Supplementary MaterialsSupplementary material mmc1. morphology and filopodia formation. A high variety of filopodia numerous branching factors is undoubtedly an indicator of security function typically, whereas a circular amoeboid shape is certainly an indicator of reactivity. BV-2 cells were still left treated or neglected with 50?ng/ml LPS, 200?m crocin or both together as well as the f-actin cytoskeleton was stained using Phalloidin-TRITC (Fig. 1). Immunofluorescence evaluation uncovered that microglia acquired some filopodia in charge circumstances (Fig. 1A) and despite some propensity to get more filopodia with crocin treatment, the transformation had not been statistically different (Fig. 1B, E). Incubation with LPS triggered an entire rounding of cells (Fig. 1C), as well as the mixed treatment with crocin reversed this amoeboid phenotype using a statistically elevated degree of ramification (Fig. 1D, E). These analyses present that crocin directly regulates filopodia formation in LPS-treated microglia clearly. Open in another home window She Fig. S/GSK1349572 1 Crocin promotes microglial filopodia development. Representative pictures of Phalloidin-TRITC/DAPI tagged BV-2 microglial cells displaying morphological adjustments in response to activation with vehicle (A), 200?M crocin (B), 50?ng/ml LPS (C), and 50?ng/ml LPS plus 200?M crocin (D). Cells were pre-treated with 200?M crocin for 30?min, followed by activation with 50?ng/ml LPS for further 24?h. Scale bar = 20?m. (E) Quantification of microglial ramification was performed using a grid-cross counting method [6]. Data show imply SEM (n = 13C18 cells/group in three impartial images), **P 0.01 for LPS + crocin- versus control, *P 0.05 for LPS + crocin- versus LPS alone, and *P 0.05 for LPS versus crocin. 3.2. Crocin induces the phagocytic capacity of microglia We next tested the effect of crocin around the phagocytic capacity, an important physiological function of microglia [22]. Fluorescently labeled dying 661?W photoreceptor-like cells were used as cargo that mimics cell debris in the S/GSK1349572 degenerating retina. BV-2 microglial cells stimulated with crocin displayed a significantly higher phagocytosis rate that control cells (Fig. 2A, B, F, 0.01). This effect of stimulated phagocytosis was also present in LPS-pretreated BV-2 cells (Fig. 2E, C, F, 0.044). These findings show that crocin promotes the ramified microglial phenotype with a high phagocytosis capacity. The exact mechanisms how crocin increases microglial phagocytosis are currently unknown. However, crocin-treated peritoneal mouse macrophages also showed increased yeast phagocytosis, corroborating our data [23]. Open in a separate windows Fig. S/GSK1349572 2 Crocin enhances the phagocytic capacity of microglia. Representative images showing phagocytic uptake of CM-DiI-stained apoptotic 661?W photoreceptor material into BV-2 cells treated with vehicle (A), 200?M crocin (B), 50?ng/ml LPS (C), or 50?ng/ml LPS plus 200?M crocin (D) for 6?h. (E) Bar graphs showing quantification of microglial phagocytosis as phagocytosis index in %. Data show imply SD (n = 9 cultures/group), ** 0.01 for crocin versus vehicle-treated, * 0.05 for LPS + crocin- versus LPS alone. Level bar = 50?m. 3.3. Crocin dampens LPS-induced pro-inflammatory gene expression and lowers nitric oxide production in microglia We then analyzed whether crocin can directly modulate pro-inflammatory gene expression in microglia. Interleukin 6 (IL6), CC-chemokine ligand 2 (CCL2) S/GSK1349572 and inducible NO synthase (iNOS) were chosen as representative markers for molecular pathways involved in acute phase response, chemotaxis and oxidative burst, respectively. LPS strongly induced IL6 (Fig. 3A), CCL2 (Fig. 3B) and iNOS (Fig. 3C) in BV-2 microglia. Co-treatment with 200?m crocin significantly reduced the LPS-induced gene transcription of IL6 (Fig. 3A, 0.021), CCL2 (Fig. 3B, 0.033), and iNOS (Fig. 3C, 0.025). To review whether S/GSK1349572 this impact was noticed on the function level also, the secretion and production of NO radicals was measured. Crocin treatment only did not impact NO secretion.