Skip to main content

Advertisement

Log in

Neferine Protects Against Brain Damage in Permanent Cerebral Ischemic Rat Associated with Autophagy Suppression and AMPK/mTOR Regulation

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

Neferine is the major alkaloid compound isolated from the seed embryos of lotus. Neferine has many pharmacological effects, such as anti-inflammatory, antioxidative stress, and antiapoptotic effects, and it maintains autophagic balance. The purpose of this study was to explore the mechanism by which neferine attenuates autophagy after permanent cerebral ischemia in rats. We performed permanent cerebral ischemia in rats by middle cerebral artery occlusion (pMCAO) for 12 h with or without administration of neferine or nimodipine, a calcium (Ca2+) channel blocker. Neuroprotective effects were determined by evaluating the infarct volume and neurological deficits. Autophagy and its signaling pathway were determined by evaluating the expression of phosphorylated AMP-activated protein kinase alpha (AMPKα), phosphorylated mammalian target of rapamycin (mTOR), beclin-1, microtubule-associated protein 1A/1B-light chain 3 class II (LC3-II), and p62 by western blotting. Autophagosomes were evaluated by transmission electron microscopy. Neferine treatment significantly reduced infarct volumes and improved neurological deficits. Neferine significantly attenuated the upregulation of autophagy-associated proteins such as LC3-II, beclin-1, and p62 as well as autophagosome formation, all of which were induced by pMCAO. Neferine exerted remarkable protection against cerebral ischemia, possibly via the regulation of autophagy mediated by the Ca2+-dependent AMPK/mTOR pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data availability

All data are available on request from authors.

References

  1. Mao Y, Yang G, Zhou L (2000) Temporary and permanent focal cerebral ischemia in the mouse: assessment of cerebral blood flow, brain damage and blood-brain barrier permeability. Chin Med J (Engl) 113(4):361–366

    CAS  Google Scholar 

  2. Hu BR, Liu CL (2017) Mechanisms of neuron death (necrosis, apoptosis, autophagy) after brain ischemia. Prim. Cerebrovasc. Dis, 2nd edn. Elsevier, United States, pp 209–215. https://doi.org/10.1016/b978-0-12-803058-5.00043-6

  3. Hossmann KA, Heiss WD (2019) Neuropathology and pathophysiology of stroke. In M. Brainin & W. Heiss (Eds.), Textbook of Stroke Medicine. Cambridge, pp. 1–32). https://doi.org/10.1017/CBO9781107239340.002

  4. Lee JM, Grabb MC, Zipfel GJ, Choi DW (2000) Brain tissue responses to ischemia. J Clin Invest 106(6):723–731. https://doi.org/10.1172/JCI11003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Balestrino M (1995) Pathophysiology of anoxic depolarization: new findings and a working hypothesis. J Neurosci Methods 59(1):99–103. https://doi.org/10.1016/0165-0270(94)00199-q

    Article  CAS  PubMed  Google Scholar 

  6. Kaminogo M, Suyama K, Ichikura A, Onizuka M, Shibata S (1998) Anoxic depolarization determines ischemic brain injury. Neurol Res 20(4):343–348. https://doi.org/10.1080/01616412.1998.11740529

    Article  CAS  PubMed  Google Scholar 

  7. Asai S, Kunimatsu T, Zhao H, Nagata T, Takahashi Y, Ishii Y (2000) Two distinct components of initial glutamate release synchronized with anoxic depolarization in rat global brain ischemia. NeuroReport 11(13):2947–2952. https://doi.org/10.1097/00001756-200009110-00023

    Article  CAS  PubMed  Google Scholar 

  8. Danton GH, Danton W (2003) Inflammatory mechanisms after ischemia and stroke. J Neuropathol Exp Neurol 62(2):127–136. https://doi.org/10.1093/jnen/62.2.127

    Article  CAS  PubMed  Google Scholar 

  9. Kawabori M, Yenari MA (2015) Inflammatory responses in brain ischemia. Curr Med Chem 22(10):1258–1277. https://doi.org/10.2174/0929867322666150209154036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sun Y, Zhu Y, Zhong X, Chen X, Wang J, Ying G (2019) Crosstalk between autophagy and cerebral ischemia. Front Neurosci 12:1022. https://doi.org/10.3389/fnins.2018.01022

