| Purpose:Under the guidance of TCM Kidney-Brain Axis theory,to observe the effect of moxibustion on hippocampal histomorphological changes of perimenopausal depression rats,to observe the effect of moxibustion on hippocampal intracellular environment and on regeneration and apoptosis of hippocampal neurons in perimenopausal depression rats,to explore the relationship between Notch signaling pathway related proteins and hippocampal neuronal regeneration and apoptosis in hippocampal neurons of perimenopausal depression rats,to study the mechanism of moxibustion therapy on perimenopausal depression,and to provide scientific theoretical basis of clinical medical research.Material and Method:1.Animal grouping and modeling60 female SPF grade Sprague-Dawley(SD)rats were provided by Beijing Wei Tong Li Hua Experimental Animal Technology Co.Animal certificate number was SCXK(Beijing)2012-0001.The treatment of animals during the course of the experiment was in accordance with the provisions of the Guidance Opinion on Treat Animals.After 3 weeks of adaptive feeding in the laboratory,60 rats were randomly divided into control group,model group,sham operation group,western medicine group and moxibustion group.Through the castration plus chronic unpredictable mild stimulation,an internationally recognized perimenopausal depression rat model was established.(1)Preparation of perimenopausal rat modelExcept the control group and sham operation group,all other rats were underwent bilateral ovariectomy.The rats of sham operation group were routinely operated but their ovaries were not removed.After the end of the castration surgery,vaginal smears of all rats were tested.The success of artificial castration perimenopausal rat model was determined by without the occurrence of estrus response in vaginal smears.The model was conformed to the internationally accepted modeling requirements.(2)Depression model preparationExcept the control group,the all other rats were subjected to preparation of the depression model,for 21 days.The model was prepared by using P.Willner's chronic unpredictable mild stimulation.The stimulation included: cold water swimming,hot water swimming,clipping,fasting,restraint,tilting cages,day reversal.The results of the general state of the rats,the results of the body weight of rats,the results of the open field test,the results of the sugar water preference test and the results of gripping force determination experiment were the crucial points to determine the successful establishment of rat depression model.The model was conformed to the internationally accepted modeling requirements.2.Treatment methodsRats in the control group were fed normally without treatment.Model group and sham operation group were not treated,but were fixed,and given grasping stimulation for 28 days.The western medicine group rats were given Clomipramine Hydrochloride Tablets via intragastric administration,for 28 consecutive days.Moxibustion group rats were given moxibustion treatment for 28 days.The acupoints "Bai Hui",“Da Zhui" and bilateral "Shen Shu" were selected as moxibustion points.3.Material and index detectionDuring the course of the experiment,two rats in the model groups and one rat in moxibustion group died after castration,two rats in the western medicine group and one rat in sham operation group died in hot water swimming,one rat in the moxibustion group died in binding test.After the end of the treatment period,the rats sacrificed during the course of the experiment were removed.Ten rats from each group were tested for the corresponding indexes.Nissl staining was used to observe the morphological changes of hippocampal neurons.Serum estrogen(E2),serum glucocorticoid(CORT),The Microtubule Associated Protein 2(MAP-2)and Apoptotic Protease 3(Caspase-3)were detected by Elisa method.The Notch signaling pathway related protein NICD,Notch-1,Jag-1,Hes-1,Mash1 were detected by Elisa method.The Notch signaling pathway related genes expression Notch-1 m RNA,Jag-1 m RNA,Hes-1 m RNA and Mash1 m RNA were detected by RT-PCR method.Results:1.The general condition and behavior test results before modelingBefore modeling,the rats were generally in good condition.Before modeling,there was no difference among the rat groups in the total moving distance of the open field test and the residence time of the central area of the open field test,no statistical difference(P>0.05).In sugar water preference test,there was no sugar consumption difference among the rat groups,no statistical difference(P>0.05).In gripping force determination experiment,there was no gripping force difference among the rat groups,no statistical