Overexpressed HspB6 Underlines a Novel Inhibitory Role in Kainic Acid-Induced Epileptic Seizure in Rats by Activating the cAMP-PKA Pathway

  • Ai-Qin Qi
  • Yan-Hui Zhang
  • Qin-De Qi
  • Ye-Hui Liu
  • Jun-Ling Zhu
Original Research


Epilepsy is a commonly occurring neurological disease that has a large impact on the patient’s daily life. Phosphorylation of heat shock protein B6 (HspB6) has been reported to protect the central nervous system. In this investigation, we explored whether HspB6 played a positive effect on epilepsy with the involvement of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway. The epileptic seizure was induced in rats by intraperitoneal injection of kainic acid (KA). The extent of HspB6 phosphorylation and expressions of HspB6, PKA, and inflammatory factors TNF-α, IL-1β, and IL-6 were quantified along with neuronal apoptosis. To further understand the regulatory mechanism of the HspB6 in the hippocampus, we altered the expression and the extent of HspB6 phosphorylation to see whether the cAMP-PKA pathway was inactivated or not in hippocampal neurons of rats post KA. Results showed that HspB6 was poorly expressed, resulting in the inactivation of the cAMP-PKA pathway in rats post KA, as well as an aggravated inflammatory response and hippocampal neuronal apoptosis. HspB6 overexpression and the cAMP-PKA pathway activation decreased the expression of inflammatory factors and inhibited hippocampal neuronal apoptosis. Additionally, HspB6 phosphorylation further augments the inhibitory effects of HspB6 on the inflammatory response and hippocampal neuronal apoptosis. The cAMP-PKA pathway activation was found to result in increased HspB6 phosphorylation. HspB6 decreased apoptosis signal-regulating kinase 1 (ASK1) expression to inhibit inflammatory response and hippocampal neuronal apoptosis. Collectively, our findings demonstrate that activation of the cAMP-PKA pathway induces overexpression and partial phosphorylation of HspB6 lead to the inhibition of ASK1 expression. This in turn protects rats against epilepsy and provides a potential approach to prevent the onset of epileptic seizure in a clinical setting.


Epilepsy Heat shock protein B6 cAMP-PKA signaling pathway Phosphorylation Inflammatory response Hippocampal neuronal apoptosis 



