Journal: Cells

Article Title: Obligatory Role of AMPK Activation and Antioxidant Defense Pathway in the Regulatory Effects of Metformin on Cellular Protection and Prevention of Lens Opacity

doi: 10.3390/cells11193021

Figure Lengend Snippet: Metformin successfully internalized into mouse eye lenses and delayed/prevented the lens opacity and mitigated ROS accumulation induced by H 2 O 2 by enhancing the Bmal1/Nrf2/antioxidant, such as Prdx6 in vitro. ( A ) Metformin penetration in the mouse lens cultured in vitro. Lenses isolated from 14 months old mouse were cultured in 48-well culture plate and treated with 3 mM Metformin for 24 h and examined for Metformin penetration using Acquity UPLC coupled with Waters triple-quad Xevo TQS analysis and Metformin peak was presented as histogram. The data represent the mean ± S.D. from three independent experiments. Untreated vs. Metformin-treated; ** p < 0.001. ( B ) Lenses isolated from 14 months old C57BL/6 mouse were used for in vitro organ culture as described previously [ , , ]. The cultured lenses were treated with 3 mM of Metformin followed by 100 µM of H 2 O 2 exposure. 44 h or 96 h later the lenses were photographed (Nikon SMZ 745T) connected with Camera and software (Nikon). Photograph is representative of three experiments: Metformin significantly prevented the H 2 O 2 -induced lens opacity by 61% and 72% (c and e) compared to H 2 O 2 (b and d) and untreated lens control (a). ( C ) Histogram indicates densitometry of lens opacity. a, 2.53 ± 0.70 (control, black bar); b, 31.81 ± 0.28 (orange bar: H 2 O 2 alone, 44 h); c, 12.50 ± 0.68 (blue-orange bar: Metformin + H 2 O 2 alone, 44 h); d, 68.14 ± 1.37 (orange bar: H 2 O 2 , 96 h); e, 19.02 ± 0.30 (blue-orange bar: Metformin + H 2 O 2 , 96 h). All histograms are presented as mean ± S.D. values-derived from three independent experiments, ** p < 0.001. ( D , E ) Lenses Isolated from 14 months old C57BL/6 mouse were cultured and treated with 3 mM of Metformin followed by H 2 O 2 exposure. 48 h later ROS level and mRNA expression of Bmal1, Nrf2 and Prdx6 were analyzed using H2-DCF-DA dye and RT-qPCR analyses, respectively as indicated in Figure, and described in Materials and Methods. All histograms are presented as mean ± S.D. values-derived from three independent experiments, ** p < 0.001.

Article Snippet: 44 h and 96 h later lenses were photographed using Nikon SMZ 745T fitted with Nikon Camera and Computer loaded with analyses software program [ , , , ].

Techniques: In Vitro, Cell Culture, Isolation, Organ Culture, Software, Derivative Assay, Expressing, Quantitative RT-PCR