MG53 Protein Delays The Senescence Of HUC-MSCs And Exerts Neuroprotective Effect In Alzheimer’ Desease Mice Combined With HUC-MSCs Transplantation

Senescence is the irreversible degradation of the structure and function accompanied with the loss of physiological functions in cells,tissues and organs of the body,which ultimately lead to the occurrence of aging-related diseases.Alzheimer’s disease(AD)is a common age-related neurodegenerative disease that seriously affects human health.Studies have shown that the number of neural stem cells in the brains of AD patients has been greatly reduced and their vitality has been severely decreased.Human umbilical cord-derived mesenchymal stem cells(h UC-MSCs)have the potential for self-renewal and multi-directional differentiation,which can promote tissues regeneration and bring hope for the treatment of neurodegenerative diseases,such as AD.However,as the number of passages increases,h UC-MSCs cultured in vitro exhibit senescent characteristics,such as decreased proliferation and differentiation,and the loss of stemness,which would seriously affect their therapeutic effects.Therefore,how to delay the senescence of stem cells and increase their activity are the keys to enhance the efficacy of stem cells.Mitsugumin53 protein(MG53)is a member of the Tripartite motif(TRIM)family,which plays an important role in cell and tissue damage repair.Studies have shown that recombinant human MG53 protein(rh MG53)can protect bone marrow mesenchymal stem cells from membrane damage induced by low density lipoprotein and improve stem cell survival rate.Our previous research found that rh MG53 could enhance the antioxidant capacity of h UC-MSCs and promote the therapeutic effect of stem cells on traumatic brain injury.However,there is no research on whether MG53could delay h UC-MSCs senescence and improve the neuroprotective effect of stem cell transplantation on AD.ObjectivesThis project intends to use replicative senescent h UC-MSCs as an aging model and MG53 as a breakthrough point to study the effects of MG53 protein on the proliferation,apoptosis,cycle,migration,aging and therapeutic efficacy of senescent h UC-MSCs through in vivo and in vitro experiments and their molecular mechanism.Meanwhile,the synergistic therapeutic effect and molecular mechanism of MG53protein combined with h UC-MSCs transplantation on AD mice were explored.MethodPart I:MG53 protein delays the senescence of h UC-MSCs in vitro and in vivo(1)Replicative senescence model of h UC-MSCs was established and then MG53 was added.CCK-8 assay,Ed U staining,transwell and flow cytometry were used to detect the survival,proliferation,migration,apoptosis and cycle of h UC-MSCs cells;β-Gal staining,DAPI staining and immunofluorescence were used to analyze the effect of MG53 on the senescence of h UC-MSCs senescence;Oxidative stress related kit were performed to examine the effect of MG53 on the oxidative stress level of aging h UC-MSCs cells;Inflammatory factor detection chips were used to measure the effect of MG53 on the release of inflammatory factors in senescent h UC-MSCs cells;Western blot and q RT-PCR were applied to examine the effect of MG53 on the expression of senescence-related genes(P16,P21,P53,p CNA and Sirt1)and Nrf2 signaling pathway related proteins(Nrf2,SOD1,NQO1 and Keap-1)in h UC-MSCs cells.(2)AD mice were randomly divided into APP~+group,P5 h UC-MSCs transplantation group(P5 MSCs group),P15h UC-MSCs transplantation group(P15MSCs group),and MG53 pretreated P15 h UC-MSCs transplantation(MG53-P15MSCs)group;Morris water maze and new object recognition were used to test the learning and memory and cognitive ability of AD mice;Forced swimming test,tail suspension test and open field test were used to evaluate the anxiety and depression of AD mice in each group;Tunel,Nissl and immunofluorescence were used to detect the number of apoptotic cells,neural corpuscles and mature neurons in the brain of AD mice 28 days after transplantation.Part II:Neuroprotective effect of MG53 protein combined with h UC-MSCs transplantation on Alzheimer’s diseases mice(1)100 AD mice were randomly divided into APP~+group,MSCs group,MG53group and MSCs+MG53 group;Morris water maze and new object recognition were used to test the learning and memory and cognitive ability of AD mice in each group;Forced swimming test,tail suspension test and open field test were used to evaluate the anxiety and depression among the four groups of AD mice;Di I-labeling assay and MAB1281 immunofluorescence were used to detect the survival of h UC-MSCs in the MG53 and MSCs+MG53 groups at 3,7 and 14 days after transplantation;Tunel and Nissl’s staining were used to detect the number of apoptotic cells and neurosomes in AD mice at 28 days after transplantation.The immunofluorescence and thioflavin S staining were used to detect inflammation and Aβdeposition in AD mice;SA-β-Gal staining was used to detect the number of senescent cells in the brains of AD mice;Immunofluorescence double-labeling method were used to detect the neuroregeneration in the brain of AD mice;Western blot was used to detect Tau phosphorylation and expression of Nrf2-related pathway proteins.