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. 2021 Feb 10;11:3518. doi: 10.1038/s41598-021-81741-3

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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ADA-409-052 administration in vivo reduced the expression levels of pro-inflammatory markers (a–f) and altered the peripheral cytokine secretion (g–j) at 1 day post-ischemia. The mRNA expression of tissue samples from the ipsi- and contralateral peri-ischemic area at 24 h was analyzed using qPCR. Expression levels of (a) Gfap and (c) Ccl2 were markedly reduced in the ipsilateral peri-ischemic area after ADA-409-052 treatment when compared to vehicle. (b) While, Aif1 remained unchanged throughout all groups, ADA-409-052 lowered (d) Arg1 and (e) Il6 mRNA expression in the ipsilateral samples when compared to contralateral. (f) Ischemic mice treated with ADA-409-052 showed reduced Hmox1 expression in the ipsilateral peri-ischemic area when compared to contralateral expression levels, to ipsilateral expression of vehicle-treated mice respectively. Minocycline decreased the expression of Hmox1 only ipsilateral when compared to contralateral. mRNA expression levels of peri-ischemic tissue samples from ipsi- and contralateral, data are expressed as mean ± s.e.m.; unpaired, two-tailed student’s t-test; n = 9–13; *p < 0.05, **p < 0.005, ***p < 0.001. (g) The secretion of the inflammation-mediating IL-6 was significantly reduced in ADA-409-052-treated mice when compared to vehicle-treated mice, as detected from the blood plasma, collected from ischemic mice at 1 dpi, by using the BD CBA mouse inflammation kit. (h,i) Both MCP-1 and TNF-α were equally low in ADA-409-052-treated mice when compared with vehicle treatment and showed a clear trend towards reduction when compared with minocycline-treated mice (changes did not reach significance). (j) The peripheral secretion of IL-10 remained unaltered by any given treatment. Data are expressed as mean ± s.e.m.; unpaired, two-tailed student’s t-test; n = 7; *p = 0.027.