Journal: International Journal of Molecular Sciences

Article Title: Ursolic Acid Alleviates Neuroinflammation after Intracerebral Hemorrhage by Mediating Microglial Pyroptosis via the NF-κB/NLRP3/GSDMD Pathway

doi: 10.3390/ijms241914771

Figure Lengend Snippet: PMA abrogates the role of UA in attenuating microglial pyroptosis. UA attenuates the activation of the NLRP3 inflammasome and the NF-κB pathway in hemin-treated BV2 cells ( A – G ). Detection of the degree of BV2 cell pyroptosis using PI staining ( H ). Quantitative analysis of the levels of IL-6 ( I ), IL-1β ( J ), and TNF-α ( K ), and by ELISA kits. Immunofluorescence analysis of GSDMD expression. ( M ) Quantitative analysis of GSDMD fluorescence intensity. ( L ). Data are expressed as mean ± SD, scale bar = 100 μm. Significant differences were analyzed using the “abcd” letter marking method of identification: first, the group means were arranged in descending order, then the largest mean was marked with an a, and that mean was compared to the remaining means. Where the difference is not significant, it is marked with the letter a, until a significant difference with the mean marked with the letter b, and marked with the letter c and d. The difference is not significant in the group marked with the same letter. The difference is significant in the group marked with a different letter. Lowercase letters denote p < 0.05, uppercase letters denote p < 0.01. The red dashed line represents the edge of the hematoma, and the white dashed line represents the needle track.

Article Snippet: BV2 microglial cells were purchased from Procell (Wuhan, China), cultured in a BV2-specific medium (Procell, Wuhan, China) at 37 °C and 5% CO 2 , and treated with drugs added at appropriate times according to the drug instructions.

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing, Fluorescence

Journal: Marine Drugs

Article Title: Marine-Steroid Derivative 5α-Androst-3β, 5α, 6β-triol Protects Retinal Ganglion Cells from Ischemia–Reperfusion Injury by Activating Nrf2 Pathway

doi: 10.3390/md17050267

Figure Lengend Snippet: TRIOL inhibits the inflammatory activation of microglia in vitro and in vivo. ( A ) Representative phase-contrast morphological images of BV2 microglia treated with or without lipopolysaccharides (LPS) or TRIOL. BV2 microglia were pretreated with 10 μM TRIOL or vehicle (20% HP-β-CD) 30 min before the treatment of 100 ng/mL LPS. Phase-contrast images were captured 12 h after LPS treatment to observe the activated morphology of microglia. Red arrows indicate activated microglia with typical amoeboid-like morphology; scale bar, 20 μm. ( B ) Number of activated microglia in independent regions of interest (ROI) of different groups were blindly calculated using Image Pro Plus software. ( C ) MRNA expression of inflammatory cytokines TNF-α and IL-1β, and inflammatory chemokines CXCL-10 and CCL2, was detected using real-time polymerase chain reaction (PCR). N = 3 independent experiments for each group. ( D ) Representative images of IHC staining by Iba-1 in the optic nerves of wt mice treated with/without TRIOL in the AIH model. Black arrowheads, activated microglia with activated morphology characterized by enlarged cell bodies, more filopodia, and dense Iba1 staining. Quantification of activated microglia shown in E . Statistical analysis of cell numbers in B and E , and relative mRNA expression in C , was performed using one-way ANOVA, followed by Dunnett’s post hoc test. Data are shown in mean ± SD. N.s. , no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: BV2 microglia (China Center for Type Culture Collection, CCTCC, Wuhan, China) cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Thermofisher Scientific, USA) with 10% FBS (Gibco, Thermofisher Scientific, Rockford, IL, USA).

Techniques: Activation Assay, In Vitro, In Vivo, Software, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

