| Objective Results of previous studies showed that the expression of miR-146b in the model of cisplatin-induced renal injury was up-regulated,while the expression level of miR-146b in MSC(mesenchymal stem cells)transplant group was lower than it in the injured group,The result suggests that MSC could protect and repair the injured cells induced by cisplatin via down-regulating the expression of miR-146b.In the meantime,the expression of hs-miR-146b-5p in patients with renal diseases was significantly higher than it in healthy people,which implies that miR-146b might be one of the early diagnostic molecules of renal injury.On the basis of these results,we will build a model of sepsis-associated acute renal injury in mice with hucMSC-exosomes mediated repair to reveal the expression change and mechanism of miR-146b in sepsis-associated acute renal injury.Meanwhile,we will detect hs-miR-146b-5p in serum of patients in early stage of acute renal injury with different clinical etiology to evaluate the feasibility and application value of hs-miR-146b-5p as a diagnostic marker of AKI early stage.Methods On one hand,constructing the model of acute renal injury associated with sepsis by cecal ligation and puncture(CLP model),we set the sham operation group(Sham group as control group,hucMSC-exosomes group received a peritoneal injection of exosomes derived from huc-MSC after CLP operation and injured group received a peritoneal injection of same amount of saline after CLP operation.Collect serum samples of each group at Oh and 24h and kidneys at 24h.Then according to the expression change of serum creatinine and urinal nitrogen and results of hematoxylin and eosin stain,we could ensure whether the CLP model was successful or not.In this research,PCNA immune histochemical staining was used to detect the proliferative ability of renal tissue cells in each group.And the expression of the apoptosis related protein--Caspase 3 was detected by Western Blot.Basing on the results of PCNA immune histochemical staining and Western Blot,we could evaluate the protective and repair effects of huc-MSC on renal tissue of sepsis-associated acute renal injury.Besides,fluorescence quantitative RT-PCR was used to detect the expression level of miR-146b in renal tissues of each group to investigate the reactivity of miR-146b in sepsis-associated acute renal injury.Meanwhile,according to a great deal of research reports,we predicted the target genes of miR-146b in sepsis-associated acute renal injury model in mice with hucMSC-exosomes mediated repair as IRAK1 and TRAF6.The expression levels of IRAK1/TRAF6 and downstream NF-?B pathway related proteins in renal tissues of each group were detected by fluorescence quantitative RT-PCR to explore the action mechanism of miR-146b sepsis-associated acute renal injury model in mice with hucMSC-exosomes mediated repair.On the other hand,we detect the expression of hs-miR-146b-5p,serum creatinine and urea nitrogen in serum samples of confirmed AKI patients in at least 3 days before diagnosis by Taq-man fluorescence quantitative RT-PCR to evaluate the clinical application of miR-146b in early diagnosis of AKI.Results The results of serum creatinine and urea nitrogen and the HE staining of renal tissues in each group showed that we successfully build a model of sepsis-associated acute renal injury in mice via CLP surgery.And results of PCNA immune histochemical staining and Western Blot showed that hucMSC-exosomes could stimulate the proliferation and regeneration and weaken the apoptosis of injured renal cells.In addition,the expression of miR-146b in the renal tissue of sepsis-associated acute renal injury had no change compared with the normal tissue,while the expression of miR-146b was significantly up-regulated in the renal tissue after repaired by hucMSC-exosomes(P<0.001).The results of fluorescence qRT-PCR showed that IRAK1 gene and TRAF6 gene increased significantly in the injured group compared to the control group,but returned to normal level after hucMSC-exosomes mediated repair,which in agreement with the result of expression changes of miR-146b in each group.Western blot showed the same results.The results of immuno histochemical staining showed that the expression level of IKB?(inhibitor of NF-?B?),pIKBa(phosphorylated inhibitor of NF-?B ?),p65(NF-KB protein)and p-p65(phosphorylated p65)increased in the injured group,and the expression of IKBa increased more during repair,while the expression of pIKBa,p65 and p-p65 decreased.The results of fluorescence qRT-PCR showed that the mRNA expression of TNF-? and IL-1? increased significantly in the injured group,but decreased in the hucMSC-exosomes group,which was still higher than that in the control group(P<0.001).Serum samples of fifty-three confirmed AKI patients with different etiology were collected.67.9%of them showed a two to more than fifty times fold change of hs-miR-146b-5p expression level,including five cases of prerenal AKI,thirty cases of renal AKI and one case of sepsis-associated AKI.The change of hs-miR-146b-5p occurs at least 24 hours earlier than changes in serum creatinine and urea nitrogen.Conclusion hucMSC-exosomes shows the same repair effect on CLP model as other acute renal injury models,but the expression trend of miR-146b in CLP model is not consistent with other acute renal injury models.It is suggest that miR-146b might have a different effect and regulatory mechanism in sepsis-associated acute renal injury compared with other models.MiR-146b inhibits inflammatory injury in hucMSC-exosomes mediated sepsis-associated acute kidney injury by inhibitingthe activation of NF-?B through inhibiting IRAK1 gene and TRAF6 gene.It is found that the expression of miR-146b may increase in varying degrees in acute renal injury caused by different etiology and the change of hs-miR-146b-5p occurs at least 24 hours earlier than those in sermm creatinine and urea nitrogen,suggesting that miR-146b can be used as a universal sensitive diagnosis marker in early stages of acute renal injury without any differences caused by etiology. |
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