| Background and objective:Cerebral ischemic damage can cause cognitive dysfunction,which is manifested as progressive memory impairment,executive function impairment,and reduced attention,and may be closely related to factors such as neurovascular unit dysfunction,neuroinflammation and oxidative stress reaction,and excessive activation of microglia.Diabetes mellitus(DM)increase the incidence rate of cognitive dysfunction.DM suffered with serious cerebral ischemia damages the health and life quality of patients,and also brings a heavy burden to the family and society.However,the mechanism of cerebral ischemia related cognitive dysfunction in DM is still unclear,and thereby the effective treatment is insufficient.Hippocampus is an important structure for the regulation of cognitive function,while hippocampal neural stem cells(h NSC)play the key role in maintaining normal hippocampal structure and function.Promotion of neural-regeneration of the endogenous h NSC is considered to be an effective therapeutic strategy for hippocampus related cognitive disorders.Exosomes(Exo)secreted by stem cells are important mediators for stem cells to perform their therapeutic functions.The exosomes secreted by mesenchymal stem cells differentiated from induced pluripotent stem cells(i PSC-MSC,iMSC)have been reported to have the effect of promoting angiogenesis and skin healing,but the function of promoting neural-regeneration is still unknown.Therefore,in this study,we applied a transient global cerebral ischemia/reperfusion model in diabetes mice(DM-t GCI/R)to simulate cerebral ischemia impairment in DM,and explored the changes and correlations of cognitive function and neural regeneration function of h NSC in mice.Subsequently,we investigated the function and related mechanisms of iMSC-Exo in promoting h NSC proliferation and neural-regeneration in DM mice suffered with cerebral ischemia impairment.Methods:1.Construction of mice diabetes model,and then their transient global ischemia/reperfusion model.Morris water maze were used to detect the cognitive function of mice in different groups;The expression level of synaptic plasticity related proteins(SYP,Gap-43)in the hippocampus of mice in different groups were detected by Western blot;The number of h NSC and newborn neurons in hippocampus in different groups were detected by immunofluorescence staining;h NSC were isolated from different groups to detect their proliferation and neuronal-differentiation function in vitro by immunofluorescence staining.2.i PSC were cultured in vitro and induced to iMSCs.iMSC-Exo was extracted by ultracentrifugation.After intervented by iMSC-Exo or PBS,the cognitive function of mice in different groups was detected by Morris water maze;The expression level of SYP and Gap-43 in the hippocampus of mice in different groups were detected by Western blot;The number of h NSC and newborn neurons in hippocampus of mice in different groups were detected by immunofluorescence staining;h NSC were isolated from different groups to detect their proliferation and neuronal-differentiation function in vitro by immunofluorescence staining.3.q PCR was used to detect the expression level of evolutionally conserved long intergenic noncoding RNA(Linc RNA)in iMSC-Exo;Construction of iMSC-Exo with normal level of target Linc RNA and low level of target Linc RNA,and intervened h NSC to detect the proliferation and neuronal-directional differentiation function in vitro.The molecular mechanism of iMSC-Exo transferred target Linc RNA to promote neural regeneration and cognitive function recovery in DM-t GCI/R mice was determined by transcriptome sequencing,q PCR and Western blot.Results:1.Changes of cognitive function and hippocampal neural-regeneration in DM-t GCI/R mice:(1)Morris water maze showed that the spatial learning and memory abilities of DM t GCI/R mice were significantly lower than those of DM or t GCI/R mice;Western blot results showed that the expression level of SYP and Gap-43 in the hippocampus of DM-t GCI/R mice was significantly lower than that of simple DM or simple t GCI/R mice.(2)The results of immunofluorescence showed that the number of h NSC and newborn neuron in the hippocampus of DM-t GCI/R mice were significantly less than those of DM or t GCI/R mice.(3)The results of immunofluorescence showed that h NSC extracted from DM-t GCI/R mice were significantly weaker than those of DM or t GCI/R mice in proliferation and neuronal-differentiation function in vitro.2.iMSC-Exo promotes neural-regeneration in hippocampus to improve cognitive impairment of DM-t GCI/R mice:(1)iMSC-Exo was extracted by ultracentrifugation,and it was observed by transmission electron microscope that iMSC-Exo was disc shaped or cup shaped;Western blot results showed that iMSC-Exo expressed CD63 and TSG101,but did not express GM130 and β-actin.The nanofluidic results showed that the diameter of iMSC-Exo was 78.3 ± 15.7 nm.(2)Morris water maze showed that iMSC-Exo intervention could improve the spatial learning and memory ability of DM-t GCI/R mice;Western blot showed that iMSC-Exo intervention could increase the expression of SYP and Gap-43 in the hippocampus of DM-t GCI/R mice.(3)The results of immunofluorescence showed that iMSC-Exo intervention could increase the total number of h NSC and the number of newborn neurons,as well as the number of proliferated h NSC and newborn neurons in the hippocampus of DM-t GCI/R mice.(4)The results of immunofluorescence showed that iMSC-Exo intervention could promote the proliferation and neuronal-differentiation function of DM-t GCI/R mice in vitro.3.iMSC-Exo transport Linc-OTX2 to regulate Fox O6-Rictor-AKT to promote neural regeneration of h NSC:(1)q PCR results showed that the expression level of Linc-OTX2 in iMSC-Exo was the highest,and it could be transported to h NSC,which increased the expression level of Linc-OTX2 in h NSC.(2)The immunofluorescence results showed that iMSC-Exo could promote the proliferation and directional differentiation of h NSC to neurons in the hippocampus of DM-t GCI/R mice by transporting Linc-OTX2.(3)Transcriptome sequencing,q PCR,Western blot and other experiments showed that iMSC-Exo could activate the expression levels of Fox O6,Rictor and P-AKT by transporting Linc-OTX2 to h NSC.Conclutions:1.The degree of cognitive impairment of DM mice with cerebral ischemia impairment was significantly higher than that of non-DM mice with cerebral ischemia impairment,which was closely related to the severe reduction of the number of h NSCs in hippocampus and the severe weakening of nerve regeneration function.2.iMSC-Exo can promote the proliferation and nerve regeneration of h NSC in the hippocampus of DM mice with cerebral ischemia impairment,thereby improving the cognitive impairment.3.iMSC-Exo contains a variety of evolutionarily conservative long chain non-coding RNAs(Linc RNAs),among which Linc-OTX2 has the highest content.4.The decrease of neural regeneration ability of h NSC in DM mice with cerebral ischemia impairment was closely related to the decrease of Fox O6 expression in cells,which led to the decrease of Rictor and P-AKT expression.5.iMSC-Exo activates Fox O6,Rictor and P-AKT in turn by transporting Linc OTX2 into h NSC,thereby promoting the proliferation and neural regeneration of h NSC in the hippocampus of DM mice with cerebral ischemia impairment. |
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