| BackgroundDiabetes is one of the most common diseases in the world.The prevalence of diabetes is on the rise.The complications of diabetes and the adverse effects of treatment greatly affect the quality of life of patients and threaten their life and health,diabetes has become a serious global public health problem,so prevention and delay of diabetes complications is an important goal of clinical treatment.Diabetic Kidney Disease(DKD)is a common chronic microvascular complication of diabetes mellitus and an important cause of end stage renal disease(ESRD).In China,the incidence of DKD is on the rise because of its complicated metabolic disorder,when it comes to ESRD,it is often trickier to treat than other kidney diseases,so delay the incidence of DKD is important.The clinical diagnosis of DKD is based on increased urinary albumin and/or decreased eGFR in diabetic patients.In recent years,there has been an increase in basic research on DKD.Podocytes are special renal parenchyma cells with highly differentiated characteristics,together with glomerular endothelial cells and basement membrane,they form an important filtration system.At the early stage of DKD,the function and structure of podocytes are destroyed,resulting in apoptosis and shedding of podocytes,damage to the integrity of podocytes,influence on glomerular filtration barrier,and proteinuria,participate in the occurrence and development of DKD.Therefore,avoiding or reducing podocyte apoptosis is not only an important factor in the prevention of DKD,but also an important method of clinical treatment.Mesenchymal stem cell cells(MSCs)are adult stem cells with the potential of self-replication and multi-directional differentiation.Its therapeutic potential and safety in regenerative therapy and immunoregulation have been recognized,and the mechanism of extracellular vesicle repair of damaged kidneys in Mesenchymal stem cell has become a research hotspot.In recent years,it has been suggested that MSCs can repair and protect organs through the action of extracellular vesicles(EVs)-mediated paracrine.Adipose-derived stem cells(ADSCs)have the characteristics of MSCs,are easy to obtain,and can secrete rich EVs.Most of experiments the reseachers extracted the EVS from ADSCs and carried out morphological verification.EVs mediate signal transduction between adjacent or distal cells by delivering microRNAs(miRNAs or miRs),proteins,long-stranded noncoding RNA,annular RNA and DNA.miRNAs are small non-coding RNAs which inhibit the translation of target messenger RNAs and participate in the regulation of organ-to-organ communication in the pathogenesis of diabetes.Based on the relationship between miRNAs in MSCs-EVs and the prevention of diabetic complications,a large number of studies have analyzed the roles and possible mechanisms of different miRNAs in diabetic complications.Mir-15b-5p is a mature miRNA spliced from the front 5' of miR-15b.MiRNA has been recognized as an important regulator of genes involved in cell cycle and apoptosis,mir-15b-5p is associated with the development of proliferative diabetic retinopathy(PDR).The circulating miR-15b is directly associated with Vascular endothelial growth factor(VEGF).VEGF is a 45kda homodimer glycoprotein.VEGFA is the most studied factor,it is the key medium of angiogenesis(the formation of new blood vessels).It is also the main angiogenic factor and mitogen of endothelial cells.It is mainly produced by podocytes in kidney.VEGFA is involved in DKD.Furthermore,Mir-15b-5p has been shown to act as a tumor suppressor in osteosarcoma by directly targeting Pyruvate dehydrogenase kinase 4(PDK4),a key regulator of cellular energy metabolism,its activation is closely related to metabolic diseases.In this study,the expression of miR-15b-5p in ADSCs was measured by extracting the EVs of ADSCs,and compared with Mir-15b-5p in mouse podocytes,the expression levels of PDK4 and VEGFA were detected under different intervention conditions,and the relationship between the changes was analyzed and the possible mechanism was explored.This study provides new insights into the molecular mechanisms of DKD.PurposeThe aim of this study was to investigate the effects and possible mechanisms of EVs-mediated miR-15b-5p secreted by ADSCs on mice renal podocytes in the presence of high glucose(HG),and to explore possible therapeutic targets for DKD,to provide a theoretical basis for further research.Methods1.EVS was isolated from ADSCs,and the structure of EVs was observed by transmission electron microscopy.The positive rate and protein expression of CD9,CD63 and CD81 were detected by Flow cytometry and Western Blot(WB)respectively.Expression of miR-15b-5p in mouse podocytes and EV by RT-qPCR.2.The proliferation rate of mouse podocytes induced by high glucose(HG)was detected by CCK-8 assay,the expression of Caspase-3 and Bcl-2 in mice podocytes induced by high glucose was detected by WB assay,and the apoptosis rate of mouse podocytes was detected by Flow cytometry assay,the expression of IL-1,IL-6 and TNF-? in HG-induced mouse podocytes was detected by WB and miR-15b-5p was detected by RT-qPCR.3.The apoptosis and inflammation of mouse podocytes induced by HG treated with ADSC-EVs or Wi38-EVs,and the expression of miR-15b-5p in ADSC-EVs were detected.4.Luciferase activity of WT-PDK4 in mouse podocytes transfected with Mir-15b-5p mimics and inhibitors was measured by double luciferase assay,RT-qPCR and WB were used to detect the overexpression of miR-15b-5p or the transcription and protein expression of PDK4 in the presence of HG or ADSC-EVs,and the luciferase