Age-related Bone Marrow Macrophages Derived Exosomal MicroRNA-191-5p Regulates Bone Marrow Mesenchymal Stem Cells Lineage Fate Switch

Background:In vitro in vitro mesenchymal stem cells,the expression of miRNA-191-5p was overexpressed,the expression of m RNA in bone differentiation indicators(Runt-related transcription factor 2,alkaline phosphatase,osteopreferoprotein,etc.)was significantly increased,and the expression of lipid differentiation indicators such as peroxisome proliferator activated receptor γ(PPAR-γ)and fatty acid binding protein 4(Fabp4)was inhibited,which was proved by cell staining.The downstream target sites of miRNA-191-5p were explored through multiple prediction websites,and the target gene SMAD2/3 involving bone metabolism and cell aging was found in the prediction results of each website with a number of elements of ten.Double luciferase experiments verified that miRNA-191-5p disappeared when the SMAD2/3 gene mutation was mutated;the Western blotting experiment showed that the overexpression of miRNA-191-5p reduced the expression of SMAD2/3 protein,and the intensity of protein band development was significantly lower than that of the NC control group;and the immunofluorescence of bone tissue was manifested as a significant decrease in the fluorescence intensity of SMAD2/3 after overexpression of miRNA-191-5p.In view of the current status of osteoporosis prevention and treatment and the bone effect of miRNA-191-5p,we have overexpressed in elderly mice in the hope of achieving clinical translation of the results.Overexpressed miRNA-191-5p exosome binding aptamer after tail vein injection into the elderly mouse,thanks to the specific targeting effect of the aptamer on the bone marrow mesenchymal stem cells,the promoting bone effect of young bone marrow macrophages was verified in the elderly mice,manifested by the increase in the bone volume fraction(BV/TV,%)of the micro-CT result,the increase in the number of osteocalcin-positive cells on the surface of the immunohistochemical stained bone trabecle and the significant decrease in the content of lipid drops in the bone marrow cavity,The low bone mass status of the elderly mice was improved.The above findings may provide an important basis for elucidating the complex pathological mechanisms of osteoporosis and provide promising drugs for the clinical treatment of osteoporosis.Conclusions:1)Young bone marrow macrophages significantly promote bone differentiation of mesenchymal stem cells,and the positive induction effect on osteogenesis differentiation of mesenchymal stem cells decreases with age.2)miRNA-191-5p-rich bone marrow macrophage exosomes mediate age-related macrophage bone mass regulation.3)Young bone marrow macrophage exosomes improve bone mass in older mice and slow down age-related bone loss.Bone marrow macrophages play a key regulatory role in the age-related bone marrow microenvironment and may be potential therapeutic targets for age-related osteoporosis.