DMSC-derived Small Extracellular Vesicles Regenerated High Glucose Senescent Fibroblasts And Promoted Wound Healing In Diabetic Mice

Objective1.To explore the effects of high glucose on human dermal fibroblasts（HDFs）and establish high glucose induced senescent HDF model.2.To investigate the effects of d MSC-CM on high glucose induced senescent HDFs.3.To explore the effects and mechanisms of d MSC-s EVs on high glucose induced senescent HDFs.4.To investigate the effects and mechanisms of d MSC-s EVs on diabetic wounds.Methods1.HDFs were obtained from human foreskin.The groups were NG group（Normal glucose,5.5 m M glucose DMEM）,HG1 group（High glucose 1,26 m M glucose DMEM）,and HG2 group（High glucose 2,35 m M glucose DMEM）.Cell proliferation rate was detected by CCK-8 kit,cell migration rate was detected by scratch assay;The level of reactive oxygen species（ROS）was detected by DCFH-DA probe;Senescence associatedβgalactoses staining（SA-β-gal）activity was detected by SA-β-gal staining kit;The expression of aging related protein p21was detected by western blot after 7 days of culture in each group.2.d MSCs were isolated from human placenta,which were identified and passed to the 5^th generation.d MSCs treated with or without s EVs inhibitor GW4869and the conditioned media was collected.The high glucose induced senescent HDFs were divided into 3 groups:HG2,HG2+d MSC-CM,and HG2+d MSC-CM+GW4869.Cell proliferation rate was observed by CCK-8,cell migration rate was observed by scratch assay.3.d MSC-s EVs were isolated from d MSC-CM by differential high-speed centrifugation,and was identified by transmission electron microscopy,nanoparticle tracking analysis technology and western blot.s EVs initialization assay was conducted to observe whether d MSC-s EVs enter HDFs.The high glucose induced senescent HDFs were divided into 5 groups:HG2,HG2+d MSC-s EVs 1.74×10¹¹particles/m L,HG2+d MSC-s EVs 3.48×10¹¹ particles/m L,HG2+d MSC-s EVs5.22×10¹¹ particles/m L and HG2+d MSC-s EVs 6.92×10¹¹ particles/m L.Then we observed the cell proliferation rate,cell cycle,cell migration,intracellular ROS level and SA-β-gal activity of HDFs.Western blot detected the expression of collagen typeⅠ（colⅠ）,alpha-Smooth muscle actin（α-SMA）and p21.Western blot was used to detect the expression of the RAGE and its downstream protein p21 RAS and Smad signaling pathway protein phosphorylated Smad2/3（p Smad2/3）.In order to clarify the role of Smad signaling pathway,p Smad2/3 inhibitor SB431542 was added in advance to the fibroblasts.The experiment was divided into 3 groups:HG2,HG2+d MSC-s EVs,HG2+d MSC-s EVs+SB431542.SA-β-gal staining was used to observe the activity of SA-β-gal activity in each group.4.A wound model of diabetic mice was established,and an equal volume of s EVs and PBS were injected around the wound edge.Wound margin skin tissues were taken at 7,14,21,and 28 days after injury.Hematoxylin-eosin staining（HE）was used for histological observation.Masson staining was used to observe collagen deposition.Immunofluorescence staining was used to observe proliferation（proliferating cell nuclear antigen,PCNA）,migration（CXCR4）,differentiation（α-SMA）and senescence（p21）of fibroblasts around the wound.Results1.The results of CCK-8 cell proliferation assay and scratch assay showed that the proliferation and migration capacity of fibroblasts could be significantly reduced in a 35 m M high-glucose environment.The results of DCFH-DA probe showed that the 35 m M high glucose environment significantly increased the level of intracellular reactive oxygen species.The results of SA-β-gal staining showed that 35 m M high glucose environment could induce the production of more senescent cells.Western blot results showed that the expression of p21 was significantly up-regulated in the 35m M high-glucose environment.2.The cells isolated from the placental decidua are fusiform.Flow cytometry showed that CD90,CD73 and CD105 were highly expressed,and CD19,CD45,CD34 and HLA-DR were low expressed.The results of directed differentiation experiments showed that d MSCs could be induced into osteoblasts,adipocytes and chondroblasts.The results of CCK-8 cell proliferation assay and scratch assay showed that d MSC-CM could significantly improve the proliferation and migration of HDFs.However,with the addition of GW4869,the effect of d MSC-CM on HDFs proliferation and migration was significantly reduced,suggesting that s EVs in d MSC-CM may be a major factor in the repair of high glucose induced senescent HDFs.3.The results of transmission electron microscopy and nanoparticle tracking analysis showed that the diameter of particles isolated from d MSC-CM was consistent with the s EV’s diameter.Western blot results showed that the particles expressed CD9,CD63 and CD81,and it was determined that the particles belonged to s EVs.s EVs internalization experiment results showed that d MSC-s EVs can interlizate into HDFs.The results cell proliferation assay and cell cycle assay showed that d MSC-s EVs significantly promoted the proliferation of high glucose senescent HDFs.The results of scratch assay showed that d MSC-s EVs can promote HDFs migration.Western blot results showed that d MSC-s EVs can significantly promote high glucose senescent HDFs high express colⅠandα-SMA,which means that d MSC-s EVs can promote HDFs differentiate into myofibroblasts.The results of ROS levels and SA-β-gal staining showed that d MSC-s EVs reduced ROS levels and the number of senescent HDFs.Western blot results showed that d MSC-s EVs could reduce the p21 expression.Detection of key proteins in the signaling pathway revealed that after the addition of d MSC-s EVs,the expression of RAGE and its downstream protein p21 RAS significantly decreased and p Smad2/3 expression were significantly increased.In order to verify the role of Smad pathway,after the addition of p Smad2/3 inhibitor SB431542,the expression of p Smad2/3 was significantly down-regulated,and the number of senescence cells was significantly increased,suggesting that Smad pathway plays a key role in d MSC-s EVs in repairing HDFs with high glucose senescence.4.Observation of the wound healing of diabetic mice in general and under the microscope confirmed that s EVs,can accelerate the healing of diabetic wounds.Masson staining showed that s EVs,can promote better HDFs collagen deposition.Immunofluorescence staining showed that d MSC-s EVs could promote the proliferation,migration and differentiation of fibroblasts around the wound surface,thus promoting wound healing.ConclusionIn this study,we established the model of high glucose induced senescent HDFs.Then,it was confirmed that d MSC-s EVs can promote the proliferation and migration capacity of HDFs with high glucose senescence and reduce the ROS level and the number of senescent cells,which are closely related to the inhibition of the RAGE pathway and the activation of the Smad pathway.Finally,we found that d MSC-s EVs accelerated the healing of diabetic wounds by promoting fibroblasts proliferation,migration and differentiation and reducing the cell senescence.This study provides a solid theoretical basis for future research on the application of d MSC-s EVs in the treatment of chronic wounds in clinical diabetes mellitus.