| Purpose:The study was under the guidance of TCM Brain-Kidney Axis theory.The purpose of this study is to observe the effect of electro-acupuncture on Nissl bodies in hippocampal CA3 region of perimenopausal depression rats;observe the absolute number of neural stem cells in hippocampal CA3 region of perimenopausal depression rats;observe the differentiation of neural stem cells in hippocampal CA3 region of perimenopausal depression rats;explore the key protein content on Wnt signaling pathway,and its relationship with proliferation and differentiation of hippocampal stem cells of perimenopausal depression rats;seek the mechanism of acupuncture treatment on perimenopausal depression;and provide the scientific basis of acupuncture treatment on perimenopausal depression.Material and method: 1.Animals and groups 60 SPF grade female SD rats were given adaptive feeding for 3 weeks.After 3 weeks of adaptive feeding,60 SPF grade female SD rats were randomly divided into blank group,sham operation group,model group,western medicine group and electro-acupuncture group.There were 12 rats in each group.During the experiment,the experimental procedure for animals was strictly compliance with the "Guidance Opinion on the Treatise of Treat Animals" 2.Modeling method The perimenopausal depression rat model is composed of two models.The first part is the perimenopausal castration model.The second part is P.Willner traditional chronic unpredictable stimulation(CUMS)improved depression model.The above two models constituted the perimenopausal depression rat model.Except the blank group rats,all other group rats were anesthetized by intraperitoneal injection of 10% chloral hydrate(0.30 ml / 100 g).Bilateral ovariectomy was performed on the model group,western medicine group and lectro-acupuncture group rats.3 days after the operation,all group rats were subjected to continuous vagina keratosis epithelial test for 5 days.Their vaginal smears were continuously collected for 5 days.The morphology and staging of vaginal smears were observed under a 200-fold microscope.The success of perimenopausal model replication could be determined by without affection cycle on vaginal smears.The P.Willner's traditional chronic unpredictable modified type of stimulation was used to produce the depression model After modeling,the behavior test was carried out by open field experiment,sugar water partial preference experiment and forced swimming experiment,which proved the success of perimenopausal depression model.3.Intervention methods The blank group rats were fed normally without any treatment.The sham operation group rats were not treated,but were fixed by bags,and given grasping stimulation.The model group rats were also not treated,but were fixed by bags,and given grasping stimulation.The western medicine group rats were given Antidepressant,Clomipramine Hydrochloride Tablets,via intragastric administration,20 mg / kg · d-1,once a day for 28 consecutive days.The electro-acupuncture were performed on electro-acupuncture group rats.The acupoints selection was conducted under the guidance of "experimental acupuncture".According to TCM Brain-Kidney Axis theory,the following acupoints were selected for this study: Bilateral "Shen Shu",bilateral "San Yin Jiao",and "Bai Hui".In the course of acupuncture treatment,the needles were inserted into the selected acupoints,and were remained in the body for 20 minutes.Acupuncture treatment once a day for a total of 28 days.Electro-acupuncture operation is as follows: two pairs of electrodes connected to the same side of the rats San Yin Jiao and Shen Shu,the third pair of negative electrode connected to the rat tail,electrode 0 ~ 18 V,load 250?,dense wave type,shuffle 4Hz,dense wave 20 Hz.The intensity of electro-acupuncture operation is according to the degree of local skin and muscle tremors..Index detection and methods The behavioral tests were performed at 3 time points.The 3 time points were before modeling,after molding,and after treatment.The behavioral tests were including the open field experiment,sugar water partial preference experiment,and the forced swimming experiment.The morphological changes of Nissl bodies in hippocampal CA3 region were observed under the condition of Nissl staining,and the total area,integral optical density and average blackness of Nissl bodies were measured.The percentage of nestin(Nestin),astrocyte specific glial fibrillary acidic protein(GFAP)and tubulin(Tuj-1)positive cells in hippocampus CA3 region were measured by immunohistochemistry.ELISA method was used to detect glycogen synthesis kinase(GSK-3?)and beta-catenin content in hippocampus.Beta-catenin and P-beta-catenin content in hippocampus were detected by Western and blots.The expression of GSK-3? m RNA and beta-catenin m RNA were detected by RT-PCR.Results: 