Bangladesh Journal of Pharmacology Volume: 20; Number 1; Year 2025 Cite this article as: Tran TT, Cuong NM, Ha NX, Nakahama T, Nguyen NT. Murrayafoline A inhibits inflamma- tory cytokines in RAW264.7 cells stimulated with LPS and poly (I:C) by targeting the aryl hydrocarbon receptor. Bangladesh J Pharmacol. 2025; 20: 35-39. Murrayafoline A inhibits inflammatory cytokines in RAW264.7 cells stimulated with LPS and poly (I:C) by targeting the aryl hydrocarbon receptor Dear Editor, The anti-inflammatory activities of murrayafoline A, an alkaloid isolated from Glycosmis stenocarpa, remain poorly understood, particularly in cytokine regulation in macrophages. In this study, the anti-inflammatory activities of murrayafoline A were further evaluated in RAW264.7 cells, which was stimulated with both lipopolysaccharide (LPS) and polyinosinic:polycyti- dylic acid (poly I:C) to mimic both bacterial and viral infections (Ding et al., 2017). This approach allowed the discovery of whether murrayafoline A, as well as other natural compounds, can inhibit inflammatory responses during both bacterial and viral infections. The co-stimulation of LPS and poly (I:C) has been used in several previous studies to enhance or limit immune res-ponses (Bogunovic et al., 2020; Ding et al., 2017; Gorskaya et al. 2020). While LPS activates TLR4, moderate activation of TLR3 by poly (I:C) seems to increase cytokine production, whereas exce- ssive TLR3 activation may lead to a reduction in inflammatory cytokine levels (Jiang et al., 2005). In this study, RAW264.7 cells were stimulated with LPS (1 µg/mL) and various concentrations of poly (I:C) (10- 50 µg/mL). The results showed that the combination of LPS (1 μg/mL) with poly (I:C) (20 μg/mL) induced peak production of the cytokines TNF-α and IL-6 (data not shown). Next, the cells were pretreated with murrayafoline A (3, 10, 30, or 60 µM) for 1 hour before being stimulated with 1 µg/mL LPS and 20 µg/ mL poly (I:C). The cells were harvested for qRT‒PCR analysis at 3, 6, and 24 hours, while the supernatants were collected for ELISA at 24 hours, as described previously (Tran et al., 2022). Murrayafoline A did not affect RAW264.7 cell viability, with viability percentages of 97 ± 5, 100 ± 5, 95 ± 2, and 92 ± 1% at concentrations of 3, 10, 30, and 60 μM, respectively (data not shown). In a previous study, synthetic murrayafoline A deriva- tives influenced LPS-stimulated inflammatory cytokine production in bone marrow-derived dendritic cells (Thuy et al., 2013). In the present study, murrayafoline A significantly suppressed the LPS- and poly (I:C)- costimulated production of IL-6, TNF-α, and IL-10 in a concentration-dependent manner in RAW264.7 cells, with IC50 values of 2.7 ± 0.7, 3.4 ± 0.5, and 3.7 ± 0.5 µM, respectively (Figure 1). The production of proinflammatory cytokines in activated macrophages is regulated by various intracellular pathways, including the NF-κB pathway, where MyD88 and NF-κB play key roles (Li et al., 2020). As shown in Figure 2, murrayafoline A significantly reduced Myd88 levels by 40, 61, 70, and 78% and Nf-κb1 levels by 32, 49, 80, and 84% at concentrations of 3, 10, 30, and 60 µM, respectively, after 6 hours compared with those in the control. It has been reported that Ahr can regulate inflammatory cytokines such as IL-6 in macrophages under LPS stimulation (Kimura et al., 2009). Recently, the alkaloid A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2025; 20: 35-39 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v20i1.80391 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor 2000 1500 1000 500 0 6000 4000 2000 0 600 400 200 0 Murrayafoline A 0 0 3 10 30 60 µM LPS - + + + + + Poly (I:C) - + + + + + IL -6 l e v e l (p g /m L ) IL -1 0 l e v e l (p g /m L ) T N F -α l e v e l (p g /m L ) a b c a b c c c c A B C Figure 1: Murrayafoline A inhibited IL-6, TNF-α, and IL-10 production in LPS- and poly (I:C)-stimulated RAW264.7 cells. The cells were pretreated with various concentrations of mur- rayafoline A followed by the presence of LPS (1 µg/mL ) and poly (I:C) (20 µg/mL) for 24 hours. Data are the mean ± SD of 3 replicate experiments. ap<0.05; bp<0.01; cp<0.005 vs the sam- ples stimulated with only LPS 36 Bangladesh J Pharmacol 2025; 20: 35-39 Murrayafoline A 0 0 3 10 30 60 0 0 3 10 30 60 µM LPS - + + + + + - + + + + + Poly (I:C) - + + + + + - + + + + + E xp re s s io n f o ld 10 8 6 4 E xp re s s io n f o ld 6 5 4 3 2 1 E xp re s s io n f o ld 6 5 4 3 2 1 0 E xp re s s io n f o ld 3 2 1 0 Myd88 Ahr Nf-kb1 Cyp1b1 A B C D Figure 2: Murrayafoline A suppressed the expression of Myd88, Nf-κb1, and enhanced the expression of Ahr in LPS- and poly (I:C)- costimulated RAW264.7 cells after 6 hours. The gene expression