Zerumbone ameliorates neuroinflammation in LPS-induced SH-SY5Y cells, an in vitro model of neuropathic pain: targeting NO, IL-6, and TNF-α
DOI:
https://doi.org/10.31117/neuroscirn.v8i3.374Keywords:
Zerumbone, Neuropathic pain, Anti-neuroinflammatory, NO, IL-6, TNF-α, LPS-induced SH-SY5Y cellsAbstract
Neuropathic pain is initiated by lesions or diseases affecting the somatosensory nervous system. The development and persistence of this condition involve complex and interconnected mechanisms, including those related to neuroinflammation and neuronal hyperexcitability. Due to an incomplete understanding of these mechanisms, conventional therapies for neuropathic pain often result in adverse effects. Recent research has proposed that zerumbone, a crystalline sesquiterpene compound extracted from Zingiber zerumbet, can attenuate neuropathic pain in animal models. Lipopolysaccharide (LPS)-induced SH-SY5Y cells were employed to allow tight control of the physiological environment, which could not be established in in vivo models, in addition to reducing the use of animals in the study of neuropathic pain. LPS induction in SH-SY5Y cells enables the observation of one of the hallmarks of neuropathic pain pathophysiology, which is the expression of pro-inflammatory mediators. This study aims to evaluate the anti-inflammatory effect of zerumbone by measuring its influence on the expression of nitric oxide (NO), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) in LPS-induced SH-SY5Y cells, an in vitro model of neuropathic pain. The anti-neuroinflammatory effect of zerumbone was first investigated through the expression level of NO, whereby the inhibitory concentration of zerumbone was determined at 8 g/ml (p<0.0001 compared to the LPS-only group). Zerumbone treatment significantly reduced the expression of IL-6 (p<0.05 compared to the LPS-only group). Although a reduction in TNF-α levels was observed, it did not reach statistical significance in the enzyme-linked immunoassay (ELISA). Data from each experiment were analysed by using the One-way Analysis of Variance (ANOVA) followed by the post hoc Tukey test, p<0.05. Zerumbone demonstrates an anti-neuroinflammatory effect in LPS-stimulated SH-SY5Y cells by suppressing the expression of key inflammatory mediators NO, IL-6, and TNF-α. These findings suggest that zerumbone is a potential therapeutic candidate for managing neuropathic pain associated with neuroinflammation.
References
Al-Zubairi, A. S. (2018). Anti-Proliferative Activity of Zerumbone Against Tumour Cell Lines. Online Journal of Biological Sciences, 18(2), 123–129. https://doi.org/10.3844/ojbsci.2018.123.129
Anaeigoudari, A., Soukhtanloo, M., Shafei, M. N., Sadeghnia, H. R., Reisi, P., Beheshti, F., Behradnia, S., Mousavi, S. M., & Hosseini, M. (2016). Neuronal nitric oxide synthase has a role in the detrimental effects of lipopolysaccharide on spatial memory and synaptic plasticity in rats. Pharmacological Reports, 68(2), 243–249. https://doi.org/10.1016/j.pharep.2015.09.004
Antoniazzi, C. T. de D., Ruviaro, N. A., Peres, D. S., Rodrigues, P., Viero, F. T., & Trevisan, G. (2024). Targeting TRPV4 Channels for Cancer Pain Relief. Cancers, 16(9), 1703. https://doi.org/10.3390/CANCERS16091703
