Mitragyna speciosa Korth. downregulates macrophage inflammatory responses by inhibiting TLR-4 and increasing IL-10 production

Abstract

Background and Aims: Mitragyna speciosa Korth. is a tropical plant native to Asia with various medicinal properties. This study examined the immunotherapeutic potential of M. speciosa methanolic extract (MSME) against lipopolysaccharide (LPS)- stimulated activation of macrophages via the expression of Toll-like receptor 4 (TLR-4) and CD14 and downstream signalling cascades leading to the activation of nuclear factor kappa B (NF-𝜅B), which potentially affects macrophage immune responses. Methods: The expression of TLR-4/CD14 and NF-𝜅B genes in macrophages was determined in total RNA by qRT-PCR. Subsequently, the macrophage phagocytic activities and secretion of immune mediators such as reactive oxygen species (ROS) and cytokines were evaluated by fluorescent latex beads uptake assay, 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), and cytometric bead array, respectively, in LPS-activated RAW264.7 cells. Results: MSME significantly reduced macrophage-mediating inflammation by inhibiting TLR-4/CD14 signalling and subsequently suppressed the NF-𝜅B expression. Inhibition of TLR-4 by MSME attenuated macrophage phagocytic activity, which consequently reduced the production of ROS and pro-inflammatory cytokines such as IL-6, MCP-1, and TNF-α. Interestingly, MSME significantly increased the production of IL-10, which supports the anti-inflammatory properties of M. speciosa. Conclusion: Our findings suggest the therapeutic potential of MSME through the suppression of macrophage inflammatory responses mediated by IL-10 secretion.

Keywords

• Mitragyna speciosa
• Macrophages; Anti-inflammatory
• IL-10
• TLR-4

References

1. Ahmad, W., Jantan, I., Kumolosasi, E., Haque, M. A., & Bukhari, S. N. A. (2018). Immunomodulatory effects of Tinospora crispa extract and its major compounds on the immune functions of RAW 264.7 macrophages. International Immunopharmacology, 60, 141-151. https://doi.org/10.1016/j.intimp.2018.04.046 google scholar
2. Annas, S., Mastura Shaik Mossadeq, W., & Abdul Kadir, A. (2020). TROPICAL AGRICULTURAL SCIENCE Antipyretic Effect of Mitragynine and Crude Methanolic Extract of Mitragyna speciosa google scholar
3. Korth. in Mice. Pertanika J. Trop. Agric. Sc, 43(2), 207-216. Retrieved from http://www.pertanika.upm.edu.my/ google scholar
4. Assanangkornchai, S., Muekthong, A., Sam-angsri, N., & Pat-tanasattayawong, U. (2007). The use of Mitragynine speciosa (‘Krathom’), an addictive plant, in Thailand. Substance Use & Misuse, 42(14), 2145-2157. https://doi.org/10.1080/ 10826080701205869 google scholar
5. Atri, C., Guerfali, F. Z., & Laouini, D. (2018). Role of Human Macrophage Polarization in Inflammation during Infectious Dis-eases. International Journal of Molecular Sciences, 19(6), 1801. https://doi.org/10.3390/ijms19061801 google scholar
6. Buckhalter, S., Soubeyrand, E., Ferrone, S. A. E., Rasmussen, D. J., Manduca, J. D., Al-Abdul-Wahid, M. S., . . . Perreault, M. L. (2021). The Antidepressant-Like and Analgesic Effects of Kratom Alkaloids are accompanied by Changes in Low Frequency Oscil-lations but not AFosB Accumulation. Frontiers in Pharmacology, 12. https://doi.org/10.3389/fphar.2021.696461 google scholar
7. Dalmas, E., Tordjman, J., Guerre-Millo, M., & Clement, K. (2012). Macrophages and Inflammation. In Adipose Tissue Biology (pp. 167-193). New York, NY: Springer New York. https://doi.org/10. 1007/978-1-4614-0965-6_6 google scholar
8. Doyle, S. E., O’Connell, R. M., Miranda, G. A., Vaidya, S. A., Chow, E. K., Liu, P. T., . . . Cheng, G. (2004). Toll-like Receptors In-duce a Phagocytic Gene Program through p38. The Journal of Experimental Medicine, 199(1), 81-90. https://doi.org/10.1084/ jem.20031237 google scholar

