| Objective:Warm ischemia reperfusion(WI/R)injury is one of the reasons for postoperative complications and organ failure.With development of DCD organ in transplantaiton,more and more researchers focus their attention on WI/R injury.Evidence accumulated reported that reactive oxide species,Ca2+ overload and cellular immunity were responsible for the acute liver injury within 6-24 hours after liver transplantaiton.However,our findings showed that liver tissue could suffer from persistent damage after the acute liver injury.This persistent damage,which was closely related with the warm ischemia time,reflected in the increasing liver enzyme and high proportion of apoptotic cells.Once the condition deteriorated,the transplant liver could suffer from irreparable damage.The phenomenon were able to recur to the clinic patient after accepted the liver transplantation of DCD organ.The liver enzyme fluctuated within 6 to 11 days after surgery.Until now no research had explained the mechanism that the transplant liver tissue originated in DCD donor suffered from acute-to-chronic injuryCalcium-calmodulin-dependent protein kinase type 2(CaMK II)is a serine/threonine protein kinase.When combined with Ca2+/CaM-binding,CaMK ? undergoes activation by autophosphorylation;The activation of CaMK ? has a hand in the regulation of cell apoptosis,memory and tumor proliferation.The recent study had been verified that the expression of CaMK ? promotes the apoptosis of myocardial after suffering from warm ischemia reperfusion injury.However,the mechanism of CaMK ? regulation of post-liver transplantation is not clear.Our research was to construct the model of SD rat liver transplantation suffering from warm ischemia reperfusion injury.Subsequently,the relationship between CaMK ?and warm ischemia reperfusion injury were analyzed,and the correlation between CaMK ? and liver function were verified.For further experiments,the signs that CaMK ? induced the hepatocyte damage and the mechanism that CaMK ?aggravated the I/R injury were displayed in vitro and in vivo.The study about CaMK? regulating the WI/R injury after liver transplantation find ways to explain the mechanism of acute-to-chronic injury in transplant liver after utilized DCD organ and provide a new treatment target for WI/R injury.Methods:1.the model of SD rat liver transplantation suffering from warm ischemia reperfusion injury was constructed.The SD rat models were divided in three groups according to the warm ischemia time(warm ischemia time 0 min,warm ischemia time 10 min,warm ischemia time 20 min).Western blot,q-PCR and Immunohistochemical(IHC)Staining were used to test the expression of CaMK ? in the transplant liver under the given warm ischemia time.The levels of AST and ALT were detected in the 1 st,3rd,7th day after liver transplantation.Finally,the correlation between the levels of liver enzyme and the expression of CaMK ? were analyzed.2.The expression of CaMK ? had an effect on the BRL-3A cell line in vitro.The CaMK ? overexpression carriers and interference carriers were constructed as required,and then infected the BRL-3A cell lines.We divided the cell lines into 5 groups(control groups,LV-sh-CaMK ?-NC groups,LV-CaMK ? groups,LV-sh-CaMK ?-NC groups and LV-sh-CaMK groups).FITC-Annexin V/Propidium Iodide Staining Assay were tested the apoptosis of the BRL-3A cells,flow cytometry were detected the cell cycle,and CCK-8 were utilized to analyze the proliferation of the BRL-3A cells.Lastly,western blot was detected the levels of protein AIF and cytochrome c3.The expression of CaMK ? had an effect on the SD rat model of liver transplantation in vivo.The CaMK ? overexpression carriers and interference carriers were injected to the SD rat model of liver transplantation.According to the experiment,we divided the SD rats into 5 groups(control groups,LV-sh-CaMK II-NC 1groups,LV-CaMK ? groups,LV-sh-CaMK ?