| [Objective]Sulforaphane, which is a naturally occurring isothiocyanate that is present in cruciferous vegetables, can alleviate renal、brain、intestinal ischemia-reperfusion injury. So far,we have not seen report about sulforaphane alleviating hepatic warm ischemia-reperfusion injury.To establish rat hepactic warm ischemia reperfusion injury model, use reagent of sulforaphane on the pretreatment of experimental animals and analyze the protein molecules related to Nrf2-ARE pathway activation、serum aminotransferase、factors related to apoptosis and hepatic histopathology after reperfusion completed. The paper discusses the alleviation of rat hepatic warm ischemia reperfusion injury mediated by sulforaphane and the possible mechanisms lies in it, which would provide us with a new idea to find a therapeutic way for hepatic ischemia reperfusion injury.[Method]1.To establish the models of70%part of hepatic ischemia-reperfusion injury (HIRI) by the operation method narrated by Nauta RJ;2.Male specific pathogen free Sprague-Dawley (SD) Rats weighing250-280g were used and divided randomly into three group:sham operation group(SO) ischemia reperfusion group(IR), sulforaphane pretreatment group (SFN),each group is divided into two equal subgroups randomly by portal reperfusion time, they are SO6h,SO24h,IR6h,IR24h,SFN6h and SFN24h (each subgroup n=8).Only laparotomy and anatomy of hepatic portal operation,without Drugs and hepatic ischemia were applied in SO group; hepatic ischemia and normal saline were applied in IR grou;Sulforaphane,25mg/Kg,dissolved in0.5ml NS, was applied through intraperitoneal injection(ip)an hour prio to HIRI.Animals of6h-subgroups and24h-subgroups were killed to obtain sample of blood(5ml) through aortaventralis and fresh liver tissue for further analysis6hours and24hours after portal opened respectively. Serum level of two aminotransferases (ALT、AST) were analyzed to evaluate hepatocellular function; Bcl-2、 Bax in liver tissue and Nrf2in nucleoprotein were detected by Western Blot. Liver tissue Caspase-3activity was analyzed with Caspase-3activity assay.Hepatocellular damage and apoptosis were analyzed by hematoxylin-eosin staining and Tunel staining respectively.[Result]1.48cases of hepatic ischemia-reperfusion injury models were successfully were established.The partial liver ischemia time was60min.2. Serum ALT and AST level:Compared with the same time point subgroup in SO group, Serum ALT and ALT level in IR6h,IR24and SFN6h were significantly higher (p<0.05), SFN24is a little higher than SO24,but the difference is not statistically significant (P>0.05); Compared with the same time point subgroup in IR group,Serum ALT and ALT level in SFN6h and SFN24h subgroups were significantly lower(p<0.05);Compared between subgroups in each group:The difference between SO6h and SO24is not statistically significant(P>0.05),6h subgroup is significantly higher than24h subgroup in IR and SFN group (p<0.05).3. Activity of Caspase-3:Compared with the same time point subgroup in SO group,IR and SFN group are significantly higher (p<0.05); Compared with the same time point subgroup in IR group, SFN6h and SFN24are significantly lower(p<0.05);Compared between subgroups in each group:The difference between SO6h and SO24is not statistically significant (P>0.05),6h subgroup is significantly higher than24h subgroup both in IR and SFN group (p<0.05).4.western Blot:(1) Nrf2in nucleoprotein:Compared with the same time point subgroup in SO group,SFN6h and SFN24h are significantly higher (p<0.05),IR6h and IR24are significantly lower (p<0.05); Compared with the same time point subgroup in IR group, SFN6h and SFN24are significantly higher(p<0.05); Compared between subgroups in each group:The differences between two subgroups of SO and IR group are not statistically significant (p>0.05),SFN6h is significantly higher than SFN24(p<0.05).(2)Bcl-2in liver tissue:Compared with the same time point subgroup in SO group:Both SFN6h and SFN24are significantly higher (p<0.05),IR6h and IR24are a little higher,but the difference between them is not significant statistically.Compared with same time point subgroup in IR group, both SFN6h and SFN24are significantly higher (p<0.05). Compared between subgroups in each group:6h subgroup is a little higher than24h subgroup both in IR and SO group, but the differences between them are not statistically significant,SFN6h is significantly higher than SFN24.(3) Bax in liver tissue:Compared with the same time point subgroup in SO group:IR6h and IR24are significantly higher (p<0.05), SFN6h and SFN24h are a little higher,but the differences between them are not statistically significantly(p>0.05); Compared with the same time point subgroup in IR group:SFN6h and SFN24h are significantly lower (p<0.05) Compared between subgroups in each group:6h subgroup is a little lower than24h subgroup in both SO and SFN group, but the differences between them are not statistically significantly(p>0.05),IR6h is significantly higher than IR24h (p<0.05)(4).Bcl-2/Bax:Compared with the same time point subgroup in SO group:IR6h and IR24are significantly lower (p<0.05), and SFN6h and SFN24are significantly higher (p<0.05) Compared with the same time point subgroup in IR group:SFN6h and SFN24are significantly higher (p<0.05).Compared between subgroups in each group:24h subgroup is a little higher than6h subgroup in SO and IR group,but the differences between them are not statistically significant (p>0.05).SFN6h is significantly higher than SFN24h (p<0.05)5.Apoptosis Index:Compared with the same time point subgroup in SO group:IR and SFN group are significantly higher (p<0.05). Compared with the same time point subgroup in IR group:SFN6h and SFN24are significantly lower (p<0.05). Compared between subgroups in each group:SO24is a little higher than SO6h, but the difference between them is not statistically significant,6h subgroup is significantly higher than24h subgroup in IR and SFN group(p<0.05). 6. Histological results:In two subgroups of SO group, the structure of hepatic lobule and liver morphology were normal,there are no cell degeneration and necrosis, there are just a few inflammatory cells infiltration around the portal area; In IR6h and IR24h, the structure of hepatic lobule were disorder, Liver cell morphology were extensive edema while different degrees of vacuolar degeneration, others were partial liver cell spotty necrosis and eosinophil increase, While in SFN6h and SFN24h, the structure of hepatic lobule and Liver cell morphology were nearly normal, hepatocellular and portal area are nearly normal, there are a few mild liver cell edema, the inflammatory cells infiltration is much less than IR group.[Conclusion]1.Rat hepatic warm ischemia reperfusion injury model is stable、reliable、simple and workable.2. Sulforaphane pretreatment can alleviate rat hepatic warm ischemia-reperfusion injury.Hepatic warm ischemia-reperfusion injury can induce Hepatocellular apoptosis, which can be alleviated by sulforaphane pretreatment. Upregulation of Bcl-2and downregulation of Bax,as a result,ratio of Bcl-2/Bax elevated,which may be the mechanism of sulforaphane mediating alleviation of hepatic ischemia reperfusion injury;3.The activation of Nrf2-ARE pathway and inhibition of apoptosis may be mechanism of sulforaphane alleviating rat hepatic warm ischemia reperfusion injury. |
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