Immunostaining against Nurr1 protein in MGCs.

The number of Nurr1 positive cells were counted in images captured from each group and their differences were analyzed. The Green color shows the Nurr1 and the blue color denotes the nuclei of cells stained with DAPI. Scale bars represent 50 μm. Data are presented as mean ± S.E.M, (**: P .01 and ****: P .0001).</p

Fig 2 -

Effect of FA on morphology (A), ramification index (B) and gene expressions in cells after treatment with different concentrations of FA. Primary MGCs were treated with different concentrations of FA (50, 100, 250, and 500 μg/ml). As represented in the schematic timeline (Fig 1), cells were treated for 24 hours with 0, 50, 100, 250 and 500 μg/mL of FA (Aa-e). Ramification indexes (RI) from different groups were measured by Sholl analysis tool in Image J software (B). Real-time qPCR was performed to evaluate the expressions of Nurr1, IL1-β and IL-10 (C). Scale bars represent 50 μm. Data are presented as mean ± S.E.M, (*: P .05, **: P .01, ***: P .001 and ****: P .0001).</p

The complete raw data (minimal data set) for all graphs in this manuscript can be found in the article’s supplementary data (S1_minimal_dataset).

The complete raw data (minimal data set) for all graphs in this manuscript can be found in the article’s supplementary data (S1_minimal_dataset).</p

Experimental design of the present study.

Microglial cells were isolated from neonate mice and at the first step they were treated with different concentrations of ferulic acid (FA) to find the most effective concentration on Nurr1 induction. Then in the second round of experiments cells were treated with FA in the presence of beta-amyloid (Aβ) stress to find the response of cells after exposure to the inflammatory signal.</p

Effect of FA on MGCs under Aβ-stress.

Changes in cell morphology (A), ramification index (B), percentage of different MGC phenotypes (C), and levels of gene expressions for Nurr1, IL1-β and IL-10 (D) were evaluated. Scale bars represent 50 μm. Data are presented as mean ± S.E.M, (*: P .05, **: P .01, ***: P .001 and ****: P .0001).</p

Primer sequence.

Microglial cells (MGCs) serve as the resident macrophages in the brain and spinal cord, acting as the first line of immune defense against pathological changes. With various phenotypes, they can shift from a homeostatic state to a reactive state or transit from a reactive to a non-inflammatory reactive state (alternative homeostatic). A well-timed transit is crucial in limiting excessive microglial reaction and promoting the healing process. Studies indicate that increased Nurr1 expression promotes anti-neuroinflammatory responses in the brain. In this study, we investigated the possible role of ferulic acid (FA) in facilitating microglia transition due to its anti-inflammatory and Nurr1-inducing effects. MGCs were extracted from the brains of male NMRI mice at postnatal day 2 (P2) and cultured with or without FA and beta-amyloid (Aβ). Real-time qRT-PCR was conducted to measure the expressions of Nurr1, IL-1β, and IL-10 genes. Immunostaining was performed to determine the number of NURR1-positive cells, and the ramification index (RI) of MGCs was calculated using Image J software. Treating MGCs with FA (50 μg/ml) induced Nurr1 and IL-10 expressions, while reducing the level of IL-1β in the absence of Aβ-stress. Further assessments on cells under Aβ-stress showed that FA treatment restored the IL-10 and Nurr1 levels, increased the RI of cells, and the number of NURR1-positive cells. Morphological assessments and measurements of the RI revealed that FA treatment reversed amoeboid and rod-like cells to a ramified state, which is specific morphology for non-inflammatory reactive microglia. To conclude, FA can provide potential alternative homeostatic transition in Aβ-reactive microglia by recruiting the NURR1 dependent anti-inflammatory responses. This makes it a promising therapeutic candidate for suppressing Aβ-induced neuroinflammatory responses in MGCs. Furthermore, given that FA has the ability to increase NURR1 levels in homeostatic microglia, it could be utilized as a preventative medication.</div

Top statistically related signaling pathways to miR-141 and miR-200a targetome obtained from different databases by means of DAVID tool.

<p>Top statistically related signaling pathways to miR-141 and miR-200a targetome obtained from different databases by means of DAVID tool.</p

Target genes of miR-141 and miR-200a specified <i>in-silico</i> were tested for further analysis and the expression level of target genes of miR-141 and miR-200a in CD4⁺ T cells of relapsing group (n = 20), remitting group (n = 20) and healthy controls (n = 10) were assessed.

<p>Results are normalized relative to expression level of reference gene, <i>18srRNA</i> (*p < 0.05 and **p < 0.01, non-parametric Mann-Whitney <i>t</i>-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).</p

Up-regulation of miR-141 and miR-200a in relapsing phase of MS patients.

<p>RT-qPCR analysis of miR-141 expression level in CD4^<b>+</b> T cells of MS patients in relapsing phase (n = 20), remitting phase (n = 20) and healthy controls (n = 10) (<i>A</i>). RT-qPCR analysis of miR-200a expression level in CD4^<b>+</b> T cells of MS patients in the same groups (<i>B</i>). Results are normalized relative to expression level of reference gene, <i>RNU48</i> (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric Mann-Whitney <i>t</i>-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).</p

Flow cytometry of FoxP3⁺ CD4⁺ T cells and RORɣt+ CD4+ T cells.

<p>CD4^<b>+</b> T cells were isolated by CD4+ Tcell isolation kit II human of Miltenyi Biotec and stained with respective antibodies and evaluated in relapsing phase (n = 20) and remitting phase (n = 20) of MS patients and healthy controls (n = 10). A forward and side scatter gate was used to select lymphocyte population and fluorescence compensation was set according to labeled lymphocytes with only green and only red fluorescent separately versus isotype control (<i>A</i>). Percentage of RORγt+ CD4+ T cells measured by Flow cytometry, shows meaningful increase in relapsing group (<i>B</i>) while percentage of FoxP3^<b>+</b> CD4^<b>+</b> T cells elevates in remitting group (<i>C</i>) (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric Mann-Whitney <i>t</i>-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).</p