Neurochemical Research

, Volume 43, Issue 8, pp 1575–1586 | Cite as

Neuroprotection of Cytisine Against Cerebral Ischemia–Reperfusion Injury in Mice by Regulating NR2B-ERK/CREB Signal Pathway

  • Peng Zhao
  • Jia-Mei Yang
  • Yong-Sheng Wang
  • Yin-Ju Hao
  • Yu-Xiang Li
  • Nan Li
  • Jing Wang
  • Yang Niu
  • Tao Sun
  • Jian-Qiang YuEmail author
Original Paper


The aim of the study was to elucidate the therapeutic effects of Cytisine (CYT) on cerebral ischemia–reperfusion injury in mice. Male ICR mice were pretreated with reagents (drug), and then subjected to 2 h focal cerebral ischemia and 24 h reperfusion. Morphologically, the histopathological impairment were estimated by the TTC, HE and TUNEL staining. The expression of GluN2B-containing NMDA receptor, phosphorylation of extracellular regulated protein kinases, total ERK, phosphorylation of cAMP-response element binding protein and total CREB were determined by immunofluorescence and Western blot assay, respectively. The mRNA expression of NR2B, ERK and CREB were quantified by the real-time RT-PCR. CYT significantly diminished the infarct size and neuronal apoptosis. Additionally, it ameliorated histopathological lesion dramatically. CYT promoted the phosphorylation of ERK, CREB and their mRNA expression. In contrast, the expression of NR2B was suppressed in concomitant with the down-regulation of genes. The overall results thus far suggest that CYT confers the neuroprotection against cerebral I/R injury by regulating the NR2B-ERK/CREB signal pathway.


Cytisine Cerebral I/R injury P-CREB P-ERK NR2B 



This work was supported by the Colleges and Universities of Science and Technology Research Projects (Grant No. NGY2013078).

