| Purpose:The study was based on the theory of "kidney and brain combination" to establish a model of perimenopausal depression,observe the changes in behavior and the morphological structure of nerve cells in rats with perimenopausal depression by electroacupuncture,and study the number,proliferation,differentiation and protein expression of neural stem cells in the hippocampal.To explore the correlation between Wnt signaling pathway and neurogenesis,and provide theoretical basis for clinical acupuncture and moxibustion in the treatment of perimenopausal depression.Material and method: 1 Animals and groups 50 SPF grade female SD rats were randomly divided into blank group,model group,sham operation group,western drug group and electroacupuncture group using random number table.There were 10 rats in each group.All animals were given adaptive breeding for 21 days after entering the laboratory.During the experiment,the experimental procedure for animals was strictly compliance with the "Guidance Opinion on the Treatise of Treat Animals".2 Modeling method According to the recognized modeling method internationally,the perimenopause depression rat model was established by selecting castration(OVX)combined with chronic unpredictability stimulation(CUMS).Blank group was not treated;In the sham operation group,only the ovaries were pulled out without ligation,and the adipose tissue beside the ovaries was removed.Bilateral ovariectomy was performed in the model group,western medicine group and electroacupuncture group.After perimenopause modeling successfully,the depression model was replicated using modified p.Willner’s chronic unpredictability stimulation modeling method.One stimulus was randomly selected every day,and the same stimulus could not be given for two consecutive days.3 Intervention methods Blank group: Rats were fed normally without any stimulation.Model group: Rats were captured and fixed with cloth bags for 20 minutes without treatment.Sham operation group: Rats were captured and fixed with cloth bags for 20 minutes without treatment.Western medicine group: Clomipramine Hydrochloride Tablets were used.The tablets were crushed and dissolved in 0.9 % normal saline.The dosage of 20 mg/kg·d-1 was given to the stomach,once a day for 28 consecutive days.Electroacupuncture group: Electroacupuncture treatment was selected as "baihui",bilateral "shenshu" and "sanyinjiao",and the acupoint positioning was conducted under the guidance of "experimental acupuncture".Shenshu was located in the back of the rat,and the ribs on both sides of the lower side of the second lumbar vertebra were opened 5mm apart,with a straight stab of 6mm.Sanyin intersection was located in the hind limbs of rats,with the tip of the medial malleolus straight for 10 mm and the spine straight for 5mm.Baihui was located in the middle of the parietal bone of rats,with a flat spine of 2mm.Rats with cloth coated and after acupuncture,connect the electroacupuncture apparatus,two pairs of electrodes by rat ipsilateral three vaginal intercourse and shen shu,a third of the anode electrode after rat tail,the cathode by will,electrode(load 250 Ω)0 ~ 18 V,the density wave type,thin wave frequency of 4 hz,the wave frequency of 20 hz,electricity intensity will be subject to local skin minor muscle tremor,retaining needle 20 min,one time a day,28 days in a row.4 Index detection and methods Before modeling,after modeling and after treatment,the general situation and estrus cycle of rats were observed,and behavioral tests were conducted,including body weight measurement,open field experiment,sugar water partial preference experiment and tail suspension experiment.The morphological and structural changes of hippocampal nerve cells were observed by nissl staining.Immunohistochemical methods were used to determine the percentage of Nestin(Nestin),astrocyte specific glial fibrillary acidic protein(GFAP),tubulin(Tuj-1)and galactocerebroside(GALC)labeled positive cells in the hippocampus.The proliferation and differentiation of neural stem cells were detected by immunofluorescence.The contents of Nestin,GFAP,tuj-1 and GALC were detected by ELISA.Contents of Wnt signaling pathway related proteins Dickkopf(DDK1),low-density lipoprotein-receptor-related protein 5(LRP-5)and low-density lipoprotein-receptor-related protein(LRP-6)in the hippocampus were detected by ELISA,and the contents of-catenin in the hippocampus were detected by Western blot.Results: 1 General situation Before modeling,the rats in each group were generally in good condition.After modeling,the rats were generally in poor condition,with listless spirit,weak limbs,crouching and prone,less movement with closed eyes,decreased appetite,and dry fur.After treatment,the general condition of rats in western medicine group and electroacupuncture group was significantly improved,the fur restored luster,mental state improved,mobility enhanced,diet acceptable,and excretion excellent.Before modeling,there was no significant difference in body weight between the groups(P > 0.05).After modeling,except the blank group,the weight of rats in each group was significantly reduced,and the difference was statistically significant(P < 0.05).After treatment,compared with the model group,the weight of rats in the western medicine group and the electroacupuncture group increased significantly(P < 0.05).The results showed that after electroacupuncture treatment,the rats’ appetite and food intake increased,and the overall state of the body was good.2 Behavior test results 2.1 Open field test Before modeling,there was no significant difference in the residence time of central area of the open field experiment and moving distance of the rats in each group for 3 minutes(P > 0.05)..After modeling,compared with the blank group,the rest of the groups reduced the residence time of central area and moveing distance(P < 0.05),which