Inhibitory Effect And Underlying Mechanisms Of AcSDKP On Murine Macrophage Cell Line

Background and Aims: Chronic hepatic inflammation is the core process involved in chronic liver injury and repair.In the injuried liver,the infiltrated macrophages and activated Kupffer cells(KCs)produce and release a large amount of inflammatory cytokines,which maintain and expand the existing liver inflammation and aggravate liver insult.The activation of the NF-κB signaling pathway is common in various inflammatory diseases.Blockade of NF-κB signaling pathway provides an important strategy for treatment of organ inflammation.AMP-activated protein kinase(AMPK)is a key factor in the regulation of energy metabolism.Phosphorylation of AMPK leads to inhibition of NF-κB signaling.N-acetyl-seryl-aspartyl-lysyl-proline(AcSDKP)is an endogenous acetylated tetrapeptide.Our previously studies showed that AcSDKP attenuates liver fibrosis induced by tetrachrolide and bile-duct ligation in rats.Those effects were related to decrease inflammatory factors both at cellular and molecular levels.However,it is not clear whether AcSDKP regulates macrophage-mediated inflammatory response directly.We hypothesize that AcSDKP has a direct anti-inflammatory effect on macrophage by activating AMPK and thus inhibition of NF-κB signaling pathway.Our research will test this hypothesis and provide new experimental basis for the application of AcSDKP in the control of chronic inflammatory diseases including the liver.Methods: Murine macrophage cell line RAW264.7 cells were cultured in vitro and divided into three groups,including control group,lipopolysaccharides(LPS)group and AcSDKP treatment group(LPS plus AcSDKP).Cells in LPS group and AcSDKP treatment group were incubated with LPS(1μg/ml)and AcSDKP treatment group also incubated with different concentrations of AcSDKP(1nmol/L,10nmol/L and 100nmol/L).The mRNA and protein levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)were detected using RT-PCR and ELISA analysis.The latex beads were used to assess the phagocytotic activity of cells.The chemotactic ability of RAW264.7 cells were detected by Transwell assay.Fluorescence microscopy and flow cytometry were performed to assess intracellular reactive oxygen species(ROS)by fluorescent probe DCFH-DA.The expression levels of proteins in NF-κB and AMPK pathway were determined by Western blotting.The impact of AMPK signaling pathway on AcSDKP function was further comfirmed by using AMPK phosphorylation inhibitor Compound C and AMPK specific siRNA.Results: In the RAW264.7 cell line,the mRNA and protein levels of TNF-α,IL-1β and IL-6 in LPS group were significantly increased when compared with control group;The mRNA and protein levels of TNF-α,IL-1β and IL-6 in cells treated with AcSDKP were significant reduced,as compared with LPS group(p<0.05);The phagocytotic activity in cells treated with AcSDKP(10nmol/L)was markedly inhibited as compared with LPS group(p<0.01);The migrated cell count across the chamber membrane in LPS group was significantly higher than that in the control group,however,the cell count in AcSDKP treatment group was significantly lower than that in the LPS group(p<0.05);DCFH-DA essay showed that AcSDKP treatment could significantly reduce the level of intracellular ROS,as compared with the LPS group and the difference was statistically significant(P<0.05).Western blot showed that AcSDKP(10nmol/L,100nmol/L)treatment significantly decreased the levels of p-IκB and p-p65 and inhibited the translocation of p65 from the cytoplasm into the nucleus.The protein level of p-AMPK in AcSDKP treatment group was significantly higher than that in the LPS group(p<0.05);After blocking AMPK signaling pathway with Compound C or siRNA in cells treated with AcSDKP and LPS,the expression of inflammatory factors and the levels of p-IκB and p-p65 were all increased significantly when compared with those in cells treated with AcSDKP alone(p<0.01).Conclusions: AcSDKP exhibits a significant inhibitory effect on the proinflammatory response induced by LPS in mouse Raw264.7 macrophage in vitro;This inhibitory effect of AcSDKP may be related to increased phosphorylation of AMPK and resulted inhibition of NF-κB signaling pathway.