| Ischemia-reperfusion injury(IRI)is defined as such a phenomenon that the body organ or tissue may develop more serious damage to its structure and function under the condition of ischemia/hypoxia,rather than structure and function recovery after restoring blood flow and oxygen supply.IRI has high incidence in all organs of the body,especially in the liver.Hepatic IRI may cause liver function damage or liver failure,and even multiple organ dysfunction/failure in severe cases.Besides,IRI can also increase the rejection rate of transplanted organs and the risk of tumor recurrence and metastasis,which will directly affect the prognosis of the disease.In this regard,It is of great significance to further explore the mechanism of hepatic IRI and develop more targeted prevention and treatment measures accordingly for future development of liver surgery and interventional therapy.Transcatheter arterial embolization(TAE)is widely used as an available treatment for advanced liver cancer,liver metastasis and some benign liver tumors.Liver function damage and even liver failure remains common complications after TAE.Nevertheless,there are few studies on whether TAE can cause IRI,and it is still unclear whether hepatic injury caused by TAE is related to IRI.Theoretically,there exists the process of tissue and organ ischemia-blood flow recovery after TAE,providing evidence for the possibility of IRI.However,there is no immediate blood flow restore after TAE,which highlights the difference of its pathophysiological process from that of IRI.With respect to the above,it is indeed worthy of our attention and further study concerning the presence of IRI after TAE,and the relationship of the resulted IRI with liver function damage.Innate immune cells such as dendritic cells,macrophages and neutrophils play important roles during the occurrence and development of hepatic IRI.Dendritic cells(DCs)are important antigen-presenting cells that provide a link between innate and adaptive immune responses.DCs exist in most organs,including liver,kidney,lung,etc.Multiple studies at home and abroad support that DCs plays an important role in a variety of liver diseases.As evidenced by recent studies,liver DCs are believed to play a central role in the process of hepatic IRI as well,yet with inconsistent conclusions.It can be explained by the high heterogeneity of DC cells population.There are significant different functions of DCs from different sources,such as those derived from myelogenous or embryonic cells,and those that enter into local tissues and differentiate into different sub-populations,such as plasmacytoid degenerative cells(pDCs)and classical degenerative cells(cDCs).DCs can be divided into several subgroups,including classic DCs,monocyte derived DCs and plasmacytoid DCs.Classic DCs can further differentiate CD103+DCs in non-lymphoid organs according to distinct phenotypes and biological functions.CD103+DCs settle in the parenchymal organs such as liver,kidney,lung,etc.,and its proportion constitutes the majority in the total population of locally settled DCs.Numerous previous studies have pointed out that it participates in the course of many diseases and plays an important role in the immune response of the body.However,there are few reports concerning CD103+DCs in hepatic IRI.Accordingly,it is a subject worthy of further study to clarify the changes and functions of CD103+DCs cells in hepatic IRI.There are some unique phenotypes in CD103+DCs,such as transcription factors(Batf3 and IRF8)and growth factor receptor(FMS-like tyrosine kinase 3,Flt3),which are different from other sub-populations of DCs in tissues.Among them,Flt3 can play an important role in the differentiation and maturation of DCs through binding to its ligand(Flt3 ligand,Flt3L)through phosphorylation activation.Besides,the development and maturity of CD103+DCs are more dependent on Flt3 compared with other subgroups.Therefore,it was speculated in our study that Flt3/Flt3L is a target that can be used to regulate the function of CD103+DCs.Regulatory T(Treg)cells constitute a distinct T lymphocyte,which plays a key role in maintaining immune homeostasis.It has been widely demonstrated that increasing Treg expression can reduce the degree of inflammation and injury.Subsequent studies also demonstrate that CD 103+DCs can induce the transformation of naive T cells into Treg to benefit the remission of the disease on the basis of its protective immunomodulatory role.However,current research on the activation of Treg cells by CD103+DCs to alleviate