Efonidipine Inhibits JNK and NF-κB Pathway to Attenuate Inflammation and Cell Migration Induced by Lipopolysaccharide in Microglial Cells

  • Nguyen, Ngoc Minh (College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul) ;
  • Duong, Men Thi Hoai (College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul) ;
  • Nguyen, Phuong Linh (College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul) ;
  • Bui, Bich Phuong (College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul) ;
  • Ahn, Hee-Chul (College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul) ;
  • Cho, Jungsook (College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul)
  • Received : 2022.06.02
  • Accepted : 2022.06.20
  • Published : 2022.09.01


Efonidipine, a calcium channel blocker, is widely used for the treatment of hypertension and cardiovascular diseases. In our preliminary study using structure-based virtual screening, efonidipine was identified as a potential inhibitor of c-Jun N-terminal kinase 3 (JNK3). Although its antihypertensive effect is widely known, the role of efonidipine in the central nervous system has remained elusive. The present study investigated the effects of efonidipine on the inflammation and cell migration induced by lipopolysaccharide (LPS) using murine BV2 and human HMC3 microglial cell lines and elucidated signaling molecules mediating its effects. We found that the phosphorylations of JNK and its downstream molecule c-Jun in LPS-treated BV2 cells were declined by efonidipine, confirming the finding from virtual screening. In addition, efonidipine inhibited the LPS-induced production of pro-inflammatory factors, including interleukin-1β (IL-1β) and nitric oxide. Similarly, the IL-1β production in LPS-treated HMC3 cells was also inhibited by efonidipine. Efonidipine markedly impeded cell migration stimulated by LPS in both cells. Furthermore, it inhibited the phosphorylation of inhibitor kappa B, thereby suppressing nuclear translocation of nuclear factor-κB (NF-κB) in LPS-treated BV2 cells. Taken together, efonidipine exerts anti-inflammatory and anti-migratory effects in LPS-treated microglial cells through inhibition of the JNK/NF-κB pathway. These findings imply that efonidipine may be a potential candidate for drug repositioning, with beneficial impacts on brain disorders associated with neuroinflammation.



This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (NRF-2018R1A5A2023127 and 2020R1F1A1075835 to J.C. and NRF-2018R1D1A1B07050975 and 2021R1F1A1063558 to H.-C.A.), Korea.


  1. Ashburn, T. T. and Thor, K. B. (2004) Drug repositioning: identifying and developing new uses for existing drugs. Nat. Rev. Drug Discov. 3, 673-683.
  2. Bogoyevitch, M. A. (2006) The isoform-specific functions of the c-Jun N-terminal Kinases (JNKs): differences revealed by gene targeting. BioEssays 28, 923-934.
  3. Boraschi, D., Cifone, M. G., Falk, W., Flad, H., Tagliabue, A. and Martin, M. (1998) Cytokines in inflammation. Eur. Cytokine Netw. 9, 205-212.
  4. Brough, D. and Rothwell, N. J. (2007) Caspase-1-dependent processing of pro-interleukin-1β is cytosolic and precedes cell death. J. Cell Sci. 120, 772-781.
  5. Bui, B. P., Oh, Y., Lee, H. and Cho, J. (2020) Inhibition of inflammatory mediators and cell migration by 1,2,3,4-tetrahydroquinoline derivatives in LPS-stimulated BV2 microglial cells via suppression of NF-κB and JNK pathway. Int. Immunopharmacol. 80, 106231.
  6. Carson, M. J., Thrash, J. C. and Walter, B. (2006) The cellular response in neuroinflammation: the role of leukocytes, microglia and astrocytes in neuronal death and survival. Clin. Neurosci. Res. 6, 237-245.
  7. Choi, W. S., Abel, G., Klintworth, H., Flavell, R. A. and Xia, Z. (2010) JNK3 mediates paraquat- and rotenone-induced dopaminergic neuron death. J. Neuropathol. Exp. Neurol. 69, 511-520.
  8. Cui, J., Zhang, M., Zhang, Y. Q. and Xu, Z. H. (2007) JNK pathway: diseases and therapeutic potential. Acta Pharmacol. Sin. 28, 601-608.
  9. Dawson, T. M. and Dawson, V. L. (2018) Nitric oxide signaling in neurodegeneration and cell death. Adv. Pharmacol. 82, 57-83.
  10. Deng, Z., Yuan, C., Yang, J., Peng, Y., Wang, W., Wang, Y. and Gao, W. (2018) Behavioral defects induced by chronic social defeat stress are protected by Momordica charantia polysaccharides via attenuation of JNK3/PI3K/AKT neuroinflammatory pathway. Ann. Transl. Med. 7, 6.
  11. DiSabato, D. J., Quan, N. and Godbout, J. P. (2016) Neuroinflammation: the devil is in the details. J. Neurochem. 139 Suppl 2, 136-153.
