Human Umbilical Cord Mesenchymal Stem Cell Exosomes Improved Depression-like Behavior In Cums Mice By Mediating Hippocampal Microglia Polarization

Objective:1.To investigate the relationship between hippocampal microglia polarization and depression-like behavior in a depressed mouse model built by Chronic Unpredictable Mild Stress(CUMS).2.The changes in depression-like behavior and microglia polarization status in CUMS mice treated with human umbilical cord mesenchymal stem cells exosomes(huc MSC-Exo)were further investigated.Methods:1.18 male C57BL/6J mice were selected and randomly divided into 3 groups,6 in each group.Two of the groups were meanwhile structured as depression models,and the remaining one group was the control group.12 mice were randomly split up into 2 groups after modeling,one group was treated with exosomes(CUMS+Exo group)after stereotactic injection of huc MSC-Exo in the hippocampus,and the other group was the model group(CUMS group).micro PET/CT(Micropositron Emission Tomography/Computed Tomography)scanning technology was used to detect the expression of inflammatory signals in the hippocampus of control and CUMS mice.Tissue immunofluorescence was used to detect the proportion of M2-type(CD206~+)microglia/microglia(Iba1)and M1-type(i NOS~+)microglia/microglia(Iba1)in the hippocampus of control and CUMS mice.The t-test was used to assess the statistical significance between control and CUMS groups,while Pearson correlation analysis was used to analyze the micro PET/CT scan results([18F]DPA-714 uptake values)with behavioral indicators were further explored.2.huc MSCs were isolated,cultured,and the 5th generation huc MSC-Exo was extracted by ultracentrifugation and identified.Western blot was used to detect biomarkers of huc MSC-Exo,including CD9 and CD63.We used related behavioral tests(opening field test(OFT)and forced swimming test(FST))to monitor changes of depression-like behavior in control,CUMS and CUMS+Exo groups of mice.We used tissue immunofluorescence to observe changes in the proportion of polarized microglia in the hippocampus of each group.We usedβ3-tubulin immunofluorescence staining to detect the length changes of neuronal axons in the depressed cell model constructed with corticosterone(CORT).We used Ed U staining to check neuronal proliferation.We used TUNEL staining to observe the apoptosis of neurons in the hippocampus of mice.Apoptosis of neurons in mouse hippocampal tissue was observed using TUNEL staining.Results:1.Micro PET/CT scan imaging experiments showed increased[18F]DPA-714 uptake values in the CUMS group of mice compared with the control group.The acquired data were covered in the analysis,and the results showed that although the difference between the control and CUMS groups in[18F]DPA-714 uptake values was statistically significant(P<0.05),the correlation between[18F]DPA-714 uptake values and behavioral indices was weak(percent central path length r=-0.833,P=0.040;central path time spent r=-0.829,P=0.041;total distance of movement r=-0.449,P=0.371;time of immobility r=0.826,P=0.043).2.Analysis of tissue immunofluorescence assay data showed statistically significant differencesin the proportions of M2-type(CD206~+)microglia/microglia(Iba1)and M1-type(i NOS~+)microglia/microglia(Iba1)in the hippocampus of control and CUMS mice(P<0.05),and correlated strongly with behavioral indices(i NOS~+/Iba1 r=0.877,P=0.022;CD206~+/Iba1 r=-0.868,P=0.025).3.After huc MSC-Exo treatment,the results of polarization state after treatment showed that there was statistically significant differences in the ratio of CD206~+/Iba1 and i NOS~+/Iba1 among the control,CUMS and CUMS+Exo groups(all P<0.05)4.After huc MSC-Exo treatment,the results of behavioral tests in the CUMS and CUMS+Exo groups showed statistically significant differences,as shown by an increase in the percentage of central path length and the time spent on the central path on the"bull’s-eye diagram"in the open field experiment in the CUMS+Exo group compared to the CUMS group(both P<0.05),while there was no difference in the total distance of movement(P＞0.05).In the forced swimming test,the immobility time of mice in CUMS+Exo group was shorter than that in the CUMS group(P<0.05).5.huc MSC-Exo reduced apoptosis of neurons in the hippocampus of depressed mice.β3-tubulin immunofluorescence results showed an increase in neuronal axon length in the CORT+Exo group compared with the CORT group.Ed U staining showed an increase in proliferation rate in the CORT+Exo group compared with the CORT group.In the TUNEL staining,compared with CUMS group,the apoptosis rate of neurons in the hippocampus of mice in the CUMS+Exo group was reduced.Conclusion:1.Microglia polarization in the hippocampus of CUMS mice showed a more significant correlation with behavioral indices than[18F]DPA-714 uptake values and behavioral indices.2.huc MSC-Exo treatment improved the polarization status of CUMS mice,which may provide a relevant strategy for the precise prevention and treatment of depression.