activity of wt-PDK4 in the presence of HG and low glucose.5.HG-induced mouse podocytes were transfected with siRNA-PDK4 to detect the proliferation rate,apoptosis rate and inflammatory reaction.RT-qPCR and Western blot were used to detect the effect of siRNA-PDK4-1/2/3 on the expression of PDK4 at mRNA and protein levels.CCK-8 was used to detect HG-induced podocyte proliferation in mice after PDK4 was knocked out,hg-induced podocyte apoptosis and expression of inflammation-related proteins in mice were detected by WB and Flow cytometry.6.VEGF expression in mouse podocytes with different glucose concentrations was detected by western blotting,and PDK4 and miR-15b-5p were detected after VEGFA expression was inhibited by siRNA,the apoptosis,inflammation and increment rate of podocyte in mice were observed,and the effect of PDK4 overexpression and knock-down on VEGFA expression was detected by WB.Mir-15b-5p simulants alone or in combination with ADSC-EVs,oe-VEGFA,ADSC-EVs and oe-VEGFA were transfected into mouse podocytes to detect the expression of miR-15b-5p,VEGFA and PDK4 induced by HG.CCK-8 assay was used to detect the cell proliferation rate after co-transfection with oe-VEGFA and miR-15b-5p simulants or ADSC-EVs,and WB assay was used to detect the expression of apoptotic and inflammatory markers in mouse podocytes co-transfected with oe-VEGFA and miR-15b-5p simulants or sc-adevs,the apoptosis of podocytes in mice induced by high glucose was detected by using Flow cytometry and WB in the presence of oe-VEGFA and miR-15b-5p simulants or oe-VEGFA and ADSC-EV.Results1.EVs was extracted from ADSCs.EVS was observed by transmission electron microscopy in round or oval shape.Flow cytometry and Western blot showed that EVs markers CD9,CD63 and CD81 were highly expressed.The expression of miR-15b-5p in podocytes was down-regulated compared with extracellular vesicle EVs.Compared with EVs,the expression of miR-15b-5p in mouse podocytes was decreased(p<0.05).2.In HG-induced mouse podocytes,the proliferation rate decreased,the number of Caspase-3 increased(p<0.001),BCL-2 decreased(p<0.001),the apoptosis rate increased,and the expression of IL-1,IL-6 and tnf-? increased significantly(p<0.001)The expression of miR-15b-5p in HG-induced mouse podocytes was significantly decreased(p<0.001).3.Only when ADSC-EVs was added to mouse podocytes,but Wi38-EVs was not added to mouse podocytes.ADSC-EVs concentration was 2x(25 ?g/mL)in cell culture medium,the proliferation rate of mouse podocytes was increased,the podocytes had the highest activity.The apoptosis and inflammation of podocytes decreased after ADSC-EVs was added.Mir-15b-5p was overexpressed in ADSC-EVs and up-expressed in HG+ADSC-EVs.Overexpression of miR-15b-5p increased cell proliferation,decreased apoptosis and inflammation.Mir-15b-5p inhibitor transfected cells with ADSC-EVs could counteract the protective effect of ADSC-EVs.4.Double luciferase report:the activity of luciferase decreased after co-transformation of WT-PDK4 with MIR-15B-5P mimics,and increased after co-transformation of WT-PDK4 with miR-15b-5p inhibitor,however,the activity of luciferase did not change when 3'-UTR Mut-PDK4 was co-transformed with miR-15b-5p mimics or miR-15b-5p inhibitor.qRT-PCR and Western blot:PDK4 expression increased in high glucose environment,and overexpression of miR-15b-5p or addition of ADSC-EVs in high glucose environment could decrease the level of PDK4 gene transcription and protein expression.5.siRNA,PDK4-1/2/3 can down-regulate the expression of PDK4,and siRNA-PDK4-1 is more effective.Hg-induced podocyte proliferation in mice was increased after PDK4 was knocked out.WB and Flow cytometry:Hg induced podocyte apoptosis and reduced expression of inflammatory related proteins in mice after removal of PDK4.6.The overexpression of VEGFA and VEGFR2 in mouse podocytes induced by HG was higher than that induced by NG,and the overexpression of miR-15b-5p or ADSC-EVs significantly decreased VEGFA expression in mouse podocytes induced by HG.The expression of PDK4 and miR-15b-5p did not change when VEGFA was knocked down by siRNA,and the expression of PDK4 and miR-15b-5p was knocked down by siRNA.The expression of PDK4 was positively correlated with the expression of VEGFA,and VEGFA did not affect the expression of PDK4.In HG environment,compared with HG+miR-15b-5p mimic,HG + ADSC-EVs,miR-15b-5p mimic+oe-VEGFA or ADSC-EVs+oe-VEGFA had no significant changes in miR-15b-5p expression,VEGFA protein level increased significantly and PDK4 expression did not change significantly.CCK8 showed that co-transfection with oe-VEGFA and miR-15b-5p simulants or ADSC-EVs significantly reduced cell proliferation.Increase the expression of apoptosis and inflammatory factors in mouse podocytes.The proportion of apoptotic cells in oe-VEGFA cotransfected cells was significantly increased.Conclusion1.ADSCs-EVs mediated miR-15b-5p,and miR-15b-5p expression in mouse podocytes was decreased in high glucose environment.2.High glucose can damage the renal podocyte,Mir-15b-5p can protect the renal podocyte.High glucose can down-regulate the expression of miR-15b-5p and promote the apoptosis and inflammation of the podocyte.3.The possible mechanism of ADSCs-EVs mediated miR-15b-5p protective effect on mouse podocytes in high glucose environment is that PDK4 is the target gene of miR-15b-5p,which is directly regulated by Mir-15b-5p in mouse podocytes.This study explored the protective effect of DKD at the molecular mechanism level and provided a new perspective for the prevention and control of DKD. |
PDF Full Text Request