Exosomes Secreted By MLO-Y4 Induced By Mechanical Stimulation Promotes The Osteogenic Differentiation Of Ligamentum Flavum Cells Through PI3K/AKT Pathway

BackgroundOssification of the ligamentum flavum(OLF)is a chronic degenerative disease,which mainly occurs in the lower thoracic vertebrae.OLF can compress the spinal cord,nerve roots,conus medullaris or cauda equina,causing various types of neurological symptoms.OLF is characterized by progressive heterotopic ossification of the spinal accessory ligament.It is common in East Asian countries,especially China and Japanese.The pathogenesis of OLF is unclear,which is closely related to age,metabolism,heredity and lifestyle.Because the initial symptoms of OLF are hidden and gradually progressing,they are often serious when found,which requires surgical treatment.Which brings a heavy burden to the family and society.Exosomes are extracellular vesicles with double-layer membrane structure,with a diameter of about 40-160nm.Exosomes contain small molecules such as protein,DN A,RNA,lipids and enzymes.Exosomes,as a hot research topic in recent years,play a role in all fields of life activities.When cells receive mechanical stimulation,the content and types of small molecular substances in exosomes will change,and the effect on recipient cells will also change.In this study,the mechanical stimulation-exosome(MSExosome)secreted by bone cell MLO-Y4 was extracted by periodic tension stimulation,and acted on Human Ligamentum Flavum Cells(hLFC)to observe its effects on the proliferation and osteogenic differentiation of hLFC.Furthermore,the mechanism of MS-Exo promoting osteogenic differentiation of hLFC was discussed,which provided new ideas for the study of the pathogenesis of OLF.Methods1.Normal ligamentum flavum samples were collected,and hLFC was extracted by collagenase I digestion and tissue block method.Vimentin and S100A4 were identified by Western Blot and immunofluorescence as markers.It was verified by Western Blot that it had osteogenic differentiation ability.2.Stimulating MLO-Y4 by periodic tension stimulation to obtain cell supernatant,and obtaining supernatant through normal culture.MS-Exo and Exosome were extracted by ultracentrifugation.The exosomes were identified by Transmission Electron Microscope(TEM),Dynamic light scattering(DLS)and Western Blot.Then the exosomes were labeled by PKH26,and the endocytosis experiment was carried out to verify that the exosomes acted by entering the cells.3.Dexamethasone and exosomes act on hLFC alone or together.The experiment was divided into Control group,Dexamethasone group,Dexamethasone+Exo group and Dexamethasone+MS-Exo group.The effect on the proliferation of hLFC was detected by CCK-8 and EdU.The effect on osteogenic differentiation of hLFC was detected by ALP staining,Alizarin red staining and Western Blot4.We conducted high-throughput sequencing on MS-Exo and Exosome.We choose PI3K inhibitor-LY294002.The experiment was divided into Control group,Dexamethasone group,Dexamethasone+Exo group,Dexamethasone+MS-Exo group and Dexamethasone+MS-Exo+LY294002 group.The effect on osteogenic differentiation of hLFC was detected by Alizarin red staining and Western BlotResults1.HLFC was successfully extracted by collagenase digestion+tissue block method.It was shown to be fibroblast-like and spindle-shaped adherent cells under light microscope.Both of them expressed Vimentin and S100A4 by Western Blot and immunofluorescence.After osteogenic differentiation,the expression of osteogenic indexes was increased by Western Blot.2.MS-Exo and Exosome were obtained by ultracentrifugation.They express CD9 and TSG101,and negatively express Calenxin detected by Western Blot.TEM showed that the exosomes were round-like bilayer lipid membrane structure,DLS showed that the diameter of exosomes was 10-100nm,and the peak value was 20-30nm.Cytophagy experiment showed that exosomes entered hLFC cells.3.CCK-8 and EdU showed that dexamethasone reduced the proliferation of hLFC,but MS-Exo reversed the effect of dexamethasone and promoted the proliferation of hLFC.ALP and alizarin red staining showed that dexamethasone reduced the formation of calcium nodules,while MS-Exo promoted the formation of calcium nodules.The results of Western Blot were consistent with the results of alizarin red staining,and both of them promoted the osteogenic differentiation of hLFC.4.Exosome sequencing showed that there was a difference in phosphatidylinositol signal pathway between MS-Exo and Exosome.Alizarin red staining showed that dexamethasone reduced the formation of calcium nodules,and MS-Exo promoted its formation.When LY294002 was added,the calcium nodules decreased again.Western Blot showed that dexamethasone reduced the expression of osteogenic related genes,and MS-Exo reversed the inhibitory effect of dexamethasone.When LY294002 was added,the expression of osteogenic related genes decreased again.ConclusionMS-Exo obtained by periodically stimulating MLO-Y4 under tension stimulation promoted the proliferation of hLFC,and promoted the osteogenic differentiation of hLFC through PI3K/AKT pathway.