was statistically significant.After treatment,compared with the model group,the residence time of central area and moving distance of rats in the central area in the western medicine group and the electroacupuncture group were prolonged(P < 0.05),which was statistically significant.The results showed that after electroacupuncture treatment,the residence time of central area and moving distance increased,the tension decreased,the behavior helplessness decreased,and the inquiry behavior increased.2.2 Sugar preference test Before modeling,the sugar water preference of rats in each group was at a high level(P > 0.05),with no statistical significance.After modeling,compared with the blank group,the sugar water preference of the other groups was significantly decreased(P < 0.05),which was statistically significant.After treatment,compared with the model group,the rats in the western medicine group and the electroacupuncture group had significantly increased glycemic water preference(P < 0.05),which was statistically significant.Compared with model group,western medicine group and electroacupuncture group had similar degree of sugar water preference in rats,and the difference was not statistically significant(P > 0.05).The results showed that after electroacupuncture treatment,the deviation of rats increased and anhedonia was alleviated.2.3 Tail suspension test Before modeling,there was no significant difference(P > 0.05)in the suspended tail immobility time of rats in each group,with no statistical significance.After modeling,compared with the blank group,the suspended tail immobility time of the other groups was significantly increased,and the time of behavior desperation state was significantly increased(P < 0.05),which was statistically significant.After treatment,compared with the model group,the suspended tail immobility time of rats in the western medicine group and the electroacupuncture group was reduced,and the time of behavior desperation state was significantly reduced(P < 0.05),which was statistically significant.The results showed that the despair state of the rats was reduced after treatment.3 Morphological indicators of observation and detection 3.1 Hippocampal CA3 area of neuronal cell morphology changes under the Nissl staining observation In the Nissl staining,abundant nissl was observed in the cytoplasm of hippocampal neurons of the blank group under light microscope,showing a purple-blue color,regular cell arrangement,complete neuron structure,normal morphology of cytoplasm and nucleus,uniform and clear staining.After modeling,the hippocampal structure of rats in the model group was significantly damaged,the number of cells was reduced,the cell gap was enlarged,the arrangement was loose and uneven,the cell level was significantly diluted,the cell layer was disrupted,the nucleus disappeared,the cytoplasm atrophy,part of the cells were shed,the structure of axons and dendrites was incomplete,and even the cell structure disappeared.In the sham group,western medicine group and electroacupuncture group,the morphological changes of rat hippocampal nerve cells were between the blank group and the model group,and the structure was basically complete.In the western medicine group and the electroacupuncture group,the number of neurons in the hippocampal area increased,the cell structure was restored,and the morphological changes of the hippocampal nerve cells were significantly improved.3.2 Detection of Nestin-positive cell rate by immunohistochemistry Compared with the blank group,the expression rate of Nestin positive cells in the model group was significantly increased(P<0.01),which was statistically significant.After treatment,the expression rate of Nestin positive cells in the western medicine group and the electroacupuncture group increased,which was statistically significant compared with the model group and the sham operation group(P< 0.01).The results showed that the number of Nestin positive cells increased after treatment.The results show that EA treatment can promote the proliferation of neural stem cells.3.2 Detection of tubulin(Tuj-1)positive cell rate by immunohistochemistry Compared with the blank group,the expression rate of Tuj-1 positive cells in the model group was significantly decreased(P < 0.01).After treatment,the expression rate of Tuj-1 positive cells in the western medicine group and the electroacupuncture group was significantly increased,which was statistically significant compared with the model group(P < 0.01).The results showed that the number of tuj-1 positive cells increased after treatment.3.4 Detection of glial fibrillar acidic protein(GFAP)positive cell rate by Immunohistochemis-try Compared with the blank group,the expression rate of GFAP positive cells in the model group was significantly decreased(P<0.01).After treatment,the expression rate of GFAP positive cells in the western medicine group and the electroacupuncture group was significantly increased,which was statistically significant compared with the model group(P < 0.01).The results showed that the number of GFAP positive cells increased after treatment.3.5 Detection of galactocerebroside(GALC)positive cell rate by immunohistochemistry Compared with the blank group,the expression rate of GALC positive cells in the model group was significantly increased(P < 0.01).After treatment,the expression rate of GALC positive cells in the western medicine group and the electroacupuncture group was significantly increased(P < 0.01).The results showed that the number of GALC positive cells increased after treatment.3.6 Detection of proliferation of neural stem cells by immunofluorescence In the experiment,1 day,7 days,14 days and 28 days after the injection of Brdu,positive cells of Brdu/Nestin expression were observed in the hippocampus of the blank group,model group,sham operation group,western medicine group