Fig 2: Antioxidant effect of andrographolide via Nrf2 activation in MPP+-induced SH-SY5Y cells. (A) The immunoblots of Nrf2 in nuclear and cytoplasmic fractions, KEAP1 and HO-1. (B) Quantification of band intensity normalized by β-actin. (C) The antioxidant mRNA levels of SOD1, CAT, GSTP1, and HO-1 were evaluated by RT-qPCR assay. SH-SY5Y cells were incubated with or without 1.5 µM of Andro or 2 mM NAC for 12 h, followed by incubation of MPP+ 1.5 mM for 8 h. (D) The antioxidant activities of SOD and GPx enzymes in SH-SY5Y cells treated with or without 1.5 µM of Andro or 2 mM of NAC for 24 h prior to incubated with 1.5 mM of MPP+ for 16 h. * p < 0.05, ** p < 0.01, **** p < 0.0001-statistical significance versus MPP+-treated group. # p < 0.05, ## p < 0.01, ### p < 0.001-statistical significance versus control group (data are mean ± SD, n = 3).

Fig 3: Analysis of the oxidative stress response. The activities of GLXR and GPX were compared in mice hearts (A,B). The ratio of GSSG/GSH was measured in hearts (C). Immunoblot analysis of protein s-glutathionylation is shown (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 5) (D). The columns represent the average ratios of relative protein s-glutathionylation in the control and irradiated samples after background correction and normalisation to VDAC expression (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 3) (E). The amount of lipid peroxidation as a marker of oxidative stress was measured in mice hearts using ELISA (one-way ANOVA and Tukey test for multiple comparisons; * adjusted p value < 0.05; n = 5) (F). The error bars represent the standard error of the mean (±SEM).

Fig 4: GPx1 knockout increases susceptibility to H2O2-induced cytotoxicity.Healthy control monocyte-derived macrophages were transfected with siRNA against GPx1 for 48 h and efficiency of knockdown (KD) was assessed by qPCR (A) and immunoblotting (B) (nsSiRNA, non-specific control SiRNA). GPx1 KD macrophages were treated with H2O2 (500 μM, 2 h) and cell death (C), lipid peroxidation (D; 50 cells per donor were analyzed from 3 healthy) and qPCR of ferroptosis markers (E) was assessed. Data are means ± SEM. Dashed lines represent the media and dotted lines the quartiles (*p < 0.05, **p < 0.005).

Fig 5: Superoxide dismutase (SOD) (a), glutathione peroxidase (GPx) (b), and malondialdehyde (MDA) (c) levels after treatment with ASX (20 μM), CPF (400 μM), pretreatment (ASX for 6 h followed by CPF for 24 h), and co-treatment (ASX + CPF for 24 h) in A549 cells. SOD (U/mg protein) and GPx (U/mg protein) activities were measured as antioxidant defense markers, while MDA levels (mmol/mg) were assessed as an indicator of lipid peroxidation and oxidative damage. Values represent means ± SD of at least three experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Bars indicated by different letters show significant differences: (CPF compared to the control; p = 0.0026 for SOD; p = 0.00285 for GPx; p = 0.0019 for MDA) (pretreatment compared to the CPF; p = 0.0048 for SOD, p = 0.00463 for GPX, p = 0.0021 for MDA) (co-treatment compared to the CPF; p = 0.0082 for SOD, p = 0.00782 for GPx, p = 0.0052 for MDA). ASX: Astaxanthin, CPF: Chlorpyrifos.