Scavenging of reactive oxygen species by astaxanthin inhibits epithelial–mesenchymal transition in high glucose-stimulated mesothelial cells

<div><p>Background</p><p>High glucose concentrations influence the functional and structural development of the peritoneal membrane. We previously reported that the oral administration of astaxanthin (AST) suppressed peritoneal fibrosis (PF) as well as inhibited oxidative stress, inflammation, and epithelial–mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in a chlorhexidine-induced PF rat model. This suggests that oxidative stress induction of EMT is a key event during peritoneal damage. The present study evaluated the therapeutic effect of AST in suppressing EMT, in response to glucose-induced oxidative stress.</p><p>Methods</p><p>Temperature-sensitive mesothelial cells (TSMCs) were cultured in the presence or absence of AST and then treated with 140 mM glucose for 3 or 12 hours. Expression levels of TNF-α, TGF-β, and VEGF were determined at the mRNA and protein levels, and nuclear factor kappa B (NF-κB) activity was evaluated. We measured NO₂⁻/NO₃⁻ concentrations in cellular supernatants and determined 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in mitochondrial and nuclear DNA. The expressions of E-cadherin and alpha-smooth muscle actin (α-SMA) were evaluated by double immunofluorescence and protein levels.</p><p>Results</p><p>High glucose concentrations induced overproduction of reactive oxidative species (ROS), increasing 8-OHdG mitochondrial DNA and cytokine levels. The NF-κB pathway was activated in response to high glucose concentrations, whereas <i>de novo</i> α-SMA expression was observed with decreased E-cadherin expression. AST treatment attenuated ROS production, inflammatory cytokine production, NF-κB activation, and EMT.</p><p>Conclusion</p><p>The findings of the present study indicate that AST may have an anti-EMT effect due to anti-oxidative and anti-inflammatory activities by scavenging glucose-induced ROS from mitochondria in PMCs. AST may be an efficacious treatment for PF.</p></div

Effect of high glucose stimulation, AST, and AA to TSMCs as for ROS.

<p>(A) NO₂⁻/NO₃⁻ concentration in medium supernatant of each group. (B) Intracellular ROS levels of each group. (C) 8-OHdG ratio in mitochondrial DNA of each group. (D) 8-OHdG ratio in nuclear DNA of each group. AST concentration: 5 μM, AA concentration: 100 μM. NS: no significant change. *: p < 0.05. **: p < 0.01. ***: p < 0.0005. ****: p < 0.0001. Error bars represent SD. (E) ROS fluorescence of each group (1) C3h group, (2) C12h group, (3) AST3h group, (4) AST12h group, (5) AA3h group, (6) AA12h group, (7) G3h group, (8) G12h group, (9) AST-G3h group, (10) AST-G12h group, (11) AA-G3h group, and (12) AA-G12h group. Fluorescence solution was 2′, 7′-dichlorodihydrofluorescin diacetate (DCFH-DA).</p

Effect of high glucose stimulation to TSMCs as for NF-κB pathway.

<p>(A) NF-κB p65 protein subunit expression in TSMCs in each group. *: p < 0.05. **: p < 0.01. ***: p < 0.0005. ****: p < 0.0001. Error bars represent SD. (B) NF-κB p65 subunit immunofluorescence. (1) C3h group, (2) C12h group, (3) AST3h group, (4) AST12h group, (5) AA3h group, (6) AA12h group, (7) G3h group, (8) G12h group, (9) AST-G3h group, (10) AST-G12h group, (11) AA-G3h group, and (12) AA-G12h group. Alexa 488 green were used as secondary antibodies.</p

Study groups and experimental design.

<p>Unstimulated control groups (Control group): Incubated in M199 medium alone for 3 h (C3h group) or 12 h (C12h group). G groups: Incubated in M199 medium with 140 mM glucose for 3 h (G3h group) or 12 h (G12h group). Ascorbic acid (AA; AA group.): Incubated in M199 medium with 140 mM glucose and 100 μM AA for 3 h (AA-G3h group) or 12 h (AA-G12h group). AST pre-treated groups (AST group): Incubated in M199 medium alone for 3 h (AST3h group) or 12 h (AST12h group) after incubated in M199 medium with 5μM AST for 24h. AST-G group: Incubated in M199 medium with 140 mM glucose for 3 h (AST-glucose [AST-G] 3h group) or for 12 h (AST-G12h group) after incubated in M199 medium with 5μM AST for 24h.</p

Effect of high glucose stimulation, AST and AA to TSMCs as for EMT.

<p>(A) E-cadherin mRNA and protein expression levels in each groups. (B) α-SMA mRNA and protein expression levels in each group. :p < 0.05. **: p < 0.01. ***: p < 0.0005. ****: p < 0.0001. Error bars represent SD. (C) Effect of high glucose stimulation, AST, and AA to TSMCs as for EMT in double immunofluorescence. (1) C3h group, (2) C12h group, (3) AST3h group, (4) AST12h group, (5) AA3h group, (6) AA12h group, (7) G3h group, (8) G12h group, (9) AST-G3h group, (10) AST-G12h group, (11) AA-G3h group, and (12) AA-G12h group. Alexa 488 green (E-cadherin) and Alexa 555 red (α-SMA) were used as secondary antibodies. DAPI was used for nuclear staining.</p