Piceatannol markedly upregulates heme oxygenase-1 expression and alleviates oxidative stress in skeletal muscle cells

Abstract

Piceatannol (PIC), a phytochemical, is abundant in passion fruit (Passiflora edulis) seeds. In this study, we investigated the effects of PIC on the expression levels of antioxidant enzymes in C2C12 skeletal muscle cells and compared its effects with those of PIC analogues and polyphenols. We also evaluated its effects on hydrogen peroxide-induced accumulation of reactive oxygen species in C2C12 myotubes. Treatment with PIC led to dose-dependent upregulation of heme oxygenase-1 (Ho-1) and superoxide dismutase 1 (Sod1) mRNA expression in C2C12 myotubes. PIC was the most potent inducer of Ho-1 among the PIC analogues and major polyphenols tested. In addition, treatment with PIC suppressed the hydrogen peroxide-induced increase in intracellular reactive oxygen species levels. Our results suggest that PIC protects skeletal muscles from oxidative stress by activating antioxidant enzymes such as HO-1 and SOD1 and can therefore help prevent oxidative stress-induced muscle dysfunction such as muscle fatigue and sarcopenia.

Keywords: Antioxidant enzymes; HO-1; Oxidative stress; Piceatannol; Skeletal muscle cell.

Graphical abstract

Fig. 1

Effects of PIC on Ho-1 and Sod1 mRNA expression. C2C12 myotubes were incubated with the DMSO control (CON; white bar) or 10–50 μM piceatannol (black bars) for 6 h, and Ho-1 (A) and Sod1 (B) mRNA expression was analyzed by real-time PCR. Values have been expressed in terms of the fold change compared with the control, which was arbitrarily set to 1. Results have been provided as mean + S.D. values from at least three separate experiments. Different alphabets represent significant difference at p < 0.05; the analysis involved ANOVA with the Games–Howell post-hoc test (A) or Tukey post-hoc test (B).

Fig. 2

Effects of PIC and other compounds on Ho-1 and Sod1 mRNA expression. C2C12 myotubes were stimulated with the DMSO control (CON; white bar) or stimulants (50 μM; black bars) for 6 h, and Ho-1 (A) and Sod1 (B) mRNA expression was analyzed by real-time PCR. The stimulants were piceatannol (PIC), resveratrol (RES), oxyresveratrol (OXY), rhapontigenin (RHA), isorhapontigenin (ISOR), 3,3',4,5'-tetramethoxypiceatannol (TMP), quercetin (QUE), epigallocatechin gallate (EGCG), and sesamin (SESA). Values have been expressed in terms of the fold change compared with the control, which was arbitrarily set to 1. Results have been provided as mean + S.D. values from four separate experiments. Different alphabets represent significant difference at p < 0.05; the analysis involved ANOVA with the Games–Howell post-hoc test.

Fig. 3

Effects of PIC on H₂O₂-induced intracellular ROS levels. The cells were incubated with the DMSO control, 1mM N-acetylcysteine (NAC), or 20 μM piceatannol (PIC) and loaded with 3 μM CM-H₂DCFDA for 30 min. ROS accumulation was determined on treatment with or without 50 μM H₂O₂. The graph shows the fluorescence intensity produced by ROS. Values have been expressed in terms of the fold change compared with the DMSO control with H₂O₂, which was arbitrarily set to 1. Results have been provided as mean + S.D. values from at least four separate experiments. Different alphabets represent significant difference at p < 0.05; the analysis involved ANOVA with the Tukey post-hoc test. The bottom panel depicts representative fluorescence microscopy images showing the fluorescence intensity produced by ROS. The scale bar is 100 μm.