Journal: Investigative Ophthalmology & Visual Science
Article Title: NMN Supplementation Inhibits Endothelial Cell ROS-Mediated Src/Pi3k/Akt Signaling Pathway to Protect High-Altitude Blood-Retinal Barrier
doi: 10.1167/iovs.66.4.51
Figure Lengend Snippet: NMN intervention reduces retinal oxidative stress and DNA damage, and inhibition of ROS protects the retina. Mice were placed in a simulated high-altitude hypobaric hypoxia chamber at an altitude of 7 km for 24 hours. Mice in the NMN intervention group began receiving NMN at 100 mg/kg/day mixed in drinking water 5 days before entering the hypoxic chamber. Mice in the NAC intervention group (N-Acetyl-L-cysteine, an ROS scavenger) received NAC at 150 mg/kg/day mixed in drinking water starting 5 days before the hypoxic exposure. Both groups were then exposed to the hypoxic chamber along with the 7 km 24 hours group mice. Subsequent experimental procedures were the same. ( A, B, C ) The DHE probe was used to detect ROS levels in retinal sections of mice ( n = 3, biological replicates). In panel A , DHE (Dihydroethidium, red ) indicated intracellular superoxide anions. The white arrows point to cross-sections of retinal blood vessels. Scale bar = 20 µm. The average DHE fluorescence intensity of retinal vessels and the whole retinal layer was analyzed, with three slices selected per eye and three eyes per group. ( D, E, F ) Immunofluorescence staining was used to detect 8-Oxoguanine ( red ) expression in retinal sections of mice ( n = 3, biological replicates). Blue represents DAPI. The merged image combined the red and blue channels. The white arrows point to cross-sections of retinal blood vessels. Scale bar = 20 µm. The yellow boxes show high-magnification views of vascular endothelial cells. The average fluorescence intensity of 8-Oxoguanine in retinal blood vessels and across the full thickness of the retina was analyzed. Three slices selected per eye and three eyes per group. ( G, I , J, K ) Western blot analysis and quantification of phosphorylated and total VE-cadherin, Bax and Bcl-2, and Cleaved-caspase3 and Caspase-3 in retinal tissues of mice under different interventions ( n = 3, biological replicates). Phosphorylated VE-cadherin signals were normalized to total VE-cadherin, Bax signals were normalized to Bcl-2, and Cleaved-caspase3 signals were normalized to Caspase-3. Relative signals for phosphorylated VE-cadherin, Bax/Bcl-2 ratios, and Cleaved-caspase3/Caspase-3 ratios were normalized to control group levels (each control group data set as 1). β-Actin was used as a loading control. ( H, L , M, N ) Western blot analysis and quantification of phosphorylated Src, Pi3k, Akt, and total Src, Pi3k, Akt in retinal tissues of mice under different interventions ( n = 3, biological replicates). Phosphorylated Src, Pi3k, and Akt signals were normalized to total Src, Pi3k, and Akt. Relative signals for phosphorylated Src, Pi3k, and Akt were normalized to control group levels (each control group data set as 1). β-Actin was used as a loading control. Values were expressed as the mean ± SD. Panels B , C , E , F , and I to N were analyzed using a one-way analysis of variance followed by Tukey's post hoc multiple comparison tests: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
Article Snippet: The cells were washed with PBS 4 times, for 5 minutes per wash. We then diluted the superoxide anion fluorescence probe DHE (HY-D0079, MedChemExpress, China) to 1 µM in PBS.
Techniques: Inhibition, Fluorescence, Immunofluorescence, Staining, Expressing, Western Blot, Control, Comparison
