Reactive oxygen species (ROS), products of oxidative stress, contribute to the initiation and progression of the pathogenesis of various diseases. Glutathione is a major antioxidant that can help prevent the process through the removal of ROS. The aim of this study was to evaluate the protective effect of glutathione on ROS-mediated DNA damage and apoptosis caused by hydrogen peroxide, H₂O₂, in RAW 264.7 macrophages and to investigate the role of the nuclear factor erythroid 2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. The results showed that the decrease in the survival rate of RAW 264.7 cells treated with H₂O₂ was due to the induction of DNA damage and apoptosis accompanied by the increased production of ROS. However, H₂O₂-induced cytotoxicity and ROS generation were significantly reversed by glutathione. In addition, the H₂O₂-induced loss of mitochondrial membrane potential was related to a decrease in adenosine triphosphate (ATP) levels, and these changes were also significantly attenuated in the presence of glutathione. These protective actions were accompanied by a increase in the expression rate of B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax) and poly(ADP-ribose) polymerase cleavage by the inactivation of caspase-3. Moreover, glutathione-mediated cytoprotective properties were associated with an increased activation of Nrf2 and expression of HO-1; however, the inhibition of the HO-1 function using an HO-1 specific inhibitor, zinc protoporphyrin IX, significantly weakened the cytoprotective effects of glutathione. Collectively, the results demonstrate that the exogenous administration of glutathione is able to protect RAW 264.7 cells against oxidative stress-induced mitochondria-mediated apoptosis along with the activity of the Nrf2/HO-1 signaling pathway.

Spermidine, a naturally occurring polyamine, displays a wide variety of internal biological activities including cell growth and proliferation. However, the molecular mechanisms responsible for its anti-inflammatory activity have not yet been elucidated.

Spermidine is a naturally occurring polyamine compound that has recently emerged with anti-aging properties and suppresses inflammation and oxidation. However, its mechanisms of action on anti-inflammatory and antioxidant effects have not been fully elucidated. In this study, the potential of spermidine for reducing pro-inflammatory and oxidative effects in lipopolysaccharide (LPS)-stimulated macrophages and zebrafish was explored. Our data indicate that spermidine significantly inhibited the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines including tumor necrosis factor-α and interleukin-1β in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects of spermidine accompanied by a marked suppression in their regulatory gene expression at the transcription levels. Spermidine also attenuated the nuclear translocation of NF-κB p65 subunit and reduced LPS-induced intracellular accumulation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Moreover, spermidine prevented the LPS-induced NO production and ROS accumulation in zebrafish larvae and was found to be associated with a diminished recruitment of neutrophils and macrophages. Although more work is needed to fully understand the critical role of spermidine on the inhibition of inflammation-associated migration of immune cells, our findings clearly demonstrate that spermidine may be a potential therapeutic intervention for the treatment of inflammatory and oxidative disorders.

Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway.

Morroniside, a major iridoid glycoside isolated from Cornus officinalis, has a variety of beneficial pharmacological properties. Although morroniside has recently been reported to exhibit anti-inflammatory and antioxidant effects, the detailed mechanism has not yet been fully elucidated. In this study, we investigated the inhibitory effect of morroniside on inflammatory and oxidative stress activated by lipopolysaccharide (LPS) in RAW 264.7 macrophages. Our results indicated that morroniside pretreatment significantly inhibited the LPS-induced phagocytic activity and release of pro-inflammatory factors, which was associated with blocking the expression of their regulatory genes. Morroniside also markedly suppressed the expression of myeloid differentiation factor 88 as well as Toll-like receptor 4 (TLR4), and attenuated the translocation of nuclear factor-κB (NF-κB) to the nucleus in LPS-treated RAW 264.7 macrophages. Furthermore, morroniside prevented the binding of LPS to the TLR4 on the cell surface. In addition, morroniside abolished reactive oxygen species (ROS) generation, and enhanced the expression of heme oxygenase-1 (HO-1) following activation of nuclear factor-E2-related factor 2 (Nrf2) in LPS-stimulated RAW 264.7 macrophages. However, zinc protoporphyrin, a specific inhibitor of HO-1, reversed the morroniside-mediated inhibition of inflammatory response in LPS-treated RAW 264.7 macrophages. In conclusion, our findings suggest that morroniside exerts LPS-induced anti-inflammatory and antioxidant effects by targeting the TLR4/NF-κB and Nrf2/HO-1 signaling pathways in RAW 264.7 macrophages. Taken together, our findings suggest that morroniside interacted structurally and electrochemically with TLR4/MD2 complex, consequently can be a potential functional agent to prevent inflammatory and oxidative damage.