| ObjectiveThe study was designed to investigate the expression and localization alteration of SGK-1 in IRI process after rat heart transplantation, and to explore the role of SGK-1 playing in cardiomyocytes apoptosis caused by IRI after heart transplantation. In addition, we wanted to know whether Dexa can increase the expression of SGK-1 and minimize cardiomyocyte damage or not.Methods1?Allogeneic heart transplantation(AHT) model was performed from Wistar(WF: RT1u)into Lewis(Lew: RT11)rats, and syngeneic heart transplantation(SHT) model was from Lewis(Lew: RT11) into Lewis(Lew: RT11) rats. In some groups, part donors were treated with Dexa 2 h prior at doses of 0.05, 0.5 and 2mg/BWkg, respectively.2?RT-PCR was performed to investigate the expression of SGK-1 mRNA after AHT/SHT.3?In IRI process after AHT/SHT, immunohischemistry, western blot analysis and double immunofluorescent staining were employed to investigate the expression, tissue distribution and localization of SGK-1.4?Double immunofluorescent staining was performed to observe the co-localization of SGK-1 with different markers as follows: ?-actinin, CD4, VCAM-1 and active caspase-3.5?The cardiac tissue were immersed in 10% formalin, fastened in paraffin, sectioned at 7 ?m, stained with hematoxylin and eosin(HE) for light microscopy.6?All of the data were analyzed with the Stata 7.0 statistical software. All of the values were expressed as the mean ± SEM.Results1?SGK-1 mRNA and protein expression were markedly increased in grafted heart 6-12 hours post transplantation in both the allogenic and isogenic models by RT-PCR and western blot. SGK-1 gradually increased from post-transplantation 6 h(P<0.05), and peaked at 12 h(P<0.001). 24 h after heart transplantation, the SGK-1 mRNA and protein expression began to decrease and reached to a level similar to that of the 6 h group.2?Iimmunohischemistry and double immunofluorescent staining experiments confirmed that SGK-1 was expressed in cardiomyoctes rather than infiltrated immune cells. And the changes of SGK-1 expression in IRI process after AHT/SHT were very similar to the results of western blot analysis.3?The co-localization of SGK-1 with ?-actinin was observed in AHT/SHT. Co-localization of SGK-1/VCAM-1, SGK-1/CD4 were not observed after heart transplantation. The result of co-localization between SGK-1 and active caspase-3 was found after heart transplantation.4?Immunohischemistry, western blot analysis and double immunofluorescent staining showed that SGK-1 expression was gradually increased under different doses of Dexa(0.05 mg/BWkg; 0.5 mg/BWkg; 2 mg/BWkg) treatments.5?Furthermore, HE staining showed the donor cardiomyocyte injury was greatly minimized by Dexa treatment.Conclusions1?The expression level of SGK-1 is up-regulated in IRI process after AHT/SHT, and SGK-1 is mainly expressed in cardiomyoctes.2?All results suggested that SGK-1 has significant anti-apoptosis effects on cardiomyocytes apoptosis caused by IRI after heart transplantation, and the mechanism is related to NF-kB. These results provide an insight into the alterations of SGK-1 protein expression in IRI process, which supports that SGK- 1 emerges as an attractive protective agent for the prevention of IRI after heart transplantation.3?In addition, Dexa increases the expression of SGK-1 and minimizes cardiomyocyte damage. It will provide a new theoretical basis for reducing cardiomyocytes apoptosis, and SGK-1 will be the novel therapeutic target in IRI process after heart transplantation. |
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