The Effects Of Melatonin Regulating Lipopolysaccharide-Stimulated Inflammatory Cytokines

Increasing evidences demonstrated that melatonin had an anti inflammatory effect and reduced the inflammatory injury significantly. Nevertheless, the molecular mechanisms remain obscure. In order to understand better the mechanism by which melatonin negatively regulates LPS-induced production of proinflammatory cytokine, we investigate the effect of melatonin on activation of inflammatory cytokine signaling pathways in RAW264.7mouse macrophage cell line.RAW264.7cells were grown in Nunc flasks in Dulbecco s modified Eagle’s medium (DMEM) supplemented with100U/mL of penicillin,100μg/mL streptomycin,10mmol/l HEPES,2mmol/l L-glutamine,0.2%NaHCO3, and10%[v/v] heat-inactivated FBS in a humidified chamber with5%CO2/95%air at37℃The cells were seeded into culture plates at a density of1×106cells/plate and incubated for at least12hr to allow them to adhere to the plates. After removing the old medium, the cells were incubated with LPS (2.0μg/mL) for different times in the absence or presence of different concentration of melatonin. The cells were washed with chilled PBS for three times and then harvested for real-time RT-PCR and immunoblots.The effects of melatonin on LPS-induced proinflammatory cytokines in RAW264.7cells was determined. In response to LPS, the expression of several proinflammatory cytokines, such as TNF-α, IL-1β,IL-6, and IL-8were significantly increased at2hr after LPS treatment. The expression of these proinflammatory cytokines remained elevated at6hr after LPS treatment, whereas LPS had little effect on the expression of IL-17and IFN-γ in RAW264.7cells. Melatonin significantly attenuated LPS-evoked upregulation of proinflammatory cytokines. The level of anti-inflammatory cytokines IL-10mRNA was obviously increased at2hr after LPS treatment, whereas LPS had little effect on the expression of IL-4in RAW264.7cells. Melatonin significantly attenuated LPS-evoked upregulation of IL-10in RAW264.7cells.The effects of melatonin on LPS-induced expression of iNOS were then investigated. The level of iNOS mRNA was significantly increased at2hr after LPS treatment. The expression of iNOS mRNA remained elevated at6hr after LPS treatment. Interestingly, melatonin significantly attenuated LPS-evoked upregulation of iNOS mRNA. Correspondingly, melatonin significantly attenuated LPS-induced elevation of iNOS protein in RAW264.7cells. Next, we analyzed the effects of melatonin on LPS-induced expression of COX-2in RAW264.7cells. The level of COX-2mRNA was slightly increased at2hr after LPS treatment. Further analysis showed that the expression of COX-2mRNA was significantly upregulated at6hr after LPS treatment. Interestingly, melatonin briefly attenuated LPS-evoked upregulation of COX-2mRNA. Interestingly, melatonin almost completely inhibited LPS-induced elevation of COX-2protein in RAW264.7cells.To investigate the effects of melatonin on LPS-induced MAPK signaling, the levels of pJNK, pp38, and pERK were measured in LPS-stimulated RAW264.7cells. As expected, the levels of pJNK, pp38, and pERK were significantly increased in LPS-treated RAW264.7cells in a time-dependent manner. Unexpectedly, melatonin had no effect on LPS-induced JNK phosphorylation. In addition, melatonin did not inhibit LPS induced p38and ERK phosphorylation in RAW264.7cells. Surprisingly, melatonin alone induced the phosphorylation of JNK, p38, and ERK in RA W264.7cells.To investigate the effects of melatonin on LPS-induced NF-κB activation, the levels of IκB, nuclear NF-κB p65and p50were measured using immunoblots in LPS-stimulated RAW264.7cells. The level of IκB was significantly decreased in LPS-stimulated macrophages. By contrast, the levels of nuclear NF-κB p65and p50were significantly increased in LPS-stimulated macrophages. Melatonin significantly attenuated LPS-induced IκB degradation. In addition, melatonin significantly inhibited LPS-induced nuclear translocation of NF-κB p65and p50.The effects of melatonin on LPS-induced Akt phosphorylation were analyzed. Results showed that neither LPS nor melatonin regulated the expression of AKT in RAW264.7cells. LPS significantly increased the level of pAKT in a time-dependent manner. Melatonin almost completely inhibited LPS-induced elevation of pAKT in RAW264.7cells.The effects of melatonin on the expression of TLR4and MyD88were analyzed in RAW264.7cells. Results showed that LPS alone had no effect on the expression of TLR4mRNA. Interestingly, melatonin slightly upregulated the expression of TLR4mRNA at6hr after LPS treatment. The effects of melatonin on the level of MyD88protein were analyzed. Melatonin had little effect on LPS-induced elevation of MyD88protein at2hr after LPS treatment. Interestingly, melatonin significantly alleviated LPS-induced elevation of MyD88protein at6hr after LPS treatment.Finally, the effects of melatonin on the expression of IFN-regulated factor-3 (IRF3), IRF7, and IFN-β were analyzed in LPS-stimulated RAW264.7cells. LPS significantly upregulated the expression of IRF7mRNA, whereas the level of IRF3mRNA was not significantly increased at6hr after LPS treatment. Interestingly, melatonin significantly attenuated LPS-induced elevation of IRF7mRNA in macrophages. The level of IFN-β mRNA was significantly increased in LPS-stimulated macrophages. Melatonin significantly attenuated LPS-induced expression of IFN-β mRNA.The present study indicates that melatonin inhibits TLR4-mediated inflammatory genes in macrophages. In addition, melatonin alleviates TLR4-mediated NF-κB and Akt activation in macrophages. Importantly, melatonin inhibits not only MyD88, the key signaling adaptor for MyD88-dependent signaling pathway, but also IRF7, which is involved in TRIF-dependent signaling pathway, in LPS-stimulated macrophages. The present study suggests that melatonin modulates TLR4-mediated inflammatory genes through not only MyD88-dependent but also TRIF-dependent signaling pathway. Thus, melatonin may have potential preventive and therapeutic utilities for protecting against TLR4-mediated inflammation.