Author Contributions

Jian-Qiang Yu contributed to the conception of the study and approved the final version. Peng Zhao and Yue Liu significantly performed the experiments and drafted the manuscript preparation. Yin-Ju Hao and Yu-Xiang Li performed the data analyses. Nan Li and Jing Wang contributed reagents and materials. Yang Niu and Tao Sun revised the manuscript. We thank Drs. Lu-Ning Cui for his critical review and subsequent editing of this manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Lai TW, Zhang S, Wang YT (2014) Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 115:157–188CrossRefPubMedGoogle Scholar
  2. 2.
    Wang Y, Qin ZH (2010) Molecular and cellular mechanisms of excitotoxic neuronal death. Apoptosis 15(11):1382–1402CrossRefPubMedGoogle Scholar
  3. 3.
    Knox R, Zhao C, Miguel-Perez D, Wang S, Yuan J, Ferriero D, Jiang X (2013) Enhanced NMDA receptor tyrosine phosphorylation and increased brain injury following neonatal hypoxia-ischemia in mice with neuronal Fyn Overexpression. Neurobiol Dis 51:113–119CrossRefPubMedGoogle Scholar
  4. 4.
    Köhr G (2006) NMDA receptor function: subunit composition versus spatial distribution. Cell Tissue Res 326(2):439–446CrossRefPubMedGoogle Scholar
  5. 5.
    Zhang Z, Wang CZ, Wen XD, Shoyama Y, Yuan CS (2013) Role of saffron and its constituents on cancer Chemoprevention. Pharm Biol 51(7):920–924CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Léveillé F, Gaamouch E, Gouix F, Lecocq E, Lobner M, Nicole D, Buisson O (2008) A. Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors. FASEB J 22(12):4258–4271CrossRefPubMedGoogle Scholar
  7. 7.
    Chen M, Lu TJ, Chen XJ, Zhou Y, Chen Q, Feng XY, Xu L, Duan WH, Xiong ZQ (2008) Differential roles of NMDA receptor subtypes in ischemic neuronal cell death and ischemic tolerance. Stroke 39(11):3042–3048CrossRefPubMedGoogle Scholar
  8. 8.
    Hardingham GE, Bading H (2010) Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat Rev Neurosci 11(10):682–696CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Manning NW, Campbell BC, Oxley TJ, Chapot R (2014) Acute ischemic stroke: time, penumbra, and reperfusion. Stroke 45(2):640–644CrossRefPubMedGoogle Scholar
  10. 10.
    Thompson-Evans TP, Glover MP, Walker N (2011) Cytisine’s potential to be used as a traditional healing method to help indigenous people stop smoking: a qualitative study with Māori. Nicotine Tob Res 13(5):353–360CrossRefPubMedGoogle Scholar
  11. 11.
    Igari M, Alexander JC, Ji Y, Qi X, Papke RL, Bruijnzeel AW (2014) Varenicline and cytisine diminish the dysphoric-like state associated with spontaneous nicotine withdrawal in rats. Neuropsychopharmacology 39(2):455–465CrossRefPubMedGoogle Scholar
  12. 12.
    Li YJ, Yang Q, Zhang K, Guo YY, Li XB, Yang L, Zhao MG, Wu YM (2013) Cytisine confers neuronal protection against excitotoxic injury by down-regulating GluN2B-containing NMDA receptors. Neurotoxicology 34:219–225CrossRefPubMedGoogle Scholar
  13. 13.
    McCullough LD, Tarabishy S, Liu L, Benashski S, Xu Y, Ribar T, Means A, Li J (2013) Inhibition of calcium/calmodulin-dependent protein kinase kinase β and calcium/calmodulin-dependent protein kinase IV is detrimental in cerebral ischemia. Stroke 44(9):2559–2566CrossRefPubMedGoogle Scholar
  14. 14.
    Vakili A, Sharifat S, Akhavan MM3, Bandegi AR (2014) Effect of lavender oil (Lavandula angustifolia) on cerebral edema and its possible mechanisms in an experimental model of stroke. Brain Res 1548:56–62CrossRefPubMedGoogle Scholar
  15. 15.
    Luo Y, Yang YP, Liu J, Li WH, Yang J, Sui X, Yuan X, Nie ZY, Liu YQ, Chen D, Lin SH, Wang YA (2014) Neuroprotective effects of madecassoside against focal cerebral ischemia reperfusion injury in rats. Brain Res 1565:37–47CrossRefPubMedGoogle Scholar
  16. 16.
    Wang TF, Lei Z, Li YX, Wang YS, Wang J, Wang SJ, Hao YJ, Zhou R, Jin SJ, Du J, Li J, Sun T, Yu JQ (2013) Oxysophoridine protects against focal cerebral ischemic injury by inhibiting oxidative stress and apoptosis in Mice. Neurochem Res 38(11):2408–2417CrossRefPubMedGoogle Scholar
  17. 17.
    Eltzschig HK, Eckle T (2011) Ischemia and reperfusion—from mechanism to translation. Nat Med 17(11):1391–1401CrossRefPubMedGoogle Scholar
  18. 18.
    Lee RHC, Lee MHH, Wu CYC, Couto E, Silva A, Possoit HE, Hsieh TH, Minagar A, Lin HW (2018) Cerebral ischemia and neuroregeneration. Neural Regen Res 13(3):373–385CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Perez-Alvarez MJ, Villa Gonzalez M, Benito-Cuesta I, Wandosell FG (2018) Role of mTORC1 controlling proteostasis after brain ischemia. Front Neurosci 12:60CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Szeto V, Chen NH, Sun HS, Feng ZP (2018) The role of KATP channels in cerebral ischemic stroke and diabetes. Acta Pharmacol Sin 9(5):683–694CrossRefGoogle Scholar
  21. 21.
    Ma Z, Xin Z, Di W, Yan X, Li X, Reiter RJ, Yang Y (2017) Melatonin and mitochondrial function during ischemia/reperfusion injury. Cell Mol Life Sci 74(21):3989–3998CrossRefPubMedGoogle Scholar
  22. 22.
    Sopjani M, Thaçi S, Krasniqi B, Selmonaj M, Rinnerthaler M, Dërmaku-Sopjani M (2017) Regulation of ion channels, cellular carriers and Na(+)/K(+)/ATPase by Janus Kinase 3. Curr Med Chem 24(21):2251–2260CrossRefPubMedGoogle Scholar
  23. 23.