disease progress emphasizes on the field of autoimmune diseases,with little knowledge about the role of inflammatory diseases.Consequently,it deserves to be further explored with respect to the role of CD103+DCs in hepatic IRI with inflamumation as the main pathogenesis and its mechanism,so as to determine whether it is feasible for targeted regulation of CD103+DCs in the treatment of hepatic IRI.Objective1.To monitor indexes related to hepatic IRI,such as SOD,MDA,IL-6,ALT,AST and LDH dynamically before and after TAE of liver cancer,to analyze the correlation between ischemia-reperfusion index and liver function,to evaluate the existence of IRI after TAE preliminarily,and to discuss the correlation between liver function damage and IRI;2.To observe the relationship between the course of hepatic IRI and CD103+DCs by detecting liver tissue injury,liver function,inflammatory cytokines and the expression of DC cells and the subsets of CD103+DCs in liver tissue at different time points of reperfusion;3.To detect the changes of liver function,tissue damage,inflammatory cytokines and the number and proportion of CD 103+DCs after intervention via infusion the pre-sorted CD103+DCs of the model mice by flow cytometry into to the model mice in advance,and thus to explore the role of CD103+DCs in hepatic IRI;4.To regulate the function of CD103+DCs by intervening the expression of Flt3/Flt3L on CD103+DCs in hepatic IRI mice model with the use of Flt3 inhibitor,Flt3 factor and si-Flt3 transfection;to detect the indexes of liver function,tissue damage,inflammatory cytokines and apoptotic protein expression by animal experiment,and then to explore the mechanism of regulating CD103+DCs by Flt3/Flt3L to affect the course of hepatic IRI;5.To verify the effect of CD103+DCs on the proliferation and function of Treg on the basis of mixed lymphocyte culture of CD103+DCs and T cells after the intervention of Flt3 inhibitor,Flt3 factor and si-Flt3 in vitro;and to detect the apoptosis of hepatocytes via intervening the hypoxia/reoxygenation model of hepatocytes using CD 103+DCs in vitro,so as to explore the mechanism of CD 103+DCs in improving the degree of hepatic IRI.MethodsThe objects of study were patients who received transcatheter arterial chemoembolization(TACE)for liver cancer in accordance with pre-set inclusion and exclusion criteria.Peripheral venous blood samples were collected from all patients in the morning before TACE and 1,2 and 3 days after TACE.Test indicators included LDH,liver function indexes of ALT and AST,oxidative stress index of MDA and SOD,and inflammatory cytokine IL-6.Further comparison was performed focusing on the changes of each index at different time points and the differences of liver function damage index,oxidative stress index and inflammation index under different embolic doses.A total of 168 SPF male C57BL/6 mice with body weight of 20-25g were used to establish the experimental model.Phase ? experiment(animal model construction)consisted of the following groups:control group,sham group and model groups(4 subgroups at different time points of Oh,6h,12h and 24h).Phase ? experiment included control group,sham group,positive control group(hepatic IRI model),PBS-treated negative control group(hepatic IRI model treated by PBS),and intervention groups(CD103+DCs adoptive transfer pretreatment group,Flt3 inhibitor pretreatment group,Flt3 inhibitor+CD103+DCs adoptive transfer pretreatment group,and Flt3 factor pre-treatment group),with 8 mice in each group.The mice were kept in a clean environment at room temperature of about 24? for 12 h light/12 h dark cycles.For establishing experimental model,noninvasive clamp was used to block the blood vessels in and out of the left lobe and middle lobe of the liver to achieve 70%of the liver ischemic.In the sham group,only the hepatic artery and portal vein were isolated with no further clamping.There was no intervention in the control group.Mice in intervention groups were pretreated according to experimental schemes.Except the model mice,mice in all the other groups were sacrificed 6h after liver ischemia.Peripheral blood samples were collected to detect liver function with enzyme-linked immunosorbent assay(ELISA).The liver samples were used for histopathological detection,tissues frozen at-80? were used for molecular biological detection.The rest of the liver samples were used for flow cytometry detection and sorting.ELISA was performed to test serum levels of liver function indexes(ALT,AST and LDH),inflammatory cytokines(TNF-?,IL-1? and IL-6),Treg