Neuroprotective Effects Of Bone Marrow Mesenchymal Stem Cells Regulated By Lin28B In Alzheimer's Disease Model

BackgroundAlzheimer's disease(AD)is a prevalent age-related degenerative disease of the central nervous system,as well as the sixth most common cause of death in humans.Although a large number of studies and prclinical trails have been conducted to explore the pathogenesis and potential therapeutic targets of this disease,there is still a lack of effective treatment strategies.Over the last few decades,mesenchymal stem cells(MSCs)transplantation have been regarded as a potential therapeutic approach for AD.However,the poor proliferation capacity and low survival rate of engrafted MSCs in the hostile microenvironment of AD limit their therapeutic efficiency.Lin28B is a conserved RNA-binding protein associated with cell self-renewal and survival in various of stem cells,but the effects of lin28B on MSCs in AD have not yet been elucidated.AimThe purpose of the present study was to explore whether lin28B might improve the survival of implanted MSCs and strengthen their neuroprotective potential in AD.Material and Methods1.MSCs were extracted from the femurs of 6-week-old C57BL/6J male mice and cultivated by whole bone marrow culture assay.For our experiments,MSCs were used at passages 4-10.2.Lin28B upregulated lentivirus and vector lentivirus were constructed respectively,and then were transfected into MSCs,respectively.MSCs were divided into three groups:lin28B MSCs(MSCs treated with lin28B upregulated lentivirus),vector MSCs(MSCs treated with vector lentivirus),and control MSCs(untreated MSCs).qRT-PCR and western-blot assay were used to detect the expression of lin28B mRNA and lin28B protein in each group.3.The Cell Counting Kit-8(CCK-8)was utilized to test the proliferation ability of the MSCs,and wound-healing and transwell assay were applied to detect the migration capacity of MSCs.4.MSCs were exposed to A?1-42 for 48 h,and the proportion of apoptotic cells in each group were examined using Annexin V/APC staining.The expression of caspase 3 and cleaved caspase 3 protein were detected by western blot.5.The level of IGF-2 were detected in all groups,and then small interfering RNA(SiRNA)was employed to knowdown the expression of IGF-2 in MSCs.After that,the proliferation and migration capacity of MSCs were examined.Besides,the apoptosis of MSCs treated with A?1-42 were also tested.6.Lin28B MSCs,vectors MSCs,and control MSCs were injected into the right lateral ventricle of ten-month-old amyloid precursor protein(APP)/presenilin 1(PS1)transgenic mice,respectively.The mice were divided into five subgroups:control group,PBS group,control-MSCs group,lin28B-MSCs group and vector-MSCs group.7.We used in vivo bioluminescence imaging(BLI)to monitor the survival and proliferation of the transplanted MSCs.BLI was performed in these mice at day 1,3,7,14,21,and 28 after the cells injection.8.At day 30-35 after injection,Morris water maze tests were carried out to assess the spatial learning capacity of the mice.9.APP/PS1 mice were sacrificed after behavioral testing and their brain paraffin sections were further processed for immunohistochemistry analysis.Thioflavin-S staining was utilized to assess the distribution of amyloid plaques.Tissue cell death analysis was performed using terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL),and the activation of microglia cells in brain tissue was detected by Iba-1 antibody.Results1.After upregulating the level of lin28B protein by lentivirus in vitro,the proliferation and migration capacity of lin 28B MSCs was enhanced compared with that of control MSCs group and vector MSCs group.Besides,the proportion of apoptotic cells of lin28B MSCs was less after A?1-42 treatment,and the levels of caspase 3 and cleaved caspase 3 were lower than those of the other two groups.2.The up-regulation of lin28B in MSCs can promote the expression of IGF-2 protein.When the level of IGF-2 was knocked down by siRNA,the proliferation,migration and anti-apoptotic ability of MSCs were weakened.3.BLI indicated that the proliferation and survival of MSCs were enhanced by lin28B compared to vector MSCs in APP/PS1 mice.4.Morris water maze test indicates that the mice in lin28B MSCs group took the shortest time to find the platform,the most times to cross the platform and the longest time in the target quadrant compared to other groups.5.Immunohistochemical analysis showed that the transplanted MSCs promoted the clearance of amyloid plaque,decreased the activation of microglia,as well as reduced the death of neuronal cell in vivo,especially in the group of lin28B MSCs.Conclusion1.The overexpression of lin28B promoted the proliferation and migration capacity of MSCs and inhibited A?1-42 induced apoptosis through regulating IGF-2 in vitro.2.In vivo,administration of the lin28B upregulated MSCs mitigated cognitive deficits,promoted amyloid plaque clearance,decreased the activation of microglia,as well as reduced neuronal cell death,and thereby enhancing the protective effects of MSCs against AD.