    Larsen BR, Stoica A, MacAulay N (2016) Managing brain extracellular K(+) during neuronal activity: the physiological role of the Na(+)/K(+)-ATPase subunit isoforms. Front Physiol 7:141CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kulbe JR, Mulcahy Levy JM, Coultrap SJ, Thorburn A, Bayer KU (2014) Excitotoxic glutamate insults block autophagic flux in hippocampal neurons. Brain Res 1542:12–19CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Puyal J, Ginet V, Clarke PG (2013) Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: a challenge for neuroprotection. Prog Neurobiol 105:24–48CrossRefPubMedGoogle Scholar
  26. 26.
    Rostas JAP, Spratt NJ, Dickson PW, Skelding KA (2017) The role of Ca2+-calmodulin stimulated protein kinase II in ischaemic stroke—a potential target for neuroprotective therapies. Neurochem Int 107:33–42CrossRefPubMedGoogle Scholar
  27. 27.
    Sun Y, Cheng X, Hu J, Gao Z (2018) The role of GluN2A in cerebral ischemia: promoting neuron death and survival in the early stage and thereafter. Mol Neurobiol 55(2):1208–1216CrossRefPubMedGoogle Scholar
  28. 28.
    Blackstone NW (2015) The impact of mitochondrial endosymbiosis on the evolution of calcium signaling. Cell Calcium 57(3):133–139CrossRefPubMedGoogle Scholar
  29. 29.
    Yurkewicz L, Weaver J, Bullock MR, Marshall LF (2005) The effect of the selective NMDA receptor antagonist traxoprodil in the treatment of traumatic brain injury. J Neurotrauma 22(12):1428–1443CrossRefPubMedGoogle Scholar
  30. 30.
    Li Y, Yu M, Zhao B, Wang Y, Zha Y, Li Z, Yu L, Yan L, Chen Z, Zhang W, Zeng X, He Z (2018) Clonidine preconditioning improved cerebral ischemia-induced learning and memory deficits in rats via ERK1/2-CREB/ NF-κB-NR2B pathway. Eur J Pharmacol 818:167–173CrossRefPubMedGoogle Scholar
  31. 31.
    Lee S, Yoon S, Kim DH, Gynecol Onco (2007) A high nuclear basal level of ERK2 phosphorylation contributes to the resistance of cisplatin-resistant human ovarian cancer cells. Gynecol Oncol 104(2):338–344CrossRefPubMedGoogle Scholar
  32. 32.
    Lee YJ, Choi SY, Yang JH (2014) NMDA receptor-mediated ERK 1/2 pathway is involved in PFHxS-induced apoptosis of PC12 cells. Sci Total Environ 491(49):227–234CrossRefPubMedGoogle Scholar
  33. 33.
    López-Valdés HE, Clarkson AN, Ao Y, Charles AC, Carmichael ST, Sofroniew MV, Brennan KC (2014) Memantine enhances recovery from stroke. Stroke 45(7):2093–2100CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Saver JL, Albers GW, Dunn B, Johnston KC, Fisher M, STAIR VI Consortium (2009) Stroke Therapy Academic Industry Roundtable (STAIR) recommendations for extended window acute stroke therapy trials. Stroke 40(7):2594–2600CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Kahle KT, Simard JM, Staley KJ, Nahed BV, Jones PS, Sun D (2009) Molecular mechanisms of ischemic cerebral edema: role of electroneutral ion transport. Physiology 24:257–265CrossRefPubMedGoogle Scholar
  36. 36.
    Chang C, Zhao Y, Song G, She K (2018) Resveratrol protects hippocampal neurons against cerebral ischemia-reperfusion injury via modulating JAK/ERK/STAT signaling pathway in rats. J Neuroimmunol 315:9–14CrossRefPubMedGoogle Scholar
  37. 37.
    Huang B, Chen P, Huang L, Li S, Zhu R, Sheng T, Yu W, Chen Z, Wang T (2017) Geniposide attenuates post-ischaemic neurovascular damage via GluN2A/AKT/ ERK-dependent mechanism. Cell Physiol Biochem 43(2):705–716CrossRefPubMedGoogle Scholar
  38. 38.
    Shioda N, Han F, Fukunaga K (2009) Role of Akt and ERK signaling in the neurogenesis following bra brain ischemia. Int Rev Neurobiol 85:375–387CrossRefPubMedGoogle Scholar
  39. 39.
    Kitagawa K (2007) CREB and cAMP response element-mediated gene expression in the ischemic brain. FEBS J 274(13):3210–3217CrossRefPubMedGoogle Scholar
  40. 40.
    Chen S, Yin W, Bi K, Lu B (2018) MicroRNA497 attenuates cerebral infarction in patients via the TLR4 and CREB signaling pathways. Int J Mol Med. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Zhang W, Song JK, Yan R, Li L, Xiao ZY, Zhou WX, Wang ZZ, Xiao W, Du GH (2018) Diterpene ginkgolides protect against cerebral ischemia/reperfusion damage in rats by activating Nrf2 and CREB through PI3K/Akt signaling. Acta Pharmacol Sin. CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang ZH, Fang XB, Xi GM, Li WC, Ling HY, Qu P (2010) Calcitonin gene-related peptide enhances CREB phosphorylation and attenuates tau protein phosphorylation in rat brain during focal cerebral ischemia/reperfusion. Biomed Pharmacother 64(6):430–436CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Peng Zhao
    • 1
  • Jia-Mei Yang
    • 1
  • Yong-Sheng Wang
    • 1
    • 2
  • Yin-Ju Hao
    • 1
  • Yu-Xiang Li
    • 3
  • Nan Li
    • 1
  • Jing Wang
    • 1
  • Yang Niu
    • 4
  • Tao Sun
    • 5
  • Jian-Qiang Yu
    • 1
    • 6
    Email author
  1. 1.Department of PharmacologyNingxia Medical UniversityYinchuanPeople’s Republic of China
  2. 2.Department of PharmacyFuzhou Second Hospital of Xiamen UniversityFuzhouPeople’s Republic of China
  3. 3.College of NursingNingxia Medical UniversityYinchuanPeople’s Republic of China
  4. 4.Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of EducationNingxia Medical UniversityYinchuanPeople’s Republic of China
  5. 5.Key Laboratory of Craniocerebral Diseases of Ningxia Hui Autonomous RegionNingxia Medical UniversityYinchuanPeople’s Republic of China
  6. 6.Ningxia Hui Medicine Modern Engineering Research Center and Collaborative Innovation CenterNingxia Medical UniversityYinchuanPeople’s Republic of China

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