Advertisement

NeuroMolecular Medicine

, Volume 18, Issue 3, pp 241–252 | Cite as

Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells

  • Grace Y. Sun
  • Runting Li
  • Jiankun Cui
  • Mark Hannink
  • Zezong Gu
  • Kevin L. Fritsche
  • Dennis B. Lubahn
  • Agnes Simonyi
Original Paper

Abstract

Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8 h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways.

Keywords

Ashwagandha Withaferin A Withanolide A Microglia LPS NO HO-1 Nrf2 

Notes

Acknowledgments

This work was partially supported by grant P50AT006273 from the National Center for Complementary and Alternative Medicine (NCCAM), the Office of Dietary Supplements (ODS), and the National Cancer Institute (NCI). The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NCCAM, ODS, NCI, or the NIH.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there is no conflict of interest for this study.

References

  1. Ashkenazi, S., Plotnikov, A., Bahat, A., Ben-Zeev, E., Warszawski, S., & Dikstein, R. (2016). A novel allosteric mechanism of NF-κB dimerization and DNA binding targeted by an anti-inflammatory drug. Molecular and Cellular Biology, 36(8), 1237–1247.CrossRefPubMedGoogle Scholar
  2. Brandenburg, L. O., Kipp, M., Lucius, R., Pufe, T., & Wruck, C. J. (2010). Sulforaphane suppresses LPS-induced inflammation in primary rat microglia. Inflammation Research, 59(6), 443–450.CrossRefPubMedGoogle Scholar
  3. Calabrese, V., Cornelius, C., Dinkova-Kostova, A. T., Calabrese, E. J., & Mattson, M. P. (2010). Cellular stress responses, the hormesis paradigm, and vitagenes: novel targets for therapeutic intervention in neurodegenerative disorders. Antioxidants and Redox Signaling, 13(11), 1763–1811.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chen, Z., & Trapp, B. D. (2016). Microglia and neuroprotection. Journal of Neurochemistry, 136(Suppl 1), 10–17.CrossRefPubMedGoogle Scholar
  5. Chen, J. C., Ho, F. M., Pei-Dawn Lee, C., Chen, C. P., Jeng, K. C., Hsu, H. B., et al. (2005). Inhibition of iNOS gene expression by quercetin is mediated by the inhibition of IκB kinase, nuclear factor-kappa B and STAT1, and depends on heme oxygenase-1 induction in mouse BV-2 microglia. European Journal of Pharmacology, 521(1–3), 9–20.CrossRefPubMedGoogle Scholar
  6. Chuang, D. Y., Chan, M. H., Zong, Y., Sheng, W., He, Y., Jiang, J. H., et al. (2013). Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells. Journal of Neuroinflammation, 10, 15.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chuang, D. Y., Simonyi, A., Kotzbauer, P. T., Gu, Z., & Sun, G. Y. (2015). Cytosolic phospholipase A2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway. Journal of Neuroinflammation, 12, 199.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dar, N. J., Hamid, A., & Ahmad, M. (2015). Pharmacologic overview of Withania somnifera, the Indian Ginseng. Cellular and Molecular Life Sciences, 72(23), 4445–4460.CrossRefPubMedGoogle Scholar
  9. Durg, S., Dhadde, S. B., Vandal, R., Shivakumar, B. S., & Charan, C. S. (2015). Withania somnifera (Ashwagandha) in neurobehavioural disorders induced by brain oxidative stress in rodents: A systematic review and meta-analysis. Journal of Pharmacy and Pharmacology, 67(7), 879–899.CrossRefPubMedGoogle Scholar
  10. Foresti, R., Bains, S. K., Pitchumony, T. S., de Castro Bras, L. E., Drago, F., Dubois-Rande, J. L., et al. (2013). Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells. Pharmacological Research, 76, 132–148.CrossRefPubMedGoogle Scholar
