| Objective: To investigate the protective effect of human umbilical cord mesenchymal stem cells derived exosomes(huc MSC-EXOs)on renal ischemia reperfusion injury and clarify the important role and regulatory mechanism of transient receptor potential canonical 6(TRPC6)/ poly adenosine-diphosphate-ribose polymerase 1(PARP1)signaling pathway in this process.Methods:(1)Huc MSCs of 3-8 generations with good growth were selected and cultured in DMEM/F12 medium(containing 10% fetal bovine serum and 1%penicillin-streptomycin solution).When the cells reached 80%-90% degree of fusion,the serum-free medium was changed for culture,and the culture supernatant was collected after 24-48 h culture.The collected supernatant was placed in a centrifugal tube for gradient centrifugation extraction of exosomes by overspeed centrifugal method.The obtained exosomes were identified by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and Western blot analysis.(2)Thirty SPF-grade healthy male SD rats with body weight of 220g-250 g were randomly divided into 5 groups: sham operation group(S group),sham operation plus TRPC6 inhibitor SKF96365 group(SS group),renal ischemia reperfusion group(IRI group),renal ischemia reperfusion plus exosome group(EXO group),exosome plus TRPC6 inhibitor SKF96365 group(ES group),with 6 rats in each group.The renal IRI model of rats was established by clamping bilateral renal pedicles for 45 min and reperfusion for 24 h.In EXO group and ES group,20 μg exosome solution was randomly injected into five sites of left kidney 15 min before clamped bilateral renal pedicles,and the other groups were injected with equal volume PBS.The ES group was injected 1.5mg/kg of TRPC6 inhibitor SKF96365 through the tail vein 5 min before clamped renal pedicles,and the SS group was injected the same amount of SKF96365 through the tail vein 5 min before free bilateral renal pedicles.The rats were killed 24 h after reperfusion.The serum and renal tissue samples were collected,and the pathological changes of renal tissue were observed by optical microscope after HE staining and Paller score was performed.The changes of renal function were evaluated by creatinine(Cr)and blood urea nitrogen(BUN).Western blot was used to determine the expression levels of receptor-interacting protein kinase(RIPK)1,RIPK3,mixed lineage kinase domain like protein(MLKL),TRPC6,PARP1 expression levels in kidney tissue.Results:(1)The results of exosome identification showed that huc MSC-EXOs were small vesicles with double plasma membrane,which were saucer-type or hemispherical with one side depression by TEM.NTA results confirmed that the extracted exosomes were 30-150 nm in particle size.Western blot analysis of exosomes showed that the positive expression of marker proteins CD9,CD63 and CD81,which was consistent with the characteristics of exosomes.(2)Compared with S group,SS group had no kidney tissue damage,Paller score,Cr and BUN levels were normal(P>0.05).IRI group had obvious renal tissue damage,Paller score,Cr and BUN levels were increased,RIPK1,RIPK3,MLKL expression was up-regulated,TRPC6,PARP1 expression was down-regulated(P<0.05);Compared with IRI group,Paller score,Cr and BUN levels were decreased,the expressions of RIPK1,RIPK3 and MLKL were down-regulated,and TRPC6 and PARP1 were up-regulated in EXO group(P<0.05);Compared with EXO group,Paller score,Cr and BUN levels were increased,the expressions of RIPK1,RIPK3 and MLKL were up-regulated,and TRPC6 and PARP1 were down-regulated in ES group(P<0.05).Conclusion: Huc MSC-EXOs can alleviate necroptosis induced by renal ischemia reperfusion injury in rats,and its protective mechanism is related to activation of TRPC6/PARP1 pathway. |
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