Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 1.736
5-Year Impact Factor – 2.135
Index Copernicus  – 168.52
MEiN – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

Download original text (EN)

Advances in Clinical and Experimental Medicine

Ahead of print

doi: 10.17219/acem/157071

Publication type: original article

Language: English

License: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

Download citation:

  • BIBTEX (JabRef, Mendeley)
  • RIS (Papers, Reference Manager, RefWorks, Zotero)

Cite as:

Li Y, Sun L, Gao G, Wang X, Zhang X. Protective effect of basic helix-loop-helix family member e40 on cerebral ischemia/reperfusion injury: Inhibition of apoptosis via repressing the transcription of pleckstrin homology-like domain family A, member 1 [published online as ahead of print on March 7, 2023]. Adv Clin Exp Med. 2023. doi:10.17219/acem/157071

Protective effect of basic helix-loop-helix family member e40 on cerebral ischemia/reperfusion injury: Inhibition of apoptosis via repressing the transcription of pleckstrin homology-like domain family A, member 1

Li Sun1,A,B,C,D,E, Guangsheng Gao1,A,C,E, Xingsheng Wang2,B,C,D, Xinxin Zhang2,B,C,D, Yun Li1,A,E,F

1 Department of Critical Medicine, Jinan Central Hospital, Shandong University, China

2 Department of Emergency Medicine, Graduate School, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China


Background. During ischemic stroke treatment, cerebral ischemia/reperfusion (I/R) injury results in neuronal cell death and neurological dysfunctions in brain. Previous studies indicate that basic helix-loop-helix family member e40 (BHLHE40) exerts protective effects on the pathology of neurogenic diseases. However, the protective function of BHLHE40 in I/R is unclear.
Objectives. This study aimed to explore the expression, role and potential mechanism of BHLHE40 after ischemia.
Material and Methods. We established models of I/R injury in rats and of oxygen-glucose deprivation/reoxygenation (OGD/R) in primary hippocampal neurons. Nissl and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to detect neuronal injury and apoptosis. Immunofluorescence was used to detect BHLHE40 expression. Cell viability and cell damage measurements were conducted using Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) assay. The regulation of BHLHE40 to pleckstrin homology-like domain family A, member 1 (PHLDA1) was assessed using the dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay.
Results. Cerebral I/R rats exhibited severe neuronal loss and apoptosis in hippocampal cornu Ammonis 1 (CA1) region, accompanied by downregulated BHLHE40 expression at both mRNA and protein levels, indicating that BHLHE40 may regulate the apoptosis of hippocampal neurons. The function of BHLHE40 in neuronal apoptosis during cerebral I/R was further explored by establishing an OGD/R model in vitro. Low expression of BHLHE40 was also observed in neurons treated with OGD/R. The OGD/R administration inhibited cell viability and enhanced cell apoptosis in hippocampal neurons, whereas BHLHE40 overexpression reversed those changes. Mechanistically, we demonstrated that BHLHE40 could repress PHLDA1 transcription by binding to PHLDA1 promoter. The PHLDA1 is a facilitator of neuronal damage in brain I/R injury and its upregulation reversed the effects caused by BHLHE40 overexpression in vitro.
Conclusion. The transcription factor BHLHE40 may protect against brain I/R injury through repressing cell damage via regulating PHLDA1 transcription. Thus, BHLHE40 may be a candidate gene for further study of molecular or therapeutic targets for I/R.