difference(P>0.05).2.Perimenopausal rat modelAfter bilateral ovariectomy,except control group and sham operation group,there was no estrous cycle in the vaginal smears of all other groups.It proves the success modeling of perimenopausal model.3.Depression rat modelAfter 21 days of chronic unpredictable mild stimulation,the general condition,the rat's weight,and behavioral test results of rats were as follows:(1)After the castration and 21 days of chronic unpredictable mild stimulation modeling,the general conditions of model group,western medicine group,and moxibustion group rats were poor.Sham operation group rats,although were not had castration surgery,but after 21 days of chronic unpredictable mild stimulation modeling,the general condition was also not good.Compared with the control group,the body weight of all other rats were significantly decreased(P<0.05).(2)The results of the open field experiment showed,compared with the control group,the 3 minutes moving distance of all other group rats was dramaticlly shortened(P<0.05).Compared with the control group,the 3 minutes residence time in open field center area of all other group rats was significantly reduced(P<0.05).(3)Sugar water preference test results showed,after 21 days of chronic unpredictable mild stimulation,compared with the control group,the sugar consumption rate of all other groups of rats was significantly decreased(P< 0.05).(4)The gripping force determination experiment result showed,after 21 days of chronic unpredictable mild stimulation,compared with the control group,the gripping force of all other group rats was significantly lower(P<0.05).All of above results prove that the depression rat model was successfully established.4.General condition and behavior test results after treatmentAfter 28 days of moxibustion and antidepressant treatment,the general condition and behavioral test results of the rats are as follows:After 28 days of moxibustion and antidepressant treatment,compared with the control group,the general condition of the model group was still poor.Compared with the model group,the general condition of western medicine group and moxibustion group was improved dramatically.Compared with the sham operation group,the general condition of the western medicine group and the moxibustion group was also improved obviously.The general condition of western medicine group and moxibustion group was similar.After 28 days of moxibustion and antidepressant treatment,compared with the control group,the body weight of the model group decreased significantly(P<0.05).Compared with the model group,the body weight of the western medicine group and the moxibustion group increased significantly(P<0.05).Compared with the sham operation group,the body weight of the western medicine group and the moxibustion group also improved significantly(P<0.05).The weight of western medicine group and moxibustion group was similar,no statistical difference(P > 0.05).After 28 days of moxibustion and antidepressant treatment,compared with the control group,the 3 minutes total moving distance of the open field test in model group was significantly reduced,the 3 minutes residence time in open field center area of model group was significantly shortened,the sugar consumption rate of model group was decreased significantly,the gripping force of model group was significantly decreased(P<0.05).Compared with the model group,the 3 minutes total moving distance of the open field tests in western medicine group and moxibustion group was increased significantly,the 3 minutes residence time of the central area of western medicine group and moxibustion group was significantly increased,the rats' sugar consumption rate of western medicine group and moxibustion group was increased significantly,the grasping force of western medicine group and moxibustion group was increased obviously(P < 0.05).Compared with the sham operation group,the 3 minutes total moving distance of the open field test in western medicine group and moxibustion group was increased obviously,the 3 minutes residence time in open field center area of western medicine group and moxibustion group was also increased obviously,the rat sugar water consumption rate of western medicine group and moxibustion group was increased obviously,the grasping force of western medicine group and moxibustion group was increased obviously(P<0.05).The 3 minutes total moving distance of the open field of western medicine group and the 3 minutes total moving distance of the open field of moxibustion group were similar;the 3 minutes residence time in open field center area of western medicine group and the 3 minutes residence