and electroacupuncture group,suggesting the existence of neural stem cells in the proliferating cells in the hippocampus of the perimenopmenopausal depression rats.Seven days after the injection of Brdu,the stress reactivity of Brdu/Nestin double labeled fluorescence increased in the model group,while the Brdu/Nestin double labeled fluorescence decreased in the electroacupuncture group.After 14 days and 28 days,the Brdu/Nestin fluorescence double labeling in the model group gradually decreased while the Brdu/Nestin double labeled fluorescence increased in the electroacupuncture group.3.7 Detection of differentiation of neural stem cells by immunofluorescence The Tuj-1 staining was observed in the cytoplasm by immunofluorescence.The cells were round or oval in size and stained green.Nestin staining in the nucleus,the cell body is small,stained red.In the blank group,model group,sham operation group,western medicine group and electroacupuncture group,Nestin/Tuj-1 expressing positive cells were found in the hippocampus.Compared with the blank group,the number of Nestin/Tuj-1 cells in the model group decreased.Compared with the model group,the number of Nestin/Tuj-1 cells in the western medicine group and the electroacupuncture group was significantly increased.The results showed that electroacupuncture could promote the differentiation of neural stem cells into neurons.GFAP staining was observed in cytoplasm by immunofluorescence assay.The cells were round or oval in size and stained green.Nestin staining in the nucleus,the cell body is small,stained red.In the blank group,model group,sham operation group,western medicine group and electroacupuncture group,Nestin/GFAP co-expressing positive cells were found in the hippocampus.Compared with the blank group,the number of Nestin/GFAP cells in the model group decreased.Compared with the model group,the number of Nestin/GFAP cells in the western medicine group and the electroacupuncture group was significantly increased.The results showed that electroacupuncture could promote the differentiation of neural stem cells into mature astrocytes.GALC staining was observed in the cytoplasm by immunofluorescence.The cells were round or oval in size and stained green.Nestin staining in the nucleus,the cell body is small,stained red.In the blank group,model group,sham operation group,western medicine group and electroacupuncture group,Nestin/GALC co-positive cells were found in the hippocampus.Compared with the blank group,the number of Nestin/GALC cells in the model group decreased.Compared with the model group,the number of Nestin/GALC cells increased in the western medicine group and the electroacupuncture group.The results showed that electroacupuncture could promote the differentiation of neural stem cells into oligodendrocytes.4 Detection of molecular biological indicators 4.1 Detection of DDK1,LRP-5 and LRP-6 In the hippocampus by ELISA Compared with the blank group,DKK1 expression was significantly decreased in the model group,while LRP-5 and LRP-6 expression were increased,and the difference was statistically significant(P<0.05).After treatment,DKK1 expression was increased in the drug group and the electroacupuncture group,while LRP-5 and LRP-6 expression were decreased.Compared with the model group,the difference was statistically significant(P<0.05).4.2 Detection of Nestin,GFAP,Tuj-1 and GALC protein expression In the hippocampus by ELISA Compared with the blank group,Nestin protein content in the model group was significantly increased(P<0.05),which was statistically significant.After treatment,Nestin protein content in the western medicine group and the electroacupuncture group decreased,which was statistically significant compared with the model group and the sham operation group(P<0.05).Compared with the blank group,the content of GFAP protein in the model group decreased,while the content of tuj-1 and GALC protein increased,which was statistically significant(P < 0.05).After treatment,GFAP and tuj-1 protein content decreased and GALC protein content increased in the western medicine group and the electroacupuncture group,which was statistically significant compared with the model group(P < 0.05).4.3 Detection of β-catenin in the hippocampus by Western blot Compared with the blank group,the expression level of β-catenin protein in the model group was significantly increased(P < 0.01),which was statistically significant.After treatment,the expression of β-catenin protein in the western medicine group and the electroacupuncture group decreased(P < 0.05),which was statistically significant.The results showed that electroacupuncture affects the Wnt signal pathway by regulating β-catenin protein.Conclusion: 1 Electroacupuncture at "Baihui","Kidney Yu" and "Sanyin Jiao" can effectively improve the behavioral indicators of perimenopausal depression rat models.2 Electroacupuncture at "Baihui","Kidney Yu" and "Sanyin Jiao" can adjust the number and function of neuronal cells in the CA3 region of the hippocampus,providing a morphological basis for the proliferation and differentiation of neural stem cells.3 Electroacupuncture at "Baihui","Kidney Yu" and "Sanyin Jiao" can increase the number of neural stem cells in the hippocampus model of perimenopausal depression and promote neurostem cells to neuronal cells,astrocytes and oligodendrocytes.Differentiation.4 The effects of electroacupuncture at "Baihui","Kidney Yu" and "Sanyin Jiao" on the rat model of perimenopausal depression may be related to the adjustment of the Wnt signal pathway by electroacupuncture therapy.5 In this study,renal deficiency in perimenopausal depression rats was identified,and the intervention of “brain and kidney with Leniency” electroacupuncture therapy on perimenopausal depression was demonstrated through the neurostem cell proliferation and differentiation mediated by Wnt /β-catenin. |
