Effects Of Interleukin 22 Fusion Protein On Liver Regeneration With A Pre-damaged Condition In Mice After Partial Hepatectomy

Beckground Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world and surgical resection is still considered to be the first choice for its treatment now.The lack of regenerative capacity and liver ischemia reperfusion injury(IRI)are the two main challage for these patients who performed partial hepatectomy(PHx).The majority of HCC patients with pre-damaged liver injury may died of acute liver failure after PHx because of the lacking the ability to regenerate.Although the hepatoprotective and pro-regenerative effects of IL-22 in mice after single PHx has been well documented,there is still lack the research on the effect of IL-22 on liver regeneration after PHx in mice with a pre-damaged liver condition.Most of the IL-22 used in the experiments of liver regeneration were recombinant human interleukin 22(rh-IL-22),until now there are still no research on interleukin 22 fusion protein(IL-22-FP)in liver regeneration.This project was designed to mimic the actual situation of clinical patients,and to explore the roles of IL-22-FP on liver regeneration and injury in mice with a pre-damaged liver condition after PHx.The results shown here will provide a new approach for the treatment of HCC patients with surgery.Objectives To study the roles of IL-22-FP on liver regeneration in mice undergone2/3 partial hepatectomy with a pre-damaged liver conditionMethods1.PHx was performed in mice following by Concanavalin A(Con A)and Carbon tetrachloride(CCl4)injection.The mice were treated with IL-22-FP or placebo 30minutes before PHx and were euthanized at different time points(32H,40 H,48h,1W)after PHx.Serum and liver tissue were collected at these different timepoint after PHx.In the Con A plus PHx experiment,the mice e were randomly divided into 4 groups as follows:1.Con A group;2.PHx group;3.Con A+PHx group;4.Con A+PHx+IL-22-FP group.Similarly,In the CCl4 plus PHx experiment,the mice were randomly divided into 4 groups as follows: 1.CCl4 group;2.PHx group;3.CCl4+PHx group;4.CCl4+PHx+IL-22-FP group.2.The liver ischemia reperfusion injury(IRI)in mice model was establised.Briefly,blood flow of 70% hepatic lobes in mice was blocked for 90 minutes,then these blood flow was restored.Mice were treated with IL-22-FP or placebo 30 minutes before the operation and were euthanized at different time points(6h,24 h,48h)after reperfusion.The mice were randomly divided into 4 groups :1.Sham group;2.IRI group;3.IRI+rh-IL-22 group;4.IRI+IL-22-FP group.3.As the indexs of the degree of liver damage,serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were determinated by an automatic biochemical analyzer.4.5-bromodeoxyuridine(Brd U)staining was performed according the protocol in the commercial kit.The positive staining cells are proliferating hepatocytes,so the level of liver regeneration can be reflected bythe ratio of positive hepatocytes to total cells.5.Observation the liver tissue of mice with HE staining,and the degree of hepatic necrosis and inflammatory cell infiltration were used to reflect the degree of liver injury.6.Western blot(WB)technique was used to detect the expression of proteins associated with liver regeneration such as proliferating cell nuclear antigen(PCNA),cell cycle proteins(Cyclin B1,Cyclin D1),p-STAT3 and STAT3 in liver tissue of mice in different groups.Results1.Mice treated with low doses(42?g/kg)of IL-22-FP had a much higher serum ALT levels and a lower Brd U positive rate compared with mice treated with medium(125?g/kg)and high doses(375?g/kg)of IL-22-FP in Con A model,however,these differences between the latter two groups were smaller.2.Treatment with IL-22-FP can effectively reduce the liver injury induced by Con A in mice.IL-22-FP could promote liver regeneration after PHx in Con A model mice,and this may be related to the activation of p-STAT3 and the expression of PCNA and Cyclin D1 protein.Compared with the rh-IL-22 treatment group and other groups,the serum ALT and AST levels in the IL-22-FP treatment group mice were significantly lower,inflammatory cell infiltration and necrosis of liver tissue were also significantly reduced in the IL-22-FP treatment group.The liver index(Liver weight / body weight,LW/BW)and Brd U positive rate of the IL-22-FP treatment group at different time points after PHx were significantly higher than those in the rh-IL-22 treatment group and other groups.Western blot results showed that compared with other groups,the expression of p-STAT3,PCNA and Cyclin D1 protein in liver tissue of mice in IL-22-FP treatment group was significantly higher at 32 h after PHx.3.IL-22-FP can significantly reduce the liver injury induced by CCl4 in mice and promote liver regeneration after PHx in CCl4 model,and this may be related to the expression of p-STAT3,Cyclin B1,Cyclin D1 and PCNA protein in liver tissue.Our experimental data show that the liver index in IL-22 group was significantly higher than that in other groups at 32,40 and 48 h after PHx in mice.Similar to this,the positive rate of Brd U in the IL-22-FP group was significantly higher than that in the other groups at 32,40 and 48 h after PHx,however,one week after the operation,all of the group were difficult to find staining positive cells.Western blot results show the expression of p-STAT3,PCNA,Cyclin D1 and Cyclin B1 protein in liver tissue ofIL-22-FP treatment group was significantly higher than that of other untreated group after PHx.4.IL-22 FP can effectively protect the liver in IRI model mice,significantly reduce liver ischemia reperfusion injury The serum AST levels of IL-22 FP treatment group was significantly lower than that of rh-IL-22 treatment group and other groups at 24 and 48 h after reperfusion in IRI model mice.The results of HE staining showed that the range of ischemic necrosis of liver tissue in IL-22-FP treatment group was smaller than that in other groups at 48 h after reperfusion.Conclusion1.The treatment of IL-22-FP can effectively protect the liver of mice,and reduce liver injury caused by Con A,CCl4 and PHx.2.Treatment with IL-22-FP can significantly improved the level of liver regeneration after PHx in Con A and CCl4 models mice.3.Administration of IL-22-FP effectively reduced hepatic ischemia reperfusion injury(IRI)in mice,and the effect is better than rh-IL-22.4.The role of IL-22-FP on hepatoprotective and pro-regeneration may be related to the activation of STAT3 pathway and the expression of related genes(such as Cyclin D1,Cyclin B1).