Study On The Mechanism Of The Brain-targeting Effect Of BMSCs-Exo In PDD Model Rats Activated By Electro-acupuncture Therapy Of "Kidney And Brain Xiangji"

Purpose:Perimenopausal depression(PDD)is one of the most common affective disorders in middle-aged and old women.The deactivation of neurons in hippocampus is an important pathological basis for the occurrence of PDD.Guided by the theory of "kidney,Tiangui and brain",the bone marrow mesenchymal stem cells(BMSCs)were observed in SD rats in vitro and in vivo Cells,BMSCs derived exosomes(-Exo)can repair hippocampal neurons,and explore the internal correlation between SDF-1/CXCR4 axis and brain targeting effect of BMSCs-Exo.The aim is to explain the mechanism of "kidney brain combination" electroacupuncture in the treatment of PDD at the cellular and molecular level,and to seek modern scientific evidence support for the theory of "kidney Tiangui brain" system in traditional Chinese medicine.Material and method:1.We isolated BMSCs from SD rats,purified and cultured by whole bone marrow adherent method.Cell morphology was observed by microscope and cell surface antigen was identified by flow cytometry.BMSCs-Exo was extracted and purified by ultrahigh speed centrifugation.Exosomes morphology was identified by transmission electron microscopy(TEM),and exosomes size was determined by nanoparticle tracking analysis(NTA)BMSCs-Exo surface antigen was identified by blot,and BMSCs-Exo was separated and enriched by molecular exclusion(SCS)method.2.30 female SD rats were randomly divided into five groups: blank group,model group,Baihui group,electroacupuncture group and Western medicine group,6 rats in each group.In addition to the blank group,the model group,Baihui group,electroacupuncture group and Western medicine group all adopted ovariectomy combined with chronic unpredictable mild stimulation to duplicate the PDD rat model.They were given acupuncture at Baihui Point,electroacupuncture of "kidney brain combination" and gastric perfusion with fluoxetine hydrochloride(Prozac)for 2 weeks,metabolic cage and sugar water preference test(SCT)and forced swimming FST was used to observe the changes of behavior and movement mode of rats in depression state,IVIS was used to detect the aggregation of BMSCs-Exo in brain,he staining was used to observe the morphological changes of hippocampal neurons,ELISA was used to detect the expression of SDF-1 in serum and brain regions,Western blot was used to detect the expression of CXCR4 in bone marrow.3.Cultured neurons in the hippocampus of SD rats in vitro,the effects of CORT at different concentrations on the morphology and viability of neurons were measured by CCK8 method;the chemotaxis of SDF-1 / CXCR4 axis were inhibited by CXCR4 antagonists at 20 ? mol / L concentration,and neurons were interfered by CORT at appropriate concentrations to simulate the damage of neurons in the hippocampus under depression.After CXCR4 was inhibited,the binding ability of BMSCs-Exo to neurons and brain targeting function were observed.Results:1.BMSCs of rats were successfully isolated and cultured,and the results of cell culture were confirmed to be in accordance with the characteristics of BMSCs by means of microscopic morphology observation and surface antigen detection.The supernatant of BMSCs was collected,and the BMSCs-Exo was separated and extracted by ultra-high speed centrifugation The surface antigens of TEM,NTA and WB were identified,and the extracted products were confirmed to be exosomes.2.The model of PDD rats was successfully reproduced.The results showed that compared with the model group,the rats in the electroacupuncture group and Western medicine group had an increase in food intake and water intake(P < 0.05),and an increase in respiratory exchange rate(RER)in the electroacupuncture group(P < 0.05).There was no significant difference between the other groups and the model group(P > 0.05).The results of SCT showed that the consumption rate of sucrose solution in EA group and Western medicine group was significantly higher than that in model group(P < 0.0001).The results of FCT showed that the immobility time of electroacupuncture group and Western medicine group was significantly lower than that of model group(P < 0.001 in electroacupuncture group,P <0.01 in western medicine group);there was no significant difference in swimming time of rats in each group;the climbing time of Baihui group,electroacupuncture group and Western medicine group was higher than that of model group(P < 0.001 in Baihui group,P < 0.0001 in electroacupuncture group and Western medicine group).It is suggested that both electroacupuncture and fluoxetine can effectively improve the depression of PDD rats.3.IVIS in vivo imaging results showed that after the injection of dir labeled BMSCs-Exo through the tail vein,the fluorescence signal in the rat gradually increased until the peak value reached at 12 h,and then decreased until disappeared at 24h;compared with other time points,the fluorescence signal in the brain of the rat was the strongest at 12h;the average fluorescence intensity analysis showed that BMSCs-Exo was injected through the tail vein After 12 hours,the average fluorescence intensity in brain area of model group was significantly lower than that of blank group(P < 0.001);the average fluorescence intensity in brain area of Baihui group and electroacupuncture group was higher than that of model group(P < 0.01,electroacupuncture group P < 0.001);the average fluorescence intensity in brain area of Western medicine group was higher than that of model group(P < 0.05),but still lower than that of Baihui group and electroacupuncture group(P < 0.05)Fluorescence intensity(P < 0.05 in Baihui group,P < 0.001 in electroacupuncture group).It is suggested that acupuncture at Baihui Point can enhance the chemotaxis of BMSCs-Exo on brain.4.The results of ELISA showed that compared with the blank group,the content of SDF-1 in the hippocampus of the model group decreased(P < 0.05),and that of the electroacupuncture group was higher than that of the model group(P < 0.05).There was no significant difference between the other groups and the model group.The results of Western blot showed that there was no significant difference in the content of CXCR4 in bone marrow cells between groups.It is suggested that SDF-1 may be an important factor to mediate the targeted convergence of BMSCs-Exo in the head,which is closely related to the theory of "kidney Tiangui brain" system in traditional Chinese medicine.5.Hippocampal neurons were cultured successfully in vitro.CCK8 method showed that CORT treatment at 25 ? mol / L concentration had a strong effect on the activity of neurons,which was similar to the morphology and activity of neurons in depressed rats,and could simulate the internal environment of neurons in depressed rats in vitro.6.The results of neuron fluorescence staining showed that BMSCs-Exo significantly reduced the binding ability of neurons after amd3100 intervention,and IVIS in vivo imaging showed that BMSCs-Exo brain targeting ability of BMSCs-Exo after amd3100 intervention wassignificantly lower than that of the control group,the difference was statistically significant(P< 0.05).It is suggested that the regulation of SDF-1 / CXCR4 axis is the key to the effect of BMSCs-Exo on the chemotaxis of neurons in the brain.Conclusion:1.Creatively put forward the construction of "Kidney-Tiangui-Brain" system,which has made a new development for the theory of "Tiangui" in traditional Chinese medicine.It provides a new theoretical support for the treatment of PDD by Electroacupuncture of "kidney brain combination".2.It was confirmed that "kidney brain combination" electroacupuncture and Prozac can effectively alleviate the depression symptoms of PDD rats,BMSCs-Exo can target to the brain area of PDD rats,and "kidney brain combination" electroacupuncture can enhance the targeted migration characteristics,Prozac has no such effect,which provides experimental basis for "kidney brain combination" electroacupuncture.3.It is confirmed that SDF-1 / CXCR4 is an important factor affecting the targeted convergence of BMSCs-Exo on neurons in the brain.This chemotaxis may be an important effective way for the electroacupuncture therapy of "kidney brain combination" in the treatment of PDD,which provides modern medical evidence support for the theory of "Kidney-Tiangui-Brain " system in traditional Chinese medicine.