Key words

BHLHE40, PHLDA1, apoptosis, cerebral I/R injury, OGD/R

Graphical abstract

Graphical abstracts

References (51)

  1. Campbell BCV, De Silva DA, Macleod MR, et al. Ischaemic stroke. Nat Rev Dis Primers. 2019;5(1):70. doi:10.1038/s41572-019-0118-8
  2. Turner RC, Dodson SC, Rosen CL, Huber JD. The science of cerebral ischemia and the quest for neuroprotection. Navigating past failure to future success: A review. J Neurosurg. 2013;118(5):1072–1085. doi:10.3171/2012.11.JNS12408
  3. Kondziella D, Cortsen M, Eskesen V, et al. Update on acute endovascular and surgical stroke treatment. Acta Neurol Scand. 2013;127(1):1–9. doi:10.1111/j.1600-0404.2012.01702.x
  4. Chamorro Á, Dirnagl U, Urra X, Planas AM. Neuroprotection in acute stroke: Targeting excitotoxicity, oxidative and nitrosative stress, and inflammation. Lancet Neurol. 2016;15(8):869–881. doi:10.1016/S1474-4422(16)00114-9
  5. Xu J, Kong X, Xiu H, Dou Y, Wu Z, Sun P. Combination of curcumin and vagus nerve stimulation attenuates cerebral ischemia/reperfusion injury-induced behavioral deficits. Biomed Pharmacother. 2018;103:614–620. doi:10.1016/j.biopha.2018.04.069
  6. Martos D, Tuka B, Tanaka M, Vécsei L, Telegdy G. Memory enhancement with kynurenic acid and its mechanisms in neurotransmission. Biomedicines. 2022;10(4):849. doi:10.3390/biomedicines10040849
  7. Spekker E, Tanaka M, Szabó Á, Vécsei L. Neurogenic inflammation: The participant in migraine and recent advancements in translational research. Biomedicines. 2021;10(1):76. doi:10.3390/biomedicines10010076
  8. Tanaka M, Tóth F, Polyák H, Szabó Á, Mándi Y, Vécsei L. Immune influencers in action: Metabolites and enzymes of the tryptophan-kynurenine metabolic pathway. Biomedicines. 2021;9(7):734. doi:10.3390/biomedicines9070734
  9. Yan HF, Tuo QZ, Yin QZ, Lei P. The pathological role of ferroptosis in ischemia/reperfusion-related injury. Zool Res. 2020;41(3):220–230. doi:10.24272/j.issn.2095-8137.2020.042
  10. Ma D, Qiao J, Qu Q, He F, Chen W, Yu B. Weighted gene co-expression network analysis to investigate the key genes implicated in global brain ischemia/reperfusion injury in rats. Adv Clin Exp Med. 2020;29(6):649–659. doi:10.17219/acem/121918
  11. Bredesen DE, Rao RV, Mehlen P. Cell death in the nervous system. Nature. 2006;443(7113):796–802. doi:10.1038/nature05293
  12. Zille M, Farr TD, Przesdzing I, et al. Visualizing cell death in experimental focal cerebral ischemia: Promises, problems and perspectives. J Cereb Blood Flow Metab. 2012;32(2):213–231. doi:10.1038/jcbfm.2011.150
  13. Qin AP, Liu CF, Qin YY, et al. Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia. Autophagy. 2010;6(6):738–753. doi:10.4161/auto.6.6.12573
  14. Naito MG, Xu D, Amin P, et al. Sequential activation of necroptosis and apoptosis cooperates to mediate vascular and neural pathology in stroke. Proc Natl Acad Sci U S A. 2020;117(9):4959–4970. doi:10.1073/pnas.1916427117
  15. Degterev A, Huang Z, Boyce M, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005;1(2):112–119. doi:10.1038/nchembio711
  16. Yuan J, Yankner BA. Apoptosis in the nervous system. Nature. 2000;407(6805):802–809. doi:10.1038/35037739
  17. Sethuraman A, Brown M, Krutilina R, et al. BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion. Breast Cancer Res. 2018;20(1):117. doi:10.1186/s13058-018-1046-3
  18. Tian J, Wu J, Chen X, et al. BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver. eLife. 2018;7:e36826. doi:10.7554/eLife.36826