1.Behavior test results 1.1 Open field test Open field test result showed: before modeling,there was no significant difference among the groups in the 180 seconds of total moving distance of the open field experiment and the 180 seconds residence time of the central area of the open field experiment(P>0.05).After the castration and 21 days of chronic unpredictable stimulation,compared with the blank group,the 180 seconds total moving distance and the 180 seconds residence time in open field center area of all other group rats were dramatically reduced,the difference was statistically significant(P<0.05).The result indicated the depression rat model was successfully produced.After 28 days of acupuncture and antidepressant treatment,compared with the blank group,the 180 second total moving distance and the 180 second residence time in open field center area of model group rats was still significantly reduced(P < 0.05).Compared with model group,the 180 second total moving distance and the 180 second residence time n open field center area of western medicine group were dramatically increased(P < 0.05).Compared with model group,the 180 second total moving distance and the 180 second residence time in open field center area of electro-acupuncture group were dramatically increased(P < 0.05).Compared with sham operation group,the 180 second total moving distance and the 180 second residence time in open field center area of western medicine group and electro-acupuncture group were dramatically increased(P < 0.05).There was no significant difference between western medicine group and electro-acupuncture group in the 180 seconds of total moving distance of the open field experiment and the 180 seconds residence time of the central area of the open field experiment(P> 0.05).1.2 Sugar preference test Sugar preference test results showed: before modeling,there was no significant difference among the groups in the sugar consumption rate of sugar preference test(P>0.05).After the castration and 21 days of chronic unpredictable stimulation,compared with the blank group,all other groups significantly decreased their sugar consumption rate of sugar preference test(P < 0.05).The result indicated the depression rat model was successfully produced.After 28 days of acupuncture and antidepressant treatment,compared with the blank group,the sugar consumption rate of model group was still low(P < 0.05).Compared with model group,the sugar consumption rate of western medicine group was dramatically increased(P < 0.05).Compared with model group,the sugar consumption rate of electro-acupuncture group was dramatically increased(P < 0.05).Compared with sham operation group,the sugar consumption rate of western medicine group and electro-acupuncture group was dramatically increased(P < 0.05).There was no significant difference between western medicine group and electro-acupuncture group in the sugar consumption rate of sugar preference test(P>0.05).1.3 Forced swimming experiment Forced swimming experiment result showed: before modeling,there was no significant difference among the groups in the 180 second immobility time of forced swimming experiment(P > 0.05).After the castration and 21 days of chronic unpredictable stimulation,compared with the blank group,the 180 second immobility time of forced swimming experiment of all other groups was significantly decreased(P<0.05).The result indicated the depression rat model was successfully produced.After 28 days of acupuncture and antidepressant treatment,compared with the blank group,the 180 second immobility time of forced swimming experiment of model group was still dramatically decreased(P < 0.05).Compared with model group,the 180 second immobility time of forced swimming experiment of western medicine group was dramatically increased(P < 0.05).Compared with model group,the 180 second immobility time of forced swimming experiment of electro-acupuncture group was dramatically increased(P < 0.05).Compared with sham operation group,the 180 second immobility time of forced swimming experiment of western medicine group and electro-acupuncture group was dramatically increased(P < 0.05).There was no significant difference between western medicine group and electro-acupuncture group in the 180 second immobility time of forced swimming experiment(P>0.05).2.Morphological indicators of observation and detection 2.1 hippocampal CA3 area of neuronal cell morphology changes under the Nissl staining observation The results of hippocampal CA3 area of neuronal cell morphology changes under the Nissl staining observation showed: the number of cells in the pyramidal cell layer of hippocampal CA3 in blank group was higher,and had orderly arrangement.Neurons in the cytoplasm are rich in Nissl bodies.The Nissl bodies were in purple blue color,Neurons