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Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM - Redox Biol (2015)

Bottom Line: Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear.A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence.Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, California University of Science & Medicine, Colton, CA 92324, USA; Department of Basic Sciences, The Commonwealth Medical College (TCMC), Scranton, PA 18509, USA. Electronic address: atamnah@calmedu.org.

No MeSH data available.


Related in: MedlinePlus

The effect of MB on NAD/NADH ratio. IMR90 cells were cultured in the presence or absence of 100 nM MB for 0 (control), 0.25, 2, and 24 h. After harvesting the cultures the cells were counted and 105 cells were used to assay NADH and NAD with the help of EnzyChrom NAD/NADH Assay Kit as described in Section 2. The level of NAD and NADH was assayed by enzymatic recycling followed by measuring the change in absorbance at 565 nm at 0 and 15 min. A. The concentration of NADH (upper panel) and NAD (center panel) were calculated using a standard curve and those were used to calculate the ratio NAD/NADH (Lower Panel). Three independent experiments are shown each was performed in triplicates. Values are Mean±sem (t-test, *P<0.02). B. A representative immunoblot depicting the effect of MB on NAMPT as well as the ratio of NAMPT to Actin calculated from immuneblots similar to the one presented.
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f0005: The effect of MB on NAD/NADH ratio. IMR90 cells were cultured in the presence or absence of 100 nM MB for 0 (control), 0.25, 2, and 24 h. After harvesting the cultures the cells were counted and 105 cells were used to assay NADH and NAD with the help of EnzyChrom NAD/NADH Assay Kit as described in Section 2. The level of NAD and NADH was assayed by enzymatic recycling followed by measuring the change in absorbance at 565 nm at 0 and 15 min. A. The concentration of NADH (upper panel) and NAD (center panel) were calculated using a standard curve and those were used to calculate the ratio NAD/NADH (Lower Panel). Three independent experiments are shown each was performed in triplicates. Values are Mean±sem (t-test, *P<0.02). B. A representative immunoblot depicting the effect of MB on NAMPT as well as the ratio of NAMPT to Actin calculated from immuneblots similar to the one presented.

Mentions: Following 15 min of treatment of IMR90 cells with MB the ratio NAD/NADH increased by 63%, which within 24 h returned to the control level (Fig. 1). MB did not affect NAMPT, a rate-limiting enzyme in NAD synthesis (Fig. 1B), indicating no de novo synthesis of NAD in MB-treated cells. The data are consistent with the transient increase in NADH oxidation in MB-treated cells (Fig. 1). The ratio NAD/NADH is a key indicator for the cellular energy and redox status [39]. Reduced intracellular energy status (high NAD/NADH ratio) results in AMPK phosphorylation [40], thus we examined the effect of MB on AMPK. MB increased the phosphorylation of AMPK at Thr-172 (pAMPK) and as a result the ratio pAMPK/AMPK gradually increased after 1 h of treatment with MB until reached significance after 24 h. pAMPK/AMPK returned to the level of the control after 48 h (Fig. 2A and B). Since pAMPK is capable of inducing PGC1α, the time-dependent effect of MB on PGC1α was also examined. MB induced PGC1α after 24 h of incubation (Fig. 3A and B). However, MB did not affect the protein level of SIRT1 or p-Ser47-SIRT1 even after 7 days of incubation (Data not shown).


Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM - Redox Biol (2015)

The effect of MB on NAD/NADH ratio. IMR90 cells were cultured in the presence or absence of 100 nM MB for 0 (control), 0.25, 2, and 24 h. After harvesting the cultures the cells were counted and 105 cells were used to assay NADH and NAD with the help of EnzyChrom NAD/NADH Assay Kit as described in Section 2. The level of NAD and NADH was assayed by enzymatic recycling followed by measuring the change in absorbance at 565 nm at 0 and 15 min. A. The concentration of NADH (upper panel) and NAD (center panel) were calculated using a standard curve and those were used to calculate the ratio NAD/NADH (Lower Panel). Three independent experiments are shown each was performed in triplicates. Values are Mean±sem (t-test, *P<0.02). B. A representative immunoblot depicting the effect of MB on NAMPT as well as the ratio of NAMPT to Actin calculated from immuneblots similar to the one presented.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588422&req=5

f0005: The effect of MB on NAD/NADH ratio. IMR90 cells were cultured in the presence or absence of 100 nM MB for 0 (control), 0.25, 2, and 24 h. After harvesting the cultures the cells were counted and 105 cells were used to assay NADH and NAD with the help of EnzyChrom NAD/NADH Assay Kit as described in Section 2. The level of NAD and NADH was assayed by enzymatic recycling followed by measuring the change in absorbance at 565 nm at 0 and 15 min. A. The concentration of NADH (upper panel) and NAD (center panel) were calculated using a standard curve and those were used to calculate the ratio NAD/NADH (Lower Panel). Three independent experiments are shown each was performed in triplicates. Values are Mean±sem (t-test, *P<0.02). B. A representative immunoblot depicting the effect of MB on NAMPT as well as the ratio of NAMPT to Actin calculated from immuneblots similar to the one presented.
Mentions: Following 15 min of treatment of IMR90 cells with MB the ratio NAD/NADH increased by 63%, which within 24 h returned to the control level (Fig. 1). MB did not affect NAMPT, a rate-limiting enzyme in NAD synthesis (Fig. 1B), indicating no de novo synthesis of NAD in MB-treated cells. The data are consistent with the transient increase in NADH oxidation in MB-treated cells (Fig. 1). The ratio NAD/NADH is a key indicator for the cellular energy and redox status [39]. Reduced intracellular energy status (high NAD/NADH ratio) results in AMPK phosphorylation [40], thus we examined the effect of MB on AMPK. MB increased the phosphorylation of AMPK at Thr-172 (pAMPK) and as a result the ratio pAMPK/AMPK gradually increased after 1 h of treatment with MB until reached significance after 24 h. pAMPK/AMPK returned to the level of the control after 48 h (Fig. 2A and B). Since pAMPK is capable of inducing PGC1α, the time-dependent effect of MB on PGC1α was also examined. MB induced PGC1α after 24 h of incubation (Fig. 3A and B). However, MB did not affect the protein level of SIRT1 or p-Ser47-SIRT1 even after 7 days of incubation (Data not shown).

Bottom Line: Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear.A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence.Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, California University of Science & Medicine, Colton, CA 92324, USA; Department of Basic Sciences, The Commonwealth Medical College (TCMC), Scranton, PA 18509, USA. Electronic address: atamnah@calmedu.org.

No MeSH data available.


Related in: MedlinePlus