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. 2022 Apr 26:16:888836.
doi: 10.3389/fncel.2022.888836. eCollection 2022.

14,15-Epoxyeicosatrienoic Acid Protect Against Glucose Deprivation and Reperfusion-Induced Cerebral Microvascular Endothelial Cells Injury by Modulating Mitochondrial Autophagy via SIRT1/FOXO3a Signaling Pathway and TSPO Protein

Youyang Qu  1 , Jinlu Cao  1 , Di Wang  1 , Shu Wang  1 , Yujie Li  1 , Yulan Zhu  1
Affiliations

14,15-Epoxyeicosatrienoic Acid Protect Against Glucose Deprivation and Reperfusion-Induced Cerebral Microvascular Endothelial Cells Injury by Modulating Mitochondrial Autophagy via SIRT1/FOXO3a Signaling Pathway and TSPO Protein

Youyang Qu et al. Front Cell Neurosci. .

Abstract

Neurovascular system plays a vital role in controlling the blood flow into brain parenchymal tissues. Additionally, it also facilitates the metabolism in neuronal biological activities. Cerebral microvascular endothelial cells (MECs) are involved in mediating progression of the diseases related to cerebral vessels, including stroke. Arachidonic acid can be transformed into epoxyeicosatrienoic acids (EETs) under the catalysis by cytochrome P450 epoxygenase. We have reported that EETs could protect neuronal function. In our research, the further role of 14,15-EET in the protective effects of cerebral MECs and the potential mechanisms involved in oxygen glucose deprivation and reperfusion (OGD/R) were elucidated. In our study, we intervened the SIRT1/FOXO3a pathway and established a TSPO knock down model by using RNA interference technique to explore the cytoprotective role of 14,15-EET in OGD/R injury. Cerebral MECs viability was remarkably reduced after OGD/R treatment, however, 14,15-EET could reverse this effect. To further confirm whether 14,15-EET was mediated by SIRT1/FOXO3a signaling pathway and translocator protein (TSPO) protein, we also detected autophagy-related proteins, mitochondrial membrane potential, apoptosis indicators, oxygen free radicals, etc. It was found that 14,15-EET could regulate the mitophagy induced by OGD/R. SIRT1/FOXO3a signaling pathway and TSPO regulation were related to the protective role of 14,15-EET in cerebral MECs. Moreover, we also explored the potential relationship between SIRT1/FOXO3a signaling pathway and TSPO protein. Our study revealed the protective role and the potential mechanisms of 14,15-EET in cerebral MECs under OGD/R condition.

Keywords: 14; 15-EET; SIRT1/FOXO3a; TSPO; cerebrovascular disease; epoxyeicosatrienoic acids; mitophagy.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
14,15-EET promoted the survival rate and inhibited the apoptosis of cerebral MECs under OGD/R condition. (A) Viability of cerebral MECs. 1 μM of 14,15-EET was used to treat cerebral MECs at 30 min, followed by OGD/R. After OGD/R, cell viability was measured by using CCK-8 assay. (B) Apoptosis of cerebral MECs. Apoptotic ratio = the count of PI-negative and annexin V-positive cells/total count of the cells used in this assay. (C) Representative results of flow cytometry illustrated annexin V-FITC and PI staining. The results were expressed as average values ± standard deviation; *P < 0.05, in comparison with the control groups; #P < 0.05, in comparison with OGD/R; $P < 0.05, in comparison with 14,15-EET + OGD/R.
FIGURE 2
FIGURE 2
The regulation of mitochondrial transmembrane potential (ΔΨm) by 14,15-EET. (A) JC-1 fluorescence mitochondrial imaging. Red fluorescence/green fluorescence ratio showed the mitochondrial membrane potential. JC-1 staining assay was used to determine the change in ΔΨm after the cells were incubated for 18 h. (B) The ratio of aggregates/monomers fluorescence intensity in each group. The results were expressed as average values ± standard deviation; *P < 0.05, in comparison with the control groups; #P < 0.05, in comparison with OGD/R; $P < 0.05, in comparison with 14,15-EET + OGD/R.
FIGURE 3
FIGURE 3
Expression levels of SIRT1/FOXO3A, and autophagy-related proteins in human cerebral MECs treated by 14,15-EET under OGD/R. (A) Representative results of the densitometric ratio of SIRT1 levels in cerebral MECs undergoing different treatments. (B) Representative images of the densitometric ratio of FOXO3a expression in cerebral MECs undergoing different treatments. (C) Representative results of the densitometric ratio of LC3 in cerebral MECs under different conditions. (D) Representative results of the densitometric ratio of P62 levels in cerebral MECs undergoing different treatments. (E) Representative images of LC3 and P62 expression in cerebral MECs undergoing different treatments. The results were described as average values ± standard deviation; *P < 0.05, in comparison with the control groups; #P < 0.05, in comparison with OGD/R; $P < 0.05, in comparison with 14,15-EET + OGD/R.
FIGURE 4
FIGURE 4
Expression levels of TSPO, and autophagy-related proteins in human cerebral MECs treated by EETs under OGD/R. (A) Representative results of the densitometric ratio of TSPO levels in cerebral MECs undergoing different treatments. (B) Representative results of the densitometric ratio of LC3 in cerebral MECs undergoing different treatments. (C) Representative results of the densitometric ratio of P62 levels in cerebral MECs undergoing different treatments. (D) Representative images of LC3 and P62 expression in cerebral MECs under different conditions. The results were described as average values ± standard deviation; *P < 0.05, in comparison with the control groups; #P < 0.05, in comparison with OGD/R; $P < 0.05, in comparison with 14,15-EET + OGD/R.
FIGURE 5
FIGURE 5
The activity of SOD and content of MDA in cerebral MECs. (A) ELISA kit was applied to determine the malondialdehyde and (B) SOD levels in cerebral MECs undergoing different treatments. The results were described as average values ± standard deviation; *P < 0.05, in comparison with the control groups; #P < 0.05, in comparison with OGD/R; $P < 0.05, in comparison with 14,15-EET + OGD/R.
FIGURE 6
FIGURE 6
Identification of hub genes by STRING, MCODE and CytoHubba. (A) PPI network containing 22 nodes and 99 edges. Edge represented the interaction between two proteins. A degree was used to describe the importance of protein nodes. (B) The hub genes with a degree cut-off = 2, haircut on, node score cut-off = 0.2, k-core = 2, and max. depth = 100 were screened with MCODE. (C) The top 10 genes founded by CytoHubba. The darker the color of the node, the more critical the gene.
FIGURE 7
FIGURE 7
(A) The main pathways, enriched pathways, and their relationship with genes indicated by CluePedia map. The figure showed ten groups of GO listed above. Group 0: response to ether; Group 1: positive regulation of gluconeogenesis; Group 2: cellular response to reactive nitrogen species; Group 3: cellular response to hyperoxia; Group 4: mitochondrial genome maintenance; Group 5: skeletal muscle adaptation; Group 6: response to metformin; Group 7: smooth muscle cells (SMCs) apoptotic process; Group 8: negative regulation of post-transcriptional gene silencing; Group 9: regulation of endogenous apoptosis signaling pathway by p53 class mediator. (B) Enriched pathways of nine genes.
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