Journal: Theranostics

Article Title: Alisol A ameliorates vascular cognitive impairment via AMPK/NAMPT/SIRT1-mediated regulation of cholesterol and autophagy

doi: 10.7150/thno.112661

Figure Lengend Snippet: Modulation of NAMPT activity and its network by Alisol A restores cholesterol balance, mitophagy, and synaptic function in Ldlr -/- mice. (A) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B protein quantitation for WB, n = 3. (B) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B in cells, and quantification result was normalized against the levels of GAPDH or COXIV, n = 3; (C) Changes in MMP was monitored by staining with JC1 in HT22 cells, scale: 100 μm; The ratio of red/green fluorescence intenity was employed to assess MMP, n = 8; (D) Representative IF images of OPTN and TOM20 in HT22 cells, scale: 2 μm; Quantification of IF stain intensity using Image J, n = 8; IF and colocalization analysis. (E) WB analysis of PINK1 in HT22 cells and quantification result was normalized against the levels of GAPDH, n = 3; (F) Subsequent COIP experiments employed NAMPT antibodies to assess interactions with NAMPT and PINK1, using IgG as a control. (G) Illustrative IF images depicting hippocampal neurons located within the CA3 subregion, scale: 100 μm, enlarge scale: 10 μm, n = 5; IF was quantified for UCP2 (red); (H) The phenomenon of intercellular ROS generation was observed through the detection of intracellular ROS levels using the DCFH-DA probe following different treatment protocols, scale: 100 μm, n = 5; (I) Intracellular ROS levels were measured using flow cytometry, comparative analysis of fluorescence intensity within peak plots derived from flow cytometry, n = 5; (J) Synapses in the hippocampal CA1 and DG region were revealed by IF staining for the pre-and postsynaptic markers, Synapsin-1 (red) and PSD95 (green) respectively, scale: 10 μm, enlarge scale: 2 μm, n = 5; and quantification of IF; (K) Golgi-Cox Staining Hippocampal Golgi-Cox staining was performed as previously described, scale: 100 μm, enlarge scale: 20 μm, n = 8; Track the neural branches after Golgi staining and analysis of Sholl results and statistical data; Analysis of dendritic spine density in neurons stained with Golgi, along with the quantification of mushroom spines, scale: 2 μm; (L) Representative IF images of NMDA 2A (green) and NMDA 2B (red), scale: 2 μm; and quantification of IF, n = 5. Compared to the Ldlr -/- mice , * P < 0.05, ** P < 0.01; compared to the FK866 group, # P < 0.05, ## P < 0.01. Compared to the 1, * P < 0.05, ** P < 0.01; compared to the 3, # P < 0.05, ## P < 0.01. DMSO: HT22 +DMSO; Rosup: HT22 + Aβ 25-35 (20 μM) and complex (50 μg/ml of cholesterol) +H 2 O 2 0.1 mM ; CCCP: HT22+ CCCP 10 μM. Compared to the 5, * P < 0.05, ** P < 0.01; compared to the 7, # P < 0.05, ## P < 0.01.

Article Snippet: Anti-GFAP antibody (GB12096, Servicebio, 1:500), anti-IBA-1 antibody (GB12105, Servicebio, 1:500). anti-PSD95 antibody (20665-1-AP, Proteintech, 1:300), anti-SYNAPSIN-1 antibody (20258-1-AP, Proteintech, 1:300), anti-UCP2 antibody (GB11377, Servicebio, 1:300), anti-CD68 antibody ( GB113109 , Servicebio, 1:300), anti-α-SMA antibody ( GB111364 , Servicebio, 1:300), anti-HMGCR antibody (DF6518, Affinity, 1:200), anti-ABCA1 Polyclonal antibody (26564-1-AP, Proteintech, 1:300). anti-NAMPT/PBEF Polyclonal antibody (11776-1-AP, Proteintech, 1:300), anti-SIRT1 antibody (13161-1-AP, Proteintech, 1:300). anti-CYP46A1 Polyclonal antibody (124861-1-AP, Proteintech, 1:300). anti-OPTN antibody ( GB114324 , Servicebio, 1:300), anti-TOM20 antibody ( GB111481 , Servicebio, 1:300), anti-NMDAR2A/GRIN2A Polyclonal antibody (28525-1-AP, Proteintech, 1:300), anti-NMDA2B antibody (21920-1-AP, Proteintech, 1:300).

Techniques: Activity Assay, Protein Quantitation, Staining, Fluorescence, Control, Flow Cytometry, Derivative Assay