    Article  PubMed  PubMed Central  Google Scholar 

  11. Tian FF, Deguchi K, Yamashita T, Ohta Y, Morimoto N, Shang J, Zhang X, Liu N, Ikeda Y, Matsuura T, Abe K (2010) In vivo imaging of autophagy in a mouse stroke model. Autophagy 6(8):1107–1114. https://doi.org/10.4161/auto.6.8.13427

    Article  PubMed  Google Scholar 

  12. Huang Y, Bai Y, Zhao L, Hu T, Hu B, Wang J (2007) Pharmacokinetics and metabolism of neferine in rats after a single oral administration. Biopharm Drug Dispos 28(7):361–372. https://doi.org/10.1002/bdd.556

    Article  CAS  PubMed  Google Scholar 

  13. Dong ZX, Zhao X, Gu DF, Shi YQ, Zhang J, Hu XX, Hu MQ, Yang BF, Li BX (2012) Comparative effects of liensinine and neferine on the human ether-a-go-go-related gene potassium channel and pharmacological activity analysis. Cell Physiol Biochem 29(3–4):431–442. https://doi.org/10.1159/000338497

    Article  CAS  PubMed  Google Scholar 

  14. Wang J, Kan Q, Li J, Zhang X, Qi Y (2011) Effect of neferine on liver ischemia-reperfusion injury in rats. Transplant Proc 43(7):2536–2539. https://doi.org/10.1016/j.transproceed.2011.04.013

    Article  CAS  PubMed  Google Scholar 

  15. Lalitha G, Poornima P, Archanah A, Padma VV (2013) Protective effect of neferine against isoproterenol-induced cardiac toxicity. Cardiovasc Toxicol 13(2):168–179. https://doi.org/10.1007/s12012-012-9196-5

    Article  CAS  PubMed  Google Scholar 

  16. Chen MS, Zhang JH, Wang JL, Gao L, Chen XX, Xiao JH (2015) Anti-fibrotic effects of neferine on carbon tetrachloride-induced hepatic fibrosis in mice. Am J Chin Med 43(2):231–240. https://doi.org/10.1142/S0192415X15500159

    Article  CAS  PubMed  Google Scholar 

  17. Yu Y, Sun S, Wang S, Zhang Q, Li M, Lan F, Li S, Liu C (2016) Liensinine- and neferine-induced cardiotoxicity in primary neonatal rat cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes. Int J Mol Sci 17(2):186. https://doi.org/10.3390/ijms17020186

    Article  CAS  PubMed Central  Google Scholar 

  18. Baskarana R, Priya LB, Kalaiselvi P, Poornima P, Huang CY, Padma VV (2017) Neferine from Nelumbo nucifera modulates oxidative stress and cytokines production during hypoxia in human peripheral blood mononuclear cells. Biomed Pharmacother 93:730–736. https://doi.org/10.1016/j.biopha.2017.07.003

    Article  CAS  Google Scholar 

  19. Li J, Hu D, Song X, Han T, Gao Y, Xing Y (2017) The role of biologically active ingredients from natural drug treatments for arrhythmias in different mechanisms. Biomed Res Int 2017:4615727. https://doi.org/10.1155/2017/4615727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wicha P, Onsa-Ard A, Chaichompoo W, Suksamrarn A, Tocharus C (2020) Vasorelaxant and antihypertensive effects of neferine in rats: an in vitro and in vivo study. Planta Med 86(7):496–504. https://doi.org/10.1055/a-1123-7852

    Article  CAS  PubMed  Google Scholar 

  21. Baskaran R, Poornima P, Priya LB, Huang CY, Padma VV (2016) Neferine prevents autophagy induced by hypoxia through activation of Akt/mTOR pathway and Nrf2 in muscle cells. Biomed Pharmacother 83:1407–1413. https://doi.org/10.1016/j.biopha.2016.08.063

    Article  CAS  PubMed  Google Scholar 

  22. Chaichompoo W, Chokchaisiri R, Apiratikul N, Chairoungdua A, Yingyongnarongkul B, Chunglok W, Tocharus C, Suksamrarn A (2018) Cytotoxic alkaloids against human colon adenocarcinoma cell line (HT-29) from the seed embryos of Nelumbo nucifera. Med Chem Res 27:939–943. https://doi.org/10.1007/s00044-017-2115-3

    Article  CAS  Google Scholar 

  23. Nishimura K, Horii S, Tanahashi T, Sugimoto Y, Yamada J (2013) Synthesis and pharmacological activity of alkaloids from embryo of lotus, Nelumbo nucifera. Chem Pharm Bull 61:59–68. https://doi.org/10.1248/cpb.c12-00820

