| Background:Diabetic kidney disease(DKD)is a serious microvascular complication caused by diabetes mellitus.The pathogenesis of DKD is complex,with genetic factors,metabolic disorders,oxidative stress,inflammation,autophagy and other mechanisms,all of which are interrelated and contribute to the development of diabetic kidney disease.However,the development of DKD is still not completely delayed by basic clinical treatments such as control of hyperglycemia,hypertension,hyperlipidemia and renin-angiotensin system inhibitors.There is no more effective treatment for diabetic nephropathy,so it is necessary to find new or potential therapeutic targets to improve the efficacy.Mesenchymal stem cells(MSCs)are mesenchymal stem cells with proliferative and multidirectional differentiation functions,which make them very plastic and can be isolated from various tissues,such as bone marrow,umbilical cord,adipose,peripheral blood,liver,gingiva,etc.,among which bone marrow is the most abundant.Bone marrow mesenchymal stem cells(BMSCs)are more frequently used in experimental studies because of their easy availability and sufficient cell numbers.Exosomes secreted from MSCs have similar tissue damage repair and regenerative functions as MSCs.Exosomes secreted from MSCs have not only a therapeutic effect on diabetes but also a tissue repair role in diabetic complications.As a carrier of intercellular signaling,exosomes repair the kidney through pro-angiogenesis,trophic effects,anti-apoptosis,and immunomodulation.Mi RNA,as the main component in exosomes,plays a key regulatory role in the process of exosomes function realization,but the specific mechanism of miRNA in exosomes in the development of DKD still needs further in-depth study.Autophagy is a metabolic pathway involving lysosomes that maintain intracellular homeostasis by degrading and recycling injured organelles and macromolecules in response to various stresses.It acts as a cell protector when cells are injured.Dysfunctional autophagy plays a pathogenic role in various diseases.Diabetes-induced dysregulation of autophagy plays a key role in renal glomerular and tubulointerstitial lesions.Impaired autophagy of renal cells in high glucose state,especially in podocytes and tubular cells,is closely associated with the development of DKD.It has been found that miRNAs are involved in the pathophysiological process of DKD through different mechanisms.Mi RNAs are one of the important regulators of autophagy.Mi RNA can regulate autophagy through regulating autophagy-related signaling pathways and autophagy-related gene(ATG),thus affecting the development process of DKD.Mi RNA may be potential targets for intervention in the regulation of diabetic nephropathy.Mi R-125 b is an important miRNA that regulates cell proliferation and apoptosis.Mi R-125 b has been found to be lowly expressed in diabetic patients in recent years,and miR-125 b expression is downregulated in exosomes found in the urine of patients with diabetic nephropathy.Mi R-125 b plays an important role in the development and progression of type 2 diabetes.Mi R-125 b is also involved in DKD occurrence.Mi R-125 b derived from Hypo-Exo can induce cellular autophagy and thus inhibit the progression of myocardial infarction.The specific role of miR-125 b in exosomes in diabetic nephropathy is not well understood.In studies of high-glucose-induced podocyte injury,inhibition of the serine-threonine protein kinase(AKT)signaling pathway was found to induce autophagy and inhibit apoptosis.Activation of TRAF6(Tumor necrosis factor receptor associated-factor 6)is a key link in the activation of AKT signaling pathway.TRAF6 induces DKD progression.Inactivation of TRAF6 may lead to autophagy.TRAF6 is involved in the regulation of autophagy and can regulate autophagy by upregulating the activity of mammalian target of rapamycin(m TOR)downstream of AKT.Whether TRAF6 affects the development of DKD by regulating autophagy through modulation of AKT signaling pathway needs further investigation.BMSC-Exos is