Journal: Brain, Behavior, & Immunity - Health

Article Title: Physical activity-associated extracellular vesicles inhibit inflammagen-induced microglial activation

doi: 10.1016/j.bbih.2026.101200

Figure Lengend Snippet: Plasma derived from TMPA rats inhibits the LPS-induced activation of NF-κB in BV2 microglial cells. (A) Timeline for experiments identifying the optimal dosage of LPS treatment for BV2 cell. (B) Representative Western blot images showing phosphorylated levels of molecules involved in MAPK and NF-κB signaling in BV2 cells treated with various LPS doses at indicated time points. (C) Quantitative results of phosphorylated level of p65 in BV2 cells treated with various LPS doses at indicated time points. (D) Quantitative results of phosphorylated level of JNK in BV2 cells treated with various LPS doses at indicated time points. (E) Timeline for experiments evaluating the effects of plasma from SED and TMPA rats on p65 and JNK phosphorylation in BV2 cells treated with 100 ng/mL of LPS. (F) Quantitative results of cell viability using CCK-8 assay. (G) Representative Western blot images showing phosphorylated levels of p65 and JNK in BV2 cells treated with plasma from SED or TMPA rats followed by 100 ng/mL of LPS at indicated time points. (H) Quantitative results of phosphorylated level of p65 in BV2 cells treated with plasma from SED or TMPA rats followed by 100 ng/mL of LPS at indicated time points. (I) Quantitative results of phosphorylated level of JNK in BV2 cells treated with plasma from SED or TMPA rats followed by 100 ng/mL of LPS at indicated time points. Data in panels (C) and (D) were expressed as the median with interquartile range and analyzed with Kruskal-Wallis test followed by Dunn's multiple comparisons test. Data in panels (F) , (G) and (H) were expressed as mean ± standard deviation and analyzed with ordinary two-way ANOVA followed by Tukey's multiple comparisons test. Statistical significances from post hoc multiple comparisons were indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001. Asterisks in panels (C) and (D) indicate significant differences compared to the 0 ng/mL group. n = 4 cultures/group in assays shown in panels (B – D) and n = 9 cultures/group in assays shown in panels (F – I) .

Article Snippet: Immortalized murine microglial BV2 cells (Cat#: ABC-TC212S, AcceGen Biotechnology, Fairfield, NJ, USA; RRID: CVCL_0182) were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12, Cat#: 12500062, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) with extremely low endotoxin levels (<0.05 EU/ml; Lot#: VP2002200, Cat#: TMS‐013‐BKR, Merck‐Millipore, Burlington, MA, USA) and penicillin-streptomycin (Cat#: 15140122, Thermo Fisher Scientific).

Techniques: Clinical Proteomics, Derivative Assay, Activation Assay, Western Blot, Phospho-proteomics, CCK-8 Assay, Standard Deviation

Journal: Brain, Behavior, & Immunity - Health

Article Title: Physical activity-associated extracellular vesicles inhibit inflammagen-induced microglial activation