  19. Yu F, Sharma S, Jankovic D, et al. The transcription factor Bhlhe40 is a switch of inflammatory versus anti-inflammatory Th1 cell fate determination. J Exp Med. 2018;215(7):1813–1821. doi:10.1084/jem.20170155
  20. Ow JR, Tan YH, Jin Y, Bahirvani AG, Taneja R. Stra13 and Sharp-1, the non-Grouchy regulators of development and disease. Curr Top Dev Biol. 2014;110:317–338. doi:10.1016/B978-0-12-405943-6.00009-9
  21. Li Y, Xie M, Yang J, et al. The expression of antiapoptotic protein survivin is transcriptionally upregulated by DEC1 primarily through multiple sp1 binding sites in the proximal promoter. Oncogene. 2006;25(23):3296–3306. doi:10.1038/sj.onc.1209363
  22. Li Y, Zhang H, Xie M, et al. Abundant expression of Dec1/stra13/sharp2 in colon carcinoma: Its antagonizing role in serum deprivation-induced apoptosis and selective inhibition of procaspase activation. Biochem J. 2002;367(2):413–422. doi:10.1042/bj20020514
  23. Sato F, Kawamoto T, Fujimoto K, et al. Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation: Interaction with BMAL1. Eur J Biochem. 2004;271(22):4409–4419. doi:10.1111/j.1432-1033.2004.04379.x
  24. Qian Y, Zhang J, Jung YS, Chen X. DEC1 coordinates with HDAC8 to differentially regulate TAp73 and ΔNp73 expression. PLoS One. 2014;9(1):e84015. doi:10.1371/journal.pone.0084015
  25. Kanda M, Yamanaka H, Kojo S, et al. Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells. Proc Natl Acad Sci U S A. 2016;113(24):E3394–E3402. doi:10.1073/pnas.1604178113
  26. Nagai MA. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and cancer. Biomed Rep. 2016;4(3):275–281. doi:10.3892/br.2016.580
  27. Xu J, Bi G, Luo Q, et al. PHLDA1 modulates the endoplasmic reticulum stress response and is required for resistance to oxidative stress-induced cell death in human ovarian cancer cells. J Cancer. 2021;12(18):5486–5493. doi:10.7150/jca.45262
  28. Murata T, Sato T, Kamoda T, Moriyama H, Kumazawa Y, Hanada N. Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: Role of PHLDA1 as an apoptosis suppressor. Exp Cell Res. 2014;320(2):247–257. doi:10.1016/j.yexcr.2013.10.023
  29. Guo Y, Jia P, Chen Y, et al. PHLDA1 is a new therapeutic target of oxidative stress and ischemia reperfusion-induced myocardial injury. Life Sci. 2020;245:117347. doi:10.1016/j.lfs.2020.117347
  30. Luo YH, Huang ZT, Zong KZ, et al. miR-194 ameliorates hepatic ischemia/reperfusion injury via targeting PHLDA1 in a TRAF6-dependent manner. Int Immunopharmacol. 2021;96:107604. doi:10.1016/j.intimp.2021.107604
  31. Fornes O, Castro-Mondragon JA, Khan A, et al. JASPAR 2020: Update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2019;48(D1):D87–D92. doi:10.1093/nar/gkz1001
  32. Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC. Neuronal cell death. Physiol Rev. 2018;98(2):813–880. doi:10.1152/physrev.00011.2017
  33. Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science. 1998;281(5381):1312–1316. doi:10.1126/science.281.5381.1312
  34. Yuan J. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme. Cell. 1993;75(4):641–652. doi:10.1016/0092-8674(93)90485-9
  35. Graham SH, Chen J, Clark RSB. Bcl-2 family gene products in cerebral ischemia and traumatic brain injury. J Neurotrauma. 2000;17(10):831–841. doi:10.1089/neu.2000.17.831