known to delay the development of diabetic nephropathy.Whether the protective effect of BMSC-Exos-derived miR-125 b on high glucose(HG)-induced apoptosis in human embryonic kidney epithelial cells(HKCs)via TRAF6/AKT axis deserves further investigation.Therefore,the present study focused on the protective effect of BMSC-Exos-derived miR-125 b on high glucose-induced apoptosis in human embryonic kidney epithelial cells and its mechanism.Part I: BMSC-Exos induces autophagy and inhibits apoptosis of HG-injured HKCs via miR-125 b Objective:The expression of miR-125 b in HG-injured HKCs,in BMSC-Exos and in HG-injured HKCs co-cultured with BMSC-Exos was studied to further investigate the effect of BMSC-Exos on autophagy and apoptosis in HG-injured HKCs and the effect of BMSC-Exos transfected with miR-125 b inhibitor on autophagy and apoptosis in HG-injured HKCs,confirming that BMSC-Exos induced autophagy and inhibited apoptosis in HG-injured HKCs via miR-125 b.Methods:1.HG-injured HKCs model and HG-injured HKCs co-culture model with BMSC-Exos were constructed and randomized as HKC group,Mannitol+HKC group,L-Glu+HKC group,H-Glu+HKC group,MSC-exo group,and H-Glu+HKC+MSC-exo group.Real-time quantitative PCR(RT-q PCR)was performed to detect the expression of miR-125 b in each group.BMSCs were co-cultured with HG-injured HKCs after addition of GW4869(exosome inhibitor)to the source exosomes,and RT-q PCR was performed to detect the change of miR-125 b expression.2.Autophagy inhibitor(bafilomycin A1,Baf A1)was added in HG-injured HKCs and HG-injured HKCs co-cultured with BMSC-Exos,and the protein levels of LC3II/LC3 I and P62 in each group were detected by Western blot.Flow cytometry was used to detect the apoptosis rate of each group after Annexin V/PI staining.3.Effect of transfection of BMSC-Exos with miR-125 b inhibitor on autophagy and apoptosis of HG-injured HKCs.Transfection of inhibitor control(inhibitor NC)or miR-125 b inhibitor into BMSCs,RT-q PCR assay was used to verify the transfection efficiency;after transfection,BMSC-derived exosomes were co-cultured with HG-injured HKCs,and RT-q PCR was applied to detect the expression of miR-125 b in each group of HKCs,while Western blot to detect the protein levels of LC3II/LC3 I and P62 in each group and flow cytometry to detect the apoptosis rate of each group of HKCs after Annexin V/PI staining.Results:1.The expression of miR-125 b was significantly decreased in HG-injured HKCs and significantly increased in BMSC-Exos.miR-125 b expression was significantly increased after co-culture of HG-injured HKCs with BMSC-Exos,indicating that exosomes derived from BMSCs reversed the down-regulation of miR-125 b expression in HG-injured HKCs.The miR-125 b expression was significantly decreased after the addition of GW4869(exosome inhibitor)to BMSC-derived exosomes co-cultured with HG-injured HKCs,indicating that GW4869 reverses the upregulation of miR-125 b by BMSC-Exos.It was confirmed that miR-125 b was delivered to HKCs via BMSC-Exos.2.LC3II/LC3 I protein levels decreased and P62 protein levels increased in HG-injured HKCs;when HG-injured HKCs were co-cultured with BMSC-Exos,LC3II/LC3 I protein levels were high and P62 protein levels were low,indicating that BMSC-Exos induced autophagy in HG-injured HKCs.However,the addition of Baf A1 did not significantly improve the autophagy of HKCs even in the presence of BMSC-Exos,confirming that the autophagy-inducing effect of BMSC-Exos could be reversed by the autophagy inhibitor.the apoptosis rate of HG-injured HKCs was significantly increased,and the apoptosis rate of HG-injured HKCs was significantly reduced after co-culture with BMSC-Exos,indicating that BMSC-Exos inhibited HG injury HKCs apoptosis.3.The miR-125 b expression was significantly decreased in the group transfected with miR-125 b inhibitor,indicating that the transfection of miR-125 b inhibitor was effective.miR-125 b expression was elevated,protein levels of LC3II/LC3 I were elevated,p62 protein levels were decreased,and apoptosis rate was