| 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. |
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