doi: 10.1016/j.bbih.2026.101200

Figure Lengend Snippet: Circulating EVs derived from TMPA rats inhibits the LPS-induced activation of NF-κB in BV2 microglial cells. (A) Representative fluorescence images from the EV uptake assay in BV2 cells. The images in the rightmost column are magnified views of the areas outlined by red frames in the color-merged images. Scale bar: 50 μm. (B) Quantitative results of EV uptake assay in BV2 cells. (C) Timeline for experiments evaluating the effects of circulating EVs from SED and TMPA rats on p65 and JNK phosphorylation in BV2 cells treated with 100 ng/mL of LPS. (D) Quantitative results of cell viability using CCK-8 assay. (E) Representative Western blot images showing phosphorylated levels of p65 and JNK in BV2 cells treated with circulating EVs from SED or TMPA rats followed by 100 ng/mL of LPS at indicated time points. (F) Quantitative results of phosphorylated level of p65 in BV2 cells treated with circulating EVs from SED or TMPA rats followed by 100 ng/mL of LPS at indicated time points. (G) Quantitative results of phosphorylated level of JNK in BV2 cells treated with circulating EVs from SED or TMPA rats followed by 100 ng/mL of LPS at indicated time points. Data in panel (B) was expressed as the median with 95% confidence interval. Data in panels (D) , (F) and (G) were expressed as mean ± standard deviation and analyzed with ordinary two-way ANOVA followed by Tukey's multiple comparisons test. Statistical significances from post hoc multiple comparisons were indicated by asterisks: *** p < 0.001. n = 3 cultures/group in assays shown in panels (A) and (B) and n = 9 cultures/group in assays shown in panels (D – G) . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Immortalized murine microglial BV2 cells (Cat#: ABC-TC212S, AcceGen Biotechnology, Fairfield, NJ, USA; RRID: CVCL_0182) were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12, Cat#: 12500062, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) with extremely low endotoxin levels (<0.05 EU/ml; Lot#: VP2002200, Cat#: TMS‐013‐BKR, Merck‐Millipore, Burlington, MA, USA) and penicillin-streptomycin (Cat#: 15140122, Thermo Fisher Scientific).

Techniques: Derivative Assay, Activation Assay, Fluorescence, Phospho-proteomics, CCK-8 Assay, Western Blot, Standard Deviation

Journal: ACS Nano

Article Title: Nicotinamide-Loaded Peptoid Nanotubes for Energy Regeneration in Acute Brain Injury

doi: 10.1021/acsnano.5c19897

Figure Lengend Snippet: NAM-PNTs show high biocompatibility and improve the cellular energy and metabolic state in the energy-depleted, acutely injured brain. a,b, MTT data showing that both NAM-PNTs (a) and Thy-PNTs (b) result in negligible effects on cell viability compared to healthy controls at all three dosages. ( n = 10). c,d, BV-2 cells exposed to 10 min of OGD showed improvement in cell viability (AlamarBlue assay) after treatment with NAM-PNTs (c) and NAD+-PNTs (d) compared to NAM and NAD+ in free form. Readings were normalized to OGD controls. Statistical significance was calculated via ordinary two-way ANOVA ( n = 3–7). e,f,g, Sonication time impacts cell viability compared to nontreated OGD-exposed controls after exposure to 20 μg/mL of NAM-PNTs for 24 h (e), 48 h (f), and 72 h (g). Readings were normalized to OGD controls. Statistical significance was calculated via a Kruskal–Wallis test ( n = 3–6). h, Intracellular ATP levels of OGD-exposed BV-2 cells with the addition of free NAM and NAM-PNTs compared to the nontreated control. All data are presented as violin plots that show the median with the interquartile range. Statistical significance was calculated via a Kruskal–Wallis test. i,j, Schematic illustration of nanoparticle uptake (h) and exocytosis (l) assays in BV-2 cells. Figure created with Biorender.com. k. BV-2 cell uptake of DNS-PNT and PLGA–PEG nanoparticles at different administration dosages after 1 h of endocytosis. Data are presented as mean ± s.d. and statistical significance was calculated via ordinary two-way ANOVA ( n = 4). l,m, Confocal images showing DNS-PNTs (red) associated with lysosomes (Lysotracker, yellow) and BV-2 cells (tomato lectin, green; ToPro-3, blue) preincubated with inhibitors for 1 h and with (l) or without (m) inhibitors for 24 h.

Article Snippet: Murine microglia BV-2 cells were purchased from ATCC (CRL-2469) and cultured based on previous literature.

Techniques: Alamar Blue Assay, Sonication, Control

Journal: iScience

Article Title: Cerebral organoid exosomes reversed behavioral deficits by repressing NLRP3-mediated neuroinflammation in stress models

