Effects Of Photobiomodulation On Bone Marrow Mesenchymal Stem Cells Under High Glucose Condition Through PI3K

Photobiomodulation(PBM)is a method of using red or near-infrared light to prevent and treat diseases and promote the recovery of the body.Studies have shown that PBM has obvious effects on regulating blood glucose,improving insulin resistance,delaying retinal degeneration and other diabetes and its complications.mesenchymal stem cells(MSCs)are undifferentiated cells with self-renewal and multi-directional differentiation potential,which can be isolated and cultured from various tissues such as bone marrow,fat,umbilical cord and so on.The homing and differentiation ability,low immunogenicity and immunomodulatory effect of MSCs make them have great application potential in diabetes stem cell therapy.Diabetic hyperglycemia can cause MSCs dysfunction and its microenvironment changes.How to protect MSCs from the adverse effects of hyperglycemia environment is of great significance in diabetes stem cell therapy.Some studies have found that PBM can improve diabetic retinal vascular degeneration and increase stem cell factor(SCF)and stromal cell derived factor-1α(SDF-1α)in serum and bone marrow supernatant of diabetic model mice levels.On this basis,we took bone marrow derived bone mesenchymal stem cells(BMSCs)as the research object to investigate whether 680 nm red light could improve the function of BMSCs under high glucose condition,and further to explore the regulatory effects of PBM on BMSCs.It provides new ideas for the treatment of diabetes stem cells.1.ObjectiveIn this study,680 nm red light was used to explore the regulatory effect of PBM on the cell viability,apoptosis,migration and other biological functions of BMSCs under high glucose conditions,and to reveal the mechanism of PBM in regulating BMSCs under high glucose conditions,so as to provide ideas and methods for improving the adverse effects of high glucose environment on MSCs and improving their therapeutic ability.2.Methods(1)Identification of primary cultured BMSCs:The morphology of BMSCs was observed under a light microscope and the expression of surface markers of BMSCs was detected by flow cytometry.(2)The effects of PBM on the viability of BMSCs:CCK8 method was used to detect the effects of different doses of 680 nm red light(1 J/cm~2,2 J/cm~2,4 J/cm~2,8J/cm~2)on the viability of BMSCs under high glucose condition.(3)Flow cytometry and Western blot were used to detect the effects of PBM on the apoptosis of BMSCs under high glucose.(4)Effects of PBM on the migration of BMSCs:The effects of 680 nm red light on the migration of BMSCs under high glucose condition was detected by cell scratch test and Western blot.(5)The effects of PBM on the expression of SCF,VEGF,CXCL12(SDF-1),CXCR4 and IL-6,TGF-βm RNA in BMSCs under high glucose condition was detected by RT-PCR.Western blot was used to detect the effects of 680 nm red light on the expression of CXCR4,PI3K/AKT proteins in BMSCs under high glucose.PI3K inhibitor LY294002 was used to further verify whether PI3K/AKT pathway was involved in the biological function of BMSCs regulated by PBM.3.Results(1)Under light microscope,the primary cultured BMSCs grew in a swirl and adhered to the wall,and the cell morphology was fusiform and fibrocyte-like.Flow cytometry showed that the positive markers CD90 and CD44 of MSCs were highly expressed,and the negative markers CD45 and CD103 were low expressed,which was consistent with the immunophenotypic characteristics of MSCs.(2)The cell viability of BMSCs decreased significantly under high glucose condition,but increased significantly after 680 nm red light treatment(2 J/cm~2).(3)Flow cytometry showed that high glucose promoted the apoptosis of BMSCs,while 680 nm red light inhibited the apoptosis of BMSCs induced by high glucose.Meanwhile,western blot showed that 680 nm red light could down-regulate the increase of Bax/Bcl2 ratio induced by high glucose.(4)High glucose inhibited the migration of BMSCs,while 680 nm red light could promote the migration of BMSCs under high glucose condition,and promoted the expression of migration-related proteins MMP2,MMP9 and N-cadherin.(5)RT-PCR results showed that the m RNA expression levels of SCF,VEGF,CXCL12(SDF1),CXCR4 and TGF-βdecreased and the m RNA expression level of IL-6 increased under high glucose condition.680 nm red light could inhibit the effects of high glucose on the expression of these m RNA.(6)Western blot results showed that compared with the control group,high glucose inhibited the expression of CXCR4,PI3K and AKT proteins,while 680 nm red light could promote the expression of CXCR4,PI3K and AKT proteins under high glucose.PI3K inhibitor LY294002 not only blocked the proliferation and migration of BMSCs promoted by PBM,but also inhibited the expression of PI3K and AKT proteins in BMSCs promoted by 680 nm red light under high glucose condition.4.Conclusion(1)PBM can promote the viability and migration of BMSCs under high glucose condition,and inhibit the apoptosis of BMSCs induced by high glucose,which may be related to the inhibition of inflammatory response and the promotion of CXCL12(SDF1)and CXCR4 expression in BMSCs.(2)PBM may regulate the cell viability,apoptosis,migration and other functions of BMSCs under high glucose condition through PI3K/AKT signaling pathway.