| Diabetic nephropathy(DN)is one of the most common and serious microvascular complications of diabetes and one of the leading causes of end-stage renal failure.Glomerular mesangial cells(GMCs)and podocytes are the two most important intrinsic cells in the kidney.Their functional changes play a crucial role in the progression of DN.The previous study of the research group found that there may be interactions between GMCs and podocytes under high glucose conditions.This interaction may have important significance for the occurrence and development of DN,but the specific mechanism is not yet clear and needs further exploration.Exosomes are microvesicles secreted by cells.Studies have shown that exosomes can carry a variety of proteins,lipids,mi RNA,involved in cell communication,cell migration,angiogenesis,inflammatory response and tumor growth and other processes.Under high glucose stimulation,exosomes secreted by glomerular endothelial cells can activate GMCs and promote renal interstitial fibrosis,suggesting that the interaction between the inherent cells in the kidney can be achieved through exosomes.Phosphatidylinositol-3-kinase(PI3K)is an intracellular phosphatidylinositol kinase,which is a key signaling molecule in many life activities.Protein Kinase B(AKT)is a direct target protein downstream of PI3 K.It is a serine/threonine protein kinase and is the core of the PI3K/AKT signal transduction pathway.Berberine(BBR)is the main component of Rhizoma Coptidis Rhizome,and studies have shown that BBR has the functions of lowering blood glucose,regulating abnormal lipid metabolism,anti-inflammatory,improving insulin resistance and reducing renal damage.The above studies indicate that the interaction between podocytes and GMCs may have an important effect on the occurrence and development of DN under high glucose conditions.It is not clear how the interaction between the two kinds of cells is achieved and how it affects the podocyte and thus affects the whole process of DN.It is not clear that this subject intends to act on podocytes by isolating exosomes derived from GMCs.The specific pathways of interaction between GMCs and podocytes were studied to understand their influence on the process of DN occurrence and development mechanism.At the same time,the effect of BBR on the podocyte effect of high glucose-induced exosomes derived from GMCs was studied in order to explore the therapeutic effect of BBR on DN target cells and its possible mechanism.Aim: GMCs and podocytes were co-cultured by transwell chambers.Exosome secretion inhibitor GW4869 was used to observe whether exosomes mediate the interaction between GMCs and podocytes,determine whether exosomes can participate in the interaction between GMCs and podocytes.The exosomes derived from GMCs were isolated by ultracentrifugation and applied to podocytes.The effects of high glucose-induced exosomes derived from GMCs on podocyte function and the effects of berberine were observed.To clarify the protective effect of BBR on high glucose-induced GMCs exosomes injured podocytes.Using small interfering RNA to silence the expression of TGFβ1 on GMCs,the exosomes derived from GMCs were isolated and treated with podocytes to observe the effects on the function of podocytes and the effect of PI3K/AKT signaling pathway on protein expression.Further investigation was made on the source of high glucose-induced GMCs induced by BBR.The specific mechanisms and important molecular targets of the protective effect of exosomes on podocyte injury provide theoretical basis for the rational development and utilization of BBR in the prevention and treatment of DN,and open up new research directions and ideas for exploring new therapeutic drugs for DN.Methods: GMCs and podocytes were co-cultured with transwell chambers,GMCs were placed in the upper chamber,and podocytes were placed in the lower chamber,and were divided into normal group(NG),high glucose model group(HG)and GW4869 Inhibitor group.After stimulation,the cells were co-cultured for 24 hours in the chamber,and the glomerular podocyte in the lower chamber was collected.The apoptosis of podocytes was detected by Annexin V-FITC / PI apoptosis kit,Podocyte adhesion function,use Western blot to detection the nephrin,podocin and WT-1 protein expression.The exosomes derived from GMCs were isolated by ultracentrifugation.The morphology and size of the exosomes were observed by transmission electron microscopy.The expressions of CD63,TSG101 and Calnexin were detected by western blot.The size distribution of exosomes was detected by particle size analyzer.The exosomes were labeled with PKH67 and then incubated with podocytes.The uptake of exosomes was observed by laser confocal microscopy.CCK-8 method to determine the appropriate concentration of BBR after the GMCs stimulated,the isolated exosomes were applied to podocytes,using Annexin V-FITC / PI apoptosis kit for detecting podocyte apoptosis;adhesion Podocin and WT-1 protein were detected by Western blot.The expression of transforming growth factor-β receptor(TGFβ1R)on podocytes was observed by immunofluorescence staining.To observe whether BBR could reduce the injury of podocyte induced by high glucose induced mesangial cell-derived exosomes.The small interfering RNA was used to silence the expression of transforming growth factor-β1(TGFβ1)in GMCs,and the exosomes of