Abstract
The effect of Celastrol on cerebral ischemia-reperfusion remains unknown. The study aims to explore the role of circular RNA DLGAP4 (circDLGAP4) in cerebral ischemia-reperfusion and the underlying mechanism. Ischemia-reperfusion (I/R) injury of human brain microvascular endothelial cells (HBMECs) was induced by oxygen-glucose deprivation and reoxygenation (OGD/R). Reverse transcription quantitative real-time PCR (RT-qPCR) and western blotting analysis were performed to detect the expression of circDLGAP4, microRNA-6085 (miR-6085), growth differentiation factor 11 (GDF11), B-cell lymphoma-2 (BCL2) and BCL2-associated x protein (BAX). Cell viability, proliferation, and apoptosis were analyzed by cell counting kit-8, 5-Ethynyl-2’-deoxyuridine and flow cytometry analysis. Oxidative stress was analyzed by evaluating the levels of Malondialdehyde (MDA) and Reactive Oxygen Species (ROS) and the activity of Superoxide Dismutase (SOD). The associations among circDLGAP4, miR-6085 and GDF11 were identified by dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. Celastrol reduced OGD/R-induced inhibition of circDLGAP4 expression in HBMECs. Celastrol treatment protected HBMECs from OGD/R-induced cell proliferation inhibition and apoptosis and oxidative stress promotion; however, circDLGAP4 depletion attenuated these effects. CircDLGAP4 acted as a sponge for miR-6085, and miR-6085 mimics restored circDLGAP4-mediated effects in OGD/R-stimulated HBMECs. In addition, GDF11 was identified as a targte of miR-6085, and participated in the regulation of miR-6085 to OGD/R-induced HBMEC damage. Further, circDLGAP4 absence inhibited GDF11 expression by interacting with miR-6085 under Celastrol treatment. Celastrol ameliorated OGD/R-induced HBMEC apoptosis and oxidative stress by circDLGAP4/miR-6085/GDF11 pathway, supporting the use of Celastrol as a therapeutic agent for cerebral infarction.
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The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.
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Conceptualization and Methodology: Jiahui Gu and Yingli Yu; Formal analysis and Data curation: Chunhong Liu and Yingli Yu; Validation and Investigation: Chunhong Liu and Jiahui Gu; Writing - original draft preparation and Writing - review and editing: Chunhong Liu, Jiahui Gu and Yingli Yu; Approval of final manuscript: all authors
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The present study was approved by the ethical review committee of Yantai Hospital of Traditional Chinese Medicine. Written informed consent was obtained from all enrolled patients.
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Highlights
1. Celastrol attenuated OGD/R-induced inhibition of circDLGAP4 expression
2. Celastrol protected HBMECs from OGD/R-induced damage by regulating circDLGAP4.
3. CircDLGAP4 acted as a miR-6085 sponge.
4. MiR-6085 targeted GDF11.
5. CircDLGAP4 regulated GDF11 expression by interacting with miR-6085.
Supplementary information
Fig. S1
Celastrol assuaged OGD/R-induced cell apoptosis and oxidative stress through circDLGAP4. (A-J) HBMECs were divided into control+si-con, control+si-circDLGAP4, OGD/R+si-con, OGD/R+si-circDLGAP4, OGD/R+Cel+si-con, OGD/R+Cel+ si-circDLGAP4. (A) CircDLGAP4 expression was examined by RT-qPCR. (B and C) Cell proliferation was analyzed by EdU assay. (D and E) Cell apoptosis was assessed by flow cytometry analysis. (F and G) The protein expression of BCL2 and BAX was detected by Western blotting analysis. (H) MDA level was detected by lipid peroxidation MDA assay kit. (I) ROS level was examined by Cellular ROS Assay kit. (J) SOD activity was analyzed by Superoxide Dismutase Activity Assay kit. NS: no significant difference; aP<0.05, bP<0.05, cP<0.05, dP<0.05, #P<0.05, &P<0.05. Statistical analysis in (A-B), (D) and (H-J) was conducted using one-way analysis of variance and in (G) using two-way analysis of variance (PNG 3733 kb)
Fig. S2
The proportion of viable, early apoptotic, late apoptotic and necrotic cells in related apoptosis assays in this study. NS: no significant difference; aP<0.05, bP<0.05, cP<0.05, dP<0.05, #P<0.05, &P<0.05 (PNG 575 kb)
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Liu, C., Gu, J. & Yu, Y. Celastrol assuages oxygen-glucose deprivation and reoxygenation-induced damage in human brain microvascular endothelial cells through the circDLGAP4/miR-6085/GDF11 pathway. Metab Brain Dis 38, 255–267 (2023). https://doi.org/10.1007/s11011-022-01106-1
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DOI: https://doi.org/10.1007/s11011-022-01106-1