| BackgroundHepatic ischemia-reperfusion(IR) injury occurs in a number of clinical settings, including liver surgery, transplantation, and hemorrhagic shock, leading to a high risk during the operation and severe impact of the postoperative survival. The mechanisms of IR injury have not been completely clarified, although there are many evidences that the complicated and interrelated process involves deleterious cellular responses and pathological change of tissues, and the activation of toll like receptors on Kupffer cells may play a fundamental role in the process. And the prevention of the liver damage in perioperative period became a popular topic. Recently, there is study confirmed that opiod agonist Remifentanil can through inducing iNOS effectively reduce liver ischemia reperfusion injury, which indicated that there may be internal relationship between TLR4 pathway and opiod agonist liver protective effect. Many reports show that as a primary negative regulator of G protein-coupled receptors(GPCRs), β-arrestin plays an important role not only in the Phosphorylation and internalization of opioid receptor, but also in the modulation of immune responses by binding to TRAF6. In short, β-arrestin mediates regulation of transcription in cell growth, apoptosis and modulation of immune functions. What’s more, mu opioid receptor bounds β-arrestin2 with higher affinity than other members of β-arrestin family. So it is possible thatβ-arrestin2 is an important link in the TLR4 inhibition of remifentanil preconditioning. The objective of our study was to determine whether remifentanil reduces liver ischemia reperfusion injury in mice via TLR4 signaling pathway, and if so, whether β-arrestin2 mediates this effect.Methods1、The mice were randomly invided into four groups including sham operation group(sham)、ischemia reperfusion group(IR)、 remifentanil preconditioning group(RPC) and mu opiod receptor agonist control group(agonist). A model of segmental(70%) ischemia reperfusion was used as previously described. Serum transaminase, the differences in liver phthology are measured. To detect how TLR4 act in liver protective effect of remifentanil preconditioning, the serum TNF-α and IL-6 level between TLR4 KO mice and WT mice were measured. In addition, we design LPS stimulate RAW264.7 cells to mimic liver IR-mediated kupffer cells damage in vivo, and compare the difference of TLR4 protein between remifentanil preconditioning group and LPS treated group.2、The mice were randomly invided into four groups including sham operation group(sham)、ischemia reperfusion group(IR)、 remifentanil preconditioning group(RPC) and mu opiod receptor agonist control group(agonist). A model of segmental(70%) ischemia reperfusion was used as previously described. β-arrestin2 expression in liver cells of each group were measured. Remifentanil pretreated RAW264.7 cell to detectβ-arrestin2 protein expression in cells. SiRNA targetingβ-arrestin2 was used to inhibitβ-arrestin2 expression in RAW264.7, and cell viability and apoptosis induced by LPS were measured.3 、 SiRNA targeting β-arrestin2 was used to inhibitβ-arrestin2 expression in RAW264.7. The model of LPS stimulate RAW264.7 cells was used to mimic liver IR-mediated kupffer cells damage in vivo, after remifentanil pretreating, p-ERK and p-JNK protein expression were measured. The amount of formation of β-arrestin2-TRAF6 complex was also detected.Results1 、 The serum AST and ALT level sinificantly decreased in remifentanil preconditioning group(RPC) compared with ischemia reperfusion group(IR). The same result of liver damage can be observed in above groups. The level of serum TNF-α、IL-6 increased significantly in IR group compared with sham group, and decreased in RPC group compared with IR group. However, in TLR4 KO mice, there was no significant difference between RPC group and IR group. In vivo, The protein expression of TLR4 in the group treated with LPS and remifentanil increased significantly compared with the group treated only with LPS.2、The expression of β-arrestin2 in liver cells significantly increased in RPC group compared with IR group and sham group, and to a large extent, the β-arrestin2 protein expressed in the cytoplasm. In vivo, after remifentanil pretreated cells, immunoflurencence method was used to detect the expression of β-arrestin2 in RAW264.7 cells. In sham group, the β-arrestin2 protein expressed in the cytoplasm, what’s more, after remifentanil pretreated, the β-arrestin2 protein was gathered on the cell membrane. After treating with LPS, the cell viability decreased, while the value increased in the group pretreated with remifentanil. The change of cell proliferation was observed with CCK8 colorimeter, and the results revealed that in the cells transfected with siRNA, there was no significant difference between the group pretreated with remifentanil and the group treated with LPS in the cell proliferation activity. The apoptotic cells increased after LPS stimulation. Similarly, cells receiving remifentanil pretreated had reduced LPS induced cell death, and the protective effect of remifentanil was blocked when silencing β-arrestin2 expression.3、 Remifentanil can reduce the phosphorylation of ERK1/2 and JNK induced by LPS, while this effect was blocked when interfering β-arrestin2 expression. We also detected that preincubation with remifentanil increase the amount of formation of β-arrestin2-TRAF6 complex, which had been reported to act as a negative regulator of TLR signaling.Conclusion:1、 Opiod receptor agonist remifentanil preconditioning can alleviate hepatic ischemia reperfusion injury in mice by inhibiting TLR4 pathway.2、 β-arrestin2 plays an important role in remifentanil protective effect in hepatic ischemia reperfusion injury, and this protective effect is blocked when silencing β-arrestin2.3、 β-arrestin2 contributes to the amelioration of cell function of remifentanil by limiting ERK1/2 and JNK activity. Remifentanil inhibits inflammation induced by LPS via improving the amount of formation of β-arrestin2-TRAF6 complex. |
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