Fig. 5

Malva parviflora hydroalcoholic extract regulates LPS-induced microglia activation. Microglia primary cultures were isolated from neonatal wild type animals as described in the “Methods” section. Microglia enrichment was determined as follows. a Confocal microscopy was used to examine GFAP (astrocytes) and F4/80 (microglia) expression in mixed cultures and after microglia purification (Microglia). Nuclei were visualized by DAPI staining (scale bar, 30 μm). b Microglia enrichment was determined by flow citometry using anti-CD11b antibodies. c The mRNA levels of different cell markers, microglia (F4/80); astrocytes (GFAP) and neurons (Neurofilament M; NF) were determined in the mixed cultures (MC), isolated microglia (microglia), whole brain (WB) and CHO cells by RT-PCR analysis as described in the “Methods” section. Actin levels were used as internal control. d Microglial cultures were exposed to PBS (Ctrl), LPS (100 ng/mL), MpHE (M. parviflora) (1 mg/mL) or LPS (100 ng/mL) and MpHE (1 mg/mL) (LPS + M. parviflora) for 24 h. Cells were fixed, stained with phalloidin and DAPI for F-actin (green) and nuclei (blue) detection, respectively. Microglia was imaged using the Olympus FluoView 1000 confocal multiphoton microscope (scale bar, 30 μm). The boxed areas were × 2 digitally magnified and shown as inset. e The microglia morphologies were classified as ramified, amoeboid and intermediate shapes in the different groups described in (D); MpHE (Mp). One hundred cells were measured for each experimental condition. Data (mean ± SEM) were analyzed by one-way ANOVA followed by Tukey’s post hoc test (^**p < 0.01; ^***p < 0.001). f Microglial cultures were exposed to PBS (Control) or LPS (100 ng/mL) in the presence or absence Vehicle) of MpHE (1 mg/mL) (Mp) for 24 h. The length of the protrusions (μm) of the microglia were determined using Neurite Tracer from ImageJ software (National Institutes of Health, Bethesda, MD). Data (mean ± SEM) were analyzed by two-way ANOVA followed by post hoc Bonferroni’s multiple comparisons test. This analysis revealed a significant effect for the LPS F(1,8) = 13.32, p = 0.006; not for the treatment with M. parviflora F(1,8) = 0.8054 p = 0.40 or for the LPS and M. parviflora interaction F(1,8) = 1.245 p = 0.30. g Microglial cultures were exposed to PBS (Control), MpHE (1 mg/mL) (Mp) or LPS (100 ng/mL) and MpHE (1 mg/mL) (LPS + Mp) for 24 h. The phagocytic index was calculated by multiplying the percentage of microglia with internalized latex beads by the average number of internalized latex beads per each group. Data were collected from seven random fields per group and analyzed by one-way ANOVA followed by Tukey’s post hoc test (^***p < 0.001 vs control)