  36. Deckwerth TL, Elliott JL, Knudson CM, Johnson EM, Snider WD, Korsmeyer SJ. BAX is required for neuronal death after trophic factor deprivation and during development. Neuron. 1996;17(3):401–411. doi:10.1016/S0896-6273(00)80173-7
  37. Dutta C, Day T, Kopp N, et al. BCL2 suppresses PARP1 function and nonapoptotic cell death. Cancer Res. 2012;72(16):4193–4203. doi:10.1158/0008-5472.CAN-11-4204
  38. Gunduz Z, Aktas F, Vatansev H, Solmaz M, Erdogan E. Effects of amantadine and topiramate on neuronal damage in rats with experimental cerebral ischemia-reperfusion. Adv Clin Exp Med. 2021;30(10):1013–1023. doi:10.17219/acem/138327
  39. Battaglia S, Fabius JH, Moravkova K, Fracasso A, Borgomaneri S. The neurobiological correlates of gaze perception in healthy individuals and neurologic patients. Biomedicines. 2022;10(3):627. doi:10.3390/biomedicines10030627
  40. Huynh JP, Lin CC, Kimmey JM, et al. Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection. J Exp Med. 2018;215(7):1823–1838. doi:10.1084/jem.20171704
  41. Peng Y, Liu W, Xiong J, et al. Downregulation of differentiated embryonic chondrocytes 1 (DEC1) is involved in 8-methoxypsoralen-induced apoptosis in HepG2 cells. Toxicology. 2012;301(1–3):58–65. doi:10.1016/j.tox.2012.06.022
  42. Hamilton KA, Wang Y, Raefsky SM, et al. Mice lacking the transcriptional regulator Bhlhe40 have enhanced neuronal excitability and impaired synaptic plasticity in the hippocampus. PLoS One. 2018;13(5):e0196223. doi:10.1371/journal.pone.0196223
  43. Zhu Z, Wang YW, Ge DH, et al. Downregulation of DEC1 contributes to the neurotoxicity induced by MPP+ by suppressing PI3K/Akt/GSK3β pathway. CNS Neurosci Ther. 2017;23(9):736–747. doi:10.1111/cns.12717
  44. Park CG, Lee SY, Kandala G, Lee SY, Choi Y. A novel gene product that couples TCR signaling to Fas(CD95) expression in activation-induced cell death. Immunity. 1996;4(6):583–591. doi:10.1016/S1074-7613(00)80484-7
  45. Yang F, Chen R. Loss of PHLDA1 has a protective role in OGD/R-injured neurons via regulation of the GSK-3β/Nrf2 pathway. Hum Exp Toxicol. 2021;40(11):1909–1920. doi:10.1177/09603271211014596
  46. Battaglia S, Orsolini S, Borgomaneri S, Barbieri R, Diciotti S, di Pellegrino G. Characterizing cardiac autonomic dynamics of fear learning in humans. Psychophysiology. 2022;59(12):e14122. doi:10.1111/psyp.14122
  47. Tanaka M, Spekker E, Szabó Á, Polyák H, Vécsei L. Modelling the neurodevelopmental pathogenesis in neuropsychiatric disorders: Bioactive kynurenines and their analogues as neuroprotective agents. In celebration of 80th birthday of Professor Peter Riederer. J Neural Transm. 2022;129(5–6):627–642. doi:10.1007/s00702-022-02513-5
  48. Battaglia S, Thayer JF. Functional interplay between central and autonomic nervous systems in human fear conditioning. Trends Neurosci. 2022;45(7):504–506. doi:10.1016/j.tins.2022.04.003
  49. Kubis N. Non-invasive brain stimulation to enhance post-stroke recovery. Front Neural Circuits. 2016;10:56. doi:10.3389/fncir.2016.00056
  50. Borgomaneri S, Battaglia S, Sciamanna G, Tortora F, Laricchiuta D. Memories are not written in stone: Re-writing fear memories by means of non-invasive brain stimulation and optogenetic manipulations. Neurosci Biobehav Rev. 2021;127:334–352. doi:10.1016/j.neubiorev.2021.04.036
  51. Tanaka M, Vécsei L. Editorial of Special Issue “Crosstalk between Depression, Anxiety, and Dementia: Comorbidity in Behavioral Neurology and Neuropsychiatry.” Biomedicines. 2021;9(5):517. doi:10.3390/biomedicines9050517