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Myosin IIA Regulated Tight Junction in Oxygen Glucose-Deprived Brain Endothelial Cells Via Activation of TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 Signal Transduction Pathway

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Abstract

Non-muscle myosin heavy chain IIA (NMMHC IIA), a member of Myosin II family, plays a critical role in various cellular physiological processes. Our previous research had suggested that NMMHC IIA could participate in regulating tight junction morphological changes induced by ischemia stroke. Thus, in the current study, we attempted to uncover the regulation pattern of NMMHC IIA on tight junction dysfunction in oxygen glucose-deprived (OGD) mouse brain bEND.3 endothelial cells. The regulation of NMMHC IIA on tight junction in OGD-stimulated bEND.3 cells was evaluated by western blotting assay. Morphologic change of occludin, claudin-5, and ZO-1 tight junction proteins was compared with pretreatment with NMMHC II inhibitor blebbistatin via immunohistochemical staining. Detection of activation of NMMHC IIA on OGD-mediated tight junction transduction pathway was investigated via Koch’s postulate using corresponding protein inhibitor. Our results showed that NMMHC IIA was activated in OGD-stimulated bEND.3 endothelial cells. The inhibition of NMMHC IIA could attenuate the morphologic change of occludin, claudin-5, and ZO-1 tight junction proteins. NMMHC IIA participated in regulating downstream transduction pathway TLR4, phosphatidylinositol 3-kinase (PI3K), Akt, JNK1/2, 14-3-3ε, nuclear factor kappa B (NF-кB) and matrix metalloprotein 9 (MMP9). Blocking of these pathways using indicated inhibitors demonstrated that NMMHC IIA destroyed the connection of tight junction via the activation of TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 pathway. Our study described the key role of NMMHC IIA in OGD-stimulated mouse brain bEND.3 endothelial cells, while also exhibited the molecule effect on tight junction dysfunction via TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 signal transduction pathway.

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Acknowledgements

This work was supported by Foundation Project: National Natural Science Foundation of China (81760094, 31602111, 81860020), China; Jiangxi Provincial Education Department Science and Technology Research Youth Project (No. GJJ160238); The Foundation of Jiangxi Provincial Department of Science and Technology Youth Project (No. 20171BAB215021). The funder has no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Pharmacy department, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China

    Yanni Lv, Zhaohui Ruan, Zixuan Xu & Longsheng Fu

  2. Jiangxi Provincial Institute of Traditional Chinese Medicine, Nanchang, 330006, Jiangxi, China

    Wen Liu

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All authors contributed to the research. YL conceived the experiments. YL, WL, ZR, ZX, LF conducted the experiments and analyzed the results. All authors contributed to the writing and editing of the manuscript.

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Correspondence to Yanni Lv.

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None of the authors have any conflicts of interest, including financial, personal or other relationships, with other individuals or organizations.

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All animal care and experimental procedures were conducted according to standard ethical guidelines (National Institutes of Health Guide to the use of Laboratory Animals) and approved by Institutional Animal Care and use Committee of Nanchang University. All efforts were made to minimize the number of mice used and their suffering.

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Lv, Y., Liu, W., Ruan, Z. et al. Myosin IIA Regulated Tight Junction in Oxygen Glucose-Deprived Brain Endothelial Cells Via Activation of TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 Signal Transduction Pathway. Cell Mol Neurobiol 39, 301–319 (2019). https://doi.org/10.1007/s10571-019-00654-y

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