| Background and objective: Diabetes mellitus is a global epidemic with insidious onset and widespread effects,which imposes a heavy economic burden on patients and society.Diabetes mellitus is characterized by hyperglycemia,which can lead to oxidative stress in tissues and cells through multiple pathways and thus affect normal physiological functions.Among the many complications of diabetes,chronic non-healing diabetic wounds are a serious life-threatening complication.Wound healing is a complex process in which multiple cells work together,including fibroblasts,an important component of connective tissue,which secrete extracellular matrix to promote wound healing.The oxidative stress caused by hyperglycemia can cause fibroblast apoptosis and thus severely interfere with the wound healing process.Platelet-rich plasma(PRP)is an autologous blood product characterized by a supraphysiologic concentration of platelets that contains a variety of growth factors that promote tissue regeneration and has been widely demonstrated to have a unique role in the field of tissue regeneration since its inception.Exosomes(Exos)are cell-secreted vesicles between 40-150 nm in diameter that carry various bioactive proteins and RNA and play an important role in intercellular communication.In recent years,it has been demonstrated that PRP-derived exosomes(PRP-Exos)hold great promise in the field of tissue regeneration and are more potent than PRP,but there are no studies on the role of PRP-Exos in diabetic wounds and its mechanisms.In addition,hydrogels have also made a splash in the field of tissue regeneration in recent years due to their excellent plasticity,biocompatibility,and sustained release capabilities.Therefore,the aim of this study was to investigate the role of PRP-derived exosomes in promoting diabetic wound healing and its molecular mechanism,and to verify the tissue regeneration-promoting function of PRP-Exos by co-application of hydrogels in a diabetic rat model.Methods: 1.PRP was isolated from human blood using differential centrifugation and exosomes were extracted from PRP using differential ultracentrifugation;2.The morphology,marker proteins,and particle size distribution of PRP-Exos were examined by transmission electron microscopy(TEM),western blotting,and nanoparticle tracking analysis(NTA),respectively;3.The uptake of PRP-Exos by fibroblasts was verified using PKH67;4.The effect of high glucose(HG)and PRP-Exos on fibroblast viability was investigated using the CCK-8 method;5.The effect of HG and PRP-Exos on fibroblast apoptosis was investigated using Annexin-V/PI apoptosis double-staining kit;6.The effect of HG and PRP-Exos on fibroblast migration was investigated with Transwell assay;7.Wound scratch assay was applied to investigate the effect of HG and PRP-Exos on fibroblast migration;8.The effect of HG and PRP-Exos on fibroblast reactive oxygen species(ROS)production was tested using the DCFH-DA probe kit;9.The effect of HG and PRP-Exos on fibroblast mitochondrial membrane potential was tested using the JC-1kit;10.Western blotting techniques were used to investigate the level of specific protein expression;11.q RT-PCR technique was used to investigate the m RNA expression level of specific genes;12.Streptozotocin(STZ)-induced diabetic rat model was used to verify the effect of PRP-Exos and hydrogel on wound healing;13.Wound samples were harvested at 7 and 14 days,respectively.The effect of PRP-Exos on re-epithelialization and collagen deposition in regenerated tissues was explored by HE and Masson histological staining methods;14.The effect of PRP-Exos on angiogenesis was explored using immunohistochemical and immunofluorescence methods to stain and label CD31 and α-SMA;15.Statistical analysis: all experiments were performed at least three replicate experiments,and the results were analyzed by Graph Pad software.Results: Part I: 1.We successfully extracted and characterized PRP-Exos,which has typical morphological features and expresses related marker proteins,and the growth factors PDGFBB,b FGF,and VEGF were also found in PRP-Exos;2.Fibroblasts could take up PKH-67-labeled PRP-Exos;3.PRP-Exos could promote fibroblast proliferation in a dose-dependent