    Article  CAS  Google Scholar 

  24. Zhang XX, Ma SC, Mao P, Wang Y (2016) A new bisbenzylisoquinoline alkaloid from Plumula nelumbinis. Chin Chem Lett 27:1755–1758. https://doi.org/10.1016/j.cclet.2016.06.051

    Article  CAS  Google Scholar 

  25. Chiang T, Messing RO, Chou WH (2011) Mouse model of middle cerebral artery occlusion. J Vis Exp 48:2761. https://doi.org/10.3791/2761

    Article  Google Scholar 

  26. Liu F, McCullough LD (2011) Middle cerebral artery occlusion model in rodents: methods and potential pitfalls. J Biomed Biotechnol 2011:464701. https://doi.org/10.1155/2011/464701

    Article  PubMed  PubMed Central  Google Scholar 

  27. Shahjouei S, Cai PY, Ansari S, Sharififar S, Azari H, Ganji S (2016) Middle cerebral artery occlusion model of stroke in rodents: a step-by-step approach. J Vasc Interv Neurol 8(5):1–8

    PubMed  PubMed Central  Google Scholar 

  28. Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano K (1988) Behavioural changes after focal cerebral ischemia by left middle cerebral artery occlusion in rats. Brain Res 452:323–328. https://doi.org/10.1016/0006-8993(88)90036-4

    Article  CAS  PubMed  Google Scholar 

  29. Ryck MD, Reempts JV, Borgers M, Wauquier A, Janssen PA (1989) Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 20:1383–1390. https://doi.org/10.1161/01.str.20.10.1383

    Article  PubMed  Google Scholar 

  30. Ashwal S, Tone B, Tian HR, Cole DJ, Liwnicz BH, Pearce WJ (1999) Core and penumbral nitric oxide synthase activity during cerebral ischemia and reperfusion in the rat pup. Pediatr Res 46(4):390–400. https://doi.org/10.1203/00006450-199910000-00006

    Article  CAS  PubMed  Google Scholar 

  31. Wu Z, Zou Z, Zou R, Zhou X, Cui S (2015) Electroacupuncture pretreatment induces tolerance against cerebral ischemia/reperfusion injury through inhibition of the autophagy pathway. Mol Med Rep 11(6):4438–4446. https://doi.org/10.3892/mmr.2015.3253

    Article  CAS  PubMed  Google Scholar 

  32. Li J, McCullough LD (2010) Effects of AMP-activated protein kinase in cerebral ischemia. J Cereb Blood Flow Metab 30(3):480–492. https://doi.org/10.1038/jcbfm.2009.255

    Article  CAS  PubMed  Google Scholar 

  33. Jiang J, Dai J, Cui H (2018) Vitexin reverses the autophagy dysfunction to attenuate MCAO-induced cerebral ischemic stroke via mTOR/Ulk1 pathway. Biomed Pharmacother 99:583–590. https://doi.org/10.1016/j.biopha.2018.01.067

    Article  CAS  PubMed  Google Scholar 

  34. Wang Y, Ren Q, Zhang X, Huiling L, Chen J (2017) Neuroprotective mechanisms of calycosin against focal cerebral ischemia and reperfusion injury in rats. Cell Physiol Biochem 45(2):537–546. https://doi.org/10.1159/000487031

    Article  CAS  Google Scholar 

  35. Nishizawa Y (2001) Glutamate release and neuronal damage in ischemia. Life Sci 69(4):369–381. https://doi.org/10.1016/S0024-3205(01)01142-0

    Article  CAS  PubMed  Google Scholar 

  36. Rama R, García JC (2016) Excitotoxicity and oxidative stress in acute stroke. Bernhard Schaller, IntechOpen. https://doi.org/10.5772/64991

    Article  Google Scholar 

  37. Chen W, Sun Y, Liu K, Sun X (2014) Autophagy: a double-edged sword for neuronal survival after cerebral ischemia. Neural Regen Res 9(12):1210–1216. https://doi.org/10.4103/1673-5374.135329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hou K, Xu D, Li F, Chen S, Li Y (2019) The progress of neuronal autophagy in cerebral ischemia stroke: mechanisms, roles and research methods. J Neurol Sci 15(400):72–82. https://doi.org/10.1016/j.jns.2019.03.015