Journal: Marine Drugs

Article Title: Marine-Steroid Derivative 5α-Androst-3β, 5α, 6β-triol Protects Retinal Ganglion Cells from Ischemia–Reperfusion Injury by Activating Nrf2 Pathway

doi: 10.3390/md17050267

Figure Lengend Snippet: TRIOL induces the nuclear translocation of Nrf2 in microglia. BV2 microglia were pretreated with/without 10 μM TRIOL or vehicle (HP-β-CD) 1 h before treatment of 1% O 2 hypoxia stimuli for 12 h, followed by reperfusion to normoxia (20% O 2 ) for 12 h. ( A ) Expression and nuclear localization of Nrf2 and HO-1 were measured using immunofluorescence staining with antibodies against Nrf2 (green) and HO-1 (red) in BV2 microglia. Nucleus was stained with Hoechst 33342 (blue); scale bar, 20 μm. ( B ) Relative HO-1 fluorescence intensity was measured by NIS-Elements AR. N = 4 independent ROIs for each group. ( C ) Nrf2 intensity accumulated in a single nucleus was also measured to evaluate the activation of Nrf2; N = 90 cells for each group. ( D ) Representative confocal imaging of Iba1 (red) and Nrf2 (green) in the optic nerves of wt mice in different groups. Nucleus was stained with Hoechst 33342 (blue). Fields in white boxes were enlarged in the panel below; scale bar, 25 μm. Statistical analysis of fluorescence intensity in B and C was performed using one-way ANOVA, followed by Dunnett’s post hoc test. N.s. , no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: BV2 microglia (China Center for Type Culture Collection, CCTCC, Wuhan, China) cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Thermofisher Scientific, USA) with 10% FBS (Gibco, Thermofisher Scientific, Rockford, IL, USA).

Techniques: Translocation Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Activation Assay, Imaging

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Malaysian brown macroalga Padina australis mitigates lipopolysaccharide-stimulated neuroinflammation in BV2 microglial cells

doi: 10.22038/IJBMS.2023.67835.14842

Figure Lengend Snippet: Effect of (a) PAEE and (b) LPS on the viability of BV2 microglial cells following incubation with different concentrations of PAEE and LPS for 24 hr. Asterisk (*) denotes significant difference ( P <0.05; using Games Howell for PAEE and Kruskal-Wallis for LPS) in viability relative to the negative control. (c) Effect of PAEE on the viability of BV2 microglial cells following pretreatment with different concentrations of ethanol extract for 2 hr and exposure to 1 μg/ml of LPS for 24 hr. Asterisk (*), hash (#), and alias (@) denote a significant difference ( P <0.05; Bonferroni) in viability relative to the negative control, LPS, and L-NAME, respectively

Article Snippet: BV2 microglial cell culture The BV2 murine microglial cell line (Elabscience Biotechnology, Wuhan, Hubei, China) is an immortalized cell line that demonstrates the functional and morphological characteristics of microglia.

Techniques: Incubation, Negative Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Malaysian brown macroalga Padina australis mitigates lipopolysaccharide-stimulated neuroinflammation in BV2 microglial cells

doi: 10.22038/IJBMS.2023.67835.14842

Figure Lengend Snippet: Effect of PAEE on (a) NO and (b) PGE 2 production, and (c) intracellular ROS generation in BV2 microglial cells following pretreatment with different concentrations of PAEE for 2 hr and exposure to 1 μg/ml of LPS for 24 hr. Asterisk (*), hash (#), alias (@), and section (§) denote a significant difference [ P <0.05; Bonferroni (NO) and Games-Howell (PGE 2 and intracellular ROS)] in NO and PGE 2 production, and intracellular ROS level relative to the negative control, LPS, L-NAME, and 0.25 mg/ml PAEE, respectively

Article Snippet: BV2 microglial cell culture The BV2 murine microglial cell line (Elabscience Biotechnology, Wuhan, Hubei, China) is an immortalized cell line that demonstrates the functional and morphological characteristics of microglia.

Techniques: Negative Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Malaysian brown macroalga Padina australis mitigates lipopolysaccharide-stimulated neuroinflammation in BV2 microglial cells

doi: 10.22038/IJBMS.2023.67835.14842

Figure Lengend Snippet: Effect of PAEE on the protein expression of iNOS and COX-2 in BV2 microglial cells following pretreatment with different concentrations of PAEE for 2 hr and exposure to 1 μg/ml of LPS for 24 hr. iNOS and COX-2 were evaluated by western blot analysis (a), the relative expression of iNOS (b), and COX-2 (c) were evaluated by densitometry with β-actin as an internal protein control. The bands corresponding to iNOS and COX-2 are noticeably more intense in LPS compared with that of negative control, whereas PAEE showed less intense bands compared with LPS. Asterisk (*) and hash (#) denote a significant difference [ P <0.05; Games-Howell (iNOS) and Bonferroni (COX-2)] in the expression of iNOS and COX-2 relative to the negative control and LPS, respectively

Article Snippet: BV2 microglial cell culture The BV2 murine microglial cell line (Elabscience Biotechnology, Wuhan, Hubei, China) is an immortalized cell line that demonstrates the functional and morphological characteristics of microglia.