manner;4.PRP-Exos could promote fibroblast migration in a dose-dependent manner.Part II: 1.Fibroblasts were treated with different concentrations of HG(5,15,25,35,45 and 55 m M)for 24,48 and 72 hours respectively,and we found that HG above 35 m M could inhibit fibroblast proliferation in a time and concentration-dependent manner,and this inhibitory effect was not due to the hyperosmolarity caused by HG;2.Fibroblasts were treated with different concentrations of HG(5,25,35,45 and 55 m M)for 48 h.It was found that HG concentration higher than 35 m M could induce fibroblast apoptosis in a concentration-dependent manner;3.HG could cause oxidative stress,increase ROS production,decrease mitochondrial membrane potential,and inhibit the expression of antioxidant enzymes CAT and SOD 1in fibroblasts;4.ROS antagonist NAC(N-Acetylcysteine)could inhibit intracellular ROS production,mitochondrial membrane potential decline,and reverse the expression of antioxidant enzymes CAT and SOD 1;5.HG could activate JNK and p38 MAPK pathway and promote the expression of pro-apoptotic protein Bax and Cleaved caspase-3and inhibit the expression of anti-apoptotic protein Bcl-2;6.NAC almost completely inhibited the activation of the JNK and p38 pathway and its influence on the expression of downstream apoptosis-related proteins;7.The JNK inhibitor SP600125 and the p38 inhibitor SB203580 could partly inhibit the activation of the JNK and p38 pathways and their influence on the expression of downstream apoptosis-related proteins,respectively;8.HG-induced apoptosis can be inhibited by NAC,SP600125 and SB203580;Part III: 1.PRP-Exos could alleviate the inhibition of fibroblast proliferation and migration by HG;2.PRP-Exos could inhibit HG-induced fibroblast apoptosis;3.PRP-Exos could not inhibit HG-induced ROS generation,but rescued HG-induced mitochondrial membrane potential decrease;4.PRP-Exos did not affect HG-induced JNK and p38 activation,but promoted protein and m RNA expression of the anti-apoptotic protein Bcl-2 and inhibited protein and m RNA expression of the pro-apoptotic protein Bax;5.The expression of PDGFRβ was significantly increased after PRP-Exos treatment;6.PRP-Exos could activate the JAK2/STAT3 pathway,while the PDGFRβ receptor inhibitor Imatinib could block the activation of the JAK2/STAT3 pathway by PRP-Exos;7.PRP-Exos could inhibit HG-induced apoptosis.This effect can be blocked by AG490,a specific inhibitor of JAK2,and mimicked by CA-1,an agonist of JAK2;8.HG did not affect JAK2/STAT3 pathway expression,while PRP-Exos could affect apoptosis-related protein expression and play an anti-apoptotic role by activating the JAK2/STAT3 pathway which can be blocked by AG490.CA-1 could mimic the anti-apoptotic effect of PRP-Exos in high glucose environment;Part IV: 1.HP-PEG/HA-SH hydrogels were prepared and proved to be non-toxic to cells;2.HP-PEG/HA-SH hydrogels showed sustained release ability and PRP-Exos contained in the gel could be released continuously for more than 96 h;3.STZ-induced diabetic full-thickness excisional wounds rat model was successfully established,and PRP-Exos and hydrogel alone or in combination could promote diabetic wound healing;4.HE and Masson histological staining results showed that PRP-Exos could promote wound re-epithelialization as well as collagen deposition;5.Immunohistochemical and immunofluorescence results of CD31 and α-SMA showed that PRP-Exos could promote wound angiogenesis.Conclusions: 1.PRP-Exos have the ability to promote fibroblast proliferation and migration;2.High glucose could inhibit fibroblast proliferation and migration and induce apoptosis;3.High glucose could cause oxidative stress and activate JNK and p38 MAPK pathways to further induce apoptosis through the mitochondrial pathway;4.PRP-Exos could not affect HG-induced oxidative stress and Instead,it prevents HG-induced apoptosis by preventing mitochondrial outer membrane permeabilization;5.PDGFBB in PRP-Exos could bind to PDGFRβ and alleviate HG-induced apoptosis via the JAK2/STAT3/Bcl-2 pathway;6.PRP-Exos could promote wound healing,re-epithelialization,collagen deposition and angiogenesis in a diabetic rat model. |
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