are intact and the nuclei and cytoplasm are stained evenly and clearly.In the model group,the number of cells in the hippocampal CA3 region was small,the cell gap was increased,and was arranged in loose,and even the emergence of cell layer was interrupted.The number of Nissl body reduced in neuronal cytoplasm,nucleus in neurons disappeared,cytoplasm atrophy,voids,axon dendritic structure is not complete,even cell structure disappeared.The condition of western medicine group,electro-acupuncture group and sham operation group was between the two above conditions.After 28 days of acupuncture and antidepressant treatment,compared with the blank group,the neuro body area,optical density,and mean blackness in neuro-cell of control group was dramatically decreased(P < 0.05).Compared with sham operation group,the neuro body area,optical density,and mean blackness in neuro-cell of model group was also dramatically decreased(P < 0.05).Compared with model group,the neuro body area,optical density,and mean blackness in neuro-cell of western medicine group were dramatically increased(P < 0.05).Compared with model group,the neuro body area,optical density,and mean blackness in neuro-cell of electro-acupuncture group were dramatically increased(P < 0.05).Compared with sham operation group,the neuro body area,optical density,and mean blackness in neuro-cell of western medicine group and electro-acupuncture group was dramatically increased(P < 0.05).There were no significant difference between western medicine group and electro-acupuncture group in the neuro body area,optical density,and mean blackness in neuro-cell(P>0.05).2.2 The immunohistochemistry nestin-positive cell count of perimenopausal depression rat The immunohistochemistry nestin-positive cell count of perimenopausal depression rat model indicated,after 28 days of acupuncture and antidepressant treatment,compared with the blank group,the total percentage of nestin-positive cells in CA3 region of model group was dramatically decreased(P < 0.01).Compared with model group,the total percentage of nestin-positive cells in CA3 region of western medicine group was dramatically increased(P < 0.01).Compared with model group,the total percentage of nestin-positive cells in CA3 region of electro-acupuncture group was dramatically increased(P < 0.01).Compared with sham operation group,the total percentage of nestin-positive cells in CA3 region of western medicine group and electro-acupuncture group was dramatically increased(P< 0.01).There were no significant difference between western medicine group and electro-acupuncture group in the total percentage of nestin-positive cells in CA3 region(P> 0.01).2.3 Hippocampal CA3 region immune-histochemical astrocyte specific glial fibrillary acidic protein(GFAP)positive cell count Hippocampal CA3 region immune-histochemical astrocyte specific glial fibrillary acidic protein(GFAP)positive cell count showed,after 28 days of acupuncture and antidepressant treatment,compared with the blank group,the percentage of total cells of GF3 positive cells in CA3 region of model group was dramatically decreased(P < 0.01).Compared with the blank group,the percentage of total cells of GFAP positive cells in CA3 region of sham operation group was dramatically decreased(P < 0.05).Compared with model group,the percentage of total cells in GFAP positive cells in CA3 region of western medicine group were dramatically increased(P < 0.01).Compared with model group,the percentage of total cells of GFAP positive cells in CA3 region of electro-acupuncture group was dramatically increased(P < 0.01).There were no significant difference between western medicine group and electro-acupuncture group in the percentage of total cells of GFAP positive cells in CA3 region(P> 0.05).2.4 The number of immune-histochemical tubulin(Tuj-1)positive cells in the hippocampal CA3 region The number of immune-histochemical tubulin(Tuj-1)positive cells in the hippocampal CA3 region showed,after 28 days of acupuncture and antidepressant treatment,compared with the blank group,the total cell percentage of Tuj-1 positive cells in CA3 region of model group was dramatically decreased(P<0.01).Compared with sham operation group,the total cell percentage of Tuj-1 positive cells in CA3 region of model group was also dramatically decreased(P<0.01).Compared with model group,the total cell percentage of Tuj-1 positive cells in CA3 region of western medicine group were dramatically increased(P<0.01).Compared with model group,the total cell percentage of Tuj-1 positive cells in CA3 region of electro-acupuncture group was dramatically increased(P<0.01).Compared with sham operation group,the total cell percentage of Tuj-1 positive cells in CA3 region of western medicine group was dramatically increased(P<0.01).There were no significant difference between western medicine group and electro-acupuncture group in the total cell percentage of Tuj-1 positive cells in CA3 region(P>0.05).3.Detection of molecular biology indicators 3.1 ELISA and RT-PCR detection of glycogen synthase kinase(GSK-3?),GSK-3? m RNA and ?