Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury
Abstract Background Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynami...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-08-01
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| Series: | Cell Communication and Signaling |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12964-023-01211-3 |
| _version_ | 1797557665501544448 |
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| author | Zhang-Li Yuan Yan-Zi Mo De-Li Li Lu Xie Meng-Hua Chen |
| author_facet | Zhang-Li Yuan Yan-Zi Mo De-Li Li Lu Xie Meng-Hua Chen |
| author_sort | Zhang-Li Yuan |
| collection | DOAJ |
| description | Abstract Background Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI. Methods SH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett’s T3 test (for unequal homogeneity) were used for statistical tests. Results ERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed. Conclusion Inhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model. Video Abstract |
| first_indexed | 2024-03-10T17:20:58Z |
| format | Article |
| id | doaj.art-fc7808f90e0540f3aafb14d1bbcff534 |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-03-10T17:20:58Z |
| publishDate | 2023-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-fc7808f90e0540f3aafb14d1bbcff5342023-11-20T10:22:09ZengBMCCell Communication and Signaling1478-811X2023-08-0121111810.1186/s12964-023-01211-3Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injuryZhang-Li Yuan0Yan-Zi Mo1De-Li Li2Lu Xie3Meng-Hua Chen4Department of Emergency Medicine, The Second Affiliated Hospital of Guangxi Medical UniversityIntensive Care Unit, The Second Affiliated Hospital of Guangxi Medical UniversityGuangxi Medical UniversityGuangxi Medical UniversityIntensive Care Unit, The Second Affiliated Hospital of Guangxi Medical UniversityAbstract Background Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI. Methods SH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett’s T3 test (for unequal homogeneity) were used for statistical tests. Results ERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed. Conclusion Inhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model. Video Abstracthttps://doi.org/10.1186/s12964-023-01211-3ERKDrp1Mfn2Mitochondrial dynamicsAutophagyOGD/R |
| spellingShingle | Zhang-Li Yuan Yan-Zi Mo De-Li Li Lu Xie Meng-Hua Chen Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury Cell Communication and Signaling ERK Drp1 Mfn2 Mitochondrial dynamics Autophagy OGD/R |
| title | Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury |
| title_full | Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury |
| title_fullStr | Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury |
| title_full_unstemmed | Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury |
| title_short | Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury |
| title_sort | inhibition of erk downregulates autophagy via mitigating mitochondrial fragmentation to protect sh sy5y cells from ogd r injury |
| topic | ERK Drp1 Mfn2 Mitochondrial dynamics Autophagy OGD/R |
| url | https://doi.org/10.1186/s12964-023-01211-3 |
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