Skip to main content

Advertisement

Log in

Autophagy Inhibition by ATG3 Knockdown Remits Oxygen–Glucose Deprivation/Reoxygenation-Induced Injury and Inflammation in Brain Microvascular Endothelial Cells

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Autophagy participates in the development of cerebral ischemia stroke. Autophagy-related 3 (ATG3), an important autophagy regulator, was reported to be upregulated in a rat model of cerebral ischemia/reperfusion (CI/R) injury and an oxygen–glucose deprivation/reoxygenation (OGD/R) cell model. However, the detailed role of ATG3 in CI/R injury remains elusive. An in vitro cellular model was established to mimic CI/R injury by exposing hBMECs and bEnd.3 cells to OGD/R. OGD/R-induced injury were evaluated by cell counting kit-8 (CCK-8), LDH release assay, caspase-3 activity assay and TUNEL assay. Inflammation was assessed by detecting mRNA expression and concentrations of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) using qRT-PCR and ELISA, respectively. The protein levels of ATG3, light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were determined by western blot analysis. We successfully established an in vitro OGD/R injury model using hBMECs and bEnd.3 cells. ATG3 was time-dependently upregulated and ATG3 knockdown inhibited autophagy in OGD/R-challenged brain microvascular endothelial cells. Moreover, autophagy inhibition by ATG3 interference attenuated OGD/R-induced viability inhibition and increase of LDH release, caspase-3 activity, programmed cell death, and production of IL-1β, IL-6 and TNF-α. Inhibition of autophagy by ATG3 silencing activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in OGD/R-challenged brain microvascular endothelial cells. Furthermore, inhibition of the PI3K/Akt pathway reversed the protective effects of ATG3 silencing on OGD/R-induced injury and inflammation. In conclusion, autophagy inhibition by ATG3 knockdown remitted OGD/R-induced injury and inflammation in brain microvascular endothelial cells via activation of the PI3K/Akt 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
Fig. 7
Fig. 8

Similar content being viewed by others

Data Availability

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Ribeiro PW, Cola PC, Gatto AR, da Silva RG, Luvizutto GJ, Braga GP, Schelp AO, de Arruda Henry MA, Bazan R (2015) Relationship between dysphagia, national institutes of health stroke scale score, and predictors of pneumonia after ischemic stroke. J Stroke Cerebrovasc Dis 24:2088–2094

    Article  PubMed  Google Scholar 

  2. Poisson SN, Schardt TQ, Dingman A, Bernard TJ (2014) Etiology and treatment of arterial ischemic stroke in children and young adults. Curr Treat Options Neurol 16:315

    Article  PubMed  Google Scholar 

  3. Lakhan SE, Kirchgessner A, Hofer M (2009) Inflammatory mechanisms in ischemic stroke: therapeutic approaches. J Transl Med 7:97

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Molina CA, Saver JL (2005) Extending reperfusion therapy for acute ischemic stroke: emerging pharmacological, mechanical, and imaging strategies. Stroke 36:2311–2320

    Article  PubMed  Google Scholar 

  5. Johnston SC, Mendis S, Mathers CD (2009) Global variation in stroke burden and mortality: estimates from monitoring, surveillance, and modelling. Lancet Neurol 8:345–354

    Article  PubMed  Google Scholar 

  6. Nour M, Scalzo F, Liebeskind DS (2013) Ischemia-reperfusion injury in stroke. Interv Neurol 1:185–199

    Article  PubMed  PubMed Central  Google Scholar 

  7. Chamorro A, Hallenbeck J (2006) The harms and benefits of inflammatory and immune responses in vascular disease. Stroke 37:291–293

    Article  PubMed  PubMed Central  Google Scholar 

  8. Zille M, Ikhsan M, Jiang Y, Lampe J, Wenzel J, Schwaninger M (2019) The impact of endothelial cell death in the brain and its role after stroke: a systematic review. Cell Stress 3:330–347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang G, Wang N, Seto SW, Chang D, Liang H (2018) Hydroxysafflor yellow a protects brain microvascular endothelial cells against oxygen glucose deprivation/reoxygenation injury: involvement of inhibiting autophagy via class I PI3K/Akt/mTOR signaling pathway. Brain Res Bull 140:243–257

    Article  CAS  PubMed  Google Scholar 

  10. Zhang QY, Wang ZJ, Sun DM, Wang Y (2017) Novel therapeutic effects of leonurine on ischemic stroke: new mechanisms of BBB integrity. Oxid Med Cell Longe 2017:7150376

