Study On The Preventive Effect And Mechanism Of Exosomes From Human Umbilical Cord Mesenchymal Stem Cells Activating Autophagy To HK-2 Cells Induced By Cisplatin

Objective Based on previous studies,exosomes secreted by human umbilical cord mesenchymal stem cells(hucMSC-ex)could prevent and repair acute kidney injury induced by cisplatin in rats.However,the mechanism underlying is still not clear.In the present study,the model in which hucMSC-ex pretreated human renal tubular epithelial cell line HK-2 could prevent the injury of cisplatin was established to explore the mechanism of hucMSC-ex preventing and repairing renal toxicity induced by cisplatin which provides new theoretical basis for the preventive effect of hucMSC-ex in clinical.Methods The control group was HK-2 cells cultured in normal condition.The cis group was treated with cisplatin for 16 h which concentration was 16μmol/L.The ex+cis group was HK-2 cells pretreated with 200μg/ml hucMSC-ex then treated with 16μmol/L cisplatin for 16 h.The 3-MA+ex+cis group was HK-2 cells pretreated with 200μg/ml hucMSC-ex and 100μmol/L 3-MA,then treated with 16μmol/L cisplatin for 16 h.Transmission electron microscope was used to observe the morphological features.The existence of CD9 and CD63 on hucMSC-ex surface was detected by flow cytometry imaging and Western blot.Gainand loss-of function study was used to explore the role of 14-3-3ζ.The expression level of proliferating cell nuclear antigen(PCNA)was analyzed by immunofluorescence assay.The number of apoptotic cellswas detected by TUNEL assay and flow cytometry analysis.The formation of autophagosomes was observed under super-resolution optical microscope.The expression levels of 14-3-3ζ,LC3 B,P62,PCNA and Bax was analyzed by Western blot.Results The exosomes used in the study were round or oval in shape and they were different in size.The CD9 and CD63 were expression in the surface of hucMSC-ex.Pretreating cells with hucMSC-ex could prevent the injury of cisplatin by reducing the number of apoptotic cells and increasing the expression level of PCNA.Simultaneously,the rate of LC3 B Ⅱ/LC3 B Ⅰ was up-regulated and the autophagic substrate P62 decreased which indicated that autophagic level was up-regulated.The application of autophagic inhibitor3-methyladenine(3-MA)could reverse the protective effect of hucMSC-ex.The pretreated of hucMSC-ex could up-regulate the 14-3-3ζlevel.Knock down of 14-3-3ζ in hucMSC-ex,the 14-3-3ζ decreased and the ability of inducing autophagy lost and the protection of hucMSC-ex lost.Overexpression of 14-3-3ζ in HK-2 cells enhanced the autophagic level and protected the injury of cisplatin.Knock down of 14-3-3ζ in HK-2 could reduce the autophagic level and enhance the hurt of cisplatin.Replenishing the knockdown of 14-3-3ζ with hucMSC-ex,the enhanced injury of cisplatin was reversed.Conclusion 14-3-3ζ transported by hucMSC-ex may up-regulate autophagic level in HK-2 cells,which can prevent the injury of cisplatin.This discovery provides new thought and project for the prevention of cisplatin-induced nephrotoxicity by hucMSC-ex.