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. 2019 Sep 19;12:222. doi: 10.3389/fnmol.2019.00222

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Copyright © 2019 Bao, Fan, Zhao, Xu, Liu, Chao, Liu, You, Liu, Wang and Ji.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Nec-1 and melatonin attenuated inflammatory responses by blocking high-mobility group box-1 (HMGB1) translocation from the nucleus to the cytoplasm and release. (A) Total levels of HMGB1 after CCI tested by western blot were not decreased by Nec-1 or melatonin. (B) Tissues were fractionated, and the nuclear and cytoplasmic extracts were subjected to immunoblotting. GAPDH and H3 were used as controls for the cytoplasm and nucleus, respectively. (C) Relative expression in cytoplasm and nucleus was analyzed respectively by statistical. Values are represented as means ± SEM (n = 3). (D) The expression of HMGB1 was analyzed by immunofluorescence assays with the indicated antibodies. Samples were stained with an anti-HMGB1 antibody (green) and DAPI (blue). (E) Released HMGB1 in cerebrospinal fluid (CSF) was tested by Elisa assays. Relative level in CSF was analyzed by statistical. Values are represented as means ± SEM (n = 3). All experiments were analyzed by one way ANOVA plus Tukey’s test. *P < 0.05; **P < 0.01 and ***P < 0.001 vs. sham group. ^#P < 0.05 and ^##P < 0.01 vs. CCI group.