Abstract
Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have therapeutic potential in various diseases due to their capacity to transfer bioactive cargoes such as microRNAs (miRNAs or miRs) to recipient cells. The present study isolated EVs from rat MSCs and aimed to delineate their functions and molecular mechanisms in early brain injury following subarachnoid hemorrhage (SAH). We initially determined the expression of miR-18a-5p and ENC1 in hypoxia/reoxygenation (H/R)-induced brain cortical neurons and rat models of SAH induced by the endovascular perforation method. Accordingly, increased ENC1 and decreased miR-18a-5p were detected in H/R-induced brain cortical neurons and SAH rats. After MSC-EVs were co-cultured with cortical neurons, the effects of miR-18a-5p on neuron damage, inflammatory response, endoplasmic reticulum (ER) stress, and oxidative stress markers were evaluated based on ectopic expression and depletion experiments. miR-18a-5p overexpression in brain cortical neurons co-cultured with MSC-EVs was shown to impede neuron apoptosis, ER stress and oxidative stress while augmenting neuron viability. Mechanistically, miR-18a-5p bound to the 3’UTR of ENC1 and reduced its expression, weakening the interaction between ENC1 and p62. Through this mechanism, transfer of miR-18a-5p by MSC-EVs contributed to the eventual inhibition of early brain injury and neurological impairment following SAH. Overall, miR-18a-5p/ENC1/p62 may be a possible mechanism underlying the cerebral protective effects of MSC-EVs against early brain injury following SAH.
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Funding
This work was funded by the Key Project of Health Commission of Sichuan Province (18PJ030,20ZD018), Research Project of Sichuan Medical Association (S21055), Innovation foundation of The Affiliated Hospital of Chengdu University (CDFYCX202209), Foundation of Affiliated Hospital of Chengdu University (YZD2017004 and Y2021008).
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Yamei Zhang and Junying Liu designed the study. Yamei Zhang, Yan Zhou and Zhonglan Zou collated the data, carried out data analyses and produced the initial draft of the manuscript. Junying Liu, Chenchen Xie and Li Ma contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.
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The current study was approved by the Animal Ethic Committee of Affiliated Hospital of Chengdu University and carried out according to the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health.
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441_2023_3754_MOESM1_ESM.docx
Supplementary Figure 1 Identification of the H/R-induced cell model establishment. a-a’’’, Morphology of primary cortical neurons. b-b’, NSE immunofluorescence staining of primary cortical neurons. c, Neuron viability upon H/R treatment measured by CCK-8 assay. d, Apoptosis rate upon H/R treatment measured by flow cytometry. e, Expression of TNF-α, IL-1β and IL-6 in the supernatant of H/R-treated brain cortical neurons measured by ELISA. f, Western blot of CHOP, GRP78, TXNIP and NLRP3 proteins in H/R-treated brain cortical neurons. g, ROS production in the supernatant of H/R-treated brain cortical neurons measured by ELISA. h, MDA production in the supernatant of H/R-treated brain cortical neurons measured by ELISA. * p < 0.05. Cell experiments were repeated independently three times (n = 3) (DOCX 638 KB)
441_2023_3754_MOESM2_ESM.docx
Supplementary Figure 2 Identification of the SAH model establishment. a, Mortality of sham-operated and SAH rats. b, SAH grade of sham-operated and SAH rats. c-d, Neurological function in sham-operated and SAH rats assessed by Modified Garcia Score (C) and beam balance test (D). e, Permeability of BBB in sham-operated and SAH rats assessed by Evans blue staining. f, Brain water content of sham-operated and SAH rats. g, Apoptosis rate of neurons in the brain tissues of sham-operated and SAH rats measured by TUNEL assay. h, Neurodegeneration in the brain tissue of sham-operated and SAH rats shown by FJC staining. i, Expression of TNF-α, IL-1β and IL-6 in the brain tissues of SAH rats measured by ELISA. j, Western blot of CHOP, GRP78, TXNIP and NLRP3 proteins in the brain tissue of SAH rats. k-k’, ROS and MDA production in the brain tissue of SAH rats measured by ELISA. n = 6-8 rats in each group. * p < 0.05 (DOCX 298 KB)
441_2023_3754_MOESM3_ESM.docx
Supplementary Figure 3 Assessment of miR-18a-5p binding to IRF2 and PTGFRN. a-a’, The putaitve binding sites of miR-18a-5p to IRF2 and PTGFRN predicted using TargetScan analysis. b, Binding of miR-18a-5p to IRF2 examined by dual-luciferase reporter assay. c, Binding of miR-18a-5p to PTGFRN examined by dual-luciferase reporter assay. * p < 0.05, compared to the HEK-293T cells transfected with mimic-NC. Cell experiments were repeated independently three times (n = 3) (DOCX 296 KB)
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Zhang, Y., Liu, J., Zhou, Y. et al. miR-18a-5p shuttled by mesenchymal stem cell-derived extracellular vesicles alleviates early brain injury following subarachnoid hemorrhage through blockade of the ENC1/p62 axis. Cell Tissue Res 392, 671–687 (2023). https://doi.org/10.1007/s00441-023-03754-w
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DOI: https://doi.org/10.1007/s00441-023-03754-w