Cavalli, E., Mammana, S., Nicoletti, F., Bramanti, P., & Mazzon, E. (2019). The neuropathic pain: An overview of the current treatment and future therapeutic approaches. International Journal of Immunopathology and Pharmacology, 33. https://doi.org/10.1177/2058738419838383
Chae, S. (2004). Protection of insulin secreting cells from nitric oxide induced cellular damage by crosslinked hemoglobin. Biomaterials, 25(5), 843–850. https://doi.org/10.1016/S0142-9612(03)00605-7
Chen, J.-Y., Chu, L.-W., Cheng, K.-I., Hsieh, S.-L., Juan, Y.-S., & Wu, B.-N. (2018). Valproate reduces neuroinflammation and neuronal death in a rat chronic constriction injury model. Scientific Reports, 8(1), 16457. https://doi.org/10.1038/s41598-018-34915-5
Chia, J. S. M., Izham, N. A. M., Farouk, A. A. O., Sulaiman, M. R., Mustafa, S., Hutchinson, M. R., & Perimal, E. K. (2020). Zerumbone Modulates α2A-Adrenergic, TRPV1, and NMDA NR2B Receptors Plasticity in CCI-Induced Neuropathic Pain In Vivo and LPS-Induced SH-SY5Y Neuroblastoma In Vitro Models. Frontiers in Pharmacology, 11, 92. https://doi.org/10.3389/fphar.2020.00092
Chia, J. S. M., Omar Farouk, A. A., Mohamad, A. S., Sulaiman, M. R., & Perimal, E. K. (2016). Zerumbone alleviates chronic constriction injury-induced allodynia and hyperalgesia through serotonin 5-HT receptors. Biomedicine & Pharmacotherapy, 83, 1303–1310. https://doi.org/10.1016/J.BIOPHA.2016.08.052
Das, N. D., Choi, M. R., Jung, K. H., Park, J. H., Lee, H. T., Kim, S. H., & Chai, Y. G. (2012). Lipopolysaccharide-mediated protein expression profiling on neuronal differentiated SH-SY5Y cells. BioChip Journal, 6(2), 165–173. https://doi.org/10.1007/s13206-012-6209-1
Ellis, A., & Bennett, D. L. H. (2013). Neuroinflammation and the generation of neuropathic pain. British Journal of Anaesthesia, 111(1), 26–37. https://doi.org/10.1093/bja/aet128
Fatima, A., Abdul, A. B. H., Abdullah, R., Karjiban, R. A., & Lee, V. S. (2018). Docking studies reveal zerumbone targets ß-catenin of the Wnt-ß-catenin pathway in breast cancer. Journal of the Serbian Chemical Society, 83(5), 575–591. https://doi.org/10.2298/JSC170313108F
Förstermann, U., & Sessa, W. C. (2012). Nitric oxide synthases: regulation and function. European Heart Journal, 33(7), 829–837. https://doi.org/10.1093/eurheartj/ehr304
Freire, M. A. M., Guimarães, J. S., Gomes-Leal, W., & Pereira, A. (2009). Pain modulation by nitric oxide in the spinal cord. Frontiers in Neuroscience, 3(2), 175–181. https://doi.org/10.3389/neuro.01.024.2009
Fronza, M. G., Ferreira, B. F., Pavan-Silva, I., Guimarães, F. S., & Lisboa, S. F. (2023). “NO” Time in Fear Response: Possible Implication of Nitric-Oxide-Related Mechanisms in PTSD. Molecules, 29(1), 89. https://doi.org/10.3390/MOLECULES29010089
Gerard, E., Spengler, R. N., Bonoiu, A. C., Mahajan, S. D., Davidson, B. A., Ding, H., Kumar, R., Prasad, P. N., Knight, P. R., & Ignatowski, T. A. (2015). Chronic constriction injury-induced nociception is relieved by nanomedicine-mediated decrease of rat hippocampal tumor necrosis factor. Pain, 156(7), 1320–1333. https://doi.org/10.1097/j.pain.0000000000000181
