| Objectives To explore the effect of PI3K/Akt/GSK-3β signaling pathway in sulforaphane protecting myocardial cold ischemia reperfusion injury through establishing the rats heart transplantation model.Methods 64 SD rats(adult healthy male, weight 230-250 g) were randomly divided into 4 groups(16 rats in each group,drug pretreatment are recipient rats): IRI group:recipient rats were injected physiological saline 0.3 ml via tail vein in 24 hours before heart transplantation. SFN+IRI group: recipient rats were injected SFN2.5mg/kg.bw via tail vein in 24 hours before heart transplantation. LY+IRI group: recipient rats were injected LY294002 0.3mg/Kg.bw via tail vein before 1 hours of injecting physiological saline and before 24 hours of heart transplantation, the other procedure was the same with IRI group. LY+SFN+IRI group:recipient rats were injected LY294002 0.3mg/Kg.bw via tail vein before 1 hours of injecting SFN and before 24 hours of heart transplantation, the other procedure was the same with SFN+IRI group. Heart transplantation model was established according to Li Zhanqing’s method: Donor heart placed in 4℃HTK for 9hours was transplanted into the receptor cavity(The donor heart aorta and main pulmonary artery were anastomosed with the recipient rat abdominal aorta and inferior vena cava individely), donor rats’ heart restored to beat, checked no bleeding and closed the abdominal cavity. Donor heart was removed for histology, immuohistochemistry and western blot 24 hrs after heart transplant. Part of the myocardial tissue was fixed with10% formaldehyde solution, paraffin embed, routine HE stained. Histology was observed by light microscope. The expression of Akt, P-Akt, GSK-3β, P-GSK-3β, Bcl-2 and Bax protein were checked by immunohistochemistry and western blot.Results 1 Histology: Myocardial injury was most serious in IRI group and LY+IRI group: normal structure of myocardial tissue destruction, Visible small blood vessels to dilate, hyperaemia, myocardial fiber breakage and arranged in disorder, Cell edema,myocardial interstitial a lot of inflammatory cell infiltration.However myocardial injury was the most light in SFN+IRI group: myocardial tissue structure is clear, Small blood vessels to dilate, hyperaemia of lighter, the myocardial fibers arranged in neat rows, slight fracture, cell morphology was normal, myocardial interstitial a small amount of inflammatory cell infiltration. myocardial cell injury in LY294002+SFN+IRI group was between IRI group and SFN+IRI group. 2 Immunohistochemistry and Western blot results: The positive expression of Akt, P-Akt, GSK-3β, P-GSK-3β, Bcl-2, Bax protein by immunohistochemical staining mainly located in the cytoplasm of myocardial cells,brownish yellow reaction. 1) The expression of Akt protein in each group are slightly different( P>0.05, no statistical significance). The expression of P-Akt protein in SFN+IRI group increased significantly compared with the IRI group( P<0.05, with statistical significance). After pretreatment using LY294002, the expression of P-Akt protein was significantly lower in LY+SFN+IRI group compared with LY+IRI group(P>0.05, no statistical significance). 2) The expression of GSK-3β protein in each group are slightly different( P>0.05, no statistical significance). The expression of PGSK-3β protein in SFN+IRI group increased significantly compared with the IRI group( P<0.05, with statistical significance). After pretreatment using LY294002, the expression of P-GSK-3β protein was significantly lower in LY+SFN+IRI group compared with LY+IRI group(P>0.05, no statistical significance). 3) The expression of Bcl-2 protein increased significantly and reduced Bax protein expression significantly in SFN+IRI group compared with IRI group(P<0.05, statistically significant). After pretreatment using LY294002, the expression of Bcl-2 protein decreased and Bax protein expression increased in LY+SFN+IRI group. The protein content of Bcl-2 and Bax were between SFN+IRI group and LY+IRI group and compared with SFN+IRI group and LY+IRI group are statistical significance(P<0.05).Conclusions Sulforaphane protects myocardial cold ischemia reperfusion injury though reactiving PI3K/Akt/GSK-3β signaling pathway and regulating the expression of apoptosis protein, but it is not the only signaling pathway. |
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