  12. Do, H. T. T., Bui, B. P., Sim, S., Jung, J.-K., Lee, H. and Cho, J. (2020) Anti-inflammatory and anti-migratory activities of isoquinoline-1-carboxamide derivatives in LPS-treated BV2 microglial cells via inhibition of MAPKs/NF-κB pathway. Int. J. Mol. Sci. 21, 2319.
  13. Dou, X., Huang, H., Li, Y., Jiang, L., Wang, Y., Jin, H., Jiao, N., Zhang, L., Zhang, L. and Liu, Z. (2019) Multistage screening reveals 3-substituted indolin-2-one derivatives as novel and isoform-selective c-Jun N-terminal Kinase 3 (JNK3) inhibitors: implications to drug discovery for potential treatment of neurodegenerative diseases. J. Med. Chem. 62, 6645-6664.
  14. Dou, Y., Wu, H. J., Li, H. Q., Qin, S., Wang, Y. E., Li, J., Lou, H. F., Chen, Z., Li, X. M., Luo, Q. M. and Duan, S. (2012) Microglial migration mediated by ATP-induced ATP release from lysosomes. Cell Res. 22, 1022-1033.
  15. El Khoury, J. (2010) Neurodegeneration and the neuroimmune system. Nat. Med. 16, 1369-1370.
  16. Gourmaud, S., Paquet, C., Dumurgier, J., Pace, C., Bouras, C., Gray, F., Laplanche, J.-L., Meurs, E. F., Mouton-Liger, F. and Hugon, J. (2015) Increased levels of cerebrospinal fluid JNK3 associated with amyloid pathology: links to cognitive decline. J. Psychiatry Neurosci. 40, 151-161.
  17. Gupta, S., Barrett, T., Whitmarsh, A. J., Cavanagh, J., Sluss, H. K., Derijard, B. and Davis, R. J. (1996) Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J. 15, 2760-2770.
  18. Haddad, J. J. (2002) Cytokines and related receptor-mediated signaling pathways. Biochem. Biophys. Res. Commun. 297, 700-713.
  19. Heneka, M. T. and Feinstein, D. L. (2001) Expression and function of inducible nitric oxide synthase in neurons. J. Neuroimmunol. 114, 8-18.
  20. Huang, B.-R., Chang, P.-C., Yeh, W.-L., Lee, C.-H., Tsai, C.-F., Lin, C., Lin, H.-Y., Liu, Y.-S., Wu, C. Y.-J., Ko, P.-Y., Huang, S.-S., Hsu, H.-C. and Lu, D.-Y. (2014) Anti-neuroinflammatory effects of the calcium channel blocker nicardipine on microglial cells: implications for neuroprotection. PLoS ONE 9, e91167.
  21. Jourdan, J.-P., Bureau, R., Rochais, C. and Dallemagne, P. (2020) Drug repositioning: a brief overview. J. Pharm. Pharmacol. 72, 1145-1151.
  22. Kempuraj, D., Thangavel, R., Natteru, P. A., Selvakumar, G. P., Saeed, D., Zahoor, H., Zaheer, S., Iyer, S. S. and Zaheer, A. (2016) Neuroinflammation induces neurodegeneration. J. Neurol. Neurosurg. Spine 1, 1003.
  23. Kettenmann, H., Hanisch, U.-K., Noda, M. and Verkhratsky, A. (2011) Physiology of microglia. Physiol. Rev. 91, 461-553.
  24. Kloss, C. U. A., Bohatschek, M., Kreutzberg, G. W. and Raivich, G. (2001) Effect of lipopolysaccharide on the morphology and integrin immunoreactivity of ramified microglia in the mouse brain and in cell culture. Exp. Neurol. 168, 32-46.
  25. Lepiarz, I. and Olajide, O. (2019) The human microglia (HMC-3) as a cellular model of neuroinflammation. IBRO Rep. 6, S92.
  26. Li, Y., Hu, X., Liu, Y., Bao, Y. and An, L. (2009) Nimodipine protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation. Neuropharmacology 56, 580-589.
  27. Liang, X., Wu, L., Wang, Q., Hand, T., Bilak, M., McCullough, L. and Andreasson, K. (2007) Function of COX-2 and prostaglandins in neurological disease. J. Mol. Neurosci. 33, 94-99.
  28. Lopez-Castejon, G. and Brough, D. (2011) Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev. 22, 189-195.
  29. Nagai, A., Nakagawa, E., Hatori, K., Choi, H. B., McLarnon, J. G., Lee, M. A. and Kim, S. U. (2001) Generation and characterization of immortalized human microglial cell lines: expression of cytokines and chemokines. Neurobiol. Dis. 8, 1057-1068.
  30. Nguyen, P. L., Bui, B. P., Duong, M. T. H., Lee, K., Ahn, H. C. and Cho, J. (2021a) Suppression of LPS-induced inflammation and cell migration by azelastine through inhibition of JNK/NF-κB pathway in BV2 microglial cells. Int. J. Mol. Sci. 22, 9061.
  31. Nguyen, P. L., Bui, B. P., Lee, H. and Cho, J. (2021b) A novel 1,8-naphthyridine-2-carboxamide derivative attenuates inflammatory responses and cell migration in LPS-treated BV2 cells via the suppression of ROS generation and TLR4/Myd88/NF-κB signaling pathway. Int. J. Mol. Sci. 22, 2527.