time in open field center area of moxibustion group were similar;the sugar consumption rate of western medicine group and the sugar consumption rate of moxibustion group were similar;the grasping force of western medicine group and the grasping force of moxibustion group were similar,no statistical difference(P> 0.05).5.Morphological observation results of hippocampal CA3 area neuronal morphologyAfter 28 days of moxibustion and antidepressant treatment,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 control 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 lower,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,moxibustion group and sham operation group was between above two groups.6.The test results of Estrogen(E2)and Cortisol(CORT)in hippocampal intracellular environmentAfter 28 days of moxibustion and antidepressant treatment,compared with the control group,the Estrogen(E2)in hippocampal intracellular environment of model group was significantly low,the Cortisol(CORT)in hippocampal intracellular environment of model group was significantly higher(P<0.05).Compared with the model group,the Estrogen(E2)in hippocampal intracellular environment of western medicine group and moxibustion group was increased significantly,and the Cortisol(CORT)in hippocampal intracellular environment of western medicine group and moxibustion group was decreased significantly(P<0.05).Compared with the sham operation group,the Estrogen(E2)in hippocampal intracellular environment of western medicine group and moxibustion group was still lower,and the Cortisol(CORT)in hippocampal intracellular environment of western medicine group and moxibustion group was still higher(P<0.05).Compared with the western medicine group,The Estrogen(E2)in hippocampal intracellular environment of moxibustion group was still increased,the Cortisol(CORT)in hippocampal intracellular environment of moxibustion group was also increased(P<0.05).7.The test results of Microtubule Associated Protein 2(MAP-2),the Apoptotic Protease 3(Caspase-3)in hippocampusAfter 28 days of moxibustion and antidepressant treatment,compared with the control group,the Microtubule Associated Protein 2(MAP-2)in hippocampus of model group was significantly decreased and the Apoptotic Protease 3(Caspase-3)in hippocampus of model group was significantly increased(P < 0.05).Compared with the model group,the Microtubule Associated Protein 2(MAP-2)in the hippocampus of western medicine group and moxibustion group was increased significantly,the Apoptotic Protease 3(Caspase-3)in hippocampus of western medicine group and moxibustion group was decreased significantly(P<0.05).Compared with the sham operation group,the Microtubule Associated Protein 2(MAP-2)in the hippocampus of western medicine group and moxibustion group was decreased,and the Apoptotic Protease 3(Caspase-3)in hippocampus of western medicine group and moxibustion group was increased(P<0.05).Compared with the western medicine group,the Microtubule Associated Protein 2(MAP-2)in the hippocampus of moxibustion group was increased,the Apoptotic Protease 3(Caspase-3)in hippocampus of moxibustion group was decreased(P<0.05).8.The detection results for related proteins on Notch signaling pathway in hippocampusAfter 28 days of moxibustion and antidepressant treatment,Elisa method was used to detect NICD,Notch-1,Jag-1,Hes-1,and Mash1 in hippocampus.The result showed,compared with the control group,the NICD and Notch-1 in the hippocampus of model group were significantly lower,the Jag-1,Hes-1,and Mash1 in the hippocampus of model group were significantly high.Compared with the model group,the NICD and Notch-1 in the hippocampus of western medicine group and moxibustion group were recovered significantly,the Jag-1,Hes-1,and Mash1 in the hippocampus of western medicine group and moxibustion group were significantly decreased(P<0.05).Compared with sham operation group,the NICD and Notch-1 in the hippocampus of western medicine group and moxibustion group were relatively low,and the Jag-1,Hes-1 and Mash1 in the hippocampus of western medicine group and moxibustion group were relatively high(P<0.05).Compared with western medicine group,the Notch-1 in the hippocampus of moxibustion group was relatively high,and the Jag-1,Hes-1 and Mash1 in the hippocampus of moxibustion group were relatively low(P<0.05).The NICD in the hippocampus of western medicine group and the NICD in the hippocampus of moxibustion group were similar,no statistical difference(P> 0.05).Notch signal pathway corresponding gene PCR detection results and Elisa test results are not exactly the same,but there are similarities.The result showed,after 