Journal: Neural Regeneration Research

Article Title: Exosome-transported lncRNA H19 regulates insulin-like growth factor-1 via the H19/let-7a/insulin-like growth factor-1 receptor axis in ischemic stroke

doi: 10.4103/1673-5374.357901

Figure Lengend Snippet: Expression of IGF1R and lncRNA H19 in neurons and astrocytes treated by OGD/R, and effect of neuronal IGF1R and lncRNA H19 inhibition on levels of astrocytic IGF1R and lncRNA H19. (A, B) Western blot bands and quantification of IGF1R protein expression in neurons. (C, D) Western blot bands and quantification of IGF1R expression in astrocytes. (E) Expression of lncRNA H19 induced by oxygen glucose deprivation/reperfusion (OGD/R) in neurons. (F) Expression of lncRNA H19 induced by OGD/R in astrocytes. (G) Effect of neuronal exosome lncRNA H19 inhibition on expression of lncRNA H19 in astrocytes. (H) Effect of neuronal exosome lncRNA H19 inhibition on expression of let-7a in astrocytes. (I, J) Western blots (I) and quantification (J) of IGF1R expression in astrocytes induced by neuronal exosome lncRNA H19 inhibition. * P < 0.05, vs . normal control group; # P < 0.05, vs . OGD/R group; & P < 0.05, vs . OGD/R and exosome co-culture system. All experiments repeated three times. Data presented as mean ± standard deviation and analyzed by Student’s t -test (A–F) or one-way analysis of variance followed by Tukey’s post hoc test (G–J). IGF1R: Insulin-like growth factor-1 receptor; N-A: normal astrocyte; N-N: normal neuron.

Article Snippet: Cell lysates were subjected to 10% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated proteins were transferred to a nitrocellulose membrane (Millipore Sigma), blocked with 5% skimmed milk powder in 0.1% Tween 20 (Ding Guo, Bejing, China) in Tris buffer solution for 1 hour at room temperature, and incubated overnight at 4°C with the following antibodies: rabbit monoclonal IGF1R antibody (1:1000, ab182408; Abcam) and mouse monoclonal β-actin antibody (1:2000, sc81178; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with a horseradish peroxidase-conjugated antibody (goat anti-rabbit IgG, 1:2000, ab150077; Abcam; goat anti-mouse IgG, 1:2000, ab150133; Abcam).

Techniques: Expressing, Inhibition, Western Blot, Co-Culture Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Exosome-transported lncRNA H19 regulates insulin-like growth factor-1 via the H19/let-7a/insulin-like growth factor-1 receptor axis in ischemic stroke

doi: 10.4103/1673-5374.357901

Figure Lengend Snippet: Effects of lncRNA H19 and let-7a inhibition on expression of IGF1R. (A, B) Western blot bands and quantification of IGF1R protein expression in normal control astrocytes, oxygen glucose deprivation/reperfusion (OGD/R) astrocytes, OGD/R astrocytes treated with exosomes secreted by neurons, OGD/R astrocytes treated with exosomes secreted by lncRNA H19-inhibited neurons, and OGD/R astrocytes treated with exosomes secreted by lncRNA H19- and let-7a-inhibited neurons. * P < 0.05, vs . normal control group; # P < 0.05, vs . OGD/R group; && P < 0.01, vs . OGD/R and exosome co-culture group; $ P < 0.05, vs . OGD/R and lncRNA H19-inhibited exosome co-culture group. All experiments repeated three times. Data are presented as mean ± standard deviation and were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. IGF1R: insulin-like growth factor-1 receptor; N-A: normal astrocyte.

Article Snippet: Cell lysates were subjected to 10% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated proteins were transferred to a nitrocellulose membrane (Millipore Sigma), blocked with 5% skimmed milk powder in 0.1% Tween 20 (Ding Guo, Bejing, China) in Tris buffer solution for 1 hour at room temperature, and incubated overnight at 4°C with the following antibodies: rabbit monoclonal IGF1R antibody (1:1000, ab182408; Abcam) and mouse monoclonal β-actin antibody (1:2000, sc81178; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with a horseradish peroxidase-conjugated antibody (goat anti-rabbit IgG, 1:2000, ab150077; Abcam; goat anti-mouse IgG, 1:2000, ab150133; Abcam).

