Sesquiterpenes from Artemisia princeps regulate inflammatory responses in RAW 264.7 macrophages

Abstract Four sesquiterpenoids were isolated from an ethyl acetate-soluble fraction of A. princeps ethanolic extract: seco-tanapartholide B (5-epi-seco-tanapartholide A) (1), 4-epi-seco-tanapartholide A (2), 11,13-dehydrodesacetylmatricarin (3) and desacetylmatricarin (4). Compounds 1 − 3 dose-dependently inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages. These compounds also decreased mRNA and protein expression levels of inducible NO synthase and cyclooxygenase-2 as well as mRNA levels of pro-inflammatory cytokines (interleukin-1β and tumour necrosis factor-α) in LPS-stimulated RAW 264.7 macrophages. Moreover, compound 3 effectively enhanced the expression of heme oxygenase-1 (HO-1) in macrophages in the presence or absence of LPS. Additionally, the exocyclic methylene of α-methylene-γ-lactone moiety of compound 3 was found to be essential for the activation of the NF erythroid 2-related factor 2 (Nrf2)/HO-1 pathway. Here, we firstly report the isolation of compounds 3 and 4 from A. princeps and the anti-inflammatory activity of compound 3 by up-regulation of Nrf2/HO-1 pathway. Graphical Abstract


Introduction
Inflammation is a complex physiological response and the main defense mechanism against noxious stimuli, such as physical or chemical injuries and infections. However, inflammation also plays a key role in the development of pathological processes such as inflammatory bowel disease, cancer, rheumatoid arthritis, gastritis and metabolic disorders (Dhingra and Chopra 2020). Macrophage regulates inflammatory responses through the production of pro-inflammatory mediators. Activated macrophages secrete a large amount of cytokines (including interleukin (IL)-1b and tumour necrosis factor (TNF)-a) in inflammatory conditions and induce pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Valledor et al. 2010). Nuclear factor erythroid 2-related factor 2/heme-oxygenase-1 (Nrf2/HO-1) pathway is responsible for physiological homeostasis of cellular responses to stress and inflammation (Saha et al. 2020). Therefore, regulating the Nrf2/HO-1 pathways represents a promising strategy to treat inflammatory disorders.