-NC and LV-sh-CaMK ? groups).After the 7th day of liver transplantation,TUNEL was use to detect the apoptotic proportion and H&E was used to observe the pathological change.The levels of AST and ALT in blood were tested at the 3rd and 7th day after surgery.To illuminate the mechanism of WI/R injury,Flou-3 was utilized to detect the concentration of Ca2+,JC-10 was used to test the change of mitochondrial membrane potential(MMP),and transmission electron microscope was used to observe the morphologic change of mitochondrial membrane and mitochondrial cristae.Similarly,western blot was detected the levels of protein AIF and cytochrome c in the liver transplant models.Finally,protein chip was used to detect the differential expression of protein in the LV-sh-CaMK ? groups vs control groups.Results:1.we firstly constructed the I/R models with prolonged WIT.Compare to the 1st and 3rd day of post-operation,the expression of CaMK ? was largely increased(p<0.05),when the WIT was prolonged to 10 min and 20 min,CaMK ? protein level was largely increased when compared with the normal group(p<0.05).The level of aminotransferase increased in the first day after operation,decresed in the third day,and increased again in the seventh day.Further more,the expression of CaMK ? have a significant linear correlation with the levels of AST(p=0.001,r=0.748)and ALT(p=0.043,r=0.495)after the 3rd and 7th day of liver transplantation.2.To analyze the effects of CaMK ? on hepatocyte,Flow cytometry assay maked us know that the apoptosis frequency of BRL-3A cells were increased in the LV-CaMK ? group,and decreased in LV-sh-CaMK ? group compared with control group(p<0.05).The S phase of BRL-3A cells were prolonged in LV-sh-CaMK ?group and shortened in LV-CaMK ? group when compared to the control group(p<0.05).Simultaneously,the cell viability was increased after inhibiting the CaMK ?(p<0.05).CaMK ? induction appears to reduce BRL-3A cells survival,and CaMK ?inhibited increase BRL-3A cells proliferation.the levels of protein AIF and cytochrome c were relevantly increasing in the LV-CaMK ? group compared to the control group(p<0.05).3.The results in vivo were as followed:1.TUNEL staining showed that the liver tissues appeared to have more cell apoptosis in the LV-CaMK ? group than the control group(p<0.05),and there is a lower apoptosis rate in LV-sh-CaMK ? group(p<0.01).2.The result of H&E staining indicates that there is an acidophilic change in 3 control groups,and spotty necrosis was found in the LV-CaMK ? group.The LV-sh-CaMK ? group represents the cell division phase.3.The liver function test showed that the AST and ALT in serum were increased over time,and both AST and ALT were increased in the LV-CaMK ? group at D7.On the contrary,the level of AST significantly declined at D7 in the LV-sh-CaMK ? group compared to the control group(p<0.01).4.The measurement of the concentration of Ca2+ indicated that there was no discrepancy among the 4 groups.5.Detection of mitochondrial membrane potential showed that after the overexpression of CaMK ?,the ratio of red/green cell was decreased compared with the control group(p<0.05).Contrastingly,the ratio of red/green cell appeared to ascend in the LV-sh CaMK ?group significantly(p<0.01).6.Transmission electron microscope observed that mitochondrial membrane integrity was destroyed,and the mitochondrial ridge was disordered in the LV-CaMK ? group,compared to the control group.7.WB analysis revealed after overexpressing CaMK ?,the expression of cytochrome c and AIF was increased significantly.When interfering with the expression of CaMK II,the level of cytochrome c and AIF has decreased accordingly(p<0.01).8.The protein chip showed that the ERK1/2 pathway was activated after interfering the expression of CaMK ?.Conclusions:1.Warm ischemia reperfusion injury is able to induce the delayed liver injury after suffer from liver transplantaiton.the expression of CaMK ? have a significant linear correlation with the degree of liver injury.In addition,the expression of CaMK ? is correlated with the warm ischemia time.2.Cell apoptosis is responsible for the WI/R injury induced by the expression of CaMK ?.overexpressing CaMK ? is able to increase the production of apoptosis,on the contrary,inhibiting CaMK ? is able to promote the cell proliferation.3.CaMK ? regulates the cell apoptosis by inducing mitochondrial apoptosis.The mian pathway is that CaMK ? induces the opening of mitochondrial permeability transition pore,which will cause the mitochondrial permeability transition and mitocial edema,and AIF and cytochrome c are released from the inner mitochondria,which aggravates the cell apoptosis.On the contrary,down-regulating the expression of CaMK ? is able to activate the ERK1/2 pathway,which can induce the proliferation of hepatocyte. |
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