Generally, exposure to a high concentration of hydrogen peroxide (H2O2) can cause oxidative stress in RAW 264.7 macrophages.24,25 Our data align with these findings, as we observed cell death in a dose-dependent manner upon hydrogen peroxide exposure. Numerous research studies have reported the dose-dependent nature of hydrogen peroxide-induced oxidative stress.26,27 This oxidative stress is associated with various diseases, including cancer, diabetes mellitus, and cardiovascular diseases.6,7 Apart from that, elevation of ROS particularly hydrogen peroxide is also part of inflammatory responses by macrophages and plays important roles in pathogen killing. However, excessive accumulation and duration of hydrogen peroxide in macrophages can cause detrimental effects.28 A study conducted by Robert N. Goddu et al. suggested that chronic exposure of RAW 264.7 macrophage cells to hydrogen peroxide enables them to survive oxidative stress due to the elevated expression of the endogenous enzyme catalase.25 Therefore, we assume that macrophages can adapt to progressive environmental stress but may not survive sudden acute changes in the environment.

We also assessed the cytotoxicity activity of fargesin in this study. The findings revealed that high concentrations of the compound (more than 25 μM) reduced the percentage of cell viability in macrophages. This result is consistent with our previous study, where we reported an IC50 of 173.5μM for fargesin in RAW264.7 cells.29 Previous research has also reported that fargesin isolated from Z. planispinum roots exhibited moderate cytotoxic activity against Hela cancer cells, with an IC50 of 15.00±1.50 μg/mL.30 These cytotoxic properties, supported by previous studies, are promising for the treatment of cancer through the inhibition of ERK1/2 kinases, which retard the release of inflammatory mediators such as interleukin-6 (IL-6), Tumour Necrosis Factor-α (TNF-α), and Reactive Oxygen Species (ROS).31,32

Our findings indicate that fargesin exhibits a protective effect against cell death induced by hydrogen peroxide, possibly due to its anti-inflammatory and antioxidant activities. Previous studies have shown that, beside ROS, hydrogen peroxide can induce cell death through activation of the inflammatory pathway via the MAPK pathway and the expression of genes related to inflammation, including interleukins, TNF-α, and NF-κB. Research suggests that fargesin is a bioactive lignan with anti-inflammatory properties.32 Recent studies have shown that fargesin exerts anti-inflammatory actions on human monocytic cells (THP-1) by preventing NF-kB signaling activation.

Figure 3:
Figure 3: Cell death protective activity of fargesin in RAW 264.7 cells. Effects of fargesin on % cell viability in (A) 1mM and (B) 2 mM H2O2-induced RAW264.7 cells. (C) Morphological changes of the cells after treatment with fargesin and hydrogen peroxide. Data are presented as mean SD values of three replications. *p<0.05 indicates significant differences from the hydrogen peroxide alone group value. #p<0.05 indicates significant differences from the control group value.

NF-kappa B plays a key role in the transcriptional induction of pro-inflammatory mediator genes. When activated, NF-kappa B translocate from the cytosol to the nucleus, facilitating the transcription of inflammatory cascades. Therefore, treatment with dose-dependent fargesin prevents the translocation of the NF-kappa B transcription factor.23 Additionally, an in vitro study on the anti-inflammatory activity of lignans from M. fargesii demonstrated that the compound suppresses NF-kappa B, reducing the expression of induced Nitric Oxide Synthase (iNOS) and Cyclooxygenase-2 (COX-2). The overproduction of nitric oxide stimulates the production of prostaglandins by activating Cyclooxygenase-2 (COX-2).33 Prostaglandins are potent inflammatory mediators derived from the conversion of arachidonic acid. Hence, the inhibition of induced Nitric Oxide Synthase (iNOS) and Cyclooxygenase-2 (COX-2) by the lignans from M. fargesii has been shown to reduce inflammation in treated cells.34 Other studies have also demonstrated fargesin’s anti-inflammatory effect in chemically induced inflammatory bowel disease in mice, significantly reducing the expression of pro-inflammatory cytokines IL-1B, IL-15, TNF-α, and IFN-γ, while increasing the expression of the anti-inflammatory cytokine IL-10 in the colon.15

Fargesin has been shown to increase the activities of some endogenous antioxidants, including superoxide dismutase, catalase, and glutathione peroxidase, while suppressing Malondialdehyde (MDA), which is a marker for oxidative stress. This reduction in MDA indicates a decrease in the release of intracellular reactive oxygen species.35 These findings explain the possible pathways of fargesin’s protective activities and its anti-inflammatory effect. Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), and Reductase (GRx) are the main antioxidant enzymes involved in the neutralization of reactive oxygen species. SOD acts as the first-line defense against ROS, as it catalyzes the dismutation of the superoxide anion radical into hydrogen peroxide through a reduction process.36 Subsequently, hydrogen peroxide is transformed into water and diatomic oxygen (O2) by Catalase (CAT) or Glutathione (GPx). The GPx enzyme then removes hydrogen peroxide by catalyzing its oxidation to the reduced form of glutathione, GSH, converting it to its oxidized form, GSSH.8–14