Mechanistic Study Of Umbilical Cord Mesenchymal Stem Cellsexosomes In The Treatment Of Intervertebral Disc Degeneration By Relieving Oxidative Stress Of Degenerative Nucleus Pulposus Cells

Objective:To investigate the effect of human umbilical cord mesenchymal stem cells-exosomes on intervertebral disc degeneration and further reveal the internal mechanism of its therapeutic effect on IDD.Methods:Umbilical cord mesenchymal stem cells and nucleus pulposus cells were extracted from neonatal umbilical cord tissue and nucleus pulposus tissue,respectively.UC-MSCs were amplified and cultured.The exosomes in the supernatant of UC-MSCs were extracted by ultracentrifugation.The morphology of the particles in the extract was observed by transmission electron microscopy.Zeta View Nanoparticle Tracking Analysis were used to track the particle size and Western blot was used to detect the exosome marker protein.After the extracted UC-MSCs-Exos were applied to NPCs,the cell proliferation ability and extracellular matrix content were detected by Western blot and immunofluorescence.The effects of UC-MSCs-Exos on H₂O₂-induced reactive oxygen species in NPCs were detected by DCFH-DA immunofluorescence and flow cytometry.Mito SOX immunofluorescence and flow cytometry were used to detect mitochondrial superoxide levels.Mito Bright LT Deep Red fluorescence staining was used to observe mitochondrial morphology.The SD rat tail disc degeneration model was established,and UC-MSCs-Exos was injected into the degenerated rat tail disc by rat tail disc injection technique.The therapeutic effect of UC-MSCs-Exos on the degenerated tail disc of SD rats was detected by MRI and immunohistochemical staining.Results:The surface antigen detection of UC-MSCs by flow cytometry showed that the positive rates of UC-MSCs positive antigens CD73,CD90 and CD105 were 82.41%,99.97%and 90.50%,respectively.The positive rates of UC-MSCs negative antigens CD34,CD45 and HLA-DR were 5.29%,5.51%and 5.29%,respectively.Immunohistochemical staining showed that the type II collagen IHC staining of adherent cells isolated from human nucleus pulposus tissue was darker and the cells were brown.Safranin O staining（chondrocytes）showed that the staining was light pink;UC-MSCs-Exos were observed by transmission electron microscopy as a bilayer membrane vesicle structure.Zeta View nanoparticle tracking analysis showed that the concentration of UC-MSCs-Exos was 10¹¹particles/ml and the average diameter was 100 nm.Western blot results showed that Alix,CD63 and TSG101 were highly expressed in the extract,and GAPDH was lowly expressed.After UC-MSCs-Exos acted on NPCs,the proliferation rate of NPCs was increased under light microscope,and the positive rate of Ki67in NPCs was increased by immunofluorescence.Western blot and immunofluorescence showed that UC-MSCs-Exos up-regulated the expression of COL2A1 and down-regulated the expression of MMP13 in NPCs treated with H₂O₂.The results of flow cytometry and immunofluorescence showed that UC-MSCs-Exos decreased the positive rate of DCFH-DA in NPCs induced by H₂O₂.Mito SOX positive rate decreased,and can reduce H₂O₂-induced NPCs mitochondrial structural abnormalities.MRI results suggest that UC-MSCs-Exos injection can increase the signal intensity of the punctured intervertebral disc in the SD rat tail disc degeneration model.Immunohistochemical staining showed that UC-MSCs-Exos could treat the unclear intervertebral disc structure and narrowed intervertebral space caused by SD rat tail disc puncture.The content of proteoglycan in tissues increased.Conclusion:In vitro experiments showed that UC-MSCs-Exos could enhance the proliferation of NPCs and increase ECM content.This may be related to UC-MSCs-Exos reducing ROS and mitochondrial superoxide production in NPCs,inhibiting oxidative stress,and reducing the destruction of mitochondrial structure.In vivo experiments showed that UC-MSCs-Exos could slow down the IDD process in SD rat tail disc degeneration model.