Journal: Nutrients

Article Title: Nicotinamide Prevents Diabetic Brain Inflammation via NAD+-Dependent Deacetylation Mechanisms

doi: 10.3390/nu15143083

Figure Lengend Snippet: Effect of NAM on the brain NAD+ content and acetylated form of NFκB (p65) in brains of STZ-induced diabetic mice. ( a ) Brain NAD+ content. ( b ) Relative protein abundance of the acetylated NFκB (p65) obtained by western blot analysis and representative images of brain acetylated NFκB (p65) in each condition. ( c ) Relative protein abundance of the acetylated NFκB (p65) obtained by IHC analysis and representative images of brain acetylated NFκB (p65) in each condition. Abbreviations used: NAD+, oxidized form of nicotinamide adenine dinucleotide; NAM LD, low-dose, NAM-treated, diabetic mice; NAM HD, high-dose, NAM-treated, diabetic mice; non-T1D, group of mice without T1D; T1D, group of mice with T1D. Data are expressed as the mean (standard deviation) of 4–5 mice/group. Images of 10 randomly selected fields were used for quantification. The scale bar shown in the images represents 100 µm. Statistically significant differences among groups for each variable were determined using a parametric ANOVA test followed by Tukey’s posttest. Differences were considered significant when p -value < 0.05. Specifically, * p -value < 0.05 vs. non-diabetic group; † p -value < 0.05 vs. diabetic group.

Article Snippet: NAD+ levels were determined in brain tissue using an NAD/NADH colorimetric determination kit (cat# ab65348 Abcam) according to the manufacturer’s instructions.

Techniques: Western Blot, Standard Deviation

Journal: Nutrients

Article Title: Nicotinamide Prevents Diabetic Brain Inflammation via NAD+-Dependent Deacetylation Mechanisms

doi: 10.3390/nu15143083

Figure Lengend Snippet: Relationship between relative NAD+ content and biomarkers of inflammation and microglial activation in brains from diabetic mice. ( a ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain NFκB (p65). ( b ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain acetyl-NFκB (p65). ( c ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain IBA-1. ( d ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain BDKRB-1. ( e ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain TNFα. ( f ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain F4/80. The relationship between parameters was tested using a parametric Pearson’s correlation test. The relative levels of brain NAD+ were calculated taking as 1 the mean of individual values for this parameter in untreated diabetic mice. Mice of all groups were considered for analysis.

Article Snippet: NAD+ levels were determined in brain tissue using an NAD/NADH colorimetric determination kit (cat# ab65348 Abcam) according to the manufacturer’s instructions.

Techniques: Activation Assay

Journal: Journal of Neuroinflammation

Article Title: A selective inhibitor of the NLRP3 inflammasome as a potential therapeutic approach for neuroprotection in a transgenic mouse model of Huntington’s disease

doi: 10.1186/s12974-022-02419-9

Figure Lengend Snippet: MCC950 markedly reduces cytotoxicity in striatal progenitor cells and BV2 microglial cells. A , B BV2 microglia were incubated for 4 h with LPS (1 μg/mL) followed by incubation with MCC950 (1 μM) for 2 h. The cells were then incubated with ATP (1 mM, 24 h). Total lysates of BV2 microglial cells were assessed by Western blot analysis to determine the levels of the NLRP3 and actin proteins. The molecular mass is indicated in kilodaltons. C , D BV2 microglia were incubated for 4 h with LPS (1 μg/mL) followed by incubation with MCC950 (1 μM) for 2 h. The cells were then incubated with ATP (1 mM for 24 h). Cell survival ( C ) and IL-1β expression levels ( D ) were measured using the CCK-8 assay and ELISA, respectively. The values of the indicated cells were normalized to those of untreated BV2 cells. * P < 0.05 compared to LPS/ATP treated cells ( n = 3). E , F ST Hdh Q109 cells were incubated for 24 h with MCC950 (1 μM). Total lysates of ST Hdh Q7 and ST Hdh Q109 cells were assessed using Western blot analysis. G ST Hdh Q7 and ST Hdh Q109 cells were incubated for 24 h with MCC950 (1 μM). Cell death was quantified using the CCK-8 assay; the values of the indicated cells were normalized to those of untreated ST Hdh Q7 cells. The data are presented as the mean ± SEM from three independent experiments. * P < 0.05, ST Hdh Q7 vs. ST Hdh Q109 cells; # P < 0.05 vs. untreated ST Hdh Q109 cells. H BV2 cells were incubated with LPS (1 µg/mL for 4 h) with or without MCC950 for 2 h before stimulation with ATP (1 mM for 24 h). The BV2 medium was then collected and used to culture the ST Hdh Q109 cells for an additional 24 h. ST Hdh Q7 and ST Hdh Q109 cell viability was determined by CCK-8 assay. The data are presented as the mean ± SEM from three independent experiments. * P < 0.05, ST Hdh Q7 vs. ST Hdh Q109 cells; # P < 0.05 vs. untreated ST Hdh Q109 cells. I BV2 cells were treated with or without MCC950 (1 μM) and 3-NP (5, 10, and 20 mM) for 24 h. The cell viability was determined using the CCK-8 assay. Data are presented as the mean ± SEM from three independent experiments. * P < 0.05 compared with controls ( n = 3)

Article Snippet: BV2 cells (mouse, C57BL/6; brain, microglial cells) were purchased from the American Type Culture Collection (Rockville, MD).

Techniques: Incubation, Western Blot, Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay