| Background and Aims:Transplantation and liver resection are effective therapies for benign liver disease and primary liver cancer at an early stage.But,these two therapies will lead to liver ischemic reperfusion(I/R)injury after surgery inevitably.Liver damage induced by I/R will influence the recovery of liver function in the period of perioperation,even leading to acute liver failure in some cases with poor reservation of liver function.Thus,it remains potential clinical implication to explore the mechanism of liver I/R injury and identify the potential targets for intervention.URI had been proved to be involved in regulating various biological processes,such as glucose metabolism,genetic transcription,apopsis and cancer metastasis.Recently,it was reported that URI could facilitate the transcription of IL-6 in the process of HCC metastasis by regulating the activation of NF-?B signaling pathway.NF-?B is an important signaling pathway in the innate immune response,which is common in the activation of macrophage.So,our previous study had shown URI could regulate the activation of NF-?B and MAPKs signaling pathway.As in the process of liver I/R,KC and innate immune response are also activated.Whether URI can regulate the function of KC still remains unclear.Thus,the present study aims to explore whether URI can affect liver I/R injury by regulating the activation of KC and its molecular mechanism.Methods: Clinical data of HCC(3-5cm)patients undergoing liver resection at our hospital between 2007 and 2012 were collected retrospectively.The postoperative serum levels of ALT and AST at the 1,3,5,7 day were compared between patients with and without liver inflow occlusion.Peritoneal macrophage and KC from myeloid URI deficency mouse were isolated to test the efficacy of kickout.Then,this model was used to conduct the I/R model with 70% liver undergoing ischemia for 1.5h and followed by reperfuion for 6h.At the same time,the sham operation group was also conducted as the negative control.Then,liver tissue and blood serum of the I/R group and the sham operation group were collected to carry out subsequent tests.HE,IHC,Tunel test,serum level of ALT and AST,serum level of IL-1? and TNF-? were tested to evaluate the extent of liver injury undergoing I/R both for WT and KO mice.Similarly,the sham operation group was tested as the negative control.After that,WB was used to test the activation of NF-?B and MAPKs signaling pathways,and RT-PCR was used to test the transcription of related pro-inflammatory factors and chemokine.Moreover,KC was isolated to compare the transcription difference of pro-inflammatory factors and chemokine under stimulation with LPS between WT and KO mice.Then,KCs of WT and KO mice undergoing I/R were isolated to compare the transcription differences of pro-inflammatory factors and chemokine.Whereafter,the change of URI itself was detected under short-term and long-term stimulation with LPS in macrophage cell line.Furthermore,small molecule inhibitors of important protein in the NF-?B and MAPKs signaling pathways were used to screen these proteins,which maybe bind to URI.Then,IP was employed to confirm the binding interaction between URI and some other proteins,and our study revealed URI could bind to IKK?.Thus,truncations of URI and IKK? were designed to reveal the specific binding position between URI and IKK? by IP tests.After that,the change of the binding status between URI and IKK? were tested when URI was phosphorylated.AT last,the binding status between URI and IKK? were tested when the phosphorylation site were mutated under stimulation with TNF-?.Results:(1)The postoperative serum levels of ALT and AST at the 1,3,5 and 7day in the occlusion group were higher than patients without occlusion.Similarly,patients undergoing occlusion less than 15 min obtained a lower serum levels of ALT and AST compared with patients occluding more than 15 min.(2)In our myeloid URI deficiency mouse model,transcription level of URI in the peritoneal macrophage and Kupffer cell was knocked out effectively and steadily.(3)HE test revealed that KO mouse underwent more severe I/R injury compared with WT mouse.The Suzuki scores for KO mice were also higher than WT mouse(5.50±1.05 Vs.3.83±0.75,p=0.01).Whereas,the sham operation group showed no evidence of liver injury.IHC test for F4/80 and CD11 b showed that more macrophages and neutrophils inflitrated to the impaired liver tissue in the KO mouse group.Tunel test revealed that KO mouse undergoing I/R appeared more apoptotic cells than WT mouse.(4)Serum tests for ALT and AST showed that KO mice obtained higher levels of ALT and AST than WT mice after I/R.Whereas,the sham operation group retained a relative low levels of ALT and AST.Elisa tests for IL-1? and TNF-? showed that the serum levels of these two pro-inflammation factors in the KO group were higher than the WT group.(5)WB test for liver tissue undergoing I/R showed that the NF-?B and MAPKs signaling pathways were more active in the KO group.The phosphorylation levels of P65,P38 and Erk were higher than WT mice.RT-PCR test for liver tissue undergoing I/R showed that the transcription levels of IL-1?,TNF-? and CCL2 were higher in the KO group.(6)KCs isolated from WT and KO mice were stimulated with LPS for 4h,and RT-PCR test showed that the transcription levels of IL-1?,TNF-? were higher in the KO group.KCs were isolated from WT and KO mouse undergoing I/R,RT-PCR test showed that the transcription levels of IL-1?,TNF-? and Il-6 were higher in the KO group.(7)Upon short-term LPS stimulation,NF-?B and MAPKs signaling pathway were activated in 264.7 cell line.In this process,we revealed that URI were modified at the same time which manifested as the shifting up of its stripe.Upon long-term LPS stimulation in 264.7cell line,we found that the transcription and expression level of URI were down-regulated.(8)Associated with the results of stimulation with ?-PP,mass spectrometry detection and early study reports,we confirmed URI were phosphorylated in the process of the activation of NF-?B and MAPKs signaling pathways.Small molecule inhibitors screening tests revealed that the inhibitors of IKK?,IKK compounds and P38 could inhibit the phosphorylation of URI in the process of the activation of NF-?B and MAPKs signaling pathways.These results implicated that URL may bind to IKK?,IKK compounds and P38.(9)IP tests were used to detect the binding interation between URI and IKK?,IKK?,AKT,P38.Our results showed that URI could bind to IKK?.(10)Truncation plasmids of URI were designed,and IP tests between IKK? and truncations of URI showed IKK? could bind to the c-terminal of URI.Truncation plasmids of IKK? were designed,and IP tests between URI and truncations of IKK? showed URI could bind to the kinase domain of IKK?.(11)Under phosphorylation of URI,result of IP test showed that the binding interaction between URI and IKK? became weaken.The phosphorylation sites were mutated in our mutated URI plasmid.Then,we found the binding interaction between mutation of URI and IKK? was reinforced upon TNF-? stimulation.(12)Previous studies reported that PP1 could bind to URI and IKK?.Thus,we postulated that PP1,URI and IKK? could form a compound.But,When URI was phosphorylated,it could release PP1 to suppress the phosphorylation of IKK?.Thus,URI could regulate the activation of NF-?B and MAPKs signaling pathways negatively in the process of activation of macrophage,which included KC.Conclusion:Myeloid expression level of URI could affect liver I/R injury negatively by regulating the activation of Kupffer cell.URI and NF-?B signaling pathway could regulate the other mutually.Upon the activation of NF-?B and MAPKs signaling pathways,URI was phosphorylated.The c-terminal of URI could bind to the kinase domain of IKK?.Upon phosphorylation of URI,the binding interaction between URI and IKK? became weaken.When the phosphorylation sites of URI was mutated,the binding interaction between URI and IKK? became reinforced.Maybe,When URI was phosphorylated,it could release PP1 to suppress the phosphorylation of IKK?. |
PDF Full Text Request