The Mechanism And Impact Of Amyloid β_1-42on Proliferation And Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective:With the problem of aging tendency becoming increasingly serious all over the world,the morbidity of Alzheimer’s disease（AD）,the most common progressive neurodegeneration disease which is characterized by cognitive function decline,behavioral disorders,and even the loss of self-care ability clinically,increases year by year,and brings severe burdens to families and our society.Accumulating clinical evidence has shown that such patients are more likely to suffer from hip bone mass loss and hip fracture,which further improves the cost for treatment.Whether such osteoporotic changes are pathological features of Alzheimer’s disease or attached to a disuse osteoporosis secondary to motor dysfunction caused by neurofunction decline remains poorly understood.As is known,the maintaining of human bone mass depends on the dynamic balance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts,while the osteoblasts mainly come from their progenitor cells-bone marrow mesenchymal stem cells,whose quantity and ability of osteogenic differentiation are closely correlated with the genesis and development of osteoporosis.Because both Alzheimer’s disease and osteoporosis are the combined effect of mulity-factor,it is difficult to identify the cause of Alzheimer’s disease-related osteoporosis.As a characteristic pathological product of Alzheimer’s disease,Amyloidβ_1-42(Aβ_1-42)plays an important role in the decelopment of Alzheimer’s disease.Our previous study based on APP/PS1 transgenic mice,an animal model of Alzheimer’s disease,has showed that overexpressed Aβ_1-42 could be detected in bone tissue,along with a significant decrease in bone mass,suggesting that such bone mass loss might be associated with the excess Aβ_1-42.It has been reported that Aβ_1-42-42 could increase the bone resorption by activating the osteoclasts,but the impact of Aβ_1-42 on osteogenic effect,especially the bone marrow mesenchymal stem cells,has not been reproted.Therefore,the main purpose of this study is to determine whether Aβ_1-42 can affect the proliferation and osteogenic differentiation ability of bone marrow mesenchymal stem cells,and to explore the relevant mechanisms to provide more theoretical basis for our further understanding of Alzheimer’s disease-related osteoporosis and lay a theoretical foundation for proposing preventing and treating measures for Alzheimer’s disease-related osteoporosis.Methods:Part one:Bone marrow mesenchymal stem cells（BMSCs）of sprague dawley rat were used in this study,to clarify the effect of diferent concentrations of Aβ_1-42-42 on the proliferation of BMSCs after 48 h culture,cell counting kit-8（CCK-8）was employed to evaluate the cell viability of BMSCs,the expression of proliferation cell nuclear antigen（PCNA）of BMSCs was evaluated by westen blotting,flow cytometry was used to detect the percentage of cells which stayed at S-phase in the cell cycle.EdU staining was also used to determine the proportion of cells in the DNA synthesis phase.Part two:based on the results that the Aβ_1-42 could inhibit the proliferation of BMSCs in part one.We detected the level of intercellular reactive oxygen species（ROS）by flow cytometry,at the same time,groups devided into control,200μM/L H₂O₂,5μM/L Aβ_1-42and 5μM/L Aβ_1-42+2.5mM/L N-AC were set to determine the relationship between ROS and the proliferation of BMSCs.Then we employed westen blotting,transmission electron microscope（TEM）and immunofluorescence to detect the autophagy level of BMSCs,subsequently,three time point including 0 h,4 h,8 h were set,westen blotting and immunofluorescence were used to detect precedence relationship between ROS and autophagy.Based on 5μM/L Aβ_1-42,autophagy inducer rapamycin（RAPA）and inhibitor3-methyladenine（3-MA）were used to further induce and inhibit the autophagy of BMSCs,then intercellular ROS detected by flow cytometry,cell viability evaluated by CCK-8 assay,expression of PCNA determined by westen