| Objective To observe the effect of Nrf2-ARE pathway on cold ischemia-reperfusion injury in rat heart transplantation and to explore the related mechanism of sulforaphane(SFN) on protecting donor heart in rats’ heart transplantation.Methods 1 Forty health Sprague-Dawley rats were randomly divided into 3 groups: Sham group(n=8), I/R group(ischemia reperfusion group, n=16), I/R+SFN group(sulforaphane preconditioning group, n=16). Sham group: open and close abdomen was only done. 0.3ml normal saline was injected via tail vein 24 hours before operation; I/R group: 0.3ml normal saline was injected via recipient tail vein 24 hours before transplantation; I/R + SFN group: 0.3ml normal saline with sulforaphane(SFN2.5 mg / kg)was injected via recipient tail vein 24 hours before transplantation. Donor heart refrigerating at 4℃HTK solution 9hrs was transplanted into recipient intraperitoneal in I/R group and I/R+SFN group, so rat heterotopic heart transplantation model was established in both groups. Grafts were taken 24 hours after operation. Myocardial enzyme : Blood was taken from the recipient rats canthus ophthalmic vein or vena cava for lactate dehydrogenase(LDH), creatine kinase(CK-MB), and troponin(Tn T) at 6hrs and 24 hrs after reperfusion.Myocardial tissue(LPO) content and SOD activity were detected by thiobarbituric acid method(TBARS) method, xanthine oxidase. Histological changes by HE staining were observed in each group. Nrf2, HO-1and NQO1 protein expression levels of donor heart in each group 24 hrs after heart transplantation were observed using immunohistochemistry(En Vision Two-step method) and Western blotting methods.Results Myocardial enzyme: Serum LDH, CK-MB, and TnT were significantly higher I/R group and SFN+I/R group compared with sham group(P<0.05); Compared with the I/R group, serum LDH, CK-MB, and Tn T in SFN+I/R group were significantly lower(P <0.05) 6hrs after reperfusion;Tn T, CK-MB, and LDH activity significantly lower(P <0.05)24hrs after reperfusion.Myocardial tissue LPO content and SOD activity: Myocardial tissue SOD activity was significantly lower(P<0.05), but LPO was significantly higher(P<0.05) in I/R group and I/R+SFN group compared with sham group; compared with the I/R group, myocardial tissue SOD activity did not change, LPOwas significantly lower(P<0.05) in I/R+SFN group. Myocardial histology: Sham group: myocardial fibers arranged in neat rows, clear structure, no damage to the muscle fibers, interstitial no exudation of inflammatory cells, nuclei and clear. I/R group: myocardial edema, disorganized, myocardial fiber breakage, a large number of neutrophil infiltration; I/R+SFN Group:cardiac structure more clearly, myocardial fibers arranged in neat, myocardial tissue centers scattered sarcoplasmic neutrophils infiltration. Myocardial tissue Nrf2 and its downstream molecular HO-1 and NQO1 protein expression: myocardial tissue Nrf2 nucleoprotein, Nrf2, HO-1 and NQO1 protein levelswere significantly increased(P<0.01) in I/R+SFN group compared with I/R group, however myocardial histology and myocardial tissue Nrf2 nuclear protein, Nrf2, HO-1 and NQO1 Protein levels are still higher than normal(P<0.01) compared with the Sham group.Conclusions 1 Heart transplantation cold ischemia-reperfusion injury can activate Nrf2-ARE pathway. 2 Sulforaphane protects cold ischemia-reperfusion injury in heart transplantation, which may be through activation of Nrf2-ARE pathway, up-regulation of downstream gene HO-1, NQO1 expression, and increase of myocardial cells against oxidative stress defense capability. |
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