We would like to express our sincerest appreciation and gratitude to the reviewers for their helpful comments and insight on this article.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aaberg KM, Bakken IJ, Lossius MI, Lund Soraas C, Tallur KK, Stoltenberg C, Chin R, Suren P (2018) Short-term seizure outcomes in childhood epilepsy. Pediatrics 141(6):e20174016CrossRefPubMedGoogle Scholar
  2. Atkins D, Reiffen KA, Tegtmeier CL, Winther H, Bonato MS, Storkel S (2004) Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem 52:893–901CrossRefPubMedGoogle Scholar
  3. Ba M, Singer CA, Tyagi M, Brophy C, Baker JE, Cremo C, Halayko A, Gerthoffer WT (2009) HSP20 phosphorylation and airway smooth muscle relaxation. Cell Health Cytoskelet 2009(1):27–42PubMedPubMedCentralGoogle Scholar
  4. Beall A, Bagwell D, Woodrum D, Stoming TA, Kato K, Suzuki A, Rasmussen H, Brophy CM (1999) The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation. J Biol Chem 274:11344–11351CrossRefPubMedGoogle Scholar
  5. Fan GC, Kranias EG (2011) Small heat shock protein 20 (HspB6) in cardiac hypertrophy and failure. J Mol Cell Cardiol 51:574–577CrossRefPubMedGoogle Scholar
  6. Fan GC, Chu G, Mitton B, Song Q, Yuan Q, Kranias EG (2004) Small heat-shock protein Hsp20 phosphorylation inhibits beta-agonist-induced cardiac apoptosis. Circ Res 94:1474–1482CrossRefPubMedGoogle Scholar
  7. Fisher RS, Acevedo C, Arzimanoglou A, Bogacz A, Cross JH, Elger CE, Engel J Jr, Forsgren L, French JA, Glynn M, Hesdorffer DC, Lee BI, Mathern GW, Moshe SL, Perucca E, Scheffer IE, Tomson T, Watanabe M, Wiebe S (2014) ILAE official report: a practical clinical definition of epilepsy. Epilepsia 55:475–482CrossRefGoogle Scholar
  8. Flynn CR, Komalavilas P, Tessier D, Thresher J, Niederkofler EE, Dreiza CM, Nelson RW, Panitch A, Joshi L, Brophy CM (2003) Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle. FASEB J 17:1358–1360CrossRefPubMedGoogle Scholar
  9. Gao B, Wu Y, Yang YJ, Li WZ, Dong K, Zhou J, Yin YY, Huang DK, Wu WN (2018) Sinomenine exerts anticonvulsant profile and neuroprotective activity in pentylenetetrazole kindled rats: involvement of inhibition of NLRP1 inflammasome. J Neuroinflammation 15:152CrossRefPubMedPubMedCentralGoogle Scholar
  10. Guo L, Breakspear A, Zhao G, Gao L, Kistler HC, Xu JR, Ma LJ (2016) Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides. Mol Plant Pathol 17:196–209CrossRefPubMedGoogle Scholar
  11. Heirbaut M, Beelen S, Strelkov SV, Weeks SD (2014) Dissecting the functional role of the N-terminal domain of the human small heat shock protein HSPB6. PLoS ONE 9:e105892CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hsieh CL, Chen MF, Li TC, Li SC, Tang NY, Hsieh CT, Pon CZ, Lin JG (1999) Anticonvulsant effect of Uncaria rhynchophylla (Miq) Jack. in rats with kainic acid-induced epileptic seizure. Am J Chin Med 27:257–264CrossRefPubMedGoogle Scholar
  13. Ishiura H, Doi K, Mitsui J, Yoshimura J, Matsukawa MK, Fujiyama A, Toyoshima Y, Kakita A, Takahashi H, Suzuki Y, Sugano S, Qu W, Ichikawa K, Yurino H, Higasa K, Shibata S, Mitsue A, Tanaka M, Ichikawa Y, Takahashi Y, Date H, Matsukawa T, Kanda J, Nakamoto FK, Higashihara M, Abe K, Koike R, Sasagawa M, Kuroha Y, Hasegawa N, Kanesawa N, Kondo T, Hitomi T, Tada M, Takano H, Saito Y, Sanpei K, Onodera O, Nishizawa M, Nakamura M, Yasuda T, Sakiyama Y, Otsuka M, Ueki A, Kaida KI, Shimizu J, Hanajima R, Hayashi T, Terao Y, Inomata-Terada S, Hamada M, Shirota Y, Kubota A, Ugawa Y, Koh K, Takiyama Y, Ohsawa-Yoshida N, Ishiura S, Yamasaki R, Tamaoka A, Akiyama H, Otsuki T, Sano A, Ikeda A, Goto J, Morishita S, Tsuji S (2018) Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy. Nat Genet 50:581–590CrossRefPubMedGoogle Scholar
  14. Klein P, Tyrlikova I (2017) Prevention of epilepsy: should we be avoiding clinical trials? Epilepsy Behav 72:188–194CrossRefPubMedGoogle Scholar
  15. Labudda K, Illies D, Herzig C, Schroder K, Bien CG, Neuner F (2017) Current psychiatric disorders in patients with epilepsy are predicted by maltreatment experiences during childhood. Epilepsy Res 135:43–49CrossRefPubMedGoogle Scholar
  16. Li F, Xiao H, Zhou F, Hu Z, Yang B (2017a) Study of HSPB6: insights into the properties of the multifunctional protective agent. Cell Physiol Biochem 44:314–332CrossRefPubMedGoogle Scholar
  17. Li L, Fan X, Zhang XT, Yue SQ, Sun ZY, Zhu JQ, Zhang JH, Gao XM, Zhang H (2017b) The effects of Chinese medicines on cAMP/PKA signaling in central nervous system dysfunction. Brain Res Bull 132:109–117CrossRefPubMedGoogle Scholar
  18. Lovering F, Morgan P, Allais C, Aulabaugh A, Brodfuehrer J, Chang J, Coe J, Ding W, Dowty H, Fleming M, Frisbie R, Guzova J, Hepworth D, Jasti J, Kortum S, Kurumbail R, Mohan S, Papaioannou N, Strohbach JW, Vincent F, Lee K, Zapf CW (2018) Rational approach to highly potent and selective apoptosis signal-regulating kinase 1 (ASK1) inhibitors. Eur J Med Chem 145:606–621CrossRefPubMedGoogle Scholar
  19. Mazarati AM, Lewis ML, Pittman QJ (2017) Neurobehavioral comorbidities of epilepsy: role of inflammation. Epilepsia 58 Suppl 3:48–56CrossRefGoogle Scholar
