| Purpose: Diabetic kidney disease（DKD）,the most serious microvascular complication of diabetes,is easy to develop into end-stage renal disease,with rapid development,difficult to control,high morbidity,high mortality and low awareness of the characteristics of social public health cause has brought a heavy burden.But at present,there is no effective treatment for the disease.Therefore,in view of the pathogenesis of DKD,it is particularly urgent and important to find a new and effective way to prevent,delay or even reverse DKD.Adipocyte derived exosomes（ADSCs）can reduce podocyte damage.However,its function and mechanism in DKD have not been fully elucidated.We therefore studied the effects of ADSC-derived exosomes loaded with miRNA-145a-5p（ADSCs-Exo-miR-145a-5p）on DKD.Methods: 1.To verify that miRNA-145a-5p inhibits podocyte injury induced by high glucose by regulating autophagy induced by BNIP3 in vitro : In the high glucose environment,the expression level of BNIP3 in each group was detected by WB,the expression level of miR-145a-5p was detected by qRT-PCR,Then,podocytes in each group were treated with BNIP3 interference and miRNA145a-5p mimics transfection.WB,CCK8,Flow cytometry and other methods were used to detect the expression of BNIP3,P62 and LC3 II/I apoptosis proteins,cell viability and apoptosis.The binding regulation of miR 145a-5p and BCL2 interacting protein 3（BNIP3）was detected by dual luciferase reporter gene.2.Construction and identification of ADSCs-Exo and ADSCs-Exo-miR145a-5p ADSCs transfected with miR145a-5p were constructed and extracted to obtain ADSCs-Exo and ADSCs-Exo-miR145a-5p,respectively.NTA and electron microscopy were used to count,evaluate morphology and number of exosomes.and WB weas used to confirm exosome marker proteins.3.To verify the improvement effect of ADSC-Ex O-miRNA145a-5p on DKD mice in vivo experiments:（1）Model construction:8-week db/db and db/m mice were used in this study,and db/m mice were used in the control group.15 db/db mice were randomly divided into DKD model group,DKD+ ADSCs-Exo-NC,DKD+ADSCs-exo-miR145a-5p,with 5 mice in each group.When db/db mice showed kidney damage after feeding to 12 week,the four groups of mice were injected into tail vein respectively: PBS 200 μL,PBS 200 μL,PBS solution containing ADSCs-Exo（protein 100 μg）,PBS solution containing ADSCs-Exo-miR145a-5p（protein 100 μg）,once a week,for 8 weeks.（2）Efficacy observation: The content of urinary microalbumin,blood urea nitrogen and serum creatinine were detected by ELISA,the expressions of apoptotic proteins such as BNIP3,P62 and LC3II/I were detected by WB,and the expression of miR 145a-5p was detected by qRT-PCR.The tissues of kidney,liver,heart and lung were evaluated by immunofluorescence,hematoxylin and eosin staining.Results: In vitro,the expression of BNIP3 in podocyte treated with high glucose was significantly increased compared with the control group,and the content of miRNA145a-5p was significantly decreased.Podocytes were interfered BNIP3 expression and transfected with miRNA145a-5p,respectively.It was found that p62 protein expression decreased,LC3II/I increased,autophagy significantly increased,cell viability increased,and apoptosis decreased in podocytes treated with high glucose induction.ADSCs-Exo and ADSCs-Exo-miR-145a-5p were successfully constructed and confirmed by identification.In vivo,it was found that ADSCs-Exo-miR-145a-5p injection can reduce urinary microalbumin,blood urea nitrogen and blood creatinine in DKD mice,significantly inhibit the expression of BNIP3,promote autophagy,reduce apoptosis and renal pathological damage in DKD mice,and have no significant influence on the body weight of DKD mice.Besides,it help improve the injury in tissue of heart,lung and liver.Conclusion: 1.MiRNA145a-5p can inhibit high glucose-induced podocyte injury by regulating BNIP3;2.ADSCs-exo-miR145a-5p can improve DKD in mice by inhibiting the expression of BNIP3;3.ADSCs-Exo injection has no obvious toxic and side effects on mice,it is safe and effective. |
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