Techniques: Inhibition, Expressing, Western Blot, Co-Culture Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Fingolimod protects against neurovascular unit injury in a rat model of focal cerebral ischemia/reperfusion injury

doi: 10.4103/1673-5374.353500

Figure Lengend Snippet: Fingolimod (FTY-720) attenuates ischemia/reperfusion-induced glial cell activation and IL-17A expression. (A) Representative immunofluorescence staining for IL-17A (red) and GFAP (green). (B) Representative immunofluorescence staining for IL-17A (red) and Iba-1 (green). Scale bars: 50 μm. (C) Representative western blot bands for IL-17A. (D) Quantification of IL-17A protein expression ( n = 5 rats per group, * P < 0.05, vs . DMSO group, one-way analysis of variance followed by Bonferroni post hoc test). The experiment was repeated five times. DMSO: Dimethyl sulfoxide; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium-binding adapter molecule 1; IL: interleukin.

Article Snippet: The frozen brain sections (20-μm thick) were blocked for 1 hour with 5% normal goat serum, followed by overnight incubation at 4°C with the following primary antibodies: rabbit anti-occludin antibody (tight junction protein, 1:100; Cat# 40-4700, Thermo Fisher Scientific), rabbit anti-claudin-5 polyclonal antibody (tight junction protein, 1:100; Cat# PA5-99415, Thermo Fisher Scientific); rabbit monoclonal anti-S1PR1 antibody (1:100; Cat# 55133-1-AP, Proteintech, Chicago, IL, USA); mouse monoclonal anti-CD31 antibody (a marker for microvascular endothelial cells; 1:50; Cat# sc-376764, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-NeuN (neuronal nuclei) antibody (a marker for neurons; 1:100; Cat# 834501, Biolegend, San Diego, CA, USA); mouse monoclonal anti-GFAP (glial fibrillary acidic protein) antibody (a marker for astrocytes; 1:100; Cat# 80788, CST, Danvers, MA, USA); rabbit monoclonal anti-Iba1 (ionized calcium-binding adapter molecule 1) antibody (a marker for microglia; 1:100; Cat# ab178846, Abcam, Cambridge, MA, USA) and mouse monoclonal anti-IL-17A antibody (a proinflammatory cytokine; 1:100; Cat#PA5-106856, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot, Binding Assay

Journal: Biomedical Reports

Article Title: Ameliorative effects of 6‑gingerol in cerebral ischemia are mediated via the activation of antioxidant and anti‑inflammatory pathways

doi: 10.3892/br.2023.1608

Figure Lengend Snippet: Schematic diagram of the experimental design use in the present study. Rt.MCAO, right middle cerebral artery occlusion; BW, body weight; TTC, 2,3,5-triphenyltetrazolium chloride; MDA, malondialdehyde; COX-2, cyclooxygenase-2; IL-6, interleukin 6.

Article Snippet: Equal quantities of protein (40 µg protein) were subjected to 10% SDS-polyacrylamide gel electrophoresis, transferred to a Hybond-P (PVDF) membrane (Cytiva) and then incubated with rabbit monoclonal anti-COX-2 (1:1,000, cat. no. ab179800, Abcam), mouse monoclonal anti-IL-6 (1:2,000, cat. no. ab9324, Abcam) and rabbit monoclonal anti-β-actin (1:5,000, cat. no. AC026, ABclonal Biotech Co., Ltd.) antibodies at 4˚C overnight.

Techniques:

Journal: Biomedical Reports

Article Title: Ameliorative effects of 6‑gingerol in cerebral ischemia are mediated via the activation of antioxidant and anti‑inflammatory pathways

doi: 10.3892/br.2023.1608

Figure Lengend Snippet: Effects of 6-gingerol on COX-2 and IL-6 expression in the cerebral cortex and hippocampus of rats determined using western blot analysis. (A) Representative western blots of COX-2 (74 kDa) and IL-6 (25 kDa) from the cerebral cortex. β-actin (41 kDa) was used as a loading control. (B) Quantitative analysis of the COX-2 band density normalized to β-actin. (C) Quantitative analysis of IL-6 band density normalized to β-actin. Data are expressed as the mean ± SEM (n=5). * P<0.05, compared to the control; # P<0.05, compared to the Rt.MCAO + vehicle group. Rt.MCAO, right middle cerebral artery occlusion; BW, body weight; Gin, 6-gingerol; COX-2, cyclooxygenase-2; IL-6, interleukin 6.

Article Snippet: Equal quantities of protein (40 µg protein) were subjected to 10% SDS-polyacrylamide gel electrophoresis, transferred to a Hybond-P (PVDF) membrane (Cytiva) and then incubated with rabbit monoclonal anti-COX-2 (1:1,000, cat. no. ab179800, Abcam), mouse monoclonal anti-IL-6 (1:2,000, cat. no. ab9324, Abcam) and rabbit monoclonal anti-β-actin (1:5,000, cat. no. AC026, ABclonal Biotech Co., Ltd.) antibodies at 4˚C overnight.

Techniques: Expressing, Western Blot