Fig 1: Effect of Crif1-knockdown on NRF2 protein level and nuclear translocation in BMMSCs after irradiation. (a) Western blot results of NRF2 in Crif1-deficient BMMSCs (Crif1-SI) and control cells (Con-EV). The representative immunoblots (upper panel) and relative levels of proteins normalized to ß-actin (lower panel) are shown. BMMSCs transfected with Crif1 shRNA are referred as Crif1-SI, and BMMSCs transfected with empty control vectors are referred as Con-EV. (b) qPCR analysis of Nrf2 in Crif1-deficient BMMSCs (Crif1-SI) and control cells (Con-EV). (c) Western blot analysis of NRF2 expression in irradiated Crif1-knockdown (Crif1-SI) and control BMMSCs (Con-EV) at the indicated time points after an irradiation dose of 9 Gy. The representative immunoblots (upper panel) and relative levels of proteins normalized to ß-actin (lower panel) are shown. (d) Cytoplasmic and nuclear fraction proteins CRIF1 and NRF2 in Crif1-deficient BMMSCs (Crif1-SI) and control cells (Con-EV) 8 h after irradiation were detected by western blot. The representative immunoblots (upper panel) and relative levels of proteins normalized to ß-actin (lower panel) are shown. (e) Immunofluorescence analysis of CRIF1 and NRF2 in Crif1-deficient BMMSCs (Crif1-SI) and control cells (Con-EV) 8h after irradiation. (f) Western blot of target proteins of NRF2 in Crif1-deficent BMMSCs (Crif1-SI) and control cells (Con-EV) at the indicated time points after an irradiation dose of 9 Gy. The representative immunoblots (upper panel) and relative levels of proteins normalized to ß-actin (lower panel) are shown. (g) qPCR results of NRF2 target genes (Ho1, Ggt1, and Gclc) in Crif1-deficent BMMSCs (Crif1-SI) and control cells (Con-EV) at the indicated time points after an irradiation dose of 9 Gy. All experiments were performed in triplicate in the same cell line, and representative images are shown. Data represent the mean ± standard error of mean (SEM). *p< 0.05, **p< 0.01, ns (not significant), compared to the control
Fig 2: Immunohistochemistry (IHC) and western blot assays of HPL-treated samples. (A) IHC revealed a disturbed morphology of uterine and renal tissues (DAB staining, scale bar, 200 µm). (B) The IHC average optional density (AOD) ratio. (C) Representative images of ERa, ERß, Ggt1, and Anpep levels detected using western blotting. (D) Representative quantitation of ERa, ERß, Ggt1, and Anpep levels detected using western blotting. (E) qRT-PCR analysis of renal levels of the Ggt1 and Anpep mRNAs. The results of the statistical analysis are presented in bar charts (mean ± s. e.m.). HPL therapy dosage: HPL-H, 300 mg/kg/d; HPL-L, 80 mg/kg/d. *p < 0.05, **p < 0.005, and ***p < 0.0005 compared with control rats; #p < 0.05, ##p < 0.005, and ###p < 0.0005 compared with OVX rats. NS, not significant.
Fig 3: The therapeutic “big picture” of “HPL-disease-gene-metabolite” networks modulated by the HPL treatment in rats with OVX-induced glutathione redox stress. OVX-induced glutathione redox stress triggered abnormalities in pathways that are presented as black solid arrows that link the main glutathione metabolism pathway, and black dotted arrows indicate indirect catalytic reactions. Significant upregulation of Ggt1 and Anpep is represented by pink and blue irregular shapes, respectively. The increased and decreased levels of quantified metabolites are represented by pink and green hexagons, respectively. Other metabolites are represented by yellow hexagons. The effects of HPL therapy on reversing the increased levels of metabolites are represented by dotted green lines, while the effects on restoring the decreased levels of metabolites are represented by solid green arrows.
Fig 4: HPL therapy reverses OVX-induced endometabolite imbalances in the glutathione metabolism pathway. (A) PLS analysis of the metabolomic profiles of urine and serum samples; the differences among control (blue dot), OVX (red square), HPH (HPL-H, green triangle), and HPL (HPL-L, yellow downward triangle) samples are shown. Each symbol on the 2D score plot represents a result for an individual. (B) Gene-metabolite KEGG pathway enrichment plot of the biomarkers in each pathway; the bar length (blue: enrichment and orange: topology) represents the significance levels of the pathways. (C) Redox GSH/GSSG ratio and relative peptide quantification for Ggt1 and Anpep. The data are representative of three experiments. The data are pooled from three experiments (n = 3, mean ± s. e.m.). (D) and (E) Quantification of metabolites from glutathione metabolism in 3, 6, and 12 weeks urine samples and 12 weeks serum samples using a liquid chromatography–tandem mass spectrometry analysis. The data were pooled from three experiments (n = 9, means ± s.d.). HPL therapy dosage: HP30, 30 mg/kg/d; HP10, 10 mg/kg/d. *p < 0.05, **p < 0.005, and ***p < 0.0005 compared with control rats; #p < 0.05, ##p < 0.005, and ###p < 0.0005 compared with OVX rats. NS, not significant.
Fig 5: Protein levels and nuclear translocation of NRF2 and CRIF1 after irradiation (a) CRIF1 and NRF2 protein expression was assessed by western blot in BMMSCs at the indicated time points after an irradiation dose of 9 Gy. The representative immunoblots (upper panel) and relative levels of proteins normalized to ß-actin (lower panel) are shown. (b) Western blot results of target proteins of NRF2 (GCLC and GGT1) in BMMSCs at the indicated time points after irradiation. The representative immunoblots (upper panel) and relative levels of proteins normalized to ß-actin (lower panel) are shown. (c) qPCR results of Gclc and Ggt1 in BMMSCs at the indicated time points after irradiation. (d) The cytoplasmic and nuclear proteins were detected at the indicated time points in irradiated BMMSCs. The representative immunoblots (upper panel) and relative levels of cytoplasmic (or nuclear) proteins normalized to ß-actin (or histone H3) (lower panel) are shown. H3 was used as nuclear protein-loading control. (e) Immunofluorescence analysis of CRIF1 and NRF2 8 h after irradiation. All experiments were performed in triplicate in the same cell line, and representative images are shown. Data represent the mean ± SEM. *p< 0.05, **p< 0.01, ns (not significant), compared to the control
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