In the immune system, macrophages play an important role in regulating immune function and maintaining homeostasis by suppressing the inflammatory reactions caused by various stimuli, such as free radicals and stress . In particular, macrophages are involved in the production of NO, a representative inflammatory mediator, which is a highly reactive biomolecule produced from L-arginine by NOS .
iNOS and COX-2 are representative pro-inflammatory cytokines. iNOS is primarily produced by stimulated macrophages, resulting in the inflammatory of NO [13, 14]. COX-2 is an enzyme that converts arachidonic acid into prostaglandins; although COX-1 is primarily found in normal cells, COX-2 is expressed at the site of inflammation and is associated with cancer . Since cytokines such as IL-1β, IL-6, and TNF-α are inflammatory cell signaling proteins , inhibition of these inflammatory mediators is a promising target for the treatment of inflammatory diseases .
In this study, the effects of RC-L on NO production, cell viability, and expression of pro-inflammatory cytokines were investigated using the RAW264.7 cell line. NF-κB is a transcription factor that participates in various processes, such as general immunity, inflammation, and cell growth regulation. Usually, it exists in an inactive state, bound to the inhibitory protein IκB in the cytoplasm . As p65 and p50, which are bound to the cell, move from the cytoplasm to the cell nucleus, NF-κB activation occurs, and inflammation is promoted . When NF-κB signaling is activated, p65 (a component of the NF-κB complex) is transferred to the nucleus , and its phosphorylation plays a major role in NF-κB activity because it regulates migration from the cytoplasm to the nucleus. Inhibition of phosphorylation is associated with the inhibition of NF-κB activity . RC-L inhibited LPS-induced NF-κB activity and intranuclear migration in RAW264.7 cells. Western blot analysis revealed that the quantity of protein transferred in the nucleus was significantly decreased in cells treated with 25 and 50 mg/mL of RC-L compared to that in the with LPS-treated control group and the LPS-only treatment group.
The MAPK family is a signal transduction mediator in cellular responses to various external stimuli and includes ERK, p38, and JNK . In this study, western blotting results showed that RC-L inhibited the LPS-induced phosphorylation of ERK, p38, and JNK in RAW264.7 cells.
HO-1 induction acts against the mechanisms that protect cellular lipids and proteins from oxidative damage, thereby weakening the inflammatory response . Nrf2 increases the expression of certain genes after binding to antioxidant response elements . Under normal conditions, there are low levels of Nrf2 in the cytoplasm because it forms a complex with Kelch-like ECH-associated protein 1 (Keap1), which degrades it. However, it can separate from Keap1 by external stimulation or oxidative stress and move to the nucleus . Of note, Nrf2 and HO-1 are closely related, and HO-1 expression is increased by Nrf2.
When RAW264.7 macrophages were treated with RC-L, Nrf2 accumulated in the nucleus in a concentration-dependent manner, and the highest value was observed after 1 h of treatment. In addition, to determine whether HO-1 plays a major role in the anti-inflammatory effect of RC-L, inhibitors such as SB203580, PD98059, SP600125, BAY11-7082, and NAC were used. As expected, treatment with these inhibitors suppressed HO-1 expression. Accordingly, the HO-1 induction ability of RC-L decreased when HO-1 inhibitors were administered. These findings confirmed that the major anti-inflammatory mechanism of RC-L is HO-1 expression. Moreover, the translocation of the transcriptional regulatory factor of the HO-1 gene into the nucleus migration of Nrf2 was also significantly increased in a concentration-specific manner. Western blotting was performed to confirm the intranuclear migration of Nrf2; it was found that the amount of Nrf2 increased in the nucleus following RC-L treatment. These findings indicated that RC-L promoted the migration of Nrf2 into the nucleus and increased the expression of HO-1.
We also investigated RC-L-induced Nrf2 expression over time and found that this expression increased after 1 h of treatment. In the group treated with RC-L for 24 h, the expression of Nrf2 showed a similar pattern to that at 0 h without treatment, indicating that treatment with RC-L for 24 h did not affect the expression of Nrf2. However, owing to the activation of Nrf2, the expression of HO-1 was highest at 24 h. We expected that HO-1 expression would increase with the expression of Nrf2, but the expression of Nrf2 and HO-1 in the group treated with RC-L for 24 h showed a contrasting trend, consistent with the results of a previous study . Similar to our results, previous studies have reported that the anti-inflammatory activity of biologically active substances derived from various natural substances is regulated by the Nrf2/HO-1 pathway . In conclusion, our results demonstrated that RC-L reduces the expression of various inflammatory genes and can regulate inflammation through various pathways, such as the NF-κB, MAPK, and Nrf2/HO-1 pathways.