Ghimire, K., Altmann, H. M., Straub, A. C., & Isenberg, J. S. (2017). Nitric oxide: what’s new to NO? American Journal of Physiology-Cell Physiology, 312(3), C254–C262. https://doi.org/10.1152/ajpcell.00315.2016
Gopalsamy, B., Farouk, A. A. O., Mohamad, T. A. S. T., Sulaiman, M. R., & Perimal, E. K. (2017). Antiallodynic and antihyperalgesic activities of zerumbone via the suppression of IL-1 beta, IL-6, and TNF-alpha in a mouse model of neuropathic pain. Journal of Pain Research, 10, 2605–2619. https://doi.org/10.2147/JPR.S143024
Guix, F. X., Uribesalgo, I., Coma, M., & Muñoz, F. J. (2005). The physiology and pathophysiology of nitric oxide in the brain. Progress in Neurobiology, 76(2), 126–152. https://doi.org/10.1016/j.pneurobio.2005.06.001
Haque, M. A., Jantan, I., & Harikrishnan, H. (2018). Zerumbone suppresses the activation of inflammatory mediators in LPS-stimulated U937 macrophages through MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways. International Immunopharmacology, 55, 312–322. https://doi.org/10.1016/j.intimp.2018.01.001
Hattangady, N., & Rajadhyaksha, M. (2009). A brief review of in vitro models of diabetic neuropathy. International Journal of Diabetes in Developing Countries, 29(4), 143. https://doi.org/10.4103/0973-3930.57344
Heir, R., & Stellwagen, D. (2020). TNF-Mediated Homeostatic Synaptic Plasticity: From in vitro to in vivo Models. Frontiers in Cellular Neuroscience, 14, 297. https://doi.org/10.3389/fncel.2020.565841
Huang, E. P. (1997). Synaptic plasticity: A role for nitric oxide in LTP. Current Biology, 7(3), R141–R143. https://doi.org/10.1016/S0960-9822(97)70073-3
Hutchinson, M. R., Loram, L. C., Zhang, Y., Shridhar, M., Rezvani, N., Berkelhammer, D., Phipps, S., Foster, P. S., Landgraf, K., Falke, J. J., Rice, K. C., Maier, S. F., Yin, H., & Watkins, L. R. (2010). Evidence that tricyclic small molecules may possess toll-like receptor and myeloid differentiation protein 2 activity. Neuroscience, 168(2), 551–563. https://doi.org/10.1016/j.neuroscience.2010.03.067
Jia, D., Wang, H., Han, B., Zhang, L., & Guo, J. (2019). Tempol Attenuates Neuropathic Pain by Inhibiting Nitric Oxide Production. Analytical Cellular Pathology, 2019, 1–5. https://doi.org/10.1155/2019/8253850
Jurga, A. M., Rojewska, E., Piotrowska, A., Makuch, W., Pilat, D., Przewlocka, B., & Mika, J. (2016). Blockade of Toll-Like Receptors (TLR2, TLR4) Attenuates Pain and Potentiates Buprenorphine Analgesia in a Rat Neuropathic Pain Model. Neural Plasticity, 2016, 5238730. https://doi.org/10.1155/2016/5238730
Kaplish, D., Vagha, J. D., Meshram, R. J., & Lohiya, S. (2024). A Comprehensive Review of Inhaled Nitric Oxide Therapy: Current Trends, Challenges, and Future Directions. Cureus, 16(2), e53558. https://doi.org/10.7759/CUREUS.53558
Kaswan, N. K., Mohd Suhaimi, N. S., Mohammed Izham, N. A., Tengku Mohamad, T. A. S., Sulaiman, M. R., & Perimal, E. K. (2020). Cardamonin inhibits nitric oxide production modulated through NMDA receptor in LPS-Induced SH-SY5Y cell in vitro model. Life Sciences, Medicine and Biomedicine, 4(9), 58. https://doi.org/10.28916/lsmb.4.9.2020.58