-catenin,?-catenin m RNA in hippocampus ELISA results showed,after 28 days of acupuncture and antidepressant treatment,compared with the blank group,the GSK-3? and beta-catenin in hippocampus of model group was dramatically decreased(P<0.05).Compared with the blank group,the GSK-3? and beta-catenin in hippocampus of sham operation group was dramatically decreased(P<0.05).Compared with the sham operation group,the GSK-3? and ?-catenin in hippocampus of western medicine group and electro-acupuncture group was dramatically increased(P<0.05).Compared with model group,the GSK-3? and ?-catenin in hippocampus of western medicine group were dramatically increased(P<0.05).Compared with model group,the GSK-3? and ?-catenin in hippocampus of electro-acupuncture group was dramatically increased(P<0.05).There were no significant difference between western medicine group and electro-acupuncture group in the GSK-3? and ?-catenin in hippocampus(P>0.05).RT-PCR detection result showed,after 28 days of acupuncture and antidepressant treatment,compared with the blank group,the GSK-3? m RNA and ?-catenin m RNA in hippocampus of model group was dramatically decreased(P<0.05).Compared with the sham operation group,the GSK-3? m RNA and beta-catenin m RNA in hippocampus of western medicine group and electro-acupuncture group was dramatically increased(P<0.05).Compared with model group,the GSK-3? m RNA and ?-catenin m RNA in hippocampus of western medicine group was dramatically increased(P<0.05).Compared with model group,the GSK-3? m RNA and ?-catenin m RNA in hippocampus of electro-acupuncture group was dramatically increased(P<0.05).There were no significant difference between western medicine group and electro-acupuncture group in the GSK-3? m RNA and ?-catenin m RNA in hippocampus(P>0.05).3.2 Western blot detection for ?-catenin and P-?-catenin content in hippocampus the western blot detection result showed,after 28 days of acupuncture and antidepressant treatment,compared with the blank group,the ?-catenin content in hippocampus of model group was dramatically decreased(P<0.01).Compared with the sham operation group,the ?-catenin content in hippocampus of western medicine group and electro-acupuncture group was dramatically increased(P < 0.01).Compared with model group,the ?-catenin content in hippocampus of western medicine group was dramatically increased(P<0.01).Compared with model group,the ?-catenin content in hippocampus of electro-acupuncture group was dramatically increased(P<0.01).There were no significant difference between western medicine group and electro-acupuncture group in the ?-catenin content in hippocampus(P>0.05).After 28 days of acupuncture and antidepressant treatment,compared with the blank group,the P-?-catenin content in hippocampus of model group was dramatically decreased(P<0.01).Compared with the blank group,the P-?-catenin content in hippocampus of sham operation group was dramatically decreased(P<0.05).There were no significant difference between western medicine group and electro-acupuncture group in the P-?-catenin content in hippocampus(P>0.05).Conclusion: 1.Both electro-acupuncture intervention and clomipramine intervention have treatment effect on perimenopausal depression model rats.2.Electro-acupuncture at Bai Hui,San Yin Jiao and Shen Shu not only can improve the neuronal cell activity of hippocampal CA3 neurons,but also can provide the material basis for the Sproliferation and differentiation of neural stem cells.3.Electro-acupuncture at Bai Hui,San Yin Jiao and Shen Shu can increase the absolute number of neural stem cells in hippocampal CA3 region of perimenopausal depression rats.Electro-acupuncture Bai Hui,San Yin Jiao and Shen Shu acu-points can promote the differentiation of neural stem cells.4.Electro-acupuncture at Bai Hui,San Yin Jiao and Shen Shu acu-points had a benign adjustment effect on hippocampal neural stem cells in perimenopausal depression rats.Electro-acupuncture Bai Hui,Shen Shu and San Yin Jiao can through this positive regulation to improve perimenopausal depressive symptoms in depression rats.The formation of this benign regulation is related to the activation of the Wnt signaling pathway.5.This experiment showed under the guidance the TCM "kidney brain theory”,electro-acupuncture therapy could activate the kidney essence,and push kidney essence toward brain.In Western medicine,this is the process of promoting the proliferation and differentiation of neural stem cells in the brain. |
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