blotting and cell percentage of S-phase in cell cycle evaluated by flow cytometry were used to assess the effect of autophagy on the intercellular ROS level as well as the proliferation of BMSCs.To clarify the mechanism how ROS regulates the autophagy level under the treatment of Aβ_1-42,four groups were set being control,200μM/L H₂O₂,5μM/L Aβ_1-42 and 5μM/L Aβ_1-42+2.5mM/L N-AC,after 12 h culture,the expression of AKT/mTOR signaling pathway-related proteins AKT,p-AKT,mTOR,p-mTOR and autophagy-related proteins were detected by westen blotting.Part three:the balance between adipogenic differentiation and osteogenic differentiation of BMSCs is one of the key factors to prevent the development of osteoporosis.In this part,we focused on the impact of different concentrations of Aβ_1-42 on the osteogenic differentiation of BMSCs,the osteogenic differentiation ability were calculated by ALP activity,protein expression of OCN and Runx2 and the formation of calcium nodules.The Wnt/β-catenin pathaway-related proteinsβ-catenin,GSK-3βand p-β-catenin were also evaluated by westen blotting.Further more,we focused on the group of 5μM/L Aβ_1-42,whose changes in osteogenic differentiation were the most remarkable,the Wnt/β-catenin pathaway agonist BML-284 and inhibitor JW74 were used and the ALP activity,expression of OCN and Runx2,formation of calcium nodules were tested again to clarify the role of Wnt/β-catenin pathaway in osteogenic differentiation of BMSCs under the treatment of Aβ_1-42.Results:Part one:After the BMSCs were treated with Aβ_1-42-42 for 48 h,cell viability of BMSCs,the protein expression of PCNA,the proportion of S-phase BMSCs and the number of BMSCs whose EdU staining were positive decreased gradually with the increase of Aβ_1-42 in our concentrations.Part two:The effect of Aβ_1-42 on the accumulation of intercellular ROS in BMSCs was positively correlated with the concentration of Aβ_1-42.The inhibitory effects of Aβ_1-42 on the proliferation of BMSCs were similar to that of the H₂O₂,a classical oxidative stress inducer,while the suage of N-AC could relieve the inhibitory effect on proliferation of BMSCs treated with Aβ_1-42.After treating with Aβ_1-42 for 48 h,we also found that the autophagy level upregulated gradually with the increasing concentration of Aβ_1-42.Further testing demonstrated that changes in ROS happened prior to the occurrence of autophagy.Rapamycin,the autophagy agonist,could reduce the accumulation of intercellular ROS of BMSCs caused by Aβ_1-42 and alleviate the inhibition on proliferation of BMSCs treated with Aβ_1-42.But on the basis of Aβ_1-42,3-MA,the autophagy inhibitor,could further increase the intercellular ROS and inhibit the proliferation of BMSCs.By detecting the key proteins of AKT/mTOR signaling pathway,the classical autophagy regulatory pathway,we found that the ratio of p-AKT/AKT and p-mTOR/mTOR in H₂O₂ group and Aβ_1-42group were much higher than that in the Aβ_1-42-42 and N-AC co-treating group after 12 h treatment.At the same time,the expression of autophagy-related proteins detected by westen blotting showed that the autophagy was associated with ROS.Part three:the ability of osteogenic differentiation of BMSCs reduced gradually with the increase of Aβ_1-42,meanwhile the Wnt/β-catenin signaling pathway was also inhibited.When the Wnt/β-catenin signaling pathway was regulated,we found that BML-284,the pathway agonist,could alleviate inhibition on osteogenic differentiation of BMSCs caused by Aβ_1-42,while JW74,the pathway inhibitor could aggravate the decline of osteogenic differentiation of BMSCs treated with Aβ_1-42.Conclutions:（1）Aβ_1-42-42 inhibit the proliferation of BMSCs by upregulating the intercellular ROS level.（2）ROS induced by Aβ_1-42 can regulate the autophagy level of BMSCs via AKT/mTOR signaling pathway,and the occurrence of autophagy undr the treatment of Aβ_1-42 antagonize the inhibition of proliferation in BMSCs.（3）Aβ_1-42inhibit the osteogenic differentiation of BMSCs via the inhibition of the Wnt/β-catenin signaling pathway.