  11. Gan, L., & Johnson, J. A. (2014). Oxidative damage and the Nrf2-ARE pathway in neurodegenerative diseases. Biochimica et Biophysica Acta, 1842(8), 1208–1218.CrossRefPubMedGoogle Scholar
  12. Gao, B., Doan, A., & Hybertson, B. M. (2014). The clinical potential of influencing Nrf2 signaling in degenerative and immunological disorders. Clin Pharmacol, 6, 19–34.PubMedPubMedCentralGoogle Scholar
  13. Grin, B., Mahammad, S., Wedig, T., Cleland, M. M., Tsai, L., Herrmann, H., et al. (2012). Withaferin a alters intermediate filament organization, cell shape and behavior. PLoS One, 7(6), e39065.CrossRefPubMedGoogle Scholar
  14. Grunz-Borgmann, E., Mossine, V., Fritsche, K., & Parrish, A. R. (2015). Ashwagandha attenuates TNF-α- and LPS-induced NF-κB activation and CCL2 and CCL5 gene expression in NRK-52E cells. BMC Complementary and Alternative Medicine, 15, 434.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Innamorato, N. G., Rojo, A. I., Garcia-Yague, A. J., Yamamoto, M., de Ceballos, M. L., & Cuadrado, A. (2008). The transcription factor Nrf2 is a therapeutic target against brain inflammation. Journal of Immunology, 181(1), 680–689.CrossRefGoogle Scholar
  16. Jazwa, A., & Cuadrado, A. (2010). Targeting heme oxygenase-1 for neuroprotection and neuroinflammation in neurodegenerative diseases. Current Drug Targets, 11(12), 1517–1531.CrossRefPubMedGoogle Scholar
  17. Jiang, J., Chuang, D. Y., Zong, Y., Patel, J., Brownstein, K., Lei, W., et al. (2014). Sutherlandia frutescens ethanol extracts inhibit oxidative stress and inflammatory responses in neurons and microglial cells. PLoS One, 9(2), e89748.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Joshi, G., & Johnson, J. A. (2012). The Nrf2-ARE pathway: A valuable therapeutic target for the treatment of neurodegenerative diseases. Recent Patents on CNS Drug Discovery, 7(3), 218–229.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kang, C. H., Choi, Y. H., Moon, S. K., Kim, W. J., & Kim, G. Y. (2013). Quercetin inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-dependent HO-1 pathway. International Immunopharmacology, 17(3), 808–813.CrossRefPubMedGoogle Scholar
  20. Kobayashi, A., Kang, M. I., Okawa, H., Ohtsuji, M., Zenke, Y., Chiba, T., et al. (2004). Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Molecular and Cellular Biology, 24(16), 7130–7139.CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kuboyama, T., Tohda, C., & Komatsu, K. (2014). Effects of Ashwagandha (roots of Withania somnifera) on neurodegenerative diseases. Biological and Pharmaceutical Bulletin, 37(6), 892–897.CrossRefPubMedGoogle Scholar
  22. Kulkarni, S. K., & Dhir, A. (2008). Withania somnifera: An Indian ginseng. Progress in Neuropsychopharmacology and Biological Psychiatry, 32(5), 1093–1105.CrossRefGoogle Scholar
  23. Kurapati, K. R., Atluri, V. S., Samikkannu, T., & Nair, M. P. (2013). Ashwagandha (Withania somnifera) reverses beta-amyloid1-42 induced toxicity in human neuronal cells: implications in HIV-associated neurocognitive disorders (HAND). PLoS One, 8(10), e77624.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kurapati, K. R., Atluri, V. S., Samikkannu, T., Garcia, G., & Nair, M. P. (2015). Natural products as anti-HIV agents and role in HIV-associated neurocognitive disorders (HAND): A brief overview. Frontiers in Microbiology, 6, 1444.PubMedGoogle Scholar
  25. Lee, J., Jo, D. G., Park, D., Chung, H. Y., & Mattson, M. P. (2014). Adaptive cellular stress pathways as therapeutic targets of dietary phytochemicals: Focus on the nervous system. Pharmacological Reviews, 66(3), 815–868.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Min, K. J., Choi, K., & Kwon, T. K. (2011). Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells. International Immunopharmacology, 11(8), 1137–1142.CrossRefPubMedGoogle Scholar