Kaye, A. D., Perilloux, D. M., Hawkins, A. M., Wester, G. C., Ragaland, A. R., Hebert, S. v., Kim, J., Heisler, M., Kelkar, R. A., Chami, A. A., Shekoohi, S., & Kaye, A. M. (2024). Tumor Necrosis Factor and Interleukin Modulators for Pathologic Pain States: A Narrative Review. Pain and Therapy, 13(3), 481–493. https://doi.org/10.1007/S40122-024-00603-8
Kiguchi, N., Kobayashi, D., Saika, F., Matsuzaki, S., & Kishioka, S. (2017). Pharmacological Regulation of Neuropathic Pain Driven by Inflammatory Macrophages. International Journal of Molecular Sciences, 18(11), 2296. https://doi.org/10.3390/ijms18112296
Kim, A., Gwon, M. H., Lee, W., Moon, H. R., & Yun, J. M. (2022). Zerumbone suppresses high glucose and LPS-induced inflammation in THP-1-derived macrophages by inhibiting the NF-κB/TLR signaling pathway. Nutrition Research, 100, 58–69. https://doi.org/10.1016/j.nutres.2022.01.002
Kim, M.-J., & Yun, J.-M. (2019). Molecular Mechanism of the Protective Effect of Zerumbone on Lipopolysaccharide-Induced Inflammation of THP-1 Cell-Derived Macrophages. Journal of Medicinal Food, 22(1), 62–73. https://doi.org/10.1089/jmf.2018.4253
Kopincová, J., Púzserová, A., & Bernátová, I. (2012). L-NAME in the cardiovascular system - nitric oxide synthase activator? Pharmacological Reports, 64(3), 511–520. https://doi.org/10.1016/s1734-1140(12)70846-0
Leung, L., & Cahill, C. (2010). TNF-alpha and neuropathic pain - a review. Journal of Neuroinflammation, 7(1), 27. https://doi.org/10.1186/1742-2094-7-27
Levy, D., & Zochodne, D. W. (2004). NO Pain: Potential Roles of Nitric Oxide in Neuropathic Pain. Pain Practice, 4(1), 11–18. https://doi.org/10.1111/j.1533-2500.2004.04002.x
Li, L., Wu, X. H., Zhao, X. J., Xu, L., Pan, C. L., & Zhang, Z. Y. (2020). Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signalling. Journal of Neuroinflammation, 17(1), 1–20. https://doi.org/10.1186/S12974-020-01744-1
Lin, H. C., Lin, T. H., Wu, M. Y., Chiu, Y. C., Tang, C. H., Hour, M. J., Liou, H. C., Tu, H. J., Yang, R. S., & Fu, W. M. (2014). 5-Lipoxygenase inhibitors attenuate TNF-α-induced inflammation in human synovial fibroblasts. PLoS One, 9(9), e107890. https://doi.org/10.1371/journal.pone.0107890
Liu, T., Zhang, L., Joo, D., & Sun, S.-C. (2017). NF-κB signalling in inflammation. Signal Transduction and Targeted Therapy, 2(1), 17023. https://doi.org/10.1038/sigtrans.2017.23
Lou, Y., Huang, Z., Wu, H., & Zhou, Y. (2022). Tranilast attenuates lipopolysaccharide induced lung injury via the CXCR4/JAK2/STAT3 signaling pathway. Molecular Medicine Reports, 26(1), 220. https://doi.org/10.3892/mmr.2022.12736
Lundberg, J. O., Weitzberg, E., & Gladwin, M. T. (2008). The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nature Reviews Drug Discovery, 7(2), 156–167. https://doi.org/10.1038/nrd2466
Ma, Y., Kang, Z., Shi, Y., Ji, W., Zhou, W., & Nan, W. (2024). The Complexity of Neuropathic Pain and Central Sensitization: Exploring Mechanisms and Therapeutic Prospects. Journal of Integrative Neuroscience, 23(5), 89. https://doi.org/10.31083/J.JIN2305089
Mattar, M., Umutoni, F., Hassan, M. A., Wamburu, M. W., Turner, R., Patton, J. S., Chen, X., & Lei, W. (2024). Chemotherapy-Induced Peripheral Neuropathy: A Recent Update on Pathophysiology and Treatment. Life, 14(8), 991. https://doi.org/10.3390/life14080991