Results and discussion
Compounds 1 À 4 were obtained via activity-guided purification of EtOAc-soluble fractions from 80% ethanolic extract of A. princeps ( Figure 1A). Their structures were identified as seco-tanapartholide B (5-epi-seco-tanapartholide A) (1), 4-epi-secotanapartholide A (2), 11,13-dehydrodesacetylmatricarin (3) and desacetylmatricarin (4) based on spectroscopic analyses and comparisons with previous data (Ohno et al. 1980;Martinez et al. 1988;Tan and Jia 1992;Kawazoe et al. 2003). A common moiety of the exocyclic methylene of the a-methylene-c-lactone was identified in compounds 1 À 3, but not in compound 4. We observed the inhibitory effects of these compounds on NO production in LPS-activated macrophages. Overproduction of NO by LPS-stimulation was decreased by treatment with compounds 1 À 3 at 10 mM, but not by compound 4 ( Figure 1B). Their IC 50 value was 11.0 mM for 1, 16.9 mM for 2 and 4.7 mM for 3 ( Figure 1C). Cell viability was not affected by treatment with compounds 1 À 3 up to 25 mM (data not shown). Since treatment with compounds 1 À 3 reduced NO production in LPS-stimulated macrophages, we next examined their effects on the expression of iNOS, an enzyme known to catalyse oxidative deamination of L-arginine to produce NO in the inflammatory response. Treatment with compounds 1 À 3 reduced the expression of iNOS protein, whereas LPS treatment markedly upregulated iNOS in RAW 264.7 cells ( Figure S1A). The expression of another representative inflammatory enzyme, COX-2, was also decreased by treatment with compounds 1 À 3 compared to that in the LPS-stimulated group ( Figure S1A). Moreover, these compounds decreased the mRNA levels of iNOS, COX-2, IL-1b and TNF-a in LPS-stimulated macrophages ( Figure S1B). Taken together, these data suggest that sesquiterpenes 1 À 3 isolated from A. princeps might have anti-inflammatory potential by suppressing NO production, downregulating pro-inflammatory enzymes iNOS and COX-2 and inhibiting the expression of cytokines IL-1b and TNF-a in activated macrophages.
Recent studies have demonstrated that overexpression of heme oxygenase-1 (HO-1) prior to stimulation with LPS can markedly inhibits the production of subsequent inflammatory mediators such as NO and IL-6. Moreover, the deficiency of HO-1 results in severe inflammation in mice (Fujioka et al. 2018;Yachie 2021). HO-1 is one of several anti-inflammatory genes expressed by macrophages. It provides beneficial effects by restoring physiological homeostasis. HO-1 exhibits key immunomodulatory functions in macrophages and plays a critical role in mediating the resolution of inflammatory responses (Vijayan et al. 2018). HO-1 induction in macrophages can functionally switch these cells to an anti-inflammatory phenotype. It can also act as an antioxidant enzyme that is induced via oxidative stress. To investigate the involvement of HO-1 expression in anti-inflammatory activity of A. princeps, we evaluated the effect of compound 3, the most potent one among the three sesquiterpenes, on HO-1 expression. Compound 3 increased HO-1 expression in LPS-activated macrophages. It also increased HO-1 expression in the absence of LPS ( Figure S2A). Additionally, the amount of HO-1 protein was reduced by treatment with N-acetyl-L-cysteine (NAC), an antioxidant, in compound 3-treated RAW264.7 cells ( Figure S2B). Previous studies have reported that the expression of HO-1 is mediated by the activation of Nrf2 which is involved in reactive oxygen species (ROS) production (Loboda et al. 2016;Saha et al. 2020). To investigate the upstream signaling pathway of HO-1 expression, the degree of ROS generation induced by compound 3 was assessed by measuring fluorescence. ROS levels were increased by treatment with 20 mM of compound 3 in RAW 264.7 cells. However, these ROS levels were reduced by co-treatment with NAC ( Figure S2C). Therefore, ROS generation might have been involved in the expression of HO-1 induced by compound 3 in RAW 264.7 cells. As shown in Figure 1 and Figure S1, compounds 1 À 3 inhibited the production of pro-inflammatory mediators and cytokines. They have a common structural moiety of exocyclic methylene in a-methylene-c-lactone, while compound 4 has a methyl moiety instead of exocyclic methylene. These structural differences might explain the structural requirement for anti-inflammatory activity of sesquiterpenes. We compared HO-1 expression levels after treatment with compound 3 and 4 in RAW 264.7 cells. HO-1 expression was observed after treatment with compound 3, but not after treatment with compound 4 ( Figure S3A). In the absence of oxidative stress, Nrf2 remains as an inactive form that can bind to Kelchlike ECH-associated protein 1 (Keap1) in the cytoplasm. In the presence of oxidative stress such as ROS, Nrf2 is activated via dissociation of a negative regulator Keap1 and translocated to the nucleus. Nrf2 can bind to antioxidant response element to induce phase II antioxidant enzymes such as HO-1 (Baird and Yamamoto 2020). In consistent with Figure S3A, we observed fluorescence of ROS upon treatment with compound 3, while no fluorescence of ROS was observed after treatment with compound 4. In addition, Nrf2 level in the nucleus was increased after treatment with compound 3, but not altered after treatment with compound 4 ( Figure S3B). These results suggest that compound 3 can induce HO-1 expression by activating Nrf2 through ROS generation. The exocyclic methylene moiety of a-methylene-c-lactone is a key component of compound 3 for Nrf2 activation. Compound 3 can modulate Nrf2/HO-1 pathway in homeostatic macrophages for mediating the resolution of inflammatory responses.

Experimental
Experimental details are provided in the supplementary materials.

Conclusion
We purified four anti-inflammatory sesquiterpenes, seco-tanapartholide B (5-epi-secotanapartholide A) (1), 4-epi-seco-tanapartholide A (2), 11,13-dehydrodesacetylmatricarin (3) and desacetylmatricarin (4), from A. princeps. Compounds 1 À 3 suppressed the production of inflammatory mediators such as NO, the expression of iNOS/COX-2 mRNA and protein and inflammatory cytokines including IL-1b and TNF-a in LPS-stimulated RAW 264.7 cells. Moreover, compound 3 induced the generation of intracellular ROS, leading to activation of Nrf2/HO-1 expression. The exocyclic methylene of a-methylene-c-lactone moiety was found to be essential for the activation of the Nrf2/ HO-1 pathway via ROS production in macrophages. Therefore, sesquiterpenes 1 À 3 obtained from A. princeps might have potential use in the management of inflammatory diseases.