(2)The Nrf2 inhibitor BRU was used to verify whether MG53 combined with h UC-MSCs exerted neuroprotective effect on AD mice by activating the Nrf2pathway.Morris water maze,open field test,forced swimming test and tail suspension test were used to evaluate the cognitive recovery and anxiety and depression of AD mice in each group.Nissl staining and immunofluorescence were used to detect neural apoptosis and nerve regeneration.ResultsPart Ⅰ:MG53 protein delays senescence of h UC-MSCs in vitro and in vivo1.With the increase of passages,h UC-MSCs exhibited decreased proliferation ability,arrested cell cycle,increased apoptosis and senescent cells,senescence-related heterochromatin accumulation(SAHF),nucleus enlargement and increased expression of senescence-related genes.Compared with the untreated group,MG53protein pretreatment promoted the proliferation and migration of h UC-MSCs,reduced G0/G1 phase arrest and inhibited apoptosis(P<0.05).MG53 reduced SA-β-Gal positive cells,SAHF formation and increased the expression of Lamin B1 by inhibiting P16,P21,P53,TNF-α,IL-6,IL-8,IL-1β,IL-1a,IL-6 expression,and increasing the expression of p CNA,Sirt1 and IL-10 release(P<0.05).In addition,MG53 reduced the content of ROS and MDA in senescent h UC-MSCs,while increased the SOD activity(P<0.05);Western blot results showed that MG53 significantly reduced the expression of Keap-1 and promoted the expressions of Nrf2,NQO1 and SOD1 in senescent h UC-MSCs(P<0.05).2.Compared with the P15MSCs group,mice in MG53-P15 MSCs group showed a decrease in the escape latency,while significant increase in the number of crossing the platform,time spent in the target quadrant and new object identification index(P<0.05).The results of forced swimming text,tail suspension text and open field text showed that transplantation of MG53-P15MSCs reduced immobility time and increased the number of feedings and distance of AD mice(P<0.05).Further research showed that compared with the P15MSCs group,MG53-P15 MSCs can increased the number of Neu N~+cells and decreased the number of Tunel~+cells(P<0.05).Part Ⅱ:Neuroprotective effect of MG53 protein combined with h UC-MSCs transplantation on Alzheimer’s diseases mice1.The results of Morris water maze,new object recognition test,novelty suppression feeding test and open field test showed that MG53,MSCs and MG53+MSCs groups showed decreased escape latency,shortened feeding incubation period,while increased crossings number,time in the target quadran,new object identification index,food intake,the distance moved and the number of uprights compared with the APP~+group,(P<0.05).And,the MG53+MSC group got the most significant effects(P<0.05).Forced swimming test,tail suspension test and sugar water preference experiment showed that the immobility time and floating rest time in the APP~+mice after MG53,MSCs or MG53+MSCs treatment were significantly reduced compared with APP~+group,and the sugar water bias was significantly increased(P<0.05).Compared with the APP~+group,the number of Nissl bodies in the hippocampal CA1 area of the MG53 group,the MSCs group and the combined group increased significantly,while the number of Tunel~+cells,GFAP~+cells and Iba-1~+cells decreased significantly,and the combined group changed most obviously(P<0.05).In addition,MG53 combined with h UC-MSCs transplantation significantly reduced serum MDA content and increased SOD activity(P<0.05),reducedβ-Gal~+cells,Aβplaque deposition and Tau protein phosphorylation in APP~+mouse brain(P<0.05).Di I staining and MAB1281 immunofluorescence showed that MG53 promoted the survival and migration of h UC-MSCs in APP~+mouse brain tissue(P<0.05).Compared with the APP~+group,the number of Ed U/DCX,Ed U/Neu N and Ed U/Nestin positive cells in each treatment group increased significantly,and the number of positive cells in the combination-treated group was the highest(P<0.05).Western blot showed that compared with the APP~+group,Keap1 expression decreased in the MG53 group,MSCs group and MG53+MSCs group,while the expression of Nrf2,NQO1,SOD1 in the nucleus significantly increased,and the combination-treated group got the most significant changes(P<0.05).However,the expression of Nrf2 in the cytoplasm was not significantly different between the groups(P<0.05).2.BRU could inhibit the Nrf2 pathway and attenuate the therapeutic effect of MG53 combined with h UC-MSCs transplantation on APP~+mice accompanied with reversed cognitive function and anxiety and depression in APP~+mice(P<0.05).Moreover,BRU significantly ameliorated the MG53+MSCs induced Nissl bodies,Aβdeposition and neurogenesis improvement(P<0.05).Thus,MG53+MSCs exerted neuroprotective effect in Alzheimer’desease mice by activating the Nrf2 pathway.Conclusion1.MG53 could promote the proliferation and migration of h UC-MSCs,inhibit apoptosis and oxidative stress,and delay the senescence in the senescent h UC-MSCs by activating the Nrf2 signaling pathway.MG53 pretreatment could improve the therapeutic effect of P15 h UC-MSCs transplantation on APP~+mice.2.MG53 could activate the Nrf2 signaling pathway and enhance the neuroprotective effect of h UC-MSCs transplantation on AD mice.