Regulating Mechanisms Of Superoxide Anion And Nitric Oxide Generation In Macrophages

Macrophages, one of the key mediators of the immune system, generate large quantities of superoxide in response to a variety of stimuli, by a coordinated sequence of biochemical reactions known as the respiratory burst. At the same time, upon stimulation by certain cytokines, the iNOS gene of macrophages is induced and large amount of NO is generated. Melatonin is a derivative of tryptophan mainly synthesized in the pineal gland of mammals, which shows hypnotization and immune-regulatory functions in vivo. Recent studies revealed that melatonin acts as a potent antioxidant in vitro. Ferulic acid and tetramethylpyrazine are two active ingredients of Chinese herbal medicine Ligusticum wallichi Franchat. Excessive production of reactive oxygen species (ROS; including superoxide, nitric oxide and peroxynitrite) by macrophages has been implicated in a number of pathological processes. Accordingly, drugs that inhibit ROS generation may have beneficial effects in the treatment of diseases due to overproduction of ROS. In the present investigation, chemiluminescence by the generation of superoxide anion and burst respiratory was related with the production of nitric oxide. The effects of melatonin on the generation of superoxide anion and nitric oxide in stimulated murine macrophages were studied by luminol-enhanced chemiluminescence and EPR techniques, and the underlying molecular mechanisms were discussed. Melatonin pretreatment significantly scavenges the superoxide anion generated by macrophages, and inhibits the generation of nitric oxide. Ferulic acid, but not tetramethylpyrazine, scavenges superoxide anion dose-dependently. Western blot and RT-PCR analysis showed that melatonin decreased LPS/IFN-γ-induced iNOS gene expression. Tetramethylpyrazine also inhibits the expression of iNOS gene, and consequently decreases the formation of nitric oxide. However, ferulic acid shows no effect on iNOS expression and NO production. Further investigation suggested that melatonin significantly inhibited the degradation of cytoplasmic IκB-α, and blocked the translocation of p65/RelA into the nuclei. The results show that melatonin is an effective modulator on macrophages, which scavenges endogenous superoxide anion dose-dependently, inhibits the expression of iNOS gene via modulating the NF-κB signaling pathway, and consequently decreases the formation of nitric oxide. Therefore, it was suggested that melatonin, ferulic acid and tetramethylpyrazine might be the potential therapeutic and defense agents for diseases associated with the over activation of macrophages. The protective effects of extracts of Ligusticum wallichi Franchat against ischemic injury might be due to the scavenging of superoxide anion and the regulation of NO production.