of Myd88, Nf-κb1, Ahr and Cyp1b1 was detected via real-time quantitative PCR. Data are presented as the mean ± SD of 3 replicate experiments. ap<0.01; bp<0.005 Figure 3: 2D and 3D interactions of murrayafoline A (A) and the reference compound indirubin (B), FICZ (C) and TCDD (D) within the active site pocket of Ahr (PDB ID: 7ZUB) Cont.. a a a a b b b b b b b b b b A B van der Waals Conventional hydrogen bond Stacked T-shaped Cation Sigma Alkyl Alkyl 9-hydroxy-canthin-6-one from Eurycoma longifolia was reported to inhibit the production of inflammatory cytokines, including IL-6, TNF-α, and IL-10, in LPS- induced RAW264.7 cells, possibly via Ahr activation (Tran et al., 2022). Interestingly, in the present study, murrayafoline A at concentrations of 3, 10, 30, and 60 μM increased Ahr expression by 2.4-, 2.5-, 5.5-, and 4.0-fold, respectively, compared with the control. In addition, murrayafoline A at concentrations of 3, 10, 30, and 60 μM also increased the expression of Cyp1b1, a downstream gene induced by Ahr activation by 2.1-, 4.5-, 4.1-, and 3.9-fold, respectively, compared with the control. These findings suggest that murraya- foline A may suppress the inflammatory response through Ahr activation in macrophages. To better understand the molecular mechanisms governing Ahr regulation and whether murrayafoline A interacts with Ahr and how this interaction affects Ahr expression, an in silico analysis was conducted and the results were compared with those of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) and 6-formylindolo [3,2-b] carbazole (FICZ), two high-affinity Ahr ligands (Ehrlich et al., 2018). Docking simulations revealed that murraya- foline A binds to Ahr (PDB ID: 7ZUB) with a binding affinity of -9.663 kcal/mol, which is stronger than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (-9.574 kcal/mol) but weaker than that of 6-formylindolo [3,2- b] carbazole (FICZ) (-12.82 kcal/mol) and indirubin (- 12.52 kcal/mol), an Ahr cocrystallized compound. The primary interactions observed for murrayafoline A were hydrophobic, including alkyl and -π alkyl interactions with Cys333, Ile325, Phe351, Val381, and Leu353, as well as π-π stacking interactions with Tyr322, Phe295, and His291 (Figure 3A). Notably, the key residues Cys333, Phe351, Tyr322, Val381, Leu353, and Phe295 are also involved in interactions with indirubin (Gruszczyk et al., 2022; Figure 3B). Similar to FICZ (Figure 3C) and TCDD (Figure 3D), Figure 3: 2D and 3D interactions of murrayafoline A (A) and the reference compound indirubin (B), FICZ (C) and TCDD (D) within the active site pocket of Ahr (PDB ID: 7ZUB) C D Bangladesh J Pharmacol 2025; 20: 35-39 37 murrayfoline A interacts with Phe351, Phe295, Cys333, Leu353, Phe324, and His291 (Hirano et al., 2015). Interestingly, these key residues are located within the ligand-binding domain of Ahr, suggesting their crucial role in ligand recognition and binding specificity. These findings support the potential of murrayafoline A as an Ahr ligand (Figure 4). Never- theless, to confirm that murrayafoline A is an Ahr ligand, further investigation is needed to assess its effects in Ahr-deficient cells. In conclusion, inflammatory cytokine production in RAW264.7 cells induced by the costimulation of the TLR4 agonist LPS and the TLR3 agonist poly (I:C) can be inhibited by murrayafoline A from Glycosmis stenocarpa, potentially by reducing the expression of Myd88 and NF-κb1 through an Ahr-related mechanism. This work was funded by the projects DL0000.05/22-23 and CT0000.07/22-24 from the Vietnam Academy of Science and Technology. Conflict of interest: The authors declare that they have no conflicts of interest. Acknowledgments: The authors highly appreciate Drs. Duc Hoang Le and Tien Vu Thi (Institute of Biotechnology, VAST) for their technical assistance. Trang Thu Tran1, Nguyen Manh Cuong2, Nguyen Xuan Ha2, Taisuke Nakahama3, and Nam Trung Nguyen1 1Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 2Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 3Department of RNA Biology and Neuroscience, Osaka University, Suita, Osaka, Japan. Corresponding authors: Nam Trung Nguyen and Nguyen Manh Cuong email: nam@ibt.ac.vn; nmcuong@vast.gov.vn References Bogunovic D, Manches O, Godefroy E, Yewdall A, Gallois A, Salazar AM, Marie I, Levy DE, Bhardwaj N. TLR4 engage- ment during TLR3-induced proinflammatory signaling in dendritic cells promotes IL-10-mediated suppression of antitumor immunity. Cancer Res. 2011; 71: 5467-76. Cuong NM, Wilhelm H, Porzel A, Arnold N, Wessjohann L. 1- O-substituted derivatives of murrayafoline A and their anti- fungal properties. Nat Prod Res. 2008; 22: 1428-32. 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