    Google Scholar 

  11. Wang Y, Cai S, Yin L, Shi K, Xia X, Zhou Y, Yu J, Zhou J (2015) Tomato HsfA1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy. Autophagy 11:2033–2047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Menzies FM, Fleming A, Rubinsztein DC (2015) Compromised autophagy and neurodegenerative diseases. Nat Rev Neurosci 16:345–357

    Article  CAS  PubMed  Google Scholar 

  13. Corti O, Blomgren K, Poletti A, Beart PM (2020) Autophagy in neurodegeneration: new insights underpinning therapy for neurological diseases. J Neurochem 154:354–371

    Article  CAS  PubMed  Google Scholar 

  14. Wang P, Shao BZ, Deng Z, Chen S, Yue Z, Miao CY (2018) Autophagy in ischemic stroke. Prog Neurobiol 163–164:98–117

    Article  PubMed  CAS  Google Scholar 

  15. Chen W, Sun Y, Liu K, Sun X (2014) Autophagy: a double-edged sword for neuronal survival after cerebral ischemia. Neural Regen Res 9:1210–1216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tekirdag KA, Korkmaz G, Ozturk DG, Agami R, Gozuacik D (2013) MIR181A regulates starvation- and rapamycin-induced autophagy through targeting of ATG5. Autophagy 9:374–385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jain K, Paranandi KS, Sridharan S, Basu A (2013) Autophagy in breast cancer and its implications for therapy. Am J Cancer Res 3:251–265

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E, Ohsumi M, Noda T, Ohsumi Y (2000) A ubiquitin-like system mediates protein lipidation. Nature 408:488–492

    Article  CAS  PubMed  Google Scholar 

  19. Murrow L, Malhotra R, Debnath J (2015) ATG12-ATG3 interacts with Alix to promote basal autophagic flux and late endosome function. Nat Cell Biol 17:300–310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kroemer G, Mariño G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang C, Liu M, Pan Y, Bai B, Chen J (2017) Global gene expression profile of cerebral ischemia-reperfusion injury in rat MCAO model. Oncotarget 8:74607–74622

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ecker N, Mor A, Journo D, Abeliovich H (2010) Induction of autophagic flux by amino acid deprivation is distinct from nitrogen starvation-induced macroautophagy. Autophagy 6:879–890

    Article  CAS  PubMed  Google Scholar 

  23. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bjørkøy G, Lamark T, Johansen T (2006) p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy 2:138–139

    Article  PubMed  Google Scholar 

  25. Breivik L, Jensen A, Guvåg S, Aarnes EK, Aspevik A, Helgeland E, Hovland S, Brattelid T, Jonassen AK (2015) B-type natriuretic peptide expression and cardioprotection is regulated by Akt dependent signaling at early reperfusion. Peptides 66:43–50

    Article  CAS  PubMed  Google Scholar 

  26. Zou H, Pan KH, Pan HY, Huang DS, Zheng MH (2015) Cerebral hemorrhage due to tuberculosis meningitis: a rare case report and literature review. Oncotarget 6:45005–45009

    Article  PubMed  PubMed Central  Google Scholar 

  27. Janardhan V, Qureshi AI (2004) Mechanisms of ischemic brain injury. Curr Cardiol Rep 6:117–123

    Article  PubMed  Google Scholar 

  28. Frugier T, Taylor JM, McLean C, Bye N, Beart PM, Devenish RJ, Crack PJ (2016) Evidence for the recruitment of autophagic vesicles in human brain after stroke. Neurochem Int 96:62–68

    Article  CAS  PubMed  Google Scholar 

  29. Wei K, Wang P, Miao CY (2012) A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury. CNS Neurosci Ther 18:879–886

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rami A, Kögel D (2008) Apoptosis meets autophagy-like cell death in the ischemic penumbra: two sides of the same coin? Autophagy 4:422–426

    Article  CAS  PubMed  Google Scholar 

  31. Wolf MS, Bayır H, Kochanek PM, Clark RSB (2019) The role of autophagy in acute brain injury: a state of flux? Neurobiol Dis 122:9–15

    Article  PubMed  Google Scholar 

  32. Zhang X, Yan H, Yuan Y, Gao J, Shen Z, Cheng Y, Shen Y, Wang RR, Wang X, Hu WW, Wang G, Chen Z (2013) Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance. Autophagy 9:1321–1333