28 days of moxibustion and antidepressant treatment,compared with the control group,the relative expression of Notch-1 m RNA in hippocampus of model group was significantly decreased,the relative expression of Jag-1 m RNA,Hes-1 m RNA and Mash1 m RNA in the hippocampus of model group was significantly increased(P<0.05).Compared with the model group,the relative expression of Notch-1 m RNA in the hippocampus of the moxibustion group was increased significantly,and the relative expression of Jag-1m RNA,Hes-1m RNA and Mash1 m RNA in the hippocampus of the moxibustion group was decreased significantly(P<0.05).Compared with the model group,the expression of Notch-1 m RNA in the hippocampus of western medicine group was increased significantly,and the relative expression of Mash1 m RNA in the hippocampus of western medicine group was decreased significantly(P<0.05).The relative expression of Jag-1m RNA and Hes-1m RNA in the hippocampus of western medicine group and the relative expression of Jag-1m RNA and Hes-1m RNA in the hippocampus of the model group were similar,no statistical difference(P> 0.05).Compared with the sham operation group,the relative expression of Notch-1m RNA in the hippocampus of western medicine group and moxibustion group was relatively low,and the relative expression of Jag-1m RNA,Hes-1m RNA and Mash1 m RNA in the hippocampus of western medicine group and the moxibustion group was relatively high(P<0.05).Compared with the western medicine group,the relative expression of Jag-1m RNA and Hes-1m RNA in the hippocampus of moxibustion group was relatively low(P<0.05).The relative expression of Notch-1 m RNA and Mash1 m RNA in the hippocampus of western medicine group and the relative expression of Notch-1 m RNA and Mash1 m RNA in the hippocampus of moxibustion group were similar,no statistical difference(P> 0.05).Conclusion:1.Moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could improve the pathologic changes of hippocampal neurons in CA3 area of perimenopausal depression rats.Moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could adjust the ecological balance in intracellular environment in hippocampus of perimenopausal depression rats.Moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could promote the regeneration of hippocampal neurons of perimenopausal depression rats.Moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could reduce the apoptosis of hippocampal neurons of perimenopausal depression rats.2.Notch signaling pathways are a few signaling systems that can repeatedly regulate neuronal cell regeneration and apoptosis.Notch signaling pathway allows neuronal regeneration and apoptosis to be controlled.Notch signaling pathway has the function that is similar to TCM "yin and yang balance" theory.Moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could strengthen this function which repeatedly regulates neuronal cell regeneration and apoptosis.3.With severe chronic stress stimulation,because of the activation of neural plasticity mechanisms,the Notch signaling pathway can be altered.Moxibustion at “Bai Hui”,“Da Zhui” and “Shen Shu” could cause the altered Notch signaling pathway back to normal by regulating the Notch signaling pathway related protein and activating the classic "side inhibition" signal pathway.4.Moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could directly up-regulated the expression of Notch-1 on the Notch signaling pathway.By activating the classic "side inhibition" signal pathway,moxibustion at "Bai Hui","Da Zhui" and "Shen Shu" could reduce the expression of Jag-1 and Mash1.The above features could promote proliferation of hippocampal neural stem cells in perimenopausal depression rats,and promote hippocampal neurogenesis in perimenopausal depression rats.5.This experiment once again proved that neurogenesis is controlled by many factors.It is the results of a variety of cytokines,a variety of signal channels interact with each other,mutual influence,mutual constraints,and mutual coordination.Through stimulating the NEI network and microcirculation,moxibustion at "Bai Hui","Da Zhui" and “Shen Shu " could directly adjust the ecological balance of hippocampal neurons intracellular environment of perimenopausal depression rats,promote perimenopausal depression rat hippocampal cell regeneration,reduce perimenopausal depression rat hippocampal cell apoptosis.Moxibustion at " Bai Hui "," Da Zhui " and “Shen Shu" could also inhibit the neuronal apoptosis and promote the neuronal regeneration by regulating the related proteins on the Notch signaling pathway in the hippocampus of perimenopausal depression rats. |
PDF Full Text Request