Fig 1: Demonstration of examined myocardial markers modulating redox state, TBARS (A), Keap1 (B), SOD1 (C), and SOD2 (D) in experimental rats. There were no changes in TBARS among the groups. The protein level of Keap1 was lower in the spontaneously hypertensive rat (S) compared to Wistar rat (W) hearts and reduced in response to ISO in W rats (Wi) while not in S (Si). Treatment with melatonin and omega-3 did not affect this parameter regardless of the strain (Wim, Wio, Sim, Sio). Protein levels of SOD1 and SOD2 were lower in S compared to W rat hearts. ISO exposure resulted in suppression of SOD isoforms in Wistar rats (Wi) but not in S (Si). There was a tendency to increase SOD1 and SOD2 in both ISO-exposed rat strains by treatment either with melatonin or omega-3. Values are the mean ± SD of 10 rats in each group. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio.
Fig 2: The effects of calcitriol (0.5 µg/kg) on Nrf2 signaling after TBI. a Representative images of Western blot staining for Keap1, cytoplasmic Nrf2, and nuclear Nrf2. b Statistical graphs of Keap1 protein expression and Nrf2 translocation. c, d Representative immunofluorescence images and statistical graphs of p62 and Keap1 co-expression (Bar = 50 µm). e, f Representative immunofluorescence images and statistical graphs of Nrf2 translocation (Bar = 50 µm). g The relative mRNA expression level of Nqo1. h The relative mRNA expression level of Gclc. i The relative mRNA expression level of HO1. Data are presented as means ± SEM (n = 5). *P < 0.05 and **P < 0.01 versus the indicated groups. (Sham: sham-operated group; TBI: TBI model group; Calcitriol: TBI + calcitriol treatment group)
Fig 3: Tomentosin exerts antioxidant activity through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. (A) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of tomentosin. DPPH (0.15 mM) was allowed to react with tomentosin and vitamin C. The data are shown as the mean ± SD of triplicates. (B) HaCaT cells were transfected with an antioxidant response element- (ARE-) luciferase reporter and incubated for 4 h. The cells were then incubated with the indicated concentrations of tomentosin for 24 h and subjected to a luciferase reporter assay. The data are presented as the mean ± SD of triplicates. TBHQ was introduced as a positive control. TBHQ, tert-Butyl hydroquinone. (C–F) HaCaT cells were incubated with 1, 5, or 10 µM tomentosin for 24 h. (C) Protein levels of NAD(P)H-quinone oxidoreductase 1 (NQO1), Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1) were analyzed by Western blot. ß-actin was introduced as the control for whole-cell lysates. (D) mRNA levels of HO-1 and NQO1 were analyzed by qRT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control. The results are shown as the mean ± SD of triplicates. ** p < 0.01 vs. untreated group. (E,F) Cells were analyzed by Western blotting (E) of nuclear and cytoplasmic fractions and by immunocytochemistry (F) to assess the level of Nrf2 nuclear translocation. a-tubulin and lamin B1 were introduced as the controls for cytoplasmic and nuclear protein extracts, respectively. NE, nuclear extracts; CE, cytosolic extracts. Scale bar = 50 µm.
Fig 4: Catalpol activated Keap1/Nrf2/ARE pathway in H2O2-treated RPE cells. (a and b) ARPE-19 cells were pretreated with or without catalpol (10, 20 and 40 µM) for 24 h, and then treated with 400 µM H2O2 for 6 h. The expressions of HO-1, NQO1, Keap1, Total-Nrf2 and Nuclear-Nrf2 were measured with Western blot assay. Histone H3 was used as an internal control for nucleoprotein, while ß-actin was used as an internal control for total protein. (c) The effect of catalpol on the formation of Keap1/Nrf2 complex was determined using co-immunoprecipitation assay. (d) Treatment with ML385, a Nrf2 inhibitor, blocked the antioxidant protective effects of catalpol on increases in the expression of Nrf2 protein in H2O2-treated ARPE-19 cells. (e) Changes in gene expression of NQO1 were assayed with qRT-PCR. Data are presented as mean ± S.D. of three independent experiments (* p < 0.05 vs. control group, # p < 0.05 vs. H2O2 (400 µM)-treated group).
Fig 5: The molecular mechanisms underlying the neuroprotective effects of calcitriol against TBI. Calcitriol maintains redox balance and protects the brain from oxidative damage through Nrf2 signaling mediated by the autophagic degradation of Keap1
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