| Objective:Cisplatin is a widely used chemotherapy drug,but the application of cisplatin is restrict by the side effects of nephrotoxicity.Our previous studies have suggested that exosomes derived from human umbilical cord mesenchymal stem cells(hucMSC-ex)can reduce cisplatin-induced renal oxidative stress and cell apoptosis in vivo and in vitro.The role of hucMSC-ex in preventing of cisplatin-induced kidney injury and cisplatin treating for cancer cells is not very clear.To investigate the prevention effect and mechanism of hucMSC-ex on nephrotoxicity during treating with cisplatin both in vivo and in vitro.To provide a new strategy for the preventing kidney injury Methods:(1)HucMSCs were isolated by adherent method and the exosomes were Extracted by exosomes kit.The size and concentration of exosomes derived from the human umbilical cord mesenchymal stem cells were detected by nanoparticle tracking analysis system.The morphology and surface markers including CD9 and CD63 were analyzed by imaging flow cytometry.(2)CM-Dil-labeled hucMSC-ex internalization into NRK-52 E cells was determined by structured illumination microscopy.To establish the preventive models of hucMSC-ex in vitro,NRK-52 E cells were divided into three groups,treated as follows: Control group,no cisplatin treatment;PBS group,equal volumes of PBS with hucMSC-ex treated for 24 h before cisplatin treatment for 16 h;hucMSC-ex group,before cisplatin treatment for 16 h,hucMSC-ex treatment for 24 h.Cell apoptosis was detected by Annexin V/PI double staining using flow cytometry.The expression of bax and Cyclin D3 proteins were detected by western blot.Immunofluorescence detected proliferating cell nuclear antigen(PCNA)expression.The formation of autophasomes in NRK-52 E cells transfected with m RFP-GFP-LC3 by structured illumination microscopy(SIM).Immunofluorescence and Western blot were used to detected the expression of LC3 B.The expression of 14-3-3? protein was analyzed by Western blot.To establish the preventive experiment animal models of hucMSC-ex during cisplatin treatment,the SD rats were divided into three groups: Control group,no cisplatin treatment;PBS group,both kidneys in one rat received a renal capsule injection of PBS for 24 h,and then intraperitoneal injection of cisplatin;hucMSC-ex group: before cisplatin treatment for 3d,rats received a renal capsule injection with hucMSC-ex.The blood were collected every day,and the sreun was dectected for blood urea nitrogen(BUN)and creatinine(Cr)levels.Rats were sacrificed after cisplatin injection 3d and kidney tissues were removed.The injury was identified by HE staining.The expression of PCNA was detected by immunohistochemistry.The apoptosis of renal tubular epithelial cells was analyzed by TUNEL stain.Westem blot analyzed the expression of apoptosis-related protein bax.The above mentioned methods were used to comprehensive evaluation of preventive effect of hucMSC-ex on acute kidney injury.Western blot was used to detected the epression of LC3 B in kidney tissue.The expression of 14-3-3? in kidney tissue was dectected by immunohistochemistry and Westem blot.(3)In vitro gastric cancer cells were pretreated with hucMSC-ex and then subjected to cisplatin treatment.The cell apoptosis and cell cycle were analyzed by flow cytometry.The migration ability of gastric cancer cells was evaluated by transwell migration assay.The growth ability of gastric cancer cells was detected by colony formation assay.(4)Adenovirus expression vectors containing 14-3-3?(Ad-14-3-3?)expression sequence,adenovirus empty vector(Ad-GFP)and lentivirus(Lenti-14-3-3? shRNA or Lenti-GFP shRNA)were transfected into hucMSCs.After transfected,collected the conditioned medium and extracted the exosomes,they were named as Ad-GFP-ex,Ad-14-3-3?-ex,shGFP-ex,sh14-3-3?-ex.The intensity of green fluorescence in hucMSC was detected by fluorescence microscope.The expression of 14-3-3? in hucMSC and hucMSC-ex was detected by Westem blot.SIM assay showed that exosomes(CM-Dil labeled)transported 14-3-3? protein(GFP-tag)to NRK-52 E cells.PBS,Ad-GFP-ex,Ad-14-3-3?-ex,shGFP-ex and sh14-3-3?-ex respectively pretreated m RFP-GFP-LC3 transfected NRK-52 E cells for 24 h,and the treated with cisplatin for 16 h,the formation of autophasomes was analyzed by structured illumination microscopy(SIM).The expression of 14-3-3? and LC3 B protein were analyzed by western blot.The expression of PCNA was detected by immunofluorescence.The cell apoptosis was analyzed by TUNEL or flow cytometry.In vivo,the expression of 14-3-3? was analyzed by immunohistochemistry in Control group,PBS group,Ad-GFP-ex group and Ad-14-3-3?-ex group.Western blot analyzed the expression of LC3 B.The sreun was analyzed for blood urea nitrogen(BUN)and creatinine(Cr)levels.The kidney injury was identified by HE staining.TUNEL stain was used to detected the apoptosis of renal tubular epithelial cells.Westem blot analyzed the expression of apoptosis-related protein caspase 3.Immunohistochemistry was used to asssy the expression of PCNA.(5)Co-precipitation technique(IP)and SIM were used to detect the interaction between 14-3-3? and ATG16 L.Results:(1)Nanosight assay showed that the diameter of hucMSC-ex was 97 nm.Imaging Flow Cytometer showed that hucMSC-ex expressed CD9 and CD63.In additional the positive rate was about 80%.(2)HucMSC-ex decreased the number of apoptotic cells and the expression of bax,increased the expression of Cyclin D3 and PCNA in NRK-52 E cells during treating with cisplatin.In vitro hucMSC-ex prevention model,exosomes were transported to target cells.In vitro pretreating with hucMSC-ex,the number of autophagosome and the expression of LC3 B were increased.In vivo,hucMSC-ex prevented the kidney injury induced by cisplatin.(3)Gastric cancer cells was treated by hucMSC-ex and then treated with cisplatin.Flow cytometry asssy showed that the cell apoptosis and the cell cycle arrest were no significantly difference.The migration and colony formation of gastric cancer cells were no significant difference.(4)LC-MS/MS assay showed that hucMSC-ex contained 14-3-3? protein and transported it to NRK-52 E cells.Both in vitro and in vivo models,the expression of 14-3-3? was increased during pretreating with hucMSC-ex.Overexpression of 14-3-3? in hucMSC-ex enhanced the formation of autophagosome and the expression of LC3 B,moreover decreased the apoptosis induced by cisplatin and promoted the cell proliferation in NRK-52 E cells.In vivo,Ad-14-3-3-ex decreased the serum level of BUN?Crea and the TUNEL-positive cells in kidney.Conversely,knock down of 14-3-3? in hucMSC-ex alleviated autophagic activity and aggravated the injury induced by cisplatin in vitro.(5)In vitro,hucMSC-ex promoted the expression of ATG16 L in NRK-52 E cells during treating with cisplatin.SIM detection showed that 14-3-3? promoted the expression and aggregation of ATG16 L.IP assay showed that 14-3-3? interacted with ATG16 L.Conclusions: HucMSC-ex-derived 14-3-3? induced autophagy and prevented AKI induced by cisplatin,at the same time it played no effect on cisplatin induced cancer cell apoptosis.14-3-3? promoted the autophagy by interacting with ATG16 L.Our findings provided a new train of thought and experimental basis for preventing cisplatin induced AKI and expanded the cisplatin clinical application. |
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