Hypoxic mesenchymal stem cell-derived exosomes decrease apoptosis and senescence by shifting M1/M2 macrophage polarization
Objective To observe whether hypoxic pretreatment can improve the efficacy of mesen-chymal stem cell extracellular vesicles in treating intervertebral disc degeneration,and explore the potential mechanisms of extracellular vesicles and macrophage polarization.Methods A non-contact co culture sys-tem for extracellular vesicles,macrophages,and nucleus pulposus cells was established.A control group,a normoxic group,and a hypoxic group were set up.The control group was given macrophages+lipopo-lysaccharide(LPS)or a co culture system for macrophages and nucleus pulposus cells.The normoxic group was given a co culture system with the addition of normoxic exosomes.The hypoxia group was given a co culture system with the addition of hypoxia induced exosomes.Through quantitative real-time polymerase chain reaction,flow cytometry,β-galactosidase staining and immunofluorescence staining,the effects of normoxic exosomes/hypoxia-induced exosomes on macrophage polarization,as well as on apoptosis and ag-ing of nucleus pulposus cells were measured.The comparison of inter group measurement data was done by t-test.Results Compared with the normoxic group,the expression level of CD86 in the hypoxic group was significantly reduced(2.78±0.31 vs.7.00±0.51,t=8.627,P＜0.05),while the expression level of CD163 was significantly increased(19.01±0.38 vs.15.54±0.44,t=9.499,P＜0.05).Flow cytome-try showed that compared with the normoxic group,the apoptosis rate of nucleus pulposus cells in the hy-poxic group was significantly reduced(14.44±0.40 vs.11.16±0.50,t=14.351,P＜0.05).The re-sults of galactosidase staining showed that compared with the normoxic group,the aging rate of nucleus pul-posus cells in the hypoxic group was significantly reduced(49.75±1.63 vs.59.71±1.63,t=4.425,P＜0.05).Immunohistochemical staining confirmed that compared with the normoxic group,the hypoxic group increased the expression of the synthetic gene COL2(1.35±0.03 vs.1.03±0.05,t=6.500,P＜0.05)and ACAN(1.66±0.05 vs.1.03±0.05,t=13.141,P＜0.05),and decreased the expression of the degradation gene matrix metalloproteinase(MMP)-13(0.80±0.04 vs.1.00±0.05,t=5.496,P＜0.05)and ADAMTS5(0.84±0.02 vs.1.00±0.05,t=6.723,P＜0.05).Conclusion Extracellular vesicles of mesenchymal stem cells reduce cell apoptosis and aging under hypoxic conditions in vitro,possi-bly through polarization of M1/M2 macrophages.
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