The Role Of Kupffer Cells On Liver Regeneration After First Stage Of ALPPS During Liver Immune Microenvironment

Objective: Hepatectomy is the main treatment for the primary liver cancer and liver metastases.However,this is limited by the quality of the functional liver after liver resection.The size and function of future liver remnant(FLR)determine the safety of liver resection.In the context,the two-step hepatectomy combined with liver partition and portal vein ligation for staged hepatectomy(ALPPS)has been proven to lead rapid and extensive liver hypertrophy,and challenge the concept of unresectable.The main obstacle to achieve R0 resection after hepatectomy is the liver failure caused by the inability to retain the sufficient remaining liver volume.At present,increasing the future liver remnant(FLR)and function of the liver after surgery is the core of hepatobiliary tumor surgery.Until 2012,ALPPS was first proposed,and the volume of FLR can be rapidly increased in a shorter time to meet the need of the next operation.The liver regeneration is a complex physiological process.During the process,the immune system will be activated,and inflammatory factors,inflammatory cells and corresponding inflammatory pathways will participate in the process of liver regeneration.At the same time,Kupffer cells play an important role in liver regeneration.In order to study the mechanism of Kupffer cells promoting liver regeneration during the change of immune mircroenvironment after ALPPS,this project intends to clarify whether M1 Kupffer cells participates in the process of liver regeneration,and confirm whether it can upregulate NF-κB,PI3K-Akt pathways and inflammatory factors IL-6,TNF-α,IL-1β to regulate liver regeneration.Methods:First,C57BL/6J mice were selected as the main experimental animals,and the mice were randomly divided into ALPPS group,portal vein ligation group(PVL)and sham operation group,6 mice in each group and surgery at the same time.The samples were taken at four time points(0h,24 h,72h,168h).By operating the changes in remaining liver mass(FLR/BW)and proliferation-related index,Ki-67 and proliferating cell nuclear antigen(PCNA),Cdk2,Cdk4 and Cyclin E1 of cell cycle-related proteins to verify whether the liver regeneration response in the ALPPS group was more obvious.The next step was to confirm whether M1 polarization Kupffer cells were at a high level in the ALPPS group by immuno-fluorescence double staining experiment F4/80 and i NOS.Then,western blotting and quantitative real-time PCR were performed on the residual liver tissue to detect the expression of inflammatory factors IL-6,TNF-α,IL-1βin the ALPPS group,and predicted whether the related cytokines of M1 kupffer cells were highly expressed in the ALPPS group.Next,the ALPPS+PBS group and the ALPPS+Wortmanin(PI3K pathway inhibitor)group were established by intravenous injection of drugs with 6 mice each group.The differences between the radio of the mass of residual liver to the mouse body weight(FLR/BW)and the protein level of p-Akt,pNF-κB,IL-6,TNF-α,IL-1β and i NOS(M1 Kupffer cell polarization marker)were used to further prove the function of M1 Kupffer cells in liver regeneration.At the same time,at the level of Raw264.7 cells(M0 macrophages),we used lipopolysaccharide(LPS)and IL-4 to induce Raw264.7 cells to differentiate into M1 polarization macrophages—high expression i NOS and M2 polarization macrophages—high expression Arg-1.The expression level of p-Akt and p-NF-κB of the two polarized cell types were determined by the cellular immuno-fluorescence and western blotting to further confirm whether M1 macrophages with higher expression p-Akt,p-NF-κB and cytokines IL-6,TNF-α and IL-1β promoted liver regeneration after ALPPS.Results: After performing different operations on C57BL/6J mice,the radio of future liver remnant mass to mouse body weight(FLR/BW)in the ALPPS group was more pronounced than the traditional PVL group and sham group,expecially on day 3 and day7,the hyperplasia was more statistically significant(P<0.05).The proliferation-related indicators Ki-67 and PCNA reached peck value on day 3(P<0.05),and fell back on day 7.The Cdk2、Cdk4 and Cyclin E1 of cyclin-related proteins peaked on day 3 and fell back on day 7(P<0.05).In the ALPPS group,the number of M1 polarized Kupffer cells was higher than the PVL and sham group on day 1 and day 3(P<0.05),and the cytokines IL-6,TNF-α and IL-1β related to the secretion of M1 Kupffer cells were higher expression level on day 1(P<0.05),then began to fall back to normal level after day 3.The protein level of p-Akt,and p-NF-κB in remnant liver tissue were higher in the ALPPS group than the other two groups,and the difference in expression was particularly significant on day 3(P<0.01).After the subsequent PI3 K pathway inhibitor injection,the liver regeneration in the ALPPS+Wortmannin group was lower than the ALPPS+PBS group,but higher than the PVL group(P<0.05).At the same time,the protein level of p-Akt,IL-6,TNF-α,IL-1β and i NOS were decreased in the inhibitor group(P<0.05)and the number of M1 Kupffer cells was also decreased(P<0.05).Then,at the cytology level,the Raw264.7 cells that were polarized by PBS had higher expression of i NOS(M1polarization)and that were induced by IL-4 had higher expression of Arg-1(M2polarization).And it can be seen that p-Akt,p-NF-κB were highly expressed in M1 polarized macrophages on cell immunofluorescence single staining.Conclusion:Above all,the results reveal that M1 polarized Kupffer cells secrete inflammatory factors IL-6,TNF-α,IL-1β and activate PI3 K,NF-κB pathways to promote rapid liver regeneration in a short time after ALPPS,which the liver regeneration and hepatocye proliferation are mediated by inflammatory immune microenvironment.