    Article  Google Scholar 

  39. Gao L, Jiang T, Guo J, Liu Y, Cui G, Gu L, Su L, Zhang Y (2012) Inhibition of autophagy contributes to ischemic postconditioning-induced neuroprotection against focal cerebral ischemia in rats. PLoS ONE 7(9):e46092. https://doi.org/10.1371/journal.pone.0046092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gu Z, Sun Y, Liu K, Wang F, Zhang T, Li Q, Shen L, Zhou L, Dong L, Shi N, Zhang Q, Zhang W, Zhao M, Sun X (2013) The role of autophagic and lysosomal pathways in ischemic brain injury. Neural Regen Res 8(23):2117–2125. https://doi.org/10.3969/j.issn.1673-5374.2013.23.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Thiebaut AM, Hedou E, Marciniak SJ, Vivien D, Roussel BD (2019) Proteostasis during cerebral ischemia. Front Neurosci 13:637. https://doi.org/10.3389/fnins.2019.00637

    Article  PubMed  PubMed Central  Google Scholar 

  42. Sugimoto Y, Furutani S, Itoh A, Tanahashi T, Nakajima H, Oshiro H (2008) Effects of extracts and neferine from the embryo of Nelumbo nucifera seeds on the central nervous system. Phytomedicine 15(12):1117–1124. https://doi.org/10.1016/j.phymed.2008.09.005

    Article  CAS  PubMed  Google Scholar 

  43. Jung HA, Jin SE, Choi RJ, Kim DH, Kim YS, Ryu JH (2010) Anti-amnesic activity of neferine with antioxidant and anti-inflammatory capacities, as well as inhibition of ChEs and BACE1. Life Sci 87:420–430. https://doi.org/10.1016/j.lfs.2010.08.005

    Article  CAS  PubMed  Google Scholar 

  44. Jung HA, Karki S, Kim JH, Choi JS (2015) BACE1 and cholinesterase inhibitory activities of Nelumbo nucifera embryos. Arch Pharm Res 38:1178–1187. https://doi.org/10.1007/s12272-014-0492-4

    Article  CAS  PubMed  Google Scholar 

  45. Tomassoni D, Lanari A, Silvestrelli G, Traini E, Amenta F (2008) Nimodipine and its use in cerebrovascular disease: evidence from recent preclinical and controlled clinical studies. Clin Exp Hypertens 30(8):744–766. https://doi.org/10.1080/10641960802580232

    Article  CAS  PubMed  Google Scholar 

  46. Das JM, Zito PM (2021) Nimodipine. Treasure Island: StatPearls. https://www.ncbi.nlm.nih.gov/books/NBK534870/. Accessed 7 May 2021

  47. Pisani A, Calabresi P, Tozzi A, D’Angelo V, Bernardi G (1998) L-type Ca2+ channel blockers attenuate electrical changes and Ca2+ rise induced by oxygen/glucose deprivation in cortical neurons. Stroke 29:196–201. https://doi.org/10.1161/01.str.29.1.196

    Article  CAS  PubMed  Google Scholar 

  48. Chen J, Qi J, Chen F, Liu JH, Wang T, Yang J, Yin CP (2007) Relaxation mechanisms of neferine on the rabbit corpus cavernosum tissue in vitro. Asian J Androl 9(6):795–800. https://doi.org/10.1111/j.1745-7262.2007.00321.x

    Article  CAS  PubMed  Google Scholar 

  49. Thornton C, Rousset CI, Kichev A, Miyakuni Y, Vontell R (2012) Baburamani AA (2012) Molecular mechanisms of neonatal brain injury. Neurol Res Int 2:506320. https://doi.org/10.1155/2012/506320

    Article  Google Scholar 

  50. Perez-Alvarez MJ, Gonzalez MV, Benito-Cuesta I, Wandosell FG (2018) Role of mTORC1 controlling proteostasis after brain ischemia. Front Neurosci 12:60. https://doi.org/10.3389/fnins.2018.00060

    Article  PubMed  PubMed Central  Google Scholar 

  51. Rami A, Langhagen A, Steiger S (2008) Focal cerebral ischemia induces upregulation of beclin 1 and autophagy-like cell death. Neurobiol Dis 29(1):132–141. https://doi.org/10.1016/j.nbd.2007.08.005

    Article  CAS  PubMed  Google Scholar 

  52. Wen YD, Sheng R, Zhang LS, Han R, Zhang X, Zhang XD, Han F, Fukunaga K, Qin ZH (2008) Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways. Autophagy 4(6):762–769. https://doi.org/10.4161/auto.6412