Mohammed Izham, N. A., Chia, J. S. M., Vidyadaran, S., Sulaiman, M. R., & Bharatham, B. H. (2018). The Effect of DMEM and DMEM:F12 Culture Media on the Growth of SH-SY5Y Cells. Life Sciences, Medicine and Biomedicine, 2(3), 3–6. https://doi.org/10.28916/lsmb.2.3.2018.23
Mohammed Izham, N. A., Chia, J. S. M., Kaswan, N. K., Sukirthalingam, K., Vidyadaran, S., Bharatham, H., Sulaiman, M. R., & Perimal, E. K. (2022). Exploring the possibilities of using in vitro model for neuropathic pain studies. Neuroscience Research Notes, 5(3), 144. https://doi.org/10.31117/neuroscirn.v5i3.144
Nashtahosseini, Z., Eslami, M., Paraandavaji, E., Haraj, A., Dowlat, B. F., Hosseinzadeh, E., Oksenych, V., & Naderian, R. (2025). Cytokine Signaling in Diabetic Neuropathy: A Key Player in Peripheral Nerve Damage. Biomedicines, 13(3), 589. https://doi.org/10.3390/BIOMEDICINES13030589
O’Neill, E., Kwok, B., Day, J. S., Connor, T. J., & Harkin, A. (2016). Amitriptyline protects against TNF-α-induced atrophy and reduction in synaptic markers via a Trk-dependent mechanism. Pharmacology Research & Perspectives, 4(2), e00195. https://doi.org/10.1002/prp2.195
Obuchowicz, E., Kowalski, J., Labuzek, K., Krysiak, R., Pendzich, J., & Herman, Z. S. (2006). Amitriptyline and nortriptyline inhibit interleukin-1 release by rat mixed glial and microglial cell cultures. The International Journal of Neuropsychopharmacology, 9(1), 27–35. https://doi.org/10.1017/S146114570500547X
Ogawa, N., Kawai, H., Terashima, T., Kojima, H., Oka, K., Chan, L., & Maegawa, H. (2014). Gene Therapy for Neuropathic Pain by Silencing of TNF-α Expression with Lentiviral Vectors Targeting the Dorsal Root Ganglion in Mice. PLoS ONE, 9(3), e92073. https://doi.org/10.1371/journal.pone.0092073
Olmos, G., & Lladó, J. (2014). Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity. Mediators of Inflammation, 2014, 861231. https://doi.org/10.1155/2014/861231
Oluwole, O. G., James, K., Yalcouye, A., & Wonkam, A. (2022). Hearing loss and brain disorders: A review of multiple pathologies. Open Medicine, 17(1), 61. https://doi.org/10.1515/MED-2021-0402
Orfali, R., Alwatban, A. Z., Orfali, R. S., Lau, L., Chea, N., Alotaibi, A. M., Nam, Y. W., & Zhang, M. (2024). Oxidative stress and ion channels in neurodegenerative diseases. Frontiers in Physiology, 15, 1320086. https://doi.org/10.3389/fphys.2024.1320086
Pandur, E., Varga, E., Tamási, K., Pap, R., Nagy, J., & Sipos, K. (2018). Effect of Inflammatory Mediators Lipopolysaccharide and Lipoteichoic Acid on Iron Metabolism of Differentiated SH-SY5Y Cells Alters in the Presence of BV-2 Microglia. International Journal of Molecular Sciences, 20(1), 17. https://doi.org/10.3390/ijms20010017
Park, C.-K., Lü, N., Xu, Z.-Z., Liu, T., Serhan, C. N., & Ji, R.-R. (2011). Resolving TRPV1- and TNF-α-mediated spinal cord synaptic plasticity and inflammatory pain with neuroprotectin D1. The Journal of Neuroscience, 31(42), 15072–15085. https://doi.org/10.1523/JNEUROSCI.2443-11.2011
Pfeiffer, S., Leopold, E., Schmidt, K., Brunner, F., & Mayer, B. (1996). Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine. British Journal of Pharmacology, 118(6), 1433–1440. https://doi.org/10.1111/j.1476-5381.1996.tb15557.x
