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Objective To investigate the mechanisms of neuroprotective roles of phosphatase and tensin homolog allele activity deleted in chromosome 10 (PTEN) inhibition on neuronal apoptosis after hypoxia-ischemia (HI)damage.Methods The cerebral cortical neurons of newbom Sprague-Dawley rats were cultured in vitro.Oxygen and glucose deprivation (OGD) model was established to imitate HI environment in vivo.Neurons were divided randomly into 3 groups:control group:neurons were treated with normal medium ; OGD group:neurons were treated with OGD for 3 h followed by reperfusion at 0.5,3.0,6.0,12.0,24.0,48.0 h ; PTEN inhibition group:before OGD treatment,neurons were pretreated with PTEN inhibitor,and then the neurons were collected at 24 h after reperfusion.Terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling staining was used to detect the apoptotic cells.Western blot was used to detect the expression of PTEN,p-PTEN,protein kinase B(Akt),p-Akt,synthesis of glucose kinase-3 betal (GSK-3β),p-GSK-3β and myeloid cell ceukemia-1 (Mcl-1).Results 1.As compared with control group,TUNEL positive cells increased after OGD observed by TUNEL staining (P ＜ 0.05).The expression of PTEN was increased,the expressions of p-PTEN,p-Akt,p-GSK-3 β,and Mcl-1 were significantly decreased after OGD (all P ＜ 0.05).However,Akt and GSK-3β remained unchanged at different time points after OGD(all P ＞ 0.05).2.As compared with OGD group,TUNEL positive cells were obviously reduced at 24 h after OGD in PTEN group (P ＜ 0.05).Although total PTEN,Akt,and GSK-3 β were not obviously changed in PTEN group(all P ＞ 0.05),the expressions of p-PTEN,p-Akt,p-GSK-3β,and Mcl-1 were significantly increased after OGD (all P ＜ 0.05).Conclusions HI can induce neuronal apoptosis,and the mechanisms of apoptosis may involve PTEN/Akt/GSK-3β/Mcl-1 pathway.The phosphorylation of Akt and GSK-3β can be increased via PTEN activity inhibition,while the ubiquitination of Mcl-1 would be decreased,and thus reduce the neuronal apoptosis.
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Objective To investigate the expression and significance of glucogen synthase kinase-3β (GSK-3β) in the pathogenesis of chronic allograft nephropathy (CAN) in rats.Methods Kidneys of Fisher (F344) rats as donors were orthotopically transplanted into Lewis (LEW) rats as recipients.The renal function and histopathological changes were observed at 4,8,12,16,and 24week post-transplantation.Phosphorylated GSK-3β (p-GSK-3β) protein and mRNA expression was determined by using immunohistological assays and RT-PCR respectively.Results Our data showed that 24-h urinary protein excretion in CAN rats was increased significantly at week 16 as compared with F344/LEW controls.Allografts showed markedly increased mononuclear cells infiltration and presented with severe interstitial fibrosis and tubular atrophy at 16 and 24 week post-transplantation.p-GSK-3β expression (protein/mRNA) was down-regulated in rat kidneys with CAN,and the decrease became more significant over time after transplantation.p-GSK-3β expression was correlated significantly with 24-h urinary protein excretion,serum creatinine levels,tubulointerstitial mononuclear cells infiltration,smooth muscle cells migration in vascular wall,and interstitial fibrosis.Conclusion It was concluded that GSK-3β down-regulation was the key event that may be involved in mononuclear cells infiltration and vascular SMCs migration at early stage,and interstitial fibrosis and allograft nephroangiosclerosis at later stage of CAN pathogenesis in rats.