decreased.BMSC-Exos transfected with miR-125 b inhibitor co-cultured with HG-injured HKCs showed a significant decrease in miR-125 b expression,a decrease in protein levels of LC3II/LC3 I,an increase in p62 protein levels and an increase in apoptosis rate.Conclusions:1.Mi R-125 b is delivered to HKCs through exosomes secreted by BMSCs.2.Autophagy of HG-injured HKCs is reduced,and BMSC-Exos can effectively restore autophagy of HG-injured HKCs,and this effect can be reversed by autophagy inhibitors.3.HG induced apoptosis of HKCs,and BMSC-Exos inhibited the apoptosis of HG-injured HKCs.4.BMSC-Exos transfected with miR-125 b inhibitor induced autophagy and inhibited apoptosis in HG-injured HKCs was reversed.In conclusion,BMSC-Exos induced autophagy and inhibited apoptosis in HG-injured HKCs via miR-125 b.Part II: Mi R-125 b in BMSC-Exos induces autophagy and inhibits apoptosis of HG-injured HKCs by targeting TRAF6Objective:To verify the target binding relationship between miR-125 b and TRAF6.miR-125 b in BMSC-Exos induce autophagy and inhibits apoptosis of HG-injured HKCs by targeting TRAF6.Methods:1.Transfected with inhibitor NC or miR-125 b inhibitor into BMSCs,and the exosomes secreted by BMSCs after transfection were co-cultured with HG-injured HKCs to observe the effect of miR-125 b on TRAF6 expression.RT-q PCR was performed to detect the expression of TRAF6 m RNA in each group,and Western blot was performed to detect the TRAF6 protein levels.2.The 3’UTR of TRAF6 gene was predicted online to be a target of miR-125 b by using a bioinformatics analysis tool(Targetscan),and the dual luciferase reporter gene assay further validated the target relationship between TRAF6 and miR-125 b.The 3’UTR of TRAF6 containing a possible binding site for miR-125 b,was cloned into the p GL3-double luciferase expression vector to construct the wild-type or mutant reporter vector TRAF6(wt/mut).TRAF6(wt/mut)was transfected into HKCs with mimic NC,inhibitor NC,miR-125 b mimic(mimic)or miR-125 b inhibitor(inhibitor),and randomly divided into mimic NC+wt-TRAF6 group,miR-125 b mimic+wt-TRAF6 group,and inhibitor NC+wt-TRAF6 group,miR-125 b inhibitor+wt-TRAF6 group,mimic NC+mut-TRAF6 group,miR-125 b mimic+mut-TRAF6 group,inhibitor NC+mut-TRAF6 group,miR-125 b inhibitor+mut-TRAF6 group.Dual luciferase reporter gene assay for luciferase activity of TRAF6 3’-UTR plasmid in HKCs.3.TRAF6 overexpression vector was constructed and transfected with vector control(vector)for HG damage HKCs respectively,and co-cultured with BMSC-Exos after transfection.RT-q PCR was performed to detect TRAF6 m RNA expression in each group,and Western blot was performed to detect TRAF6 protein level in each group.4.Western blot detected the protein levels of LC3II/LC3 I and P62 in each group,and flow cytometry detected the apoptosis rate of HKCs after Annexin V/PI staining.Results:1.After HG-injured HKCs were co-cultured with BMSC-Exos,TRAF6 m RNA expression was significantly decreased,suggesting that miR-125 b of BMSC-Exos origin inhibited TRAF6 m RNA expression;after BMSC-Exos transfected with miR-125 b inhibitor was co-cultured with HG-injured HKCs,TRAF6 m RNA expression was significantly increased,suggesting that knockdown of miR-125 b promoted TRAF6 m RNA expression.After co-culture of HG-injured HKCs with BMSC-Exos,TRAF6 protein levels were significantly decreased,suggesting that miR-125 b of BMSC-Exos origin inhibited TRAF6 protein levels;after co-culture of BMSC-Exos transfected with miR-125 b inhibitor and HG-injured HKCs,TRAF6 protein levels were significantly increased.suggesting that knockdown of miR-125 b promotes TRAF6 protein expression.2.Successful construction of wild type(wt-TRAF6)and mutant(mut-TRAF6)of TRAF6 gene.After the p GL3-luciferase reporter vector for 3’UTR,the dual luciferase reporter gene assay showed that miR-125 b mimic significantly decreased the luciferase activity of the wt-TRAF6 TRAF6 3’-UTR plasmid compared to the mutant plasmid transfection group,while miR-125 b inhibitor significantly increased