  32. Okun, E., Griffioen, K. J., Lathia, J. D., Tang, S.-C., Mattson, M. P. and Arumugam, T. V. (2009) Toll-like receptors in neurodegeneration. Brain Res. Rev. 59, 278-292.
  33. Pan, J., Wang, G., Yang, H. Q., Hong, Z., Xiao, Q., Ren, R. J., Zhou, H. Y., Bai, L. and Chen, S. D. (2007) K252a prevents nigral dopaminergic cell death induced by 6-hydroxydopamine through inhibition of both mixed-lineage kinase 3/c-Jun NH2-terminal kinase 3 (JNK3) and apoptosis-inducing kinase 1/JNK3 signaling pathways. Mol. Pharmacol. 72, 1607-1618.
  34. Park, S. E., Sapkota, K., Kim, S., Kim, H. and Kim, S. J. (2011) Kaempferol acts through mitogen-activated protein kinases and protein kinase B/AKT to elicit protection in a model of neuroinflammation in BV2 microglial cells. Br. J. Pharmacol. 164, 1008-1025.
  35. Prakash, P., Jethava, K. P., Korte, N., Izquierdo, P., Favuzzi, E., Rose, I. V. L., Guttenplan, K. A., Manchanda, P., Dutta, S., Rochet, J. C., Fishell, G., Liddelow, S. A., Attwell, D. and Chopra, G. (2021) Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time. Chem. Sci. 12, 10901-10918.
  36. Rajan, R. K. and Ramanathan, M. (2020) Identification and neuroprotective evaluation of a potential c-Jun N-terminal kinase 3 inhibitor through structure-based virtual screening and in-vitro assay. J. Comput. Aided Mol. Des. 34, 671-682.
  37. Resnick, L. and Fennell, M. (2004) Targeting JNK3 for the treatment of neurodegenerative disorders. Drug Discov. Today 9, 932-939.
  38. Saini, R. and Singh, S. (2019) Inducible nitric oxide synthase: an asset to neutrophils. J. Leukoc. Biol. 105, 49-61.
  39. Salter, M. W. and Stevens, B. (2017) Microglia emerge as central players in brain disease. Nat. Med. 23, 1018-1027.
  40. Shabab, T., Khanabdali, R., Moghadamtousi, S. Z., Kadir, H. A. and Mohan, G. (2017) Neuroinflammation pathways: a general review. Int. J. Neurosci. 127, 624-633.
  41. Singh, S. S., Rai, S. N., Birla, H., Zahra, W., Rathore, A. S. and Singh, S. P. (2020) NF-κB-mediated neuroinflammation in Parkinson's disease and potential therapeutic effect of polyphenols. Neurotox. Res. 37, 491-507.
  42. Smith, A. M. and Dragunow, M. (2014) The human side of microglia. Trends Neurosci. 37, 125-135.
  43. Soufli, I., Toumi, R., Rafa, H. and Touil-Boukoffa, C. (2016) Overview of cytokines and nitric oxide involvement in immuno-pathogenesis of inflammatory bowel diseases. World J. Gastrointest. Pharmacol. Ther. 7, 353-360.
  44. Tai, C.-H., Yang, Y.-C., Pan, M.-K., Huang, C.-S. and Kuo, C.-C. (2011) Modulation of subthalamic T-type Ca2+ channels remedies locomotor deficits in a rat model of Parkinson disease. J. Clin. Invest. 121, 3289-3305.
  45. Tanaka, H. and Shigenobu, K. (2002) Efonidipine hydrochloride: a dual blocker of L- and T-type Ca(2+) channels. Cardiovasc. Drug Rev. 20, 81-92.
  46. Trott, O. and Olson, A. J. (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455-461.
  47. Waetzig, V., Czeloth, K., Hidding, U., Mielke, K., Kanzow, M., Brecht, S., Goetz, M., Lucius, R., Herdegen, T. and Hanisch, U.-K. (2005) c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia. Glia 50, 235-246.
  48. Wang, D., Fei, Z., Luo, S. and Wang, H. (2020) MiR-335-5p inhibits β-Amyloid (Aβ) accumulation to attenuate cognitive deficits through targeting c-jun-N-terminal kinase 3 in Alzheimer's disease. Curr. Neurovasc. Res. 17, 93-101.
  49. Zhu, X., Castellani, R. J., Takeda, A., Nunomura, A., Atwood, C. S., Perry, G. and Smith, M. A. (2001) Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer disease: the 'two hit' hypothesis. Mech. Ageing Dev. 123, 39-46.
  50. Zulfiqar, Z., Shah, F.A., Shafique, S., Alattar, A., Ali, T., Alvi, A.M., Rashid, S. and Li, S. (2020) Repurposing FDA approved drugs as JNK3 inhibitor for prevention of neuroinflammation induced by MCAO in rats. J. Inflamm. Res. 13, 1185-1205.