doi: 10.1016/j.isci.2026.115069

Figure Lengend Snippet: OExo alleviates LPS-induced microglia activation by suppressing NLRP3 expression (A) Representative images of IBA-1 immunostaining reveal the uptake of PKH26-labeled OExo by IBA-1-positive BV-2 microglia after 48 h of incubation. The white arrows highlight the presence of PKH26-labeled OExo surrounding the nuclei of microglia. (B) Representative images of immunostaining for CD206 (top) and iNOS (bottom) demonstrating BV-2 microglia treated with CON (PBS), OExo (50 μg/mL), LPS (100 ng/mL), and LPS combined with OExo (LPS+OExo) for 24 h. (C and D) Quantitative analysis of the fluorescence intensity of CD206 (C) and iNOS (D). n = 6. (E) q-PCR analysis of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in BV-2 microglia treated with CON, OExo, LPS, and LPS+OExo, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene. n = 9. (F) Representative WB images displaying the expression of CD206, Arg-1, and iNOS in BV-2 cells treated with CON, OExo, LPS, and LPS+OExo, with β-actin as the internal control. Full-length blots are presented in . (G) Statistical analysis of CD206, Arg-1, and iNOS expression levels from WB. n = 6. (H) Representative WB images illustrating the NLRP3, Casp-1, and IL-1β expression levels in BV-2 cells treated with CON, OExo, LPS, LPS+OExo, nigericin, and nigericin+LPS+OExo. nigericin, a NLRP3 activator, was utilized to inhibit the rescuing effects of OExo on LPS-induced NLRP3 activation. β-actin was used as the internal control. Full-length blots are presented in . (I) Statistical analysis of NLRP3, Casp-1, and IL-1β expression levels by WB. n = 4. (J) Representative WB images showing NLRP3 levels in BV-2 cells treated with CON, MExo, OExo, LPS, LPS+MExo and LPS+OExo. β-actin was used as the internal control. Full-length blots are presented in . (K) Statistical analysis of NLRP3 expression levels in (J). n = 4. All western blot quantifications are based on at least three biological replicates, with two to three technical replicates per experiment. Scale bars, 50 μm. DAPI was used for nuclear counterstaining in all images. Data are presented as mean ± SEM. Data are subjected to one-way ANOVA followed by the Tukey’s test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: BV-2 microglia cells (Procell, Wuhan, China) were maintained and passaged in Dulbecco’s Modified Eagle’s Medium (DMEM; Sbjbio, Nanjing, China; BC- M -002) with 10% fetal bovine serum (FBS; ExCell, Suzhou, China; FDN500).

Techniques: Activation Assay, Expressing, Immunostaining, Labeling, Incubation, Fluorescence, Control, Western Blot

Journal: iScience

Article Title: Cerebral organoid exosomes reversed behavioral deficits by repressing NLRP3-mediated neuroinflammation in stress models

doi: 10.1016/j.isci.2026.115069

Figure Lengend Snippet: Alleviation of OExo on LPS-induced microglia activation impacts on astrocytes and neurons (A) Experimental design flowchart. BV-2 microglia were treated with CON, OExo, LPS, and LPS+OExo for 24 h. Subsequently, media was replaced with fresh serum-free medium, and the conditioned medium CON-CM, OExo-CM, LPS-CM, and LPS+OExo-CM was collected after 6 h of conditioning. Primary astrocyte and hippocampal neuronal cultures were incubated with respective CM for 24 h prior to analysis. (B) Representative immunofluorescence images of C3 and GFAP double-labeling in primary astrocytes treated with CM. DAPI was used for nuclear counterstaining. (C) Representative immunofluorescence images of SYN1 in primary hippocampal neuronal processes treated with CM. DAPI staining was used for nuclear visualization. (D) Quantification of C3+GFAP+/GFAP+ astrocytes (% of GFAP+ cells). n = 6. (E) Quantification of SYN1 positive puncta in neurites. n = 9. Scale bars, 100 μm in (B), 50 μm in (C), and 10 μm (bottom) in (C). Data are presented as mean ± SEM. Data are subjected to one-way ANOVA followed by the Tukey’s test. ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: BV-2 microglia cells (Procell, Wuhan, China) were maintained and passaged in Dulbecco’s Modified Eagle’s Medium (DMEM; Sbjbio, Nanjing, China; BC- M -002) with 10% fetal bovine serum (FBS; ExCell, Suzhou, China; FDN500).

Techniques: Activation Assay, Incubation, Immunofluorescence, Labeling, Staining