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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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(a) ADA-409-052 inhibits LPS-induced inflammation in BV2 microglial-like cells. The addition of 5, 10, and 20 µM ADA-409-052 significantly attenuated the release of NO, measured from the media of LPS-exposed BV2 cells using the Griess reagent, when compared to cells treated with LPS only. Data are represented as percentages of NO-release after normalization to LPS-stimulated BV2 cells ± s.e.m.; unpaired, two-tailed student’s t-test; technical n = 6; *p < 0.05, ***p < 0.0001. (b) ADA-409-052 protects N2a neuronal cells when co-cultured with LPS-stimulated RAW 264.7 macrophages. Boxplots show that LPS and INF-γ treatment of co-cultured N2a cells and RAW 264.7 macrophages decreased the viability of N2a cells about 40% as detected by the CytoFLEX S (Beckman Coulter) as a fraction of CD11b negative (CD11b^-), PI negative (PI^-), surviving cells to the total number of gated cells, normalized to the control sample. Co-treatment of ADA-409-052 and LPS/INF-γ resulted in a significant increase of the N2a cell viability, detected by the reduced percentage of CD11b^-/ PI positive (PI⁺, dead) cells when compared to LPS/INF-γ treatment only. In contrast, the proportion of CD11b^-/PI⁺ N2a cells remained unaltered by minocycline. Cell survival is displayed as individual boxplot per treatment with interquartile range, whiskers set from min to max; unpaired, two-tailed student’s t-test; technical n = 3/4; *p = 0.0314. (c–e) ADA-409-052 alters the expression of pro- and anti-inflammatory cytokine expression of BV2 microglial cells. By measuring cytokine-secretion of ADA-409-052-stimulated BV2 cells, using the BD CBA mouse inflammation kit, (c) no changes in secretion of the anti-inflammatory IL-10 were detected. (d) However, we detected a major decrease of MCP-1 upon the addition of 10 µM ADA-409-052. (e) Furthermore, TNF-α decreased upon ADA-409-052-stimulation (5 µM) significantly when compared with untreated cells (0 µM ADA-409-052). Student’s t-test, n = 4; *p < 0.05.