    Article  CAS  PubMed  Google Scholar 

  53. Lee RHC, Lee MHH, Wu CYC, Silva AC, Possoit HE, Hsieh TH, Minagar A, Lin HW (2018) Cerebral ischemia and neuroregeneration. Neural Regen Res 13(3):373–385. https://doi.org/10.4103/1673-5374.228711

    Article  PubMed  PubMed Central  Google Scholar 

  54. Lan R, Xiang J, Zhang Y, Wang GH, Bao J, Li WW, Zhang W, Xu LL, Cai DF (2013) PI3K/Akt pathway contributes to neurovascular unit protection of Xiao-Xu-Ming decoction against focal cerebral ischemia and reperfusion injury in rats. Evid Based Complement Alternat Med 2013:459467. https://doi.org/10.1155/2013/459467

    Article  PubMed  PubMed Central  Google Scholar 

  55. Huang YG, Tao W, Yang SB, Wang JF, Mei ZG, Feng ZT (2019) Autophagy: novel insights into therapeutic target of electroacupuncture against cerebral ischemia reperfusion injury. Neural Regen Res 14(6):954–961. https://doi.org/10.4103/1673-5374.250569

    Article  PubMed  PubMed Central  Google Scholar 

  56. Wang MM, Zhang M, Feng YS, Xing Y, Tan ZX, Li WB, Dong F, Zhang F (2020) Electroacupuncture inhibits neuronal autophagy and apoptosis via the PI3K/AKT pathway following ischemic stroke. Front Cell Neurosci 14:134. https://doi.org/10.3389/fncel.2020.00134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Gao X, Zhang H, Steinberg G, Zhao H (2010) The Akt pathway is involved in rapid ischemic tolerance in focal ischemia in rats. Transl Stroke Res 1(3):202–209. https://doi.org/10.1007/s12975-010-0017-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Amantea D, Tassorelli C, Russo R, Petrelli F, Morrone LA, Bagetta G, Corasaniti MT (2011) Neuroprotection by leptin in a rat model of permanent cerebral ischemia: effects on STAT3 phosphorylation in discrete cells of the brain. Cell Death Dis 8;2(12): e238. https://doi.org/10.1038/cddis.2011.125

  59. Li XG, Du JH, Lu Y, Lin XJ (2019) Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling. Neural Regen Res 14(4):721–727. https://doi.org/10.4103/1673-5374.247476

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank the Nara Institute of Science and Technology, Japan.

Funding

This research was supported by the Faculty of Medicine Research Fund (grant no. 062–2563), Center for Research and Development of Natural Products for Health, Chiang Mai University. We also gratefully acknowledge support from The Thailand Research Fund (DBG6180030) and the Center of Excellence for Innovation in Chemistry, Ministry of Higher Education, Science, Research and Innovation. Jirakhamon Sengking acknowledges the teaching assistant/research assistant financial support from the Graduate School, Chiang Mai University; the Faculty of Medicine Graduate Scholarship, Chiang Mai University; a 2018 scholarship for graduate students from The King Prajadhipok and Queen Rambhai Barni Memorial Foundation; and a 2020 graduate scholarship from the National Research Council of Thailand (NRCT).

Author information

Authors and Affiliations

Authors

Contributions

CT conceived the project; JS, PW, NY, and WC performed the experiments; JS, PW, and CT collected data; JS, JT, CT, and CO analyzed the data; CT and AS supervised the project; JS, CO, JT, AS, and CT wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Chainarong Tocharus.

Ethics declarations

Ethics Approval and Consent to Participate

All authors confirm that all experiments were performed in accordance with relevant guidelines and regulations. All experiments in this study were conducted in compliance with the Animal Ethics Committee in accordance with the guidelines for the care and use of laboratory animals, as prepared by the Faculty of Medicine, Chiang Mai University Institutional Animal Care and Use Committee.

Consent for Publication

All authors approve the manuscript for publication.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sengking, J., Oka, C., Wicha, P. et al. Neferine Protects Against Brain Damage in Permanent Cerebral Ischemic Rat Associated with Autophagy Suppression and AMPK/mTOR Regulation. Mol Neurobiol 58, 6304–6315 (2021). https://doi.org/10.1007/s12035-021-02554-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12035-021-02554-z

Keywords

Navigation