Skip to main content

Advertisement

SpringerLink
Log in

Neural Stem Cell Extracellular Vesicles Carrying YBX1 Inhibited Neuronal Pyroptosis Through Increasing m6A-modified GPR30 Stability and Expression in Ischemic Stroke

  • Research
  • Published:
Translational Stroke Research Aims and scope Submit manuscript

Abstract

Neural stem cell-derived extracellular vesicles (NSC-derived EVs) alleviated ischemic stroke (IS) by suppressing the activation of nucleotide-binding domain leucine-rich repeats family protein 3 (NLRP3) inflammasome and neuronal pyroptosis. However, the specific mechanism needs further investigation. qRT-qPCR, Western blotting, and immunofluorescence detected related gene expression. Immunofluorescent analyzed the expression of Ki-67, βIII-Tubulin (Tuj1), and GFAP. Lactate dehydrogenase (LDH) release and IL-1β and IL-18 levels were analyzed by LDH and ELISA kits. TTC staining evaluated the infarction of brain tissues. Flow cytometric analysis measured caspase-1 activity. M6A methylated RNA immunoprecipitation PCR (MeRIP-PCR) measured methylation levels of G protein-coupled receptor 30 (GPR30). RIP and Co-IP analyzed the interactions of Y box binding protein (YBX1)/GPR30, YBX1/IGF2BP1 and NLRP3/speckle-type POZ protein (SPOP), as well as the ubiquitination levels of NLRP3. NSC-derived EVs inhibited the ischemia–reperfusion (I/R) injury of rats and the neuronal pyroptosis induced by oxygen–glucose deprivation/reoxygenation (OGD/R). Knockdown of EVs carrying YBX1 or GPR30 silencing abolished these inhibiting effects. GPR30 mRNA and IGF2BP1 protein were enriched by YBX1 antibody. YBX1 enhanced the stability of m6A-modified GPR30 by interacting with IGF2BP1 and thus promoting GPR30 expression. Knockdown of IGF2BP1 suppressed the binding between YBX1 and GPR30 mRNA. GPR30 promoted NLRP3 ubiquitination by interacting with SPOP. EVs carrying YBX1 could reduce the infarction of brain tissues and inhibit neuronal pyroptosis in rats with I/R injury. NSC-derived EVs carrying YBX1 increased the stability of m6A-modified GPR30 by interacting with IGF2BP1; the upregulation of GPR30 inhibited the activation of NLRP3 inflammasome through promoting NLRP3 ubiquitination by SPOP, ultimately suppressing the neuronal pyroptosis in IS.

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

Access this article

Log in via an institution

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
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Data Availability

All data generated or analyzed during this study are included in this published article.

References

  1. Feske SK. Ischemic stroke. Am J Med. 2021;134(12):1457–64.

    Article  PubMed  Google Scholar 

  2. Farina M, Vieira LE, Buttari B, Profumo E, Saso L. The Nrf2 pathway in ischemic stroke: a review. Molecules. 2021;26(16):5001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chehaibi K, Trabelsi I, Mahdouani K, Slimane MN. Correlation of oxidative stress parameters and inflammatory markers in ischemic stroke patients. J Stroke Cerebrovasc Dis. 2016;25(11):2585–93.

    Article  PubMed  Google Scholar 

  4. Stanzione R, Cotugno M, Bianchi F, Marchitti S, Forte M, Volpe M, et al. Pathogenesis of ischemic stroke: role of epigenetic mechanisms. Genes (Basel). 2020;11(1):89.

    Article  CAS  PubMed  Google Scholar 

  5. Rabinstein AA. Update on treatment of acute ischemic stroke. Continuum (Minneap Minn). 2020;26(2):268–86.

    PubMed  Google Scholar 

  6. Shi J, Gao W, Shao F. Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci. 2017;42(4):245–54.

    Article  CAS  PubMed  Google Scholar 

  7. Yang Y, Liu PY, Bao W, Chen SJ, Wu FS, Zhu PY. Hydrogen inhibits endometrial cancer growth via a ROS/NLRP3/caspase-1/GSDMD-mediated pyroptotic pathway. BMC Cancer. 2020;20(1):28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Xu P, Hong Y, Xie Y, Yuan K, Li J, Sun R, et al. TREM-1 exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental subarachnoid hemorrhage. Transl Stroke Res. 2021;12(4):643–59.

    Article  CAS  PubMed  Google Scholar 

  9. Jiang Q, Geng X, Warren J, Eugene Paul Cosky E, Kaura S, Stone C, et al. Hypoxia inducible factor-1alpha (HIF-1alpha) mediates NLRP3 inflammasome-dependent-pyroptotic and apoptotic cell death following ischemic stroke. Neuroscience. 2020;448:126–39.