  20. Mohler H (2006) GABAA receptors in central nervous system disease: anxiety, epilepsy, and insomnia. J Recept Signal Transduct Res 26:731–740CrossRefPubMedGoogle Scholar
  21. Nagasawa T, Matsushima-Nishiwaki R, Toyoda H, Matsuura J, Kumada T, Kozawa O (2014) Heat shock protein 20 (HSPB6) regulates apoptosis in human hepatocellular carcinoma cells: direct association with Bax. Oncol Rep 32:1291–1295CrossRefPubMedGoogle Scholar
  22. Nagasawa T, Matsushima-Nishiwaki R, Yasuda E, Matsuura J, Toyoda H, Kaneoka Y, Kumada T, Kozawa O (2015) Heat shock protein 20 (HSPB6) regulates TNF-alpha-induced intracellular signaling pathway in human hepatocellular carcinoma cells. Arch Biochem Biophys 565:1–8CrossRefPubMedGoogle Scholar
  23. Ryan K, Backos DS, Reigan P, Patel M (2012) Post-translational oxidative modification and inactivation of mitochondrial complex I in epileptogenesis. J Neurosci 32:11250–11258CrossRefPubMedPubMedCentralGoogle Scholar
  24. Sakauchi C, Wakatsuki H, Ichijo H, Hattori K (2017) Pleiotropic properties of ASK1. Biochem Biophys Acta 1861:3030–3038CrossRefGoogle Scholar
  25. Sato SM, Woolley CS (2016) Acute inhibition of neurosteroid estrogen synthesis suppresses status epilepticus in an animal model. ELife 5:e12917CrossRefPubMedPubMedCentralGoogle Scholar
  26. Schraegle WA, Titus JB (2017) The influence of endophenotypic, disease-specific, and environmental variables on the expression of anxiety in pediatric epilepsy. Epilepsy Behav 75:90–96CrossRefPubMedGoogle Scholar
  27. Shinoda S, Skradski SL, Araki T, Schindler CK, Meller R, Lan JQ, Taki W, Simon RP, Henshall DC (2003) Formation of a tumour necrosis factor receptor 1 molecular scaffolding complex and activation of apoptosis signal-regulating kinase 1 during seizure-induced neuronal death. Eur J Neurosci 17:2065–2076CrossRefPubMedGoogle Scholar
  28. Song J, Cheon SY, Lee WT, Park KA, Lee JE (2015) PKA inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulfonamide) attenuates synaptic dysfunction and neuronal cell death following ischemic injury. Neural Plast 2015:374520Google Scholar
  29. Tan CC, Zhang JG, Tan MS, Chen H, Meng DW, Jiang T, Meng XF, Li Y, Sun Z, Li MM, Yu JT, Tan L (2015) NLRP1 inflammasome is activated in patients with medial temporal lobe epilepsy and contributes to neuronal pyroptosis in amygdala kindling-induced rat model. J Neuroinflammation 12:18CrossRefPubMedPubMedCentralGoogle Scholar
  30. Tombini M, Squitti R, Cacciapaglia F, Ventriglia M, Assenza G, Benvenga A, Pellegrino G, Campana C, Assenza F, Siotto M, Pacifici L, Afeltra A, Rossini PM (2013) Inflammation and iron metabolism in adult patients with epilepsy: does a link exist? Epilepsy Res 107:244–252CrossRefPubMedGoogle Scholar
  31. Wang X, Tang X, Li M, Marshall J, Mao Z (2005) Regulation of neuroprotective activity of myocyte-enhancer factor 2 by cAMP-protein kinase A signaling pathway in neuronal survival. J Biol Chem 280:16705–16713CrossRefPubMedGoogle Scholar
  32. Wang QQ, Zhu LJ, Wang XH, Zuo J, He HY, Tian MM, Wang L, Liang GL, Wang Y (2016) Chronic trigeminal nerve stimulation protects against seizures, cognitive impairments, hippocampal apoptosis, and inflammatory responses in epileptic rats. J Mol Neurosci 59:78–89CrossRefPubMedGoogle Scholar
  33. Xiao Z, Peng J, Wu L, Arafat A, Yin F (2017) The effect of IL-1beta on synaptophysin expression and electrophysiology of hippocampal neurons through the PI3K/Akt/mTOR signaling pathway in a rat model of mesial temporal lobe epilepsy. Neurol Res 39:640–648CrossRefPubMedGoogle Scholar
  34. Xie N, Wang C, Wu C, Cheng X, Gao Y, Zhang H, Zhang Y, Lian Y (2016) Mdivi-1 protects epileptic hippocampal neurons from apoptosis via inhibiting oxidative stress and endoplasmic reticulum stress in vitro. Neurochem Res 41:1335–1342CrossRefPubMedGoogle Scholar
  35. Yamamoto A, Schindler CK, Murphy BM, Bellver-Estelles C, So NK, Taki W, Meller R, Simon RP, Henshall DC (2006) Evidence of tumor necrosis factor receptor 1 signaling in human temporal lobe epilepsy. Exp Neurol 202:410–420CrossRefPubMedGoogle Scholar
  36. Yu J, Luo N, Wang Z, Lin W (2017) Current status of epilepsy treatment and efficacy of standard phenobarbital therapy in rural areas of Northern China. Int J Neurosci 127:659–666CrossRefPubMedGoogle Scholar
  37. Zhang TL, Fu JL, Geng Z, Yang JJ, Sun XJ (2012) The neuroprotective effect of losartan through inhibiting AT1/ASK1/MKK4/JNK3 pathway following cerebral I/R in rat hippocampal CA1 region. CNS Neurosci Ther 18:981–987CrossRefPubMedGoogle Scholar
  38. Zhen JL, Chang YN, Qu ZZ, Fu T, Liu JQ, Wang WP (2016) Luteolin rescues pentylenetetrazole-induced cognitive impairment in epileptic rats by reducing oxidative stress and activating PKA/CREB/BDNF signaling. Epilepsy Behav 57:177–184CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Ai-Qin Qi
    • 1
  • Yan-Hui Zhang
    • 2
  • Qin-De Qi
    • 1
  • Ye-Hui Liu
    • 1
  • Jun-Ling Zhu
    • 1
  1. 1.Department of NeurologyLaiwu Hospital Affiliated to Taishan Medical UniversityLaiwuPeople’s Republic of China
  2. 2.Department of NeurologyBeijing Haidian HospitalBeijingPeople’s Republic of China

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