the wt-TRAF6 TRAF6 3’-UTR luciferase activity was elevated.The luciferase activity of HKCs remained unchanged when miR-125 b mimic or inhibitor was cotransfected with mut-TRAF6.3.HG-injured HKCs co-cultured with BMSC-Exos showed decreased TRAF6 m RNA expression and decreased TRAF6 protein levels;HG-injured HKCs overexpressing TRAF6 co-cultured with BMSC-Exos showed increased TRAF6 m RNA expression and increased TRAF6 protein levels.4.After HG-injured HKCs were co-cultured with BMSC-Exos,the LC3II/LC3 I protein level was significantly increased,P62 protein level was decreased,and apoptosis rate was significantly decreased;after HG-injured HKCs overexpressing TRAF6 were co-cultured with BMSC-Exos,the LC3II/LC3 I protein level was decreased,P62 protein level was increased,and apoptosis was significantly increased.It indicates that BMSC-Exos induced autophagy and inhibited apoptosis of HKCs by suppressing the expression of TRAF6 in HG-injured HKCs,and after overexpression of TRAF6,the autophagy of HG-injured HKCs co-cultured with BMSC-Exos decreased and apoptosis increased.Conclusion:1.Mi R-125 b represses TRAF6 expression and miR-125 b knockdown promotes TRAF6 expression.2.TRAF6 in HKCs is a direct target of miR-125 b.3.BMSC-Exos inhibited TRAF6 expression in HG-injured HKCs.4.BMSC-Exos induces autophagy and inhibits apoptosis in HG-injured HKCs by suppressing TRAF6 expression in HG-injured HKCs,and overexpression of TRAF6 reverses the autophagy-inducing and anti-apoptotic effects of BMSC-Exos on HG-injured HKCs.Taken together,BMSC-Exos-derived miR-125 b induced autophagy and inhibited apoptosis in high glucose injured HKCs by targeting TRAF6.Part III:Protection of BMSC-Exos-derived miR-125 b against HG-induced apoptosis of HKCs via TRAF6/AKT axisObjective:To further investigate the mechanism of miR-125 b in exosomes secreted by bone marrow mesenchymal stem cells on autophagy and apoptosis of high glucose-injured HKCs,we confirmed the protection of BMSC-Exos-derived miR-125 b against high glucose-induced apoptosis in human embryonic kidney epithelial cells via TRAF6/AKT axis.Methods:1.TRAF6 overexpression vector was constructed,and transfected with vector control(vector)into HG-injured HKCs,respectively,and co-cultured with BMSC-Exos after transfection,and the protein levels of AKT and P-AKT were detected by Western blot.2.Transfected inhibitor NC or miR-125 b inhibitor was transfected into BMSCs,and the exosomes secreted by BMSCs after transfection were co-cultured with HG-injured HKCs or added MK-2206(AKT inhibitor)and randomly divided into: H-Glu group,H-Glu+MSC-exo group,H-Glu+MSC-exo+inhibitor NC group,H-Glu+MSC-exo+miR-125 b inhibitor group,H-Glu+MSC-exo+MK-2206 group,and H-Glu+MSC-exo+MK-2206+miR-125 b inhibitor group.RT-q PCR was performed to detect miR-125 b m RNA and TRAF6 m RNA expression,and Western blot to detect TRAF6,AKT,P-AKT protein expression.3.The effect of altering miR-125 b expression on TRAF6,AKT,and P-AKT,and the protein levels of TRAF6,AKT,and P-AKT were detected by Western blot.4.The effect of altering miR-125 b expression on autophagy of HKCs,Western blot to detect the protein levels of LC3II/LC3 I,P62.5.The effect of changing the expression of miR-125 b,on the apoptosis of HKCs,flow cytometry detected the apoptosis rate of HKCs after Annexin V/PI staining.Results:1.HG-injured HKCs co-cultured with BMSC-Exos showed a decrease in P-AKT/AKT protein levels;HG-injured HKCs overexpressing TRAF6 co-cultured with BMSC-Exos showed a significant increase in P-AKT/AKT protein levels,indicating that BMSC-Exos inhibits AKT phosphorylation by downregulating TRAF6 and TRAF6 overexpression promotes AKT phosphorylation.2.Compared with the H-Glu group,miR-125 b m RNA expression was significantly increased and TRAF6 m RNA expression was significantly decreased in the H-Glu+MSC-exo group;compared with the H-Glu+MSC-exo+inhibitor NC group,miR-125 b m RNA expression was significantly decreased and TRAF6 m RNA expression was significantly increased;with the addition of MK-2206,there