  27. Mirjalili, M. H., Moyano, E., Bonfill, M., Cusido, R. M., & Palazon, J. (2009). Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules, 14(7), 2373–2393.CrossRefPubMedGoogle Scholar
  28. Nair, S., Doh, S. T., Chan, J. Y., Kong, A. N., & Cai, L. (2008). Regulatory potential for concerted modulation of Nrf2- and Nfkb1-mediated gene expression in inflammation and carcinogenesis. British Journal of Cancer, 99(12), 2070–2082.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Narayan, M., Seeley, K. W., & Jinwal, U. K. (2015). Identification and quantitative analysis of cellular proteins affected by treatment with withaferin a using a SILAC-based proteomics approach. Journal of Ethnopharmacology, 175, 86–92.CrossRefPubMedGoogle Scholar
  30. Oh, J. H., Lee, T. J., Park, J. W., & Kwon, T. K. (2008). Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-κB in RAW 264.7 cells. European Journal of Pharmacology, 599(1–3), 11–17.CrossRefPubMedGoogle Scholar
  31. Paine, A., Eiz-Vesper, B., Blasczyk, R., & Immenschuh, S. (2010). Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochemical Pharmacology, 80(12), 1895–1903.CrossRefPubMedGoogle Scholar
  32. Parada, E., Buendia, I., Navarro, E., Avendano, C., Egea, J., & Lopez, M. G. (2015). Microglial HO-1 induction by curcumin provides antioxidant, antineuroinflammatory, and glioprotective effects. Molecular Nutrition and Food Research, 59(9), 1690–1700.CrossRefPubMedGoogle Scholar
  33. Patil, D., Gautam, M., Mishra, S., Karupothula, S., Gairola, S., Jadhav, S., et al. (2013). Determination of withaferin A and withanolide A in mice plasma using high-performance liquid chromatography-tandem mass spectrometry: Application to pharmacokinetics after oral administration of Withania somnifera aqueous extract. Journal of Pharmaceutical and Biomedical Analysis, 80, 203–212.CrossRefPubMedGoogle Scholar
  34. Queiroga, C. S., Vercelli, A., & Vieira, H. L. (2015). Carbon monoxide and the CNS: Challenges and achievements. British Journal of Pharmacology, 172(6), 1533–1545.CrossRefPubMedGoogle Scholar
  35. Salter, M. W., & Beggs, S. (2014). Sublime microglia: Expanding roles for the guardians of the CNS. Cell, 158(1), 15–24.CrossRefPubMedGoogle Scholar
  36. Sandberg, M., Patil, J., D’Angelo, B., Weber, S. G., & Mallard, C. (2014). NRF2-regulation in brain health and disease: Implication of cerebral inflammation. Neuropharmacology, 79, 298–306.CrossRefPubMedGoogle Scholar
  37. Scapagnini, G., Vasto, S., Abraham, N. G., Caruso, C., Zella, D., & Fabio, G. (2011). Modulation of Nrf2/ARE pathway by food polyphenols: A nutritional neuroprotective strategy for cognitive and neurodegenerative disorders. Molecular Neurobiology, 44(2), 192–201.CrossRefPubMedGoogle Scholar
  38. Shah, N., Singh, R., Sarangi, U., Saxena, N., Chaudhary, A., Kaur, G., et al. (2015). Combinations of Ashwagandha leaf extracts protect brain-derived cells against oxidative stress and induce differentiation. PLoS One, 10(3), e0120554.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sheng, W., Zong, Y., Mohammad, A., Ajit, D., Cui, J., Han, D., et al. (2011). Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA(2)-IIA expression in astrocytes and microglia. Journal of Neuroinflammation, 8, 121.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Simonyi, A., Chen, Z., Jiang, J., Zong, Y., Chuang, D. Y., Gu, Z., et al. (2015). Inhibition of microglial activation by elderberry extracts and its phenolic components. Life Sciences, 128, 30–38.CrossRefPubMedPubMedCentralGoogle Scholar
