Study On Protective Effects Of Remifentynil On Hepatocytes Against Injury Induced By Anoxia-reoxygenation

Background:With the lasting development and deeply research of organ transplantation,we realized gradually that the cell apoptosis had a special biology nuance to organ transplantation.It was reported that the cell apoptosis was one of the important machenisms leading to hepatocytes injury inducd by ischemia-reperfusion,which was the key factor determined the fate of transplanted liver.Researchers had thoroughly studied the protective effect of opioid on cadiocyte and neurocyte injury induced by ischemia-reperfusion.And it demonstrated that the protective effect was related to protein kinase C and MAPK.The protein kinase C which contains abundent serine and threonine is composed of 77-23 kd polypeptide single strand,and can adjust the nervous excitation,synapsis moulding,cellular proliferation and differentiation,gene expression,cell necrosis besides cell apoptosis.There were few reports on effect of remifentanil on hepatocytes against injury induced by ischemia-reperfusion, also the role of protein kinase C played was not clarified.Objective:To study the altered expression of protein kinase C and the influence of remifentanil on expression of protein kinase C on cultured human hepatocytes after anoxia-reoxygenation. To study the protective effect and the mechanism of remifentanil on cultured human hepatocytes against anoxia-reoxygenation injury.Methods:This study is composed of two parts.1.The effect of remifentanil on cultured human hepatocytes against injury induced by anoxia-reoxygenation.A hepatocyte anoxia-reoxygenation model was established.Cultured hepatocytes were randomly divided into 7 groups:group A received no anoxia served as control;group B received 15 hours anoxia followed by 5 hours reoxygenation;and group C1,C2,C3,C4,C5 each received 5ng/ml,10ng/ml,20ng/ml,30ng/ml,40ng/ml remifentanil before reoxygenation.The content of ALT,AST,MDA in the culture medium and the SOD viability in the mitochondria were measured at the end of the experiment.2.The relationship between the effect of remifentanil and the PKC on cultured human hepatocytes against injury induced by anoxia-reoxygenation.A hepatocyte anoxia-reoxygenation model was established.Cultured hepatocytes were randomly divided into 5 groups:group C received no anoxia served as control;groups AR,R,CH,R+CH each received 15 hours anoxia followed by 5 hours reoxygenation;group AR just exposure to anoxia and reoxygenation;group R received 5ng/ml remifentanil;group CH received 10umol/1 chelerythrine;group R+CH received 5ng/ml remifentanil and 10umol/l chelerythrine before re-oxygenation.The content of MDA in the mitochondria were measured at the end of the experiment.The rate of apoptotic cell was measured by flow cytometry.The transcription of protein kinase C-αmRNA was measured by RT-PCR. The expression of protein kinase C-αwas measured by immunohistochemistry.Results:1.Anoxia-reoxygenation caused dramatic increase in the content of ALT,AST,MDA, and decrease in SOD viability compared with those in control group.Remifentanil 5ng/ml, 10ng/ml,and 20ng/ml significantly attenuated the increases in ALT,AST,MDA content, and enhanced SOD viability caused by anoxia-reoxygenation.The result of remifentanil group beyond 30ng/ml made nodifference compared control.2.Anoxia reoxygenation caused dramatic increase in the content of MDA in group AR. The MDA content in group R and CH were between that in group C and AR.3.Anoxia re-oxygenation caused dramatic increase in the rate of apoptotic cell in group AR.The rate of apoptotic cell in group R and CH were between that in group C and AR.4.Anoxia reoxygenation caused dramatic increase in the transcription of protein kinase C-αmRNA in group AR.The transcription of protein kinase C-αmRNA in group R and CH were between that in group C and AR.The transcription of protein kinase C-αmRNA in group R+CH was less than that in group C.5.Anoxia reoxygenation caused dramatic increase in the expression of protein kinase C-αin group AR.The expression of protein kinase C-αin group R and CH were between that in group C and AR.The expression of protein kinase C-αin group R+CH was less than that in group C.Conclusions:1.Anoxia reoxygenation caused dramatic increase in the transcription of protein kinase C-αmRNA,so was the expression and the activities of protein kinase C-α.2.Chelerythrine(CH) could decrease apoptosis of cultured human hepatocyte after anoxia-reoxygenation.3.Low concentrative remifentanil could produce protective effects on cultured hepatocytes after anoxia-reoxygenation injury.4.Remifentanil could produce protective effects on cultured hepatocytes after anoxiareoxygenation injury,and one of the protective mechanisms may be by inhibiting the transcription of protein kinase C-αmRNA and expression of protein kinase C-α.