each group were isolated.The content of TGFβ1 in each exosome was detected by enzyme-linked immuno sorbent assay(ELISA).Group exosomes were applied to podocytes respectively.The apoptosis of podocytes was detected by Annexin V-FITC / PI kit.Adhesion ability was evaluated by podocyte adhesion assay.The expression of nephrin,podocin and WT-1 Western blot was used to detect the expression of PI3 K,AKT,P-AKT,P65,P-P65,TGFβ1R and related signaling pathways.Results: 1.The mesangial cell culture supernatant was successfully isolated exosomes Transmission electron microscopy showed that the isolated exosomes showed a typical cup-shaped,diameter 30-100 nm,in line with the typical characteristics of exosomes reported in the literature.Western blot results showed that the resulting exosomes expressed CD63 and TSG101,but endoplasmic reticulum chaperone calnexin did not express further evidence that the cell-free exosomes were isolated from the supernatant.Confocal microscopy showed that PKH67-labeled exosomes could be taken up by glomerular podocytes.2.High glucose induces mesangial cell-derived exosomes involved in the process of podocyte injury Compared with the normal group,the apoptosis rate of podocyte in the high glucose model group was significantly increased,the adhesion ability was significantly reduced,and the expressions of nephrin,podocin and WT-1 protein in podocytes were significantly down-regulated.Compared with the high glucose model group,GW4869 inhibitor group were significantly improved.3.BBR significantly ameliorated the injury of podocytes induced by high glucose-induced mesangial cell-derived exosomes CCK-8 cell proliferation experiments showed that hyperglycemic stimulation of GMCs over-proliferation,BBR has a significant inhibitory effect on GMCs proliferation,according to the results,set the follow-up GMCs administered concentration of 50μmol / L and 100 μmol / L.Compared with the normal group,the apoptosis rate of podocyte in the high glucose model group was significantly increased,the adhesion ability was significantly reduced,and the expressions of nephrin,podocin and WT-1 protein in podocytes were significantly down-regulated.Compared with the high glucose model group,BBR high and low concentration group can improve the above indicators to varying degrees.4.High glucose induced GMCs-derived exosomes affect podocyte function may be through the TGFβ1R-PI3K/AKT signaling pathway The expression of TGFβ1R in podocyte was observed by immunofluorescence assay.Compared with normal group,the expression of TGFβ1R in podocytes of high glucose group was significantly increased,and the level of TGFβ1R in podocytes was significantly decreased by BBR compared with high glucose group.Small interfering RNA was used to silence the expression of TGFβ1 in GMCs.The exosomes were isolated and the content of TGFβ1 in each exosome was detected by ELISA.The results showed that compared with the normal group,TGFβ1 content was significantly increased,compared with high glucose group,BBR group and TGFβ1 gene silencing group TGFβ1 content were significantly decreased,BBR and TGFβ1 gene silencing interaction TGFβ1 content decreased more significantly.Each group of exosomes were applied to podocytes respectively.The apoptosis of podocytes was detected by Annexin V-FITC / PI kit.The results showed that the apoptosis rate of podocytes in high glucose group was significantly higher than that in normal group Compared with high glucose group,the apoptosis rates of podocytes in the BBR group and the TGFβ1 silencing group were significantly decreased,and the apoptosis rate of podocytes in the combined group of BBR and TGFβ1 silencing was more significant.Adherence ability test podocyte adhesion function,the results showed that compared with the normal group,the high glucose group podocyte adhesion capacity was significantly reduced,compared with high glucose group,BBR group and TGFβ1 gene silencing group podocyte adhesion was significant Elevated,BBR and TGFβ1 gene silencing group podocyte adhesion capacity increased more significantly.Western blot was used to detect the expressions of nephrin,podocin and WT-1 protein.The results showed that compared with the normal group,the expression of nephrin,podocin and WT-1 protein in podocytes of high glucose group was significantly decreased,Compared with the expression of nephrin,podocin and WT-1protein in the podocytes of BBR group and TGFβ1 silencing group,the expressions of nephrin,podocin and WT-1 protein expression increased more significantly.The expression of PI3 K,AKT,P-AKT,P65,P-P65,TGFβR1 and other signaling pathways were detected by Western blot.The results showed that compared with the normal group,the expressions of PI3 K,P-AKT,P65,TGFβ1R expression levels were significantly increased,compared with high glucose group,BBR group and TGFβ1 silencing group podocytes PI3 K,P-AKT,P-P65,TGFβ1R expression was significantly lower,BBR and TGFβ1 Gene silencing co-treatment group podocyte PI3 K,P-AKT,P-P65,TGFβ1R expression decreased more significantly.Conclusions: 1.Exosomes can mediate the interaction between GMCs and podocytes.2.BBR protects podocytes from injury induced by high glucose-induced GMCs-derived exosomes.3.BBR may play a protective role on podocytes by regulating TGFβ1R-PI3K/AKT pathway through exosome... |
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