  41. Singh, N., Bhalla, M., de Jager, P., & Gilca, M. (2011). An overview on ashwagandha: A Rasayana (rejuvenator) of Ayurveda. African Journal of Traditional, Complementary and Alternative Medicines, 8(5 Suppl), 208–213.Google Scholar
  42. Soares, M. P., Marguti, I., Cunha, A., & Larsen, R. (2009). Immunoregulatory effects of HO-1: How does it work? Current Opinion in Pharmacology, 9(4), 482–489.CrossRefPubMedGoogle Scholar
  43. Sun, A. Y., Wang, Q., Simonyi, A., & Sun, G. Y. (2008). Botanical phenolics and brain health. Neuromolecular Medicine, 10(4), 259–274.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Sun, G. Y., Chen, Z., Jasmer, K. J., Chuang, D. Y., Gu, Z., Hannink, M., et al. (2015). Quercetin attenuates inflammatory responses in BV-2 microglial cells: Role of MAPKs on the Nrf2 pathway and induction of heme oxygenase-1. PLoS One, 10(10), e0141509.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Syapin, P. J. (2008). Regulation of haeme oxygenase-1 for treatment of neuroinflammation and brain disorders. British Journal of Pharmacology, 155(5), 623–640.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Terazawa, R., Akimoto, N., Kato, T., Itoh, T., Fujita, Y., Hamada, N., et al. (2013). A kavalactone derivative inhibits lipopolysaccharide-stimulated iNOS induction and NO production through activation of Nrf2 signaling in BV2 microglial cells. Pharmacological Research, 71, 34–43.CrossRefPubMedGoogle Scholar
  47. Vanden Berghe, W., Sabbe, L., Kaileh, M., Haegeman, G., & Heyninck, K. (2012). Molecular insight in the multifunctional activities of Withaferin A. Biochemical Pharmacology, 84(10), 1282–1291.CrossRefPubMedGoogle Scholar
  48. Ven Murthy, M. R., Ranjekar, P. K., Ramassamy, C., & Deshpande, M. (2010). Scientific basis for the use of Indian ayurvedic medicinal plants in the treatment of neurodegenerative disorders: Ashwagandha. Central Nervous System Agents in Medicinal Chemistry, 10(3), 238–246.CrossRefPubMedGoogle Scholar
  49. Vyas, A. R., & Singh, S. V. (2014). Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone. AAPS Journal, 16(1), 1–10.CrossRefPubMedGoogle Scholar
  50. Wadhwa, R., Konar, A., & Kaul, S. C. (2016). Nootropic potential of ashwagandha leaves: Beyond traditional root extracts. Neurochemistry International, 95, 109–118.CrossRefPubMedGoogle Scholar
  51. Wardyn, J. D., Ponsford, A. H., & Sanderson, C. M. (2015). Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochemical Society Transactions, 43(4), 621–626.CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhang, D. D., Lo, S. C., Cross, J. V., Templeton, D. J., & Hannink, M. (2004). Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Molecular and Cellular Biology, 24(24), 10941–10953.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Grace Y. Sun
    • 1
    • 2
    • 4
  • Runting Li
    • 1
    • 4
  • Jiankun Cui
    • 2
    • 4
  • Mark Hannink
    • 1
    • 4
  • Zezong Gu
    • 2
    • 4
  • Kevin L. Fritsche
    • 3
    • 4
  • Dennis B. Lubahn
    • 1
    • 3
    • 4
  • Agnes Simonyi
    • 1
    • 4
  1. 1.Biochemistry DepartmentUniversity of MissouriColumbiaUSA
  2. 2.Department of Pathology and Anatomical SciencesUniversity of Missouri School of MedicineColumbiaUSA
  3. 3.Department of Animal SciencesUniversity of MissouriColumbiaUSA
  4. 4.MU Center for Botanical Interaction StudiesUniversity of MissouriColumbiaUSA

Personalised recommendations