| Objective To investigate the protective effect of combined adipose-derived mesenchymal stem cells(ADSCs)with dexmedetomidine on acute ischemia-reperfusion injury(IRI)in rats.Methods Forty adult healthy SD male rats were randomly divided into five groups:Sham-operated(Sham)group,the ischemia reperfusion injury(IR)group,the dexmedetomidine-treated(IR+DEX)group,ADSCs-treated(IR+ADSCs)group and the dexmedetomidine and ADSCs-treated(IR+DEX+ADSCs)group.Isolated ADSCs were cultured,moreover,it was identified using adipogenie and osteoplastic differentiation,and using flow cytometry for determination of the phenotypes of the cultured cells.To establish the IRI model in rats(clamping the both renal pedicles for 40 min by vascular clips,and then removing the clips for kidney reperfusion),and the rats receiving cell suspension of ADSCs(1×106)through tail vein or/and intraperitoneal injection of dexmedetomidine 25μg/kg according to the above groups.(1)The plasma samples in each group were harvested for determination the level of serum creatinine(Scr)and blood urea nitrogen(BUN)at Oh(before the treatment),24h and 96h(after the experimental treatment).Meanwhile,urine samples of rats in each group were collected at the same time(0h,24h and 96h)for measurement of urine protein(UP)and urine creatinine(UC),and then calculate the ratio of urine protein/creatinine(UP/UC).The rats were euthanized and kidney specimens were collected for HE stainning at 96h after the experimental treatment,and the histopathology injury of kidney was scored.(2)At 96h after experimental treatment,the mRNA expression of TNF-a,IL-6,IL-1β and IL-10 of kidney specimens were quantified by real-time quantitative PCR,and the protein expression of it also analyzed by ELISA.The indicators related oxidative stress of kidney such as MDA,ROS(the level of oxygen radical)and SOD(the capacity of scavenging oxygen radical)were detected by Assay Kit.The expression of oxidative stress related protein such as NOX-1,NOX-2(oxidation protein),NQO-1 and HO-1(anti-oxidation protein)were analyzed by western blot.(3)By 96h after the treatment,the protein expression of rats kidney were analyzed by western blot,which including PI3K/Akt signal pathway related protein(PI3K,p-Akt),vascular endothelial related factors(VEGF,eNOS,CD31),apoptosis-related factors(Caspase-3,Bax)and anti-apoptosis factor Bcl-2.Meanwhile,the mRNA expression of VEGF,eNOS,Bcl-2 and Caspase-3 were quantified by real-time quantitative PCR-Kidney apoptosis was detected with TUNEL assay.The kidney expression of CD31 was analyzed by immunofluorescence staining,and Caspase-3 was analyzed by immunohistochemistry.Results(1)ADSCs showed fusiform and uniform shape size,and were arranged in whirlpool or radial configuration.Flow cytometry indicated that ADSCs expressed high level of CD90(91%),CD73(94%),CD44(87%),CD166(89%).ADSCs were also successfully differentiated into osteoblasts and lipocytes.The renal function index and tissue injury scores of IRI demonstrated that using either dexmedetomidine or ADSCs could attenuate renal IRI and improve renal function to different extent.However,combined dexmedetomidine with ADSCs could further enhance the above effects(P<0.05).(2)The PCR and ELISA findings of inflammatory factors showed that combination of dexmedetomidine and ADSCs could significantly inhibit inflammatory factors released,as compare with either dexmedetomidine or ADSCs treated(P<0.05).Meanwhile,it could further decrease the level of MDA,ROS and increase the activity of SOD(P<0.05).The results of Western blot showed that combining dexmedetomidine with ADSCs not only significantly de-regulated the oxidation protein expression(NOX-1,NOX-2),but also up-regulated the anti-oxidation protein expression(NQO-1,HO-1)(P<0.05).(3)As compare with dexmedetomidine or ADSCs treated group,the combined group remarkably increased the mRNA expression of VEGF,eNOS,Bcl-2,but decreased the level of Caspase-3 mRNA after renal IRI(P<0.05).The results of Western blot showed that combined dexmedetomidine with ADSCs can further increased the protein expression of PI3K,p-Akt,VEGF,eNOS,CD31 and Bcl-2 while reduced the protein expression of Caspase-3,Bax.The TUNEL assayed findings showed that combination of dexmedetomidine and ADSCs also inhibited apoptosis of kidney ischemia reperfusion injury(P<0.05).Besides,it further prominently increased the expression of CD31 immunofluorescence staining and decreased the expression of Caspase-3 immunohistochemistry(P<0.05).Conclusion In acute kidney IRI model of rats,using eithter dexmedetomidine or ADSCs could inhibit inflammation,oxidative stress,apoptosis and enhance angiogenesis to some extent;Moreover,combined dexmedetomidine with ADSCs could further strengthen the above effects and improve renal function and mitigate renal tissue injury,hence,combined dexmedetomidine with ADSCs could provide significant renal protection. |
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