0.05)；从前后变化趋势上来看,ZL大鼠血糖较为平稳,ZDF大鼠血糖实验前后比较均显著增高(P<0.01),MLT-ZDF组在第2周时表现了较好的血糖控制作用(P<0.05),但在第4周时血糖有所回升,而EA-ACR-ZDF与EA-AE-ZDF组在第3周和第4周时表现了较好的血糖控制作用(P<0.05)。3.2.2褪黑素实验后各组大鼠血浆褪黑素水平与N-ZL组比较,ZDF各组均有显著差异,ZDF组中N.EA-AE、M、X褪黑素水平显著较低(P<0.01),EA-ACR、EA-ACR-C、M-C褪黑素较低(P<0.05)；ZL组中EA-ACR褪黑素较N组高(P>0.05),EA-AE组无显著差异。ZDF组中,与N-ZDF组比较,EA-ACR、EA-ACR-C、M-C组褪黑素水平升高(P<0.05),C组褪黑素水平下降(P<0.05)。ZDF组中,EA-ACR与EA-AE、M组比较褪黑素水平较高(P＜0.05); EA-ACR-C、M-C组较C组显著较高(P<0.01)。ZL组中,N与EA-AE组无显著差异(P>0.05)；ZDF组中,N、AE-EA、M组之间无显著差异(P>0.05),EA-ACR-C与M-C之间无显著差异(P>0.05)。3.2.3糖化血红蛋白(Glycosylated hemoglobin, Gh)实验后各组间大鼠血浆糖化血红蛋白水平,与N-ZL组比较,ZDF组中N、EA-AE、M组糖化血红蛋白血浆浓度较高(P<0.05),其中ZDF中N、EA-AE显著较高(P<0.01),ZL组中EA-AE、EA-ACR组无显著差异,EA-ACR-ZDF组亦无显著差异；EA-ACR-ZDF组与N-ZDF、EA-AE-ZDF比较显著较低(P<0.01),与M-ZDF比较较高(P<0.05)。3.2.4胰岛素实验后大鼠各组血浆胰岛素水平变化,与N-ZL相比,EA-ACR-ZL组、N-ZDF组、EA-ZCR-ZDF组、M-ZDF组较高(P<0.05),其中EA-ACR干预的两组血浆胰岛素水平显著较高(P<0.01)；ZL组中,EA-ACR干预组血浆胰岛素水平较EA-AE组显著增高(P<0.01)；ZDF组中,EA-ACR干预组较其余各组均显著增高(P<0.01)；而N-ZL与EA-AE组相比无统计学差异(P>0.05), N-ZDF组、EA-AE组、M-ZDF组之间亦无统计学差异(P>0.05)。3.2.5五羟色胺(5-HT)实验后各组血浆5-HT水平,与N-ZL、N-ZDF、C-ZDF组相比,EA-ACR-C-ZDF组(P<0.01)与M-C-ZDF组(P<0.05)显著升高；EA-ACR-C-ZDF与M-C-ZDF组相比较高(P＜0.05); N-ZL、N-ZDF、C-ZDF之间没有显著差异(P>0.05)。4电针刺激对ZDF大鼠中枢相关受体表达的影响4.1动物分组同“3.1动物分组”。4.2实验结果4.2.1免疫荧光从图11、12中可以观察到,N-ZL、EA-ACR-ZL、EA-ACR-ZDF、M-ZDF组mt1受体与INSRβ受体在下丘脑部共同表达；EA-AE-ZL、EA-AE-ZDF组仅见mt1受体表达；N-ZDF组未见mt1受体与INSRP受体表达。N-ZL、 EA-ACR-C-ZDF、M-C-ZDF组mt1受体与5-HT1AR受体在脊髓腰膨大共同表达；N-ZDF组与C-ZDF组未见mt1受体与5-HT1AR受体表达。4.2.1Western blotmt1在下丘脑部的表达水平,与N-ZL相比,N-ZDF组较低(P<0.05),EA-ACR-ZL组,EA-AE-ZDF组,EA-ACR-ZDF组,M-ZDF组显著升高；与Z-ZDF相比,EA-ACR-ZL组,EA-AE-ZDF组,EA-ACR-ZDF组,M-ZDF组升高(P<0.05),其中EA-ACR-ZL组显著提高(P<0.01),ZL组中EA-ACR组比EA-AE组亦有显著提高；而N-ZL组与EA-AE-ZL组无统计学差异,ZDF干预组之间亦无统计学差异。mt1在脊髓腰膨大部的表达水平,与N-ZL相比EA-ACR-C-ZDF(P<0.01)显著提高, N-ZDF与C-ZDF表达降低(P<0.05)；与N-ZDF相比,EA-ACR-C-ZDF显著提高(P＜0.01), N-ZDF与C-ZDF表达降低(P<0.05)；与M-C-ZDF相比,EA-ACR-C-ZDF亦有显著提高(P<0.01)。INSRβ在下丘脑处的表达水平,与N-ZL和N-ZDF相比,EA-ACR-ZL, EA-ACR-ZDF, M-ZDF组显著提高(P<0.05),而EA-ACR与EA-AE组相比亦分别有显著提高。5-HT1AR在脊髓腰膨大部的表达水平,N-ZL水平与其他组相比有较高表达(P<0.05)；与C-ZDF和N-ZDF组相比EA-ACR-C-ZDF和M-C-ZDF组较高(P<0.05)。5结论本研究基于前期大量的临床及基础研究,从耳甲区-迷走神经联系学说入手,提出耳甲区电刺激促进褪黑素分泌,继而产生缓解慢性神经痛、调节血糖、降低体重等褪黑素缺乏类相关疾病的假说,并采用了Zucker Diabetes Fat (ZDF)大鼠(fa/fa)基因突变模型,进行了从行为学到生物化学到分子生物学不同层面的系统研究。通过数据统计整理分析可以发现,耳甲区电针刺激(EA-ACR),可以有效控制ZDF肥胖大鼠模型的体重,抑制高血糖,提高血浆胰岛素水平,降低了血浆糖化血红蛋白水平,提高胰岛素受体INSRP在下丘脑的表达；且缓解了ZDF慢性神经痛模型大鼠的机械刺激疼痛和热刺激疼痛,提高了血浆五羟色胺水平(5-HT),以及5-HT受体在脊髓腰膨大处的表达；上述效果以EA-ACR提高了血浆褪黑素浓度、调高了褪黑素受体(mt1)在中枢神经系统表达水平为基础,并可能通过EA-ACR兴奋了副交感神经神经促进褪黑素分泌来实现,这可能是针刺复合效应的共同机制之一。可以展望,许多褪黑素缺乏类疾病与症状,可以用EA-ACR的方法进行干预和治疗。更多还原

【Abstract】 IntroductionAs a kind of hormone, melatonin (MLT) is known that is secreted from pineal in circadian rhythm in mammal. There is nearly a centry from MLT being found to now. With further research, the understanding of MLT become more widespread in addition to the day and night, seasons, light, menstrual cycles, and even the life cycle of some physiological reactionand physiological function, the function of melatonin is also relieve pain, regulate blood sugar, weight loss, anti-depressants and other aspects of in-depth inquiry, and researchers melatonin enduring passion for research. With the very small toxic side effects, MLT is widely used in the research and development of health care products, and MLT drugs are also in-depth research and development. Someone believe MLT is not just the hormone, but a vitamin.Comparing with a centry history of MLT research, traditional Chinese medicine could be traced to2000years ago, and acupuncture therapy may be older. After the formation of