related cytokines(IL-10,TGF-?),RT-qPCR was used to detect the gene expression level of ICAM-1,TNF-? and IL-?,and the protein expression levels of inflammatory cytokines IL-1?and ICAM-1 were measured by western blot(WB).HE staining was conducted to observe the pathological changes of mouse liver.TUNEL staining was used to detect the degree of apoptosis.Furthermore,flow cytometry was performed to detect the number and proportion of CD 103+DCs and Treg cells in liver tissue,and the apoptotic ratio of hepatocytes in vitro.SPSS 17.0 statistical software package was used for analysis in this study.With the confirmation of normal distribution of measurement data,normally distributed data was expressed as mean± standard deviation(x±s).T-test was used for comparison between groups,and one-way analysis of variance(ANOVA)was used to analyze the data of three groups and above.Bonferroni test Tamhane test were used for comparison of data with homogeneity and heteroscedasticity of variance,variance analysis of repeated measurement data were used for comparison of repeated measurement data,respectively.Non-parametric test was applied for data did not conform to normal distribution.The inspection level was a=0.05.The results of immunohistochemistry and immunofluorescence were analyzed by Image J software.The results were processed and plotted with Graph pad 5.0 software.Results1.According to the eligibility criteria,a total of 43 patients with liver cancer were included,including 31 males and 12 females.ALT and AST were obviously increased in 1d,2d and 3d after TAE,peaked in 2d postoperatively,and LDH was also increased after operation,with the most significant increase in 1d postoperatively.Furthermore,MDA and IL-6 of patients after TAE were significantly higher than those before TAE,and reached the peak in Id after TAE,followed by a slow decrease but still higher than those before TAE(P<0.05).SOD decreased evidently in Id after operation(P<0.05),and still lower than the preoperative level in 2-3d after surgery.In the comparison of levels in 1d after surgery,liver function indexes of ALT and AST,IRI indexes of LDH,MDA,and IL-6 were significantly higher in lipiodol?12ml group(n=17)than those in lipiodol<12ml group(n=26),while an opposite trend was found in the level of SOD.2.For the establishment of hepatic IRI,observation on hepatic injury of model mice in each group after ischemia for 60 min and reperfusion for 0,6,12 and 24 h indicated that the histological damage of mouse liver increased with the prolongation of reperfusion time.Besides,the levels of ALT,AST and LDH increased gradually with prolonged reperfusion time,peaked at 12 h after reperfusion,and then recovered.Furthermore,the levels of inflammatory cytokines TNF-?,IL-1? and IL-6 peaked at 6h after reperfusion.A negative correlation of the changes of CD103+DCs in hepatic IRI model was found with the above detected levels of inflammatory cytokines.Meanwhile,the proportion of CD103+DCs was the lowest at 6h after reperfusion3.The changes of CD103+DCs in liver tissue were detected by flow cytometry after exogenous infusion of CD103+DCs to mice in the hepatic IRI model in advance.Corresponding results showed that the proportion of CD103+DCs in liver tissue was significantly higher than that in IRI model group.The proportion of CD103+DCs in IRI group was 8.85%of total DC cells,while that in exogenous infusion group was 14.2%.Compared with IRI model mice,the degree of hepatic injury was significantly improved,which was manifested as follows after adoptive transfer of CD103+DCs:1)liver function indexes such as ALT,AST and LDH were significantly lower than those of model mice;2)HE staining of liver tissue showed that the degree of cell edema and inflammatory cell infiltration was notably improved,associated with significantly reduced score of hepatic injury;and TUNEL staining showed no cell apoptosis in the liver tissue;and 3)there was decrease in the expression of apoptosis related genes(Fas and Bax)and proteins(Bcl-2).4.Mice were given Flt3 inhibitor(MLN518),CD103+DCs adoptive transfer(DCs infusion),MLN518 combined with CD103+DCs adoptive transfer(MLN518+DCs infusion),or Flt3L factor(Flt3L)treatment before establishing hepatic IRI model.The liver tissue samples of each group were collected 6h after reperfusion.1)In terms of the results of flow cytometry,compared with IRI group,the proportion of CD103+DCs in liver tissue of MLN518 group decreased significantly,with the detection of statistical difference(P<0.05);there was obvious increase in the proportion