    Article  CAS  PubMed  Google Scholar 

  10. Daadi MM. In vitro assays for neural stem cell differentiation. Methods Mol Biol. 2002;198:149–55.

    PubMed  Google Scholar 

  11. Lees JS, Sena ES, Egan KJ, Antonic A, Koblar SA, Howells DW, et al. Stem cell-based therapy for experimental stroke: a systematic review and meta-analysis. Int J Stroke. 2012;7(7):582–8.

    Article  PubMed  Google Scholar 

  12. Martino G, Pluchino S. The therapeutic potential of neural stem cells. Nat Rev Neurosci. 2006;7(5):395–406.

    Article  CAS  PubMed  Google Scholar 

  13. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang K, Ru J, Zhang H, Chen J, Lin X, Lin Z, et al. Melatonin enhances the therapeutic effect of plasma exosomes against cerebral ischemia-induced pyroptosis through the TLR4/NF-kappaB pathway. Front Neurosci. 2020;14:848.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Zhang G, Zhu Z, Wang H, Yu Y, Chen W, Waqas A, et al. Exosomes derived from human neural stem cells stimulated by interferon gamma improve therapeutic ability in ischemic stroke model. J Adv Res. 2020;24:435–45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Suresh PS, Tsutsumi R, Venkatesh T. YBX1 at the crossroads of non-coding transcriptome, exosomal, and cytoplasmic granular signaling. Eur J Cell Biol. 2018;97(3):163–7.

    Article  CAS  PubMed  Google Scholar 

  17. Tuerxun T, Li X, Lou F, Wang X, Ma L. YBX1 protects against apoptosis induced by oxygen-glucose deprivation/reoxygenation in PC12 cells via activation of the AKT/GSK3beta pathway. Folia Biol (Praha). 2021;67(4):150–7.

    Article  CAS  PubMed  Google Scholar 

  18. Tang H, Zhang Q, Yang L, Dong Y, Khan M, Yang F, et al. GPR30 mediates estrogen rapid signaling and neuroprotection. Mol Cell Endocrinol. 2014;387(1–2):52–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang L, Ma Y, Liu M, Ma Y, Guo H. The effects of various estrogen doses on the proliferation and differentiation of cultured neural stem cells. Gen Physiol Biophys. 2019;38(5):417–25.

    Article  CAS  PubMed  Google Scholar 

  20. Buleon M, Cuny M, Grellier J, Charles PY, Belliere J, Casemayou A, et al. A single dose of estrogen during hemorrhagic shock protects against kidney injury whereas estrogen restoration in ovariectomized mice is ineffective. Sci Rep. 2020;10(1):17240.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Qin Y, Wang C, Xu S, Wu C, Wang S, Pan D, et al. G protein-coupled receptor 30 activation protects hepatic ischemia-reperfusion injury of liver tissue through inhibiting NLRP3 in the rat model. J Histotechnol. 2021;44(1):27–36.

    Article  CAS  PubMed  Google Scholar 

  22. Zhang N, Sun P, Xu Y, Li H, Liu H, Wang L, et al. The GPER1/SPOP axis mediates ubiquitination-dependent degradation of ERalpha to inhibit the growth of breast cancer induced by oestrogen. Cancer Lett. 2021;498:54–69.

    Article  CAS  PubMed  Google Scholar 

  23. Wang B, Dai Z, Gao Q, Liu Y, Gu G, Zheng H. Spop ameliorates diabetic nephropathy through restraining NLRP3 inflammasome. Biochem Biophys Res Commun. 2022;594:131–8.

    Article  CAS  PubMed  Google Scholar 

  24. Pandey RR, Pillai RS. Counting the cuts: MAZTER-Seq quantifies m(6)A levels using a methylation-sensitive ribonuclease. Cell. 2019;178(3):515–7.

    Article  CAS  PubMed  Google Scholar 

  25. Li N, Zhan X. Identification of pathology-specific regulators of m(6)A RNA modification to optimize lung cancer management in the context of predictive, preventive, and personalized medicine. EPMA J. 2020;11(3):485–504.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Wang G, Zhang L, Chen X, Xue X, Guo Q, Liu M, et al. Formylpeptide receptors promote the migration and differentiation of rat neural stem cells. Sci Rep. 2016;6:25946.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the international society for extracellular vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Zan L, Zhang X, Xi Y, Wu H, Song Y, Teng G, et al. Src regulates angiogenic factors and vascular permeability after focal cerebral ischemia-reperfusion. Neuroscience. 2014;262:118–28.