the traditional Chinese medicine theory, the mechanism acupuncture is summarized as "Reconciling Yin and Yang", and how to reconcile the traditional theory did not give a convincing answer, and the "Yin and Yang" of the origin of the word is indeedthe ancient observation of light, followed by deduction to interpret and linked to people’s own physiological and pathological nature day and night, the seasons. Melatonin in close contact with the light adjustment as a biological signal carrier, and can produce a wide range of the role of hormones, becomes a natural rhythm of illumination change and human biological rhythms of a bond. Adapt to conditions acupuncture just a lack of class and melatonin illness coincide, this can not let bold Lenovo, regulating melatonin secretion may be one of the mechanisms that produce the composite effect of acupuncture.Our previous experiments found that the concha region of the vagus nerve ear support the distribution of afferent fibers, an appropriate level of electrical stimulation can produce the effect of vagus nerve stimulation (VNS), and then produce analgesia, regulate blood sugar, anti-epileptic, anti-depressants, anti-inflammatory,effect of lowering blood pressure, and observed the change of the nuclei stimulate conchaplasty area of central nervous system discharge and neurotransmitters and related proteins. Literature researchers Acupuncture and exogenous melatonin synergy, that exogenous melatonin and acupuncture effect mutually reinforcing. Our pre-experiment was repeated electrical stimulation of the concha area observed for the the pathological rat model analgesic, hypoglycemic and anti-depressant effects, and observed that electrical stimulation of the concha area, pathological model of rat plasma melatonin concentration increased. These clues let us think of the ear vagus nerve stimulation can ease the pain threshold of the rat model of pathological pain, lower blood sugar in diabetic rat model and promote insulin secretion, relieve the symptoms of chronic stress rat model of depression, these cases may have the participation of melatonin, and the ears of the vagus nerve electrical stimulation to promote the secretion of melatonin, to improve the plasma melatonin concentrations, thereby generating or enhancing the above-mentioned effects.In this experiment, we chose Zucker, of Diabetes Fat (ZDF) rats as a model. This rat model has obese (fa/fa) gene and hereditary gradually special feed induced spontaneous typical symptoms of high blood sugar, high cholesterol, insulin resistance and type2diabetes mellitus (T2DM), obesity, concurrent sensory nerve type2diabetic complications such as pain in the international community widely used in T2DM and its complications, as well as obesity, metabolic diseases, than traditional islet beta cells destroyed or partially damaged with high fat diet-induced type2diabetic model has an unparalleled advantage. This model reflection acupuncture effect in a series of studies in our ears-fans go the first time, ear vagus nerve regulate melatonin secretion acupuncture effect is a relatively new research ideas.MethodsExperimental AnimalsMale Zucker diabetic fatty (ZDF) rats andZucker lean (ZL) littermates were purchased from VitalRiver Laboratories International Inc.