9. Elisia, I., Pae, H. B., Lam, V., Cederberg, R., Hofs, E., & Krystal, G. (2018). Comparison of RAW264.7, human whole blood and PBMC assays to screen for immunomodulators. Journal of Im-munological Methods, 452, 26-31. https://doi.org/10.1016/j.jim. 2017.10.004 google scholar
10. Feng, G., Laijin, S., Chen, S., Teng, W., Dejian, Z., Yin, C., & Shoudong, G. (2021). In vitro and in vivo immunoregulatory ac-tivity of sulfated fucan from the sea cucumber A. leucoprocta. In-ternational Journal of Biological Macromolecules, 187(March), 931-938. https://doi.org/10.1016/j.ijbiomac.2021.08.008 google scholar
11. Finkel, T. (2011). Signal transduction by reactive oxygen species. Journal of Cell Biology, 194(1), 7-15. https://doi.org/10.1083/ jcb.201102095 google scholar
12. Firmansyah, A., Sundalian, M., & Taufiq, M. (2020). Kratom (Mi-tragyna speciosa Korth) for a New Medicinal: a Review of Phar-macological and Compound Analysis. Biointerface Research in Applied Chemistry, 11(2), 9704-9718. https://doi.org/10.33263/ BRIAC112.97049718 google scholar
13. Franchimont, D., Martens, H., Hagelstein, M.-T., Louis, E., Dewe, W., Chrousos, G. P., . . . Geenen, V. (1999). Tumor Necrosis Factor a Decreases, and Interleukin-10 Increases, the Sensitivity of Hu-man Monocytes to Dexamethasone: Potential Regulation of the Glucocorticoid Receptor. The Journal of Clinical Endocrinology & Metabolism, 84(8), 2834-2839. https://doi.org/10.1210/jcem. 84.8.5931 google scholar
14. Fushimi, T., Okayama, H., Seki, T., Shimura, S., & Shirato, K. (1997). Dexamethasone Suppressed Gene Expression and Production of lnterleukin-10 by Human Peripheral Blood Mononuclear Cells and Monocytes. International Archives of Allergy and Immunology, 112(1), 13-18. https://doi.org/10.1159/000237425 google scholar
15. Gao, W., Xiong, Y., Li, Q., & Yang, H. (2017). Inhibition of Toll-Like Receptor Signaling as a Promising Therapy for Inflammatory Dis-eases: A Journey from Molecular to Nano Therapeutics. Frontiers in Physiology, 8. https://doi.org/10.3389/fphys.2017.00508 google scholar
16. George, G., Shyni, G. L., Abraham, B., Nisha, P., & Raghu, K. G. (2021). Downregulation of TLR4/MyD88/p38MAPK and JAK/STAT pathway in RAW 264.7 cells by Alpinia galanga reveals its beneficial effects in inflammation. Journal of Ethnopharmacology, 275, 114132. https://doi.org/10.1016/j.jep. 2021.114132 google scholar
17. Gu, B. J., Sun, C., Fuller, S., Skarratt, K. K., Petrou, S., & Wiley, James. S. (2014). A quantitative method for measuring innate phagocyto-sis by human monocytes using real-time flow cytometry. Cytome-try Part A, 85(4), 313-321. https://doi.org/10.1002/cyto.a.22400 google scholar
18. Jansen, K. L. R., & Prast, C. J. (1988). Psychoactive Properties of Mitragynine (Kratom). Journal of Psychoactive Drugs, 20(4), 455-457. https://doi.org/10.1080/02791072.1988.10472519 google scholar
19. Jantan, I., Ilangkovan, M., Yuandani, & Mohamad, H. F. (2014). Cor-relation between the major components of Phyllanthus amarus and Phyllanthus urinaria and their inhibitory effects on phagocytic ac-tivity of human neutrophils. BMC Complementary and Alternative Medicine, 14(1), 429. https://doi.org/10.1186/1472-6882-14-429 google scholar
20. Juanda, E., Andayani, S., & Maftuch, M. (2019). Phytochemical Screening and Antibacterial Activity of Kratom Leaf (Mitrag-yna speciosa Korth.) Against Aeromonas hydrophilla. The Journal of Experimental Life Sciences, 9(3), 155-158. https://doi.org/10. 21776/ub.jels.2019.009.03.02 google scholar
