Mechanism of astrocyte derived exosome miR-193b-3p targeting TRPM2 in improving oxygen-glucose deprivation/reoxygenation injury in cerebral microvascular endothelial cells
10.3760/cma.j.cn115354-20240704-00394
- VernacularTitle:星形胶质细胞源性外泌体miR-193b-3p靶向TRPM2改善脑微血管内皮细胞氧糖剥夺/再氧合损伤的机制研究
- Author:
Yunfei DENG
1
;
Huihong LI
;
Wei WEI
;
Damei TAO
;
Xiaoqing ZHENG
;
Xiehua XUE
Author Information
1. 福建中医药大学康复医学院，福州 350122
- Keywords:
Exosome;
miR-193b-3p;
Astrocyte;
Brain microvascular endothelial cell;
Cerebral ischemia reperfusion injury;
TRPM2
- From:
Chinese Journal of Neuromedicine
2024;23(9):874-885
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the role and mechanism of astrocyte (AS) derived exosomes in protecting brain microvascular endothelial cells (bEnd. 3) from cerebral ischemia reperfusion injury by establishing an oxygen-glucose deprivation/re-oxygenation (OGD/R) model in vitro. Methods:(1) Dual-luciferase reporter gene assay was used to confirm the regulating effect of miR-193b-3p on transient receptor potential cation channel, subfamily M, member 2 (TRPM2). OGD/R model was established by OGD 8 h followed by reoxygenation 24 h in bEnd. 3 cells after being transfected miR-193b-3p mimics/negative sequence (OGD/R+miR-193b-3p mimics group or OGD/R+miR-193b-3p negative sequence group); real-time quantitative PCR (RT-qPCR) was used to detect the miR-193b-3p expression, Western blotting (WB) was used to detect the expressions of TRPM2, cleaved caspase-3, Bax, Bcl-2, ZO-1 and Claudin-5, and flow cytometry was used to detect the apoptosis. (2) AS was extracted from the cerebral cortex of C57BL/6 suckling mice and identified; modeling time was determined by CCK-8 and AS-derived exosomes were extracted by ultracentrifugation from cell supernatant and identified by electron microscopy, particle size analysis, and WB for marker proteins. RT-qPCR was used to detect the miR-193b-3p expression in AS and AS-derived exosomes. Low-expressed miR-193b-3p exosomes were extracted from AS after being transfected miR-193b-3p inhibitory sequence and co-incubated with OGD/R bEnd. 3 cells (group of OGD/R+AS-derived inhibitory sequence exosomes); exosomes were extracted from AS transfecting with miR-193b-3p negative sequence, and co-incubated with OGD/R bEnd.3 cells (group of OGD/R+AS-derived negative sequence exosomes); and normal exosomes were co-incubated with OGD/R bEnd. 3 cells (group of OGD/R+AS-derived exosomes). The miR-193b-3p expression in these 3 groups was detected by RT-qPCR, expressions of TRPM2, cleaved-caspase-3, Bax, Bcl-2, ZO-1, and Claudin-5 were detected by WB, cell apoptosis was detected by flow cytometry, and cell migration ability was detected by scratch test.Results:(1) Dual-luciferase reporter gene assay showed that miR-193b-3p could bind to TRPM2 mRNA. The miR-193b-3p can improve the TRPM2-mediated apoptosis and tight junction reduction in bEnd.3 cells during OGD/R: compared with OGD/R group, OGD/R+miR-193b-3p mimics group had significantly decreased TRPM2, cleaved-caspase-3, and Bax protein expressions, and statistically increased Bcl-2, ZO-1 and Claudin-5 protein expressions ( P<0.05). Flow cytometry further verified the above results: compared with OGD/R group, OGD/R+miR-193b-3p mimics group had significantly decreased cell apoptosis rate (21.34% vs. 13.93%, P<0.05). (2) The extracted exosomes exhibited lipid bilayer cup-like structure with particle size of 126.5 nm (exosome marker proteins: negative Cal and positive CD81 and SG101), indicating successful exosome extraction. After modeling, miR-193b-3p expression in AS and AS-derived exosomes was significantly decreased compared with that in the Control goup ( P<0.05). Compared with the OGD/R group, the group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly increased miR-193b-3p expression, statistically decreased TRPM2, cleaved-caspase-3 and Bax protein expressions, and significantly increased ZO-1, Claudin-5 and Bcl-2 expressions ( P<0.05); while compared with those in the OGD/R group, no significant changes in the above protein expressions in group of OGD/R+AS-derived inhibitory sequence exosomes were noted ( P>0.05). Compared with the OGD/R group (18.22%), group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly decreased apoptosis rate (14.09% and 13.79%, P<0.05), while group of OGD/R+AS-derived inhibitory sequence exosomes had no significant change (18.41%, P>0.05). Compared with the OGD/R group (13.55%), group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly increased migration ability (43.01% and 40.59%, P<0.05), while group of OGD/R+AS-derived inhibitory sequence exosomes had no significant change (16.26%, P>0.05). Conclusion:AS-derived exosomes can suppress TRPM2 protein expression in brain microvascular endothelial cells distantly by miR-193b-3p to improve the brain microvascular endothelial cell injury caused by OGD/R, and then improve OGD/R injury.