(Beijing, China). The animal room was artificially lighted from7:00A.M. to7:00P.M. Littermates from the same or foster mother were housed in one large cage until they were ready to enter the experiment procedure at8weeks of age and were separated into Zucker diabetic fatty (ZDF) and Zucker lean (ZL) groups. We used only mature (8weeks of age at the beginning of experiment) male ZDF rats for further detailed study and some ZL littermates for controlto avoid a possible confounding effect from gender differences on the endogenous melatonin level and other possible hormone variation. The experimental protocol was approved by the Institutional Animal Care and Use Committee in China Academy of Chinese Medical Sciences.Measurements of Tactile and Thermal Withdrawal LatencyAnimals were habituated to the test environment daily (a60min session) for2d before baseline testing. The testing procedure for thermal hyperalgesia was performed according to a previously published method. Temperature was set to have the baseline latency of12-14sec and a cutoff of30sec. Mechanical allodynia was examined by applying a set of von Frey filaments to the plantar surface of each hindpaw, up and down depending on the withdrawal responses of the paw. The cutoff force was26gm. All behavior testing was conducted between8:00A.M. and12P.M. before any daily treatment.ElectroacupunctureRats were exposed to daily treatments for28day continuously according to the experimental design. For electroacupuncture,under2%isoflurane inhalation anesthesia, a procedure of30min electroacupuncture at a frequency of2/15Hz alternately in a second and an intensity of2mA was administered to a rat via an electrical stimulator (HANS-100, Nanjing, China) daily in the afternoon, beginning from day0. The acupoint selected in this study was auricular conchaof both sidesand the auricular edge was used as the sham acupoint.Collection of plasmaFor analyzing the concentration of melatonin in plasma,0.1ml blood samples were collectedat eachtime pointbetween4:00PM and5:00PM (before daily treatment) to minimize the variations of melatonin level.Rats were anesthetized by inhalation of2%isoflurane in oxygen. All blood samples were collected before daily treatment (acupuncture, injection) fromone of the tail veins. The blood sample was centrifuged for10min at1000rpm, and plasma was collected. All plasma were stored at-80℃until use. The animals were sacrificed after the last collection of blood, brain samples were harvested for immunostaining and Western blot analysis.ELISAThe concentration of plasma melatonin was analyzed using enzyme linked immunosorbent assay (ELISA) kit (Lot#DZE30014,R&D System, Beijing, China) by Huanya Biomedicine Technology Co. LTD. The results were read using a microplate reader (MULTISKAN MK3, Thermo Scientific, Beijing, China) at wavelengths of450nm. The plasma melatonin concentration was calculated based on the standard curve and presented in nanogramsper liter (ng/L).Immunohistochemical stainingHalf of the rats from each group (n=3) were anesthetized with pentobarbital (60mg/kg, i.p.) and transcardially perfused with200ml of saline followed by400ml of4%paraformaldehyde in0.1m phosphate buffer (PB). The brainswere dissected, postfixed for2hr, and kept in30%sucrose in0.1m PBin cold room until sank to the bottom. Tissues were then mounted in OCT compound and frozen on dry ice. The brain (30μm) sections were cut on a cryostat, mounted serially onto microscope slides, and stored at-80℃. Immunohistochemical staining was used to detect5-HT1A R (1:500, mousemonoclonal; EMD Millipore Corporation, Billerica, MA, USA). Sections were blocked with1%goat serum in0.3%Triton x100for1hr at room temperature and incubated overnight at4℃with the primary