21. Kafo, A. S. K., Elsalami, R. M. A., Zailan, N. F. Z., Mahayidin, H., Ramasamy, R., Zaidan, U. H., & Hassan, M. (2023). Effects of Mi-tragyna Speciosa (Korth.) on macrophage immune responses. Cur-rent Trends in Biotechnology and Pharmacy, 17(4A), 121-130. https://doi.org/10.5530/ctbp.2023.4s.99 google scholar
22. Kafo, A. S. K., Mahayidin, H., Zailan, N. F. Z., Zaidan, U. H., Syed Azhar, S. N. A., Ramasamy, R., & Hassan, M. (2023). Isolation of Phytochemical and Pharmacological Bioactive Com-pounds From Mitragyna speciosa (Korth.): A Scoping Review. Malaysian Journal of Medicine and Health Sciences, 19(s16), 38-47. https://doi.org/10.47836/mjmhs.19.s16.7 google scholar
23. Kawasaki, T., & Kawai, T. (2014). Toll-Like Receptor Signaling Path-ways. Frontiers in Immunology, 5. https://doi.org/10.3389/fimmu. 2014.00461 google scholar
24. Kim, E.-A., Kim, S.-Y., Ye, B.-R., Kim, J., Ko, S.-C., Lee, W. W., . . . Heo, S.-J. (2018). Anti-inflammatory effect of Apo-9-fucoxanthinone via inhibition of MAPKs and NF-kB signaling pathway in LPS-stimulated RAW 264.7 macrophages and ze-brafish model. International Immunopharmacology, 59, 339-346. https://doi.org/10.1016/j.intimp.2018.03.034 google scholar
25. Kruegel, A. C., Uprety, R., Grinnell, S. G., Langreck, C., Pekarskaya, E. A., Le Rouzic, V., . . . Sames, D. (2019). 7-Hydroxymitragynine Is an Active Metabolite of Mitragynine and a Key Mediator of Its Analgesic Effects. ACS Central Science, 5(6), 992-1001. https: //doi.org/10.1021/acscentsci.9b00141 google scholar
26. Liao, J., Xie, X., Wang, W., Gao, Y., Cai, Y., Peng, J., . . . Wang, L. (2021). Anti-inflammatory Activity of Essential Oil from Leaves of Blumea balsamifera (L.) DC through Inhibiting TLR4/NF-kB Signaling Pathways and NLRP3 Inflammasome Activation in LPS-induced RAW264.7 Macrophage Cells. Journal of Essen-tial Oil Bearing Plants, 24(2), 160-176. https://doi.org/10.1080/ 0972060X.2021.1912645 google scholar
27. Limtrakul, P., Yodkeeree, S., Pitchakarn, P., & Punfa, W. (2015). Sup-pression of Inflammatory Responses by Black Rice Extract in RAW 264.7 Macrophage Cells via Downregulation of NF-kB and AP-1 Signaling Pathways. Asian Pacific Journal of Cancer Prevention, 16(10), 4277-4283. https://doi.org/10.7314/APJCP. 2015.16.10.4277 google scholar
28. Liu, Y., Chen, W., Zheng, F., Yu, H., & Wei, K. (2022). Xanthatin Alleviates LPS-Induced Inflammatory Response in RAW264.7 Macrophages by Inhibiting NF-kB, MAPK and STATs Activation. Molecules, 27(14), 4603. https://doi.org/10.3390/molecules27144603 google scholar
29. Meng, Z., Yan, C., Deng, Q., Gao, D., & Niu, X. (2013). Curcumin inhibits LPS-induced inflammation in rat vascular smooth muscle cells in vitro via ROS-relative TLR4-MAPK/NF-kB pathways. Acta Pharmacologica Sinica, 34(7), 901-911. https://doi.org/10. 1038/aps.2013.24 google scholar
30. Parthasarathy, S., Bin Azizi, J., Ramanathan, S., Ismail, S., Sasid-haran, S., Said, M. I. Mohd., & Mansor, S. M. (2009). Eval-uation of Antioxidant and Antibacterial Activities of Aqueous, Methanolic and Alkaloid Extracts from Mitragyna Speciosa (Ru-biaceae Family) Leaves. Molecules, 14(10), 3964-3974. https: //doi.org/10.3390/molecules14103964 google scholar
31. Qi, Z., Yin, F., Lu, L., Shen, L., Qi, S., Lan, L., . . . Yin, Z. (2013). Baicalein reduces lipopolysaccharide-induced inflamma-tion via suppressing JAK/STATs activation and ROS production. Inflammation Research, 62(9), 845-855. https://doi.org/10.1007/ s00011-013-0639-7 google scholar
32. Ryu, J. H., Sung, J., Xie, C., Shin, M.-K., Kim, C.-W., Kim, N.-G., . . . Kang, D. (2016). Aplysia kurodai -derived glycosaminoglycans increase the phagocytic ability of macrophages via the activation of AMP-activated protein kinase and cytoskeletal reorganization in RAW264.7 cells. Journal of Functional Foods, 27, 122-130. https://doi.org/10.1016/j.jff.2016.08.059 google scholar
