Mechanism Of Ischemic Preconditioning Regulating Macrophage Polarization To Alleviate Hepatic Ischemia-reperfusion Injury

Objectives1.To investigate the effects of ischemia preconditioning(IPC)on the function of grafts after liver transplantation.2.To study the mechanism by which IPC improves hepatic ischemia-reperfusion injury(IRI)by regulating the polarization of liver macrophages.Method1.Patients who were procured at the OPO and received liver transplantation at the First Affiliated Hospital of Zhengzhou University from January 2017 to December 2017 were selected as the subjects.According to whether the donor has any history of transient cardiopulmonary resuscitation before organ procurement,the 163 qualified cases were divided into cardiopulmonary resuscitation group(CPR group,n=26)and non-cardiopulmonary resuscitation group(NCPR group,n=137).The following clinical data were collected:(1)donor-related indicators,such as the age,gender,and ICU duration of the donors;(2)receptor-related indicators,such as the age and gender of the corresponding receptors;(3)surgery-related indicators,such as the operation time,cold ischemia time(CIT)and warm ischemia time(WIT)of liver transplantation.The qualitative and quantitative data were analyzed by Fisher's Exact Test and Wilcoxon Rank-Sum test,respectively,where P <0.05 indicates statistical difference.2.The mechanism by which IPC improves liver IRI was explored in animal and cell experiments.The first step: 108 male SD rats were randomly divided into 3 groups: sham group(n=36),ischemia-reperfusion(IR)group(n=36),IPC group(n=36).Each group was further divided into 6 subgroups with 6 rats in each subgroup,according to the reperfusion time of 0.5h,1h,6h,12 h,24h and 48 h.The 70% liver IR model of rat was used,with the left and middle hepatic lobe suffering from ischemia for 1 h.Blood and tissue specimens were collected at 0.5 h,1 h,6 h,12 h,24 h and 48 h after blood flow recovery,for the detection of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),TNF-?,IL-10,liver histopathology examination and expression of M1 and M2 type liver macrophages.The second step: first of all,the bone myelomoncyte of the rats were extracted,and then IL-4 was added to induce differentiation into M2 type macrophages,so as to figure out the effects of IL-4 on macrophage polarization.Subsequently,18 male SD rats were randomly divided into 3 groups: IL-4 adding medicine group(n=6),phosphate buffered saline(PBS)group(n=6),IL-4 antibody group(n=6),in which IL-4,PBS and IL-4 antibodies were added respectively.Finally,the blood and tissue specimens were collected from each group for examining the indicators after 6 hours of reperfusion.Results1.With regard to donors,no statistical differences were found in the age,gender,ICU duration,serum sodium concentration,and prothrombin time(PT)among the donors,except the history of transient cardiopulmonary resuscitation,preoperative ALT and AST.The mean serum ALT and AST before organ procurement of the donors in the CPR group were 189 U / L(33-639 U / L)and 176 U / L(18-584 U / L),respectively,higher than those in the NCPR group where the mean ALT and AST were 74 U / L(27-565 U / L)and 39(11-653 U / L),respectively.In terms of receptors,there were no statistical differences in age,gender,MELD score,preoperative ALT and AST between the two groups.However,the ALT and AST levels in the CPR group were lower than those in the NCPR group within 7 days after surgery,with statistical significance(P<0.05).2.The results of animal experiments in the first step showed that the ALT,AST,TNF-? and IL-10 values in the IR group and the IPC group were higher than those in the sham group,with a peak value achieved at 6h.The levels of ALT,AST and TNF-? in the IPC group were significantly lower than those in the IR group with 0.5h,1h,6h,12 h,24h and 48 h of reperfusion(P<0.05).Besides,IL-10 at 0.5h,1h,6h,12 h,24h and 48 h in the IPC group was higher than that in the IR group,with statistical significance(P<0.05).Liver histopathology examination showed that the injury degree in the IPC group was lighter than that in the IR group.The results of Western blotting revealed that under physiological conditions,M2 macrophages were more highly expressed in liver compared with M1 macrophages,and that the number of M1 and M2 macrophages decreased gradually at 0.5h,1h and 6h of reperfusion,and afterwards,the number of M1 macrophages gradually increased at 12 h and decreased after 48 h,while M2 macrophages gradually increased at 24 h and reached a higher level at 48 h.In the late reperfusion period(12h),the expression of M1 macrophages in the IPC group was less than that in the IR group,while the M2 macrophages at 24 h were more than those in the IR group.The results of cell experiments indicated that the expression of M2 macrophages in IL-4 group was significantly higher than that in PBS group.In the animal experiments of the second step,the ALT,AST and TNF-? values in the IL-4 adding medicine group were lower than those in the PBS group and the IL-4 antibody group,while the IL-10 values were opposite,with statistically significant difference(P<0.05).Liver histopathology examination showed that the injury of liver tissues in the IL-4 adding medicine group was lighter than that in the PBS group and the IL-4 antibody group.The expression of M1 in the IL-4 adding medicine group was lower than that in the IL-4 antibody group and PBS group,while the M2 expression was higher than that in the IL-4 antibody and PBS group.Conclusion1.IPC exerts a protective effect on the function of grafts after liver transplantation.2.IPC may improve liver IRI protection by promoting the polarization of liver macrophage to M2 type.