antibody. For controls, the primary antibody was omitted. The sections were then incubated for1hr at room temperature with corresponding Cy3-conjugated secondary antibody (1:200; JacksonlmmunoResearch, West Grove, PA). Brain sections were read using a LEXT OLS40003D Laser Measuring Microscope(Olympus), recorded using a digital camera, and processed using Adobe Photoshop.Western blotRats (n=3-4each group) were decapitated under anesthesia. Hypothalamuswas collected separately. The segments were homogenized in SDS sample buffer containing a mixture of proteinase inhibitors (Sigma). Protein samples were separated on SDS-PAGE gel (4-15%gradient gel; Bio-Rad, Hercules, CA) and transferred to polyvinylidenedifluoride filters (Millipore, Bedford, MA). The filters were blocked with3%milk and incubated overnight at4℃with MT1primary antibody (40kD, rabbit polyclonal,1:500, Millipore, Billerica, MA) and lhr at room temperature with HRP-conjugated secondary antibody (1:7000; Abcam, Cambridge, MA). The blots were visualized in ECL solution (NEN, Boston, MA) for lmin and exposed onto hyperfilms (Amersham Biosciences) for1-10min. The blots were then incubated in a stripping buffer (67.5mMTris, pH6.8,2%SDS, and0.7%β-mercaptoethanol) for30min at50℃and reprobed with a polyclonal rabbit anti-β-actin antibody (1:20,000; Alpha Diagnostic International, San Antonio, TX) as loading control. The Western analysis was made in triplicates. The density of the bands was measured with a computer-assisted imaging analysis system and normalized against loading controls.Statistical analysisBy running IBM SPSS Statistics v20.0for Windows(SSPS Inc., Chicago, IL, USA), raw data from behavior tests, ELISA and Western blots were analyzed by using repeated measures ANOVA across testing time points to detect overall differences among treatment groups and across treatment groups to examine overall differences among testing time points. Differences were considered to be statistically significant at the level of a=0.05.ConclusionThis study is based on pre-clinical and basic research, starting from the the concha area-vagus nerve contact theory, proposed the concha District electrical stimulation to promote the secretion of melatonin, which in turn produce the relief of chronic neuropathic pain, regulate blood sugar, reduce weight, lack of melatoninthe hypothesis of class-related diseases, and the use of Zucker, of Diabetes Fat (ZDF) rats (fa/fa) gene mutation model, learned from the behavior of the system study of biochemistry to molecular biology at different levels.Compiles data analysis can be found, concha area electro-acupuncture (EA-ACR), can effectively reduce the weight of the ZDF rat model of obesity, improve the symptoms of high blood sugar, increased plasma insulin levels, reduced plasma glycated hemoglobin level to improve expression of the insulin receptor INSRβ" the hypothalamus; and ease of ZDF rats with chronic neuropathic pain model of mechanical stimulation of pain and thermal stimulation pain, increased plasma serotonin level (5-HT) and5-HT receptors in the spinal cord at the lumbar enlargementexpression of; the effect EA-ACR plasma melatonin levels, an increase in melatonin receptor (MT1) expression levels in the central nervous system, and may be excited by the EA-ACR parasympathetic nervous promote fadedmelanin secretion, which may be one of the mechanisms of acupuncture combined effects of common. Looking Many lack of melatonin-like illness symptoms, EA-ACR’s intervention and treatment.更多还原