    Article  CAS  PubMed  Google Scholar 

  33. Zheng Y, Hou J, Liu J, Yao M, Li L, Zhang B, Zhu H, Wang Z (2014) Inhibition of autophagy contributes to melatonin-mediated neuroprotection against transient focal cerebral ischemia in rats. J Pharmacol Sci 124:354–364

    Article  CAS  PubMed  Google Scholar 

  34. 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 4:762–769

    Article  CAS  PubMed  Google Scholar 

  35. Lundberg M, Curbo S, Bohman H, Agartz I, Ögren SO, Patrone C, Mansouri S (2020) Clozapine protects adult neural stem cells from ketamine-induced cell death in correlation with decreased programmed cell death and autophagy. Biosci Rep 40:BSR20193156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Murrow L, Debnath J (2015) ATG12-ATG3 connects basal autophagy and late endosome function. Autophagy 11:961–962

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Shaw RJ, Cantley LC (2006) Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441:424–430

    Article  CAS  PubMed  Google Scholar 

  38. Hou S, Zhao MM, Shen PP, Liu XP, Sun Y, Feng JC (2016) Neuroprotective effect of salvianolic acids against cerebral ischemia/reperfusion injury. Int J Mol Sci 17:1190

    Article  PubMed Central  CAS  Google Scholar 

  39. Kamel EO, Hassanein EHM, Ahmed MA, Ali FEM (2020) Perindopril ameliorates hepatic ischemia reperfusion injury via regulation of NF-κB-p65/TLR-4, JAK1/STAT-3, Nrf-2, and PI3K/Akt/mTOR signaling pathways. Anat Rec 303:1935–1949

    Article  CAS  Google Scholar 

  40. Zhao H, Sapolsky RM, Steinberg GK (2006) Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke. Mol Neurobiol 34:249–270

    Article  CAS  PubMed  Google Scholar 

  41. Lu C, Liu L, Chen Y, Ha T, Kelley J, Schweitzer J, Kalbfleisch JH, Kao RL, Williams DL, Li C (2011) TLR2 ligand induces protection against cerebral ischemia/reperfusion injury via activation of phosphoinositide 3-kinase/Akt signaling. J Immunol 187:1458–1466

    Article  CAS  PubMed  Google Scholar 

  42. Chen A, Xiong LJ, Tong Y, Mao M (2013) Neuroprotective effect of brain-derived neurotrophic factor mediated by autophagy through the PI3K/Akt/mTOR pathway. Mol Med Rep 8:1011–1016

    Article  CAS  PubMed  Google Scholar 

  43. Zhang Y, Li F, Liu L, Jiang H, Jiang X, Ge X, Cao J, Wang Z, Zhang L, Wang Y (2016) Salinomycin-induced autophagy blocks apoptosis via the ATG3/AKT/mTOR signaling axis in PC-3 cells. Life Sci 207:451–460

    Article  CAS  Google Scholar 

  44. He H, Zeng Q, Huang G, Lin Y, Lin H, Liu W, Lu P (2019) Bone marrow mesenchymal stem cell transplantation exerts neuroprotective effects following cerebral ischemia/reperfusion injury by inhibiting autophagy via the PI3K/Akt pathway. Brain Res 1707:124–132

    Article  CAS  PubMed  Google Scholar 

  45. Zhang Y, He Q, Yang M, Hua S, Ma Q, Guo L, Wu X, Zhang C, Fu X, Liu J (2020) Dichloromethane extraction from Piper nigrum L. and P longum L. to mitigate ischemic stroke by activating the AKT/mTOR signaling pathway to suppress autophagy. Brain Res 1749:1047

    Article  CAS  Google Scholar 

  46. Yu S, Yu M, Bu Z, He P, Feng J (2020) FKBP5 exacerbates impairments in cerebral ischemic stroke by inducing autophagy via the AKT/FOXO3 pathway. Front Cell Neurosci 14:193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hongjuan Huang.

Ethics declarations

Conflict of interest

None.

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

Peng, Z., Ji, D., Qiao, L. et al. Autophagy Inhibition by ATG3 Knockdown Remits Oxygen–Glucose Deprivation/Reoxygenation-Induced Injury and Inflammation in Brain Microvascular Endothelial Cells. Neurochem Res 46, 3200–3212 (2021). https://doi.org/10.1007/s11064-021-03423-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-021-03423-w

Keywords

Navigation