Rahman, H. S., Rasedee, A., Chartrand, M. S., Othman, H. H., Yeap, S. K., & Namvar, F. (2014). Zerumbone Induces G2/M Cell Cycle Arrest and Apoptosis via Mitochondrial Pathway in Jurkat cell line. Natural Product Communications, 9(9), 1934578X1400900. https://doi.org/10.1177/1934578X1400900904
Raju, K., Doulias, P. T., Evans, P., Krizman, E. N., Jackson, J. G., Horyn, O., Daikhin, Y., Nissim, I., Yudkoff, M., Nissim, I., Sharp, K. A., Robinson, M. B., & Ischiropoulos, H. (2015). Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation. Science Signaling, 8(384), ra68. https://doi.org/10.1126/scisignal.aaa4312
Schäfers, M., Lee, D. H., Brors, D., Yaksh, T. L., & Sorkin, L. S. (2003). Increased sensitivity of injured and adjacent uninjured rat primary sensory neurons to exogenous tumour necrosis factor-alpha after spinal nerve ligation. The Journal of Neuroscience, 23(7), 3028–3038. https://doi.org/10.1523/JNEUROSCI.23-07-03028.2003
Shubayev, V. I., & Myers, R. R. (2002). Anterograde TNF alpha transport from rat dorsal root ganglion to spinal cord and injured sciatic nerve. Neuroscience Letters, 320(1–2), 99–101. https://doi.org/10.1016/s0304-3940(02)00010-1
Si, M., Cai, X., Liu, Y., Li, Z., Luo, X., Zhu, H. L., & Qian, Y. (2024). An antagonist-based two-photon fluorogenic probe for imaging metabotropic glutamate receptor 5 in neuronal cells. Talanta, 275, 126167. https://doi.org/10.1016/J.TALANTA.2024.126167
Tanabe, K., Matsushima-Nishiwaki, R., Yamaguchi, S., Iida, H., Dohi, S., & Kozawa, O. (2010). Mechanisms of tumour necrosis factor-α-induced interleukin-6 synthesis in glioma cells. Journal of Neuroinflammation, 7(1), 16. https://doi.org/10.1186/1742-2094-7-16
Tian, X., Wang, W. T., Zhang, M. M., Yang, Q. Q., Xu, Y. L., Wu, J. B., Xie, X. X., Wang, J. Y., & Wang, J. Y. (2024). Red nucleus mGluR1 and mGluR5 facilitate the development of neuropathic pain through stimulating the expressions of TNF-α and IL-1β. Neurochemistry International, 178, 105786. https://doi.org/10.1016/J.NEUINT.2024.105786
Wei, S., Qiu, C. Y., Jin, Y., Liu, T. T., & Hu, W. P. (2021). TNF-α acutely enhances acid-sensing ion channel currents in rat dorsal root ganglion neurons via a p38 MAPK pathway. Journal of Neuroinflammation, 18(1), 1–10. https://doi.org/10.1186/S12974-021-02151-W
Wong-Riley, M. T. T. (2012). Bigenomic regulation of cytochrome c oxidase in neurons and the tight coupling between neuronal activity and energy metabolism. Advances in Experimental Medicine and Biology, 748, 283–304. https://doi.org/10.1007/978-1-4614-3573-0_12
Yao, S. Y., Ljunggren-Rose, A., Chandramohan, N., Whetsell, W. O., & Sriram, S. (2010). In vitro and in vivo induction and activation of nNOS by LPS in oligodendrocytes. Journal of Neuroimmunology, 229(1–2), 146–156. https://doi.org/10.1016/j.jneuroim.2010.07.023
Zhang, J.-M., & An, J. (2007). Cytokines, inflammation, and pain. International Anesthesiology Clinics, 45(2), 27–37. https://doi.org/10.1097/AIA.0b013e318034194e
Zhu, B., Zhou, X., Zhou, Q., Wang, H., Wang, S., & Luo, K. (2019). Intra-Venous Lidocaine to Relieve Neuropathic Pain: A Systematic Review and Meta-Analysis. Frontiers in Neurology, 10, 954. https://doi.org/10.3389/fneur.2019.00954
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