of CD103+DCs in liver tissue of Flt3L group(P<0.05);compared with DCs infusion group,there was statistical difference that the proportion of CD103+DCs in liver tissue decreased obviously in MLN518+DCs infusion group(P<0.05).2)The results of liver function test and pathological examination showed that compared with IRI group,ALT,AST and LDH levels as well as hepatic injury scores were significantly reduced in Flt3L group(P<0.05).Besides,compared with CD103+DCs infusion group,MLN518+CD103+DCs infusion group had notably increased ALT,AST and LDH levels as well as hepatic injury scores,with statistical difference(P<0.05).3)According to the detection of apoptosis in liver,compared with IRI group,the expression of apoptotic Fas and Bax was significantly higher and that of anti-apoptotic Bcl-2 was much lower in MLN518 group(P<0.05);in Flt3 group,the expression of Fas and Bax was evidently decreased,and that of Bcl-2 was remarkably increased(P<0.05).Furthermore,compared with DCs infusion group,there were significant increase in the expression of Fas and Bax,and obvious decrease in that of Bcl-2 in MLN518+DCs infusion group(P<0.05).4)In view of the detection results of inflammatory factor expression in liver by RT-qPCR,in relative to IRI group,MLN518 group exhibited notably increased expression of TNF-?,IFN-y,ICAM-1 and IL-2 mRNA as well as IL-1? protein in the liver(P<0.05);while significant decrease was found in liver TNF-?,IFN-y,ICAM-1 and IL-2 mRNA expression as well as IL-1? protein expression in Flt3 group(P<0.05).Besides,compared with DCs infusion group,MLN518+DCs infusion had obviously increase in the expression of TNF-?,IFN-y,ICAM-1 and IL-2 mRNA as well as IL-1? protein in the liver(P<0.05).5.Prior to the establishment of liver ischemia-reperfusion model,mice were provided with different treatments,including Flt3 inhibitor(MLN518),CD103+DCs adoptive transfer(DC infusion),MLN518 combined with CD103+DCs adoptive transfer(MLN518+DC infusion),or Flt3L factor(Flt3L).The results of flow cytometry revealed that compared with IRI group,the proportion of Treg cells in the peripheral blood of MLN518 group was significantly lower,showing statistical difference(P<0.05).The proportion of Treg cells in peripheral blood of Flt3L group increased significantly(P<0.05).Compared with DCs infusion group,the Treg cell proportion were much lower in the peripheral blood and liver tissue of MLN518+DCs infusion group,with statistical difference(P<0.05).In the mixed lymphocyte reaction experiment,CD4+T cells were co-cultured with CD103+DCs under different conditions(negative control group,si-Flt3+Flt3L group,si-RNA+Flt3L and Flt3L group)to detect the proportion of Treg cells in CD4+T cells.In view of the results,compared with PBS control group,the proportion of Treg cells was significantly higher in Flt3L group(P<0.01),accompanied by obvious increase in the gene expression levels of TGF-? and IL-10.Meanwhile,compared with Flt3L group,the proportion of Treg cells was notably decreased in Flt3L+si-Flt3 group(P<0.01),and the gene expression levels of TGF-P and IL-10 were obviously decreased(P<0.01).However,there was no apparent difference in the comparison between Flt3L group and Flt3L+si-RNA group(P>0.05)6.In the established hypoxia/reoxygenation model,the apoptosis rate of hepatocytes increased significantly after post-hypoxia reoxygenation treatment.Following co-culture of L02 hepatocyte line with CD103+DCs under different treatments(negative control group,si-Flt3+Flt3L group,si-RNA+Flt3L and Flt3L group),it was found that the proportion of hepatocyte apoptosis in Flt3L group decreased significantly compared with PBS control group(P<0.01).Compared with Flt3L group,the apoptosis rate of hepatocytes was much higher in Flt3L+si-Flt3 group(P<0.01).In addition,no significant difference was detected between Flt3L group and Flt3L+si-RNA group(P>0.05).Conclusions1.IRI can occur in both embolized tumor tissue and normal liver tissue after TAE.There is a positive correlation of the degree of IRI with the degree of hepatic injury,as well as that of the degree of hepatic injury and IRI with the embolized area.2.Adoptive transfer of CD103+DCs can reduce the degree of hepatic injury in IRI model mice.3.The liver protective effect of CD 103+DCs in hepatic IRI model is achieved by regulating the surface expression of Flt3/Flt3L targets4.CD 103+DCs can promote the differentiation and proliferation of Treg in hepatic IRI model,and thus alleviate the hepatic injury and function. |
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