33. Shaik Mossadeq, W. M., Sulaiman, M. R., Tengku Mohamad, T. A., Chiong, H. S., Zakaria, Z. A., Jabit, M. L., . . . Israf, D. A. (2009). Anti-inflammatory and antinociceptive effects of Mitrag-yna speciosa Korth methanolic extract. Medical Principles and Practice, 18(5), 378-384. https://doi.org/10.1159/000226292 google scholar
34. Shin, K.-M., Kim, Y.-H., Park, W.-S., Kang, I., Ha, J., Choi, J.-W., . . . Lee, K.-T. (2004). Inhibition of Methanol Extract from the Fruits of Kochia scoparia on Lipopolysaccharide-Induced Nitric Oxide, Prostagladin E2, and Tumor Necrosis Factor-.ALPHA. Production from Murine Macrophage RAW 264.7 Cells. Bi-ological and Pharmaceutical Bulletin, 27(4), 538-543. https: //doi.org/10.1248/bpb.27.538 google scholar
35. Sornsenee, P., Chimplee, S., & Romyasamit, C. (2023). Evaluation of Antibacterial, Antibiofilm, Antioxidant, and Anti-Inflammatory Activities of Kratom Leaves (Mitragyna speciosa) Fermen-tation Supernatant Containing Lactobacillus rhamnosus GG. Probiotics and Antimicrobial Proteins. https://doi.org/10.1007/ s12602-023-10142-x google scholar
36. Srisuwan, S., Tongtawe, P., Srimanote, P., & Voravuthikunchai, S. P. (2014). Rhodomyrtone modulates innate immune responses of THP-1 monocytes to assist in clearing methicillin-resistant Staphylococcus aureus. PLoS ONE, 9(10), 1-11. https://doi.org/ 10.1371/journal.pone.0110321 google scholar
37. Tan, W. S., Arulselvan, P., Karthivashan, G., & Fakurazi, S. (2015). Moringa oleifera flower extract suppresses the activation of inflam-matory mediators in lipopolysaccharide-stimulated RAW 264.7 macrophages via NF-kB pathway. Mediators of Inflammation, 2015(1). https://doi.org/10.1155/2015/720171 google scholar
38. Tohar, N., Shilpi, J. A., Sivasothy, Y., Ahmad, S., & Awang, K. (2019). Chemical constituents and nitric oxide inhibitory ac-tivity of supercritical carbon dioxide extracts from Mitragyna speciosa leaves. Arabian Journal of Chemistry, 12(3), 350-359. https://doi.org/10.1016/j.arabjc.2016.09.005 google scholar
39. Utar, Z., Majid, M. I. A., Adenan, M. I., Jamil, M. F. A., & Lan, T. M. (2011). Mitragynine inhibits the COX-2 mRNA expression and prostaglandin E2 production induced by lipopolysaccharide in RAW264.7 macrophage cells. Journal of Ethnopharmacology, 136(1), 75-82. https://doi.org/10.1016/j.jep.2011.04.011 google scholar
40. Wang, N., Xu, C., Li, N., Wang, F., Wang, F., Li, Z., . . . Zhang, G. (2022). Synergistic anti-inflammatory effects of resveratrol and vitamin E in lipopolysaccharide-induced RAW264.7 cells. Food Science and Technology, 42. https://doi.org/10.1590/fst.24122 google scholar
41. Wang, Y., Smith, W., Hao, D., He, B., & Kong, L. (2019). M1 and M2 macrophage polarization and potentially therapeutic naturally occurring compounds. International Immunopharmacology, 70, 459-466. https://doi.org/10.1016/j.intimp.2019.02.050 google scholar
42. Wickramasinghe, R., Kumara, R. R., De Silva, E. D., Ratnasooriya, W. D., & Handunnetti, S. (2014). Inhibition of phagocytic and intracellular killing activity of human neutrophils by aqueous and methanolic leaf extracts of Ixora coccinea. Journal of Ethnopharmacology, 153(3), 900–907. https://doi.org/10.1016/j.jep.2014.03.064 google scholar
43. Zailan, N. F. Z., Sarchio, S. N. E., & Hassan, M. (2022). Evaluation of Phytochemical Composition, Antioxidant and anti-Diabetic Activities of Mitragyna speciosa Methanolic Extract (MSME). Malaysian Journal of Medicine and Health Sciences, 18(s21), 93-100. https://doi.org/10.47836/mjmhs.18.s21.15 google scholar