PPARα Agonist, MHY3200, Alleviates Renal Inflammation during Aging via Regulating ROS/Akt/FoxO1 Signaling

Abstract

PPARα is a ligand-dependent transcription factor and its activation is known to play an important role in cell defense through anti-inflammatory and antioxidant effects. MHY3200 (2-[4-(5-chlorobenzo[d]thiazol-2-yl)phenoxy]-2,2-difluoroacetic acid), a novel benzothiazole-derived peroxisome proliferator-activated receptor α (PPARα) agonist, is a synthesized PPARα activator. This study examined the beneficial effects of MHY3200 on age-associated alterations in reactive oxygen species (ROS)/Akt/forkhead box (FoxO) 1 signaling in rat kidneys. Young (7-month-old) and old (22-month-old) rats were treated with MHY3200 (1 mg/kg body weight/day or 3 mg/kg body weight/day) for two weeks. MHY3200 treatment led to a notable decrease in triglyceride and insulin levels in serum from old rats. The elevated kidney ROS level, serum insulin level, and Akt phosphorylation in old rats were reduced following MHY3200 treatment; moreover, FoxO1 phosphorylation increased. MHY3200 treatment led to the increased level of FoxO1 and its target gene, MnSOD. MHY3200 suppressed cyclooxygenase-2 expression by activating PPARα and inhibiting the activation of nuclear factor-κB (NF-κB) in the kidneys of old rats. Our results suggest that MHY3200 ameliorates age-associated renal inflammation by regulating NF-κB and FoxO1 via ROS/Akt signaling.

Keywords: Akt; MHY3200; NF-κB; ROS; aging; inflammation.

Figure 1

Biochemical features of aging. MHY3200 (1 mg/kg/day) or (3 mg/kg/day) was orally administered for 14 days in old rats. (A) triglyceride, (B) total cholesterol, (C) glucose, (D) insulin concentration in serum was quantified using the colorimetric kit. Young (6-month-old) rats, Young; Old (22-month-old) rats, Old. One-way analysis of variance (ANOVA) was used to determine the statistical result: ^## p < 0.01, ^### p < 0.001 vs. Young rats; ^** p < 0.01, ^*** p < 0.001 vs. Old rats. Young (-), non-treated young rats; Old (-), non-treated old rats; 1, MHY3200 (1mg/kg)-treated old rats; 3, MHY3200 (3mg/kg)-treated old rats.

Figure 2

Effect of MHY3200 on NOX4/Akt signaling during the aging process. (A) Western blot analysis was performed to detect NOX4, phosphorylated Akt, and total-Akt in cytoplasmic extracts from kidneys. (B) ROS production was measured in the kidneys of young and aged rats using DCFH-DA assay. (C) Western blot analysis was performed to detect the phosphorylation of FoxO1 serine256 residue and total FoxO1 in the nuclear fraction. (D) Levels of MnSOD protein in the cytoplasmic extracts from rat kidneys. TFIIB and β-actin were used as nuclear and cytosolic internal controls and graphs were quantified with CS analyzer image analysis software. One representative blot is shown from three independent experiments in each group that yielded similar results (n = 6). One-way analysis of variance (ANOVA) was used to determine the statistical result: ^## p < 0.01, ^### p < 0.001 vs. Young rats; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. Old rats. Young (-), non-treated young rats; Old (-), non-treated old rats; 1, MHY3200 (1mg/kg)-treated old rats; 3, MHY3200 (3mg/kg)-treated old rats.

Figure 3

Effect of MHY3200 on NF-κB and its downstream inflammatory signaling during aging. (A) Nuclear fraction NF-κB and (B) cytosol fraction COX-2 protein levels were determined using Western blot analysis. TFIIB and β-actin were used as the nuclear and cytosolic internal controls and graphs were quantified with CS analyzer image analysis software. One representative blot is shown from three independent experiments in each group that yielded similar results (n = 6). One-way analysis of variance (ANOVA) was used to determine the statistical result: ^## p < 0.01 vs. Young rats; ^** p < 0.01, ^*** p < 0.001 vs. Old rats. Young (-), non-treated young rats; Old (-), non-treated old rats; 1, MHY3200 (1mg/kg)-treated old rats; 3, MHY3200 (3mg/kg)-treated old rats.

Figure 4

Binding affinity between MHY3200 and PPARα and its effects on PPARα activity during aging. Docking simulation was performed to identify the interaction between MHY3200 and PPARα and the ligand binding affinity was assessed. (A) MHY3200 has similar binding affinity to the known PPARα agonist, WY14643. (B) MHY3200 interacts with PPARα via multiple interactions involving several residues, including a single hydrogen bond (shown in the red square). (C) The control compound WY14643 also interacts with PPARα via multiple residues but does not form a hydrogen bond. (D) Levels of PPARα protein in the nucleus. TFIIB was used as an internal nuclear control and graphs were quantified with CS analyzer image analysis software. One representative blot is shown from three independent experiments in each group that yielded similar results (n = 6). One-way analysis of variance (ANOVA) was used to determine the statistical result: ^# p < 0.05 vs. Young rats; ^* p < 0.05 vs. Old rats. Young (-), non-treated young rats; Old (-), non-treated old rats; 1, MHY3200 (1mg/kg)-treated old rats; 3, MHY3200 (3mg/kg)-treated old rats.

Figure 5

Possible role of MHY3200 in modulating ROS/Akt/FoxO1 signaling in kidney tissues of aged rats. MHY3200 ameliorated age-related inflammatory response by inhibiting the proinflammatory NF-κB activity regulated by ROS/Akt/FoxO1 signaling.