was no significant difference in miR-125 b m RNA expression and TRAF6 m RNA expression.It indicates that BMSC-Exos-derived miR-125 b down-regulates TRAF6 expression and inhibits miR-125 b up-regulates TRAF6 expression,so miR-125 b inhibits TRAF6 expression.3.Compared with H-Glu group,TRAF6 protein level was significantly decreased and P-AKT/AKT protein level was decreased in H-Glu+MSC-exo group;compared with H-Glu+MSC-exo+inhibitor NC group,TRAF6 protein level was significantly increased and P-AKT/AKT protein level was increased in H-Glu+MSCexo+miR-125 b inhibitor group.P-AKT/AKT protein levels were increased in the H-Glu+MSC-exo+inhibitor group compared to the H-Glu+MSC-exo+MK-2206 group;TRAF6 protein levels were significantly decreased and P-AKT/AKT protein levels were decreased in the H-Glu+MSC-exo+MK-2206 group.compared to the H-Glu+MSC-exo+miR-125 b inhibitor group,H-Glu+MSC-exo+MK-2206+miR-125 b inhibitor group showed no statistical difference in TRAF6 protein levels,but decreased P-AKT/AKT protein levels compared with H-Glu+MSC-exo+miR-125 b inhibitor group.The above results suggest that miR-125 b of BMSC-Exos origin downregulates TRAF6 expression and inhibits AKT phosphorylation,while BMSC-Exos inhibiting miR-125 b upregulates TRAF6 expression and promotes AKT phosphorylation,while MK-2206 reverses the phosphorylation of AKT caused by miR-125 b downregulation and has no effect on TRAF6 expression effect.The study confirmed that BMSC-Exos-derived miR-125 b inhibited TRAF6 expression and AKT phosphorylation.4.Compared with the H-Glu group,LC3II/LC3 I protein levels were significantly increased and P62 protein levels were significantly decreased in the H-Glu+MSC-exo+inhibitor group;compared with the H-Glu+MSC-exo+inhibitor NC group,LC3II/LC3 I protein levels were decreased and P62 protein levels were increased in the H-Glu+MSC-exo+miR-125 b inhibitor group.Compared with the H-Glu+MSC-exo+miR-125 b inhibitor group,the LC3II/LC3 I protein levels were increased and P62 protein levels were decreased in the H-Glu+MSCexo+MK-2206+miR-125 b inhibitor group;the above results suggest that BMSC-Exos-derived miR-125 b induced autophagy in HG-injured HKCs,while miR-125b-inhibiting BMSC-Exos inhibited autophagy in HKCs,and MK-2206 reversed miR-125 b downregulation leading to autophagy inhibition in HKCs.The study confirmed that BMSC-Exos-derived miR-125 b induced autophagy in HG-injured HKCs through AKT signaling pathway.5.Compared with the H-Glu group,the apoptosis rate was significantly reduced in the H-Glu+MSC-exo group;compared with the H-Glu+MSC-exo+inhibitor NC group,the apoptosis rate was significantly increased in the H-Glu+ MSC-exo +miR-125 b inhibitor group;compared with the H-Glu+MSC-exo+MK-2206 group,H-Glu+MSC-exo+MK-2206+miR-125 b inhibitor group showed a significant increase in apoptosis rate.The apoptosis rate was reduced in the H-Glu+ MSC-exo+ MK-2206+miR-125 b inhibitor group compared to the H-Glu+ MSC-exo+MK-2206+miR-125 b inhibitor group;the above results suggest that BMSC-Exos-derived miR-125 b inhibited the apoptosis of HG-damaged HKCs,and the miR-125b-derived BMSC-Exos promoted the apoptosis of HKCs,while MK-2206 reversed the miR-125 b down-regulation promoting the apoptosis of HKCs.It was confirmed that BMSC-Exos-derived miR-125 b induced autophagy and inhibited apoptosis in HG-injured HKCs by inhibiting the AKT signaling pathway.Conclusion:1.BMSC-Exos downregulates TRAF6 to inhibit AKT phosphorylation and overexpression of TRAF6 promotes AKT phosphorylation.2.Mi R-125 b inhibits TRAF6 expression.3.BMSC-Exos-derived miR-125 b inhibits TRAF6 expression and AKT phosphorylation.4.BMSC-Exos-derived miR-125 b induces autophagy in HKCs via AKT signaling pathway.5.BMSC-Exos-derived miR-125 b inhibits apoptosis of HKCs via AKT signaling pathway.Taken together,the protection of BMSC-Exos-derived miR-125 b against high glucose-induced apoptosis in human embryonic kidney epithelial cells via TRAF6/AKT axis was confirmed. |
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