| Objective: Interleukin-4(IL-4)has been reported to play a key role in alleviating ischemia-reperfusion injury(IRI)and acute rejection after liver transplantation(LT),but the exact mechanism was not well understood.The signal transducers and activators of transcription 6(STAT6)and Jumonji domain containing 3(JMJD3)both play key roles in the polarization of macrophages.This study was focused on the role of Kupffer cells(KCs)and STAT6-JMJD3 pathway in IRI after liver transplantation.Methods: Rat live transplantation model was established by the modified Kamada's "two-sleeve cannula" method.The rats were randomly divided into Sham group,LT group,LT+NS group and LT+IL-4 group.In the LT+NS group and LT+IL-4 group,the livers of donor rats were respectively infused with normal saline(NS)or IL-4 via portal vein(PV)cannulation using a syringe pump.The liver allografts and serum were collected at 6 hours after liver transplantation to test liver function,inflammation,cell apoptosis and Pathological changes.The polarization of KCs and the activation of STAT6-JMJD3 pathway were evaluated by Flow cytometry,RT-PCR and Western Blot.The siRNA was used to knockdown JMJD3 or STAT6 was inhibited by AS1517499 in KCs isolated from livers of untreated rats,then the influence of IL-4 on the polarization of KCs and STAT6-JMJD3 were evaluated in vitro.At last,KCs or JMJD3 were respectively inhibited by Clodronate liposome or GSK-J4,then IRI after liver transplantation was test again to determine whether the protection of IL-4 was depended on KCs.Results: The level of serum ALT(Sham: 50.33±8.73IU/L,LT: 1836.27±112.30IU/L,n=3,p<0.05)and AST(Sham: 52.17±9.55IU/L,LT: 2134.63±137.23IU/L,n=3,p<0.05)increased significantly and pathologic damage such as sinusoidal congestion,hepatocellular necrosis were severer after liver transplantation.IL-4 treatment could alleviate liver damage as indicated by the level of ALT(LT+NS: 1919.64±127.23IU/L,LT+IL-4: 924.18±76.2 IU/L,p<0.05),AST(LT+NS: 2101.76±179.54 IU/L,LT+IL-4: 1190±199.12 IU/L,P<0.05)and histological grading(LT+NS: 5.5±1.04,LT+IL-4: 2.33±0.86,n=3,p<0.05).The inflammation,cell apoptosis and pathologic changes were alleviated in the LT+IL-4 group compared with the LT and LT+ NS groups.IL-4 treatment also polarized KCs to M2 phenotype(Sham: 2.48±0.69%,LT: 3.39±1.20%,LT+NS: 3.22±1.11%,LT+IL-4: 37.12±5.01%,n=3,p<0.05)and activated the STAT6-JMJD3 pathway.We further detected the role of STAT6-JMJD3 pathway in vitro.JMJD3 knockdown reduced the KCs M2 polarization induced by IL-4(IL-4 vs IL-4+Si-JMJD3,42.62±2.62% vs 12.12±1.23%,n=3,p<0.05).STAT6 inhibition also suppressed the expression of JMJD3(IL-4 vs IL-4+AS1517499,0.99±0.08 vs 0.20±0.04,n=3,p<0.05),but JMJD3 knockdown had no influence on the phosphorylation of STAT6(IL-4 vs IL-4+si-JMJD3,1.14±0.07 vs 0.93±0.06,n=3,p>0.05).JMJD3 knockdown also abolished the anti-inflammatory effect of IL-4,as the decreased expressions of p-p65 and IL-1? were abolished by JMJD3 knockdown.Inhibition of KCs or JMJD3 in liver also blocked the protection of IL-4 after liver transplantation as indicated by the level of serum ALT(LT+IL-4+CL vs LT+IL-4,1483.23±75.09 IU/L vs 754.7±73.12 IU/L,n=3,p<0.05;LT+IL-4+GSK-J4 vs LT+IL-4,1511.73±118.70 IU/L vs 754.7±73.12 IU/L,n=3,p<0.05),AST(LT+IL-4+CL vs LT+IL-4,1950.26±114.30 IU/L vs 966.81±162.12 IU/L,n=3,p<0.05;LT+IL-4+GSK-J4 vs LT+IL-4,1960.75±102.83 IU/L vs 966.81±162.12 IU/L,n=3,p<0.05)and histological grading(LT+IL-4+CL vs LT+IL-4,6.03±0.71 IU/L vs 2.52±0.64 IU/L,n=3,p<0.05;LT+IL-4+GSK-J4 vs LT+IL-4,4.25±0.48 IU/L vs 2.52±0.64 IU/L,n=3 p<0.05)Conclusion: IL-4 treatment-induced KCs M2 polarization was dependent on the STAT6-JMJD3 pathway and protected liver grafts from IRI after liver transplantation. |
PDF Full Text Request