Techniques: Expressing, Western Blot, Control, Negative Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Malaysian brown macroalga Padina australis mitigates lipopolysaccharide-stimulated neuroinflammation in BV2 microglial cells

doi: 10.22038/IJBMS.2023.67835.14842

Figure Lengend Snippet: Effect of PAEE on the expressions of (a) TNF-α and (b) IL-6 in BV2 microglial cells following pretreatment with different concentrations of PAEE for 2 hr and exposure to 1 μg/ml of LPS for 24 hr. Asterisk (*) and hash (#) denote a significant difference [ P <0.05; Bonferroni (TNF-α) and Games-Howell (IL-6)] in the TNF-α and IL-6 concentrations relative to the negative control and LPS, respectively

Article Snippet: BV2 microglial cell culture The BV2 murine microglial cell line (Elabscience Biotechnology, Wuhan, Hubei, China) is an immortalized cell line that demonstrates the functional and morphological characteristics of microglia.

Techniques: Negative Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Malaysian brown macroalga Padina australis mitigates lipopolysaccharide-stimulated neuroinflammation in BV2 microglial cells

doi: 10.22038/IJBMS.2023.67835.14842

Figure Lengend Snippet: Proposed protective effects of PAEE against LPS-stimulated neuroinflammation in BV2 cells. Figure was created using BioRender (https://biorender.com/) and Microsoft PowerPoint 2017. The image of P. australis was captured at Cape Rachado, Port Dickson, Negeri Sembilan, Malaysia

Article Snippet: BV2 microglial cell culture The BV2 murine microglial cell line (Elabscience Biotechnology, Wuhan, Hubei, China) is an immortalized cell line that demonstrates the functional and morphological characteristics of microglia.

Techniques:

Journal: Science Advances

Article Title: Microglial Ffar4 deficiency promotes cognitive impairment in the context of metabolic syndrome

doi: 10.1126/sciadv.adj7813

Figure Lengend Snippet: ( A ) Experimental flow chart. i.p., intraperitoneal. ( B ) Representative trajectory of mice in OFT. ( C ) Center distance and entries into the center area traveled in the open field ( n = 8). ( D ) Representative trajectory of mice in EPM. ( E ) Time spent in the open zones of the mazes and entries into EPM. MWM analysis shows escape latency ( F ) to target in the invisible platform trials. ( G ) Representative trajectory of mice in the probe trials and target cross number in the MWM test. ( H and I ) mRNA expression levels of pro- and anti-inflammatory cytokines in isolated microglial cells from cKO and cKO/PDTC mice ( n = 6). ( J ) Expression levels of type I IFNs ( n = 6). ( K ) ELISA analysis of IFN -β content in hippocampal tissue ( n = 6). ( L ) Representative images of Iba-1–positive cells in the hippocampal DG with 3D reconstruction using Imaris. Scale bar, 50 μm. ( M ) Microglial morphology as total branch length, average branch length, branch numbers, and terminal numbers ( n = 15 from three mice per group). ( N ) Immunoblotting and statistical analysis of p–NF-κB/NF-κB, p-JAK1/JAK1, and p-STAT1/STAT1 levels in hippocampal tissue ( n = 4). ( O ) Immunoblotting and statistical analysis of SYN, PSD95, cleaved caspase-3, and Bcl-2 in hippocampal tissue ( n = 4). ( P ) Interaction between Ffar4-myc and β-arrestin-2 in BV2 cells. ( Q ) The knockdown of β -arrestin-2 increased the NF-κB activation. Immunoblotting and statistical analysis of NF-κB, p–NF-κB, and β-arrestin-2 level in BV2 cells ( n = 3). ( R ) CUT&RUN assay–validated NF-κB binds to target IFN- β promoter after Ffar4 knockdown ( n = 3). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. Detailed statistical analyses are presented in Materials and Methods.

Article Snippet: Microglia primary cell or BV2 cells (China Center for Type Culture Collection, Wuhan, China) were grown to 50 to 70% confluence and then transfected with IFN- β siRNA (50 pM) or normal control (NC)small interfering RNA (siRNA) (50 pM) using jetPRIME transfection reagent (Polyplus, 114-15) according to the manufacturer’s protocol.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Activation Assay