    Article  CAS  PubMed  Google Scholar 

  29. Zhang Y, Zhang Y, Chopp M, Pang H, Zhang ZG, Mahmood A, et al. MiR-17-92 Cluster-enriched exosomes derived from human bone marrow mesenchymal stromal cells improve tissue and functional recovery in rats after traumatic brain injury. J Neurotrauma. 2021;38(11):1535–50.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20(1):84–91.

    Article  CAS  PubMed  Google Scholar 

  31. Alibrahim A, Zhao LY, Bae CY, Barszczyk A, Sun CL, Wang GL, et al. Neuroprotective effects of volume-regulated anion channel blocker DCPIB on neonatal hypoxic-ischemic injury. Acta Pharmacol Sin. 2013;34(1):113–8.

    Article  CAS  PubMed  Google Scholar 

  32. PuzarDominkus P, Stenovec M, Sitar S, Lasic E, Zorec R, Plemenitas A, et al. PKH26 labeling of extracellular vesicles: characterization and cellular internalization of contaminating PKH26 nanoparticles. Biochim Biophys Acta Biomembr. 2018;1860(6):1350–61.

    Article  CAS  Google Scholar 

  33. Huang J, Yang J, Li J, Chen Z, Guo X, Huang S, et al. Association of long noncoding RNA H19 polymorphisms with the susceptibility and clinical features of ischemic stroke in southern Chinese Han population. Metab Brain Dis. 2019;34(4):1011–21.

    Article  CAS  PubMed  Google Scholar 

  34. Wardlaw JM, Murray V, Berge E, del Zoppo G, Sandercock P, Lindley RL, et al. Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated systematic review and meta-analysis. Lancet. 2012;379(9834):2364–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. El Amki M, Lerouet D, Garraud M, Teng F, Beray-Berthat V, Coqueran B, et al. Improved reperfusion and vasculoprotection by the poly(ADP-Ribose)polymerase inhibitor PJ34 after stroke and thrombolysis in mice. Mol Neurobiol. 2018;55(12):9156–68.

    Article  PubMed  Google Scholar 

  36. McKay R. Stem cells in the central nervous system. Science. 1997;276(5309):66–71.

    Article  CAS  PubMed  Google Scholar 

  37. Banerjee S, Williamson D, Habib N, Gordon M, Chataway J. Human stem cell therapy in ischaemic stroke: a review. Age Ageing. 2011;40(1):7–13.

    Article  PubMed  Google Scholar 

  38. Chen L, Zhang G, Gu Y, Guo X. Meta-analysis and systematic review of neural stem cells therapy for experimental ischemia stroke in preclinical studies. Sci Rep. 2016;6:32291.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kalladka D, Sinden J, Pollock K, Haig C, McLean J, Smith W, et al. Human neural stem cells in patients with chronic ischaemic stroke (PISCES): a phase 1, first-in-man study. Lancet. 2016;388(10046):787–96.

    Article  PubMed  Google Scholar 

  40. Zhang GL, Zhu ZH, Wang YZ. Neural stem cell transplantation therapy for brain ischemic stroke: Review and perspectives. World J Stem Cells. 2019;11(10):817–30.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Xin H, Li Y, Chopp M. Exosomes/miRNAs as mediating cell-based therapy of stroke. Front Cell Neurosci. 2014;8:377.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Rong Y, Liu W, Wang J, Fan J, Luo Y, Li L, et al. Neural stem cell-derived small extracellular vesicles attenuate apoptosis and neuroinflammation after traumatic spinal cord injury by activating autophagy. Cell Death Dis. 2019;10(5):340.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Webb RL, Kaiser EE, Jurgielewicz BJ, Spellicy S, Scoville SL, Thompson TA, et al. Human neural stem cell extracellular vesicles improve recovery in a porcine model of ischemic stroke. Stroke. 2018;49(5):1248–56.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Liu X, Zhang M, Liu H, Zhu R, He H, Zhou Y, et al. Bone marrow mesenchymal stem cell-derived exosomes attenuate cerebral ischemia-reperfusion injury-induced neuroinflammation and pyroptosis by modulating microglia M1/M2 phenotypes. Exp Neurol. 2021;341:113700.

    Article  CAS  PubMed  Google Scholar 

  45. Lyabin DN, Eliseeva IA, Ovchinnikov LP. YB-1 protein: functions and regulation. Wiley Interdiscip Rev RNA. 2014;5(1):95–110.

    Article  CAS  PubMed  Google Scholar 

  46. Mordovkina D, Lyabin DN, Smolin EA, Sogorina EM, Ovchinnikov LP, Eliseeva I. Y-Box Binding proteins in mRNP assembly, translation, and stability control. Biomolecules. 2020;10(4):591.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Stratford AL, Habibi G, Astanehe A, Jiang H, Hu K, Park E, et al. Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy. Breast Cancer Res. 2007;9(5):R61.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Shinkai K, Nakano K, Cui L, Mizuuchi Y, Onishi H, Oda Y, et al. Nuclear expression of Y-box binding protein-1 is associated with poor prognosis in patients with pancreatic cancer and its knockdown inhibits tumor growth and metastasis in mice tumor models. Int J Cancer. 2016;139(2):433–45.

    Article  CAS  PubMed  Google Scholar 

  49. Evans MK, Matsui Y, Xu B, Willis C, Loome J, Milburn L, et al. Ybx1 fine-tunes PRC2 activities to control embryonic brain development. Nat Commun. 2020;11(1):4060.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bai N, Zhang Q, Zhang W, Liu B, Yang F, Brann D, et al. G-protein-coupled estrogen receptor activation upregulates interleukin-1 receptor antagonist in the hippocampus after global cerebral ischemia: implications for neuronal self-defense. J Neuroinflammation. 2020;17(1):45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Chang H, Yang J, Wang Q, Zhao J, Zhu R. Role of N6-methyladenosine modification in pathogenesis of ischemic stroke. Expert Rev Mol Diagn. 2022;22(3):295–303.

    Article  CAS  PubMed  Google Scholar 

  52. Zhang Z, Wang Q, Zhao X, Shao L, Liu G, Zheng X, et al. YTHDC1 mitigates ischemic stroke by promoting Akt phosphorylation through destabilizing PTEN mRNA. Cell Death Dis. 2020;11(11):977.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang F, Gong S, Wang T, Li L, Luo H, Wang J, et al. Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice. Theranostics. 2020;10(6):2714–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Akther M, Haque ME, Park J, Kang TB, Lee KH. NLRP3 ubiquitination – a new approach to target NLRP3 inflammasome activation. Int J Mol Sci. 2021;22(16):8780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Fink SL, Cookson BT. Pyroptosis and host cell death responses during Salmonella infection. Cell Microbiol. 2007;9(11):2562–70.

    Article  CAS  PubMed  Google Scholar 

  56. Yan YQ, Fang Y, Zheng R, Pu JL, Zhang BR. NLRP3 Inflammasomes in Parkinson’s disease and their regulation by Parkin. Neuroscience. 2020;446:323–34.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by High level Talents Program of Hainan Natural Science Foundation (822RC865) and the Special project of Hainan Clinical Medical Research Center for Cerebrovascular Diseases (LCXY202206).

Author information

Authors and Affiliations

  1. Department of Neurosurgery, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Hainan Province, Haikou, 570208, People’s Republic of China

    Jun Peng, Jun He, Long Lin, You Li & Ying Xia

Authors
  1. Jun Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun He
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Long Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. You Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ying Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Jun Peng: Conceptualization, Methodology, Validation, Visualization, Writing- Original draft preparation, Investigation, Methodology, Writing- Reviewing and Editing Jun He: Data curation Long Lin: Software You Li: Data curation Ying Xia: Conceptualization, Writing- Original draft preparation, Supervision, Writing- Reviewing and Editing

Corresponding author

Correspondence to Ying Xia.

Ethics declarations

Ethics Approval and Consent to Participate

All animal experiments were permitted by the Animal Experimental Ethics Committee of Haikou Affiliated Hospital of Central South University Xiangya School of Medicine.

Consent for Publication

Not applicable.

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.

Supplementary Information

Below is the link to the electronic supplementary material.

12975_2023_1210_MOESM1_ESM.tif

Supplementary file1. Figure S1 Knockdown of NSCs-derived EVs carrying YBX1 reversed the promoting effects of exosomes on NSC proliferation and differentiation. (A and B), Immunofluorescence of Ki-67 in NSCs incubated with EV-shNC or EV-shYBX1. (C and D), Representative images of pre-mature neuronal (Tuj1) and glial (GFAP) markers in NSCs cultured with EV-shNC or EV-shYBX1. (E-H), The levels of pre-mature cell markers (βIII-tubulin and GFAP) and matured cell markers (Map2 and GS) in NSCs cultured with Exo-shNC or Exo-shYBX1 were measured by qRT-PCR. Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis was carried out by one-way ANOVA (TIF 4893 KB)

Supplementary file2 (DOCX 3746 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Peng, J., He, J., Lin, L. et al. Neural Stem Cell Extracellular Vesicles Carrying YBX1 Inhibited Neuronal Pyroptosis Through Increasing m6A-modified GPR30 Stability and Expression in Ischemic Stroke. Transl. Stroke Res. (2023). https://